FOCIS' Translational Immunology Update Compliation

TRANSLATIONAL IMMUNOLOGY UPDATE Volume 4, Issue 1 Editors: Abul K. Abbas, Carla J. Greenbaum, Andrew H. Lichtman

HIGHLIGHTS FROM RECENT LITERATURE CD8 T CELL AVIDITY IS DEPENDENT ON T REGULATORY CELLS Research paper: Pace L., et al. Regulatory T Cells Increase the Avidity of Primary CD8+ T Cell Responses and Promote Memory. Science 338; 532-36, 2012. PMID: 23112334 T regulatory cells (Tregs) are necessary to prevent aberrant immune activation that can lead to autoimmunity. Interestingly however, Tregs do not appear to prevent immune activation in response to pathogens. In fact, in two separate reports, Tregs have been shown to contribute to the clearance of L. major and herpes simplex virus (Belkaid et al., 2002; Lund et al., 2008). The precise mechanisms as to how Tregs contribute to the priming of the immune response remain unclear. Here, Pace et al. utilize FoxP3 DTR mice, in which FoxP3+ cells express the human diphtheria toxin receptor, to eliminate Tregs via an injection of diphtheria toxin. They administered the toxin before administering a foreign antigen which activates CD8 T cells, and employed MHC-multimers to measure the frequency of antigen reactive T cells during the response. Their key findings were: • •

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A low percentage of high affinity Db-Uty (male) specific CD8+ T cells were replaced by a relatively high percentage of low affinity Db-Uty specific CD8+ T cells upon depletion of Tregs from female recipients that received an i.p. injection of male splenocytes.

Under conditions more similar to a physiological infection, high multimer-binding Ova-antigen specific OT-I cells exist at a higher frequency following infection with Listeria monocytogenes that express ova (LM-OVA). However, when Tregs are depleted prior to infection, the low multimer-binding OT-I cells increase in frequency.

When Tregs are depleted from mice at the time of infection with LM-OVA, and mice are rechallenged with LM-OVA 50 days later, there are significantly more bacteria in the spleen and liver as well as a lower frequency of antigen specific CD8 T cells, suggesting a diminished memory response to LM-OVA in the absence of Tregs.

Taken together, these results paradoxically highlight the importance for Tregs in the immune response to non-self antigens and also in the formation of memory to pathogens. Not only do lowaffinity clones expand in the absence of Tregs, but the avidity of the response also decreases, highlighting the importance of Tregs in promoting efficient pathogen clearance. This study also presents another potential mechanism by which Tregs might prevent autoimmunity by inhibiting the accumulation of the low-affinity and potential self-crossreactive CD8 T cell clones that arise in the periphery due to constant exposure to self-antigen.

Reviewed by Christopher Borges and Laurence Turka, MD, Beth Israel Deaconess Medical Center and Harvard Medical School.

BACTERIAL FLAGELLIN PRESENT IN HOUSE DUST EXTRACTS (HDES) PROMOTE ASTHMA Research Paper: Wilson R.H., et al. The Toll-like Receptor 5 Ligand Flagellin Promotes Asthma by Priming Allergic Responses to Indoor Allergens. Nature Medicine 181705; 1705-11, 2012.

PMID: 23064463 Regulatory T cells (Tregs) are critical for suppressing harmful autoimmune reactions. In chronic inflammatory diseases such as multiple sclerosis, Tregs are often present but are incapable of suppressing the activity of pathogenic effector T cells. The authors studied the mechanism of Tregresistance in patients with multiple sclerosis and found that it was critically dependent on IL-6 signaling.

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Using assays of T cell suppression, the authors demonstrated that effector T cells in patients with ongoing flares of relapsing-remitting multiple sclerosis (RRMS) were resistant to Tregmediated suppression. Effector T cells from RRMS patients in remission and from normal individuals were suppressed normally by Tregs.

Previous studies have shown that IL-6 signaling and phosphorylation of STAT3 in effector T cells can contribute to effector T cell resistance to suppression.

Effector T cells in RRMS patients with active disease showed increased IL-6 receptor alpha expression and elevated IL-6 signaling as shown by increased levels of phosphorylated STAT3 (pSTAT3), as compared to patients with quiescent disease and healthy controls.

Increased levels of pSTAT3 and IL-6 receptor expression correlated with resistance to suppression in in vitro suppression assays.

Blockade of STAT3 phosphorylation in vitro in effector T cells restored the ability of these cells to be suppressed by Tregs.

Active and effective regulatory T cell function is critical for maintaining self tolerance. The authors find that effector T cells become resistant to Treg suppression during flares of MS and that this resistance is likely mediated by IL-6 signaling. These studies suggest that inhibiting IL-6 signaling in effector T cells may have the potential to enhance tolerance, suppress disease flares and slow disease progression in multiple sclerosis and possibly other autoimmune diseases as well.

Reviewed by Christopher Borges and Laurence Turka, MD, Beth Israel Deaconess Medical Center and Harvard Medical School.

DOUBLE TROUBLE: IL-17 PRODUCING TREGS PROMOTE COLON CANCER AND IMPAIR IMMUNE RESPONSES Research Paper: Blatner, N.R., et al. Human Colon Cancer Expression of RORγt Marks a Pathogenic Regulatory T Cell Subset in Human Colon Cancer. Science Translational Medicine 4, 164ra159 (2012). PMID: 23241743 Immunosuppressive regulatory T cells (Tregs) play critical roles in suppressing autoimmunity.Tregs are expanded in and/or recruited to most human cancers, where they either suppress anti-cancer immunity or, paradoxically, protect the host by suppressing pro-carcinogenic inflammation. By studying colon cancer in humans and in a mouse models, the authors found that a subset of RORγt expressing Tregs promoted colon cancer while at the same time suppressing antitumor immunity.

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Human colon cancers contained a distinct population of RORγt -expressing Tregs that retained suppressive capacities and yet appeared to be pro-inflammatory. These Tregs were not observed in normal donors.

Tregs with similar characteristics were present in the tumors of mouse models of hereditary polyposis. Ablation of the RORγt gene in FOXP3+ Tregs restored the anti-inflammatory effects of these cells, suppressed inflammation, improved anti-polyp immune responses and reduced the number of pre-cancerous polyps in these mice.

Deficiency of the IL-17A gene in these mice reduced the formation of precancerous polyps but these mice still had RORγt -expressing Tregs. Although fewer polyps were present, the remaining precancerous lesions were more likely to progress to invasive colon cancer in the absence of IL-17. This suggests IL-17 promotes polyp formation but may protect against progression to invasive cancer.

The authors observed that distinct Tregs subsets are present in human colon cancer with proinflammatory and anti-inflammatory phenotypes and functions. RORγt -expressing Tregs were proinflammatory, supported the development of pre-cancerous polyps, and reduced anti-polyp immune responses. IL-17 produced by these Tregs increased the formation of precancerous polyps but paradoxically impaired progression to invasive cancer. In summary, the authors find Tregs within cancer can be surprisingly diverse and that some of Tregs can directly promote carcinogenesis as well as suppressing anticancer responses.

Reviewed by Rachael A. Clark, MD, PhD, Department of Dermatology, Brigham and Women's Hospital.

IL-6 MEDIATES EFFECTOR T CELL RESISTANCE TO TREGS IN MULTIPLE SCLEROSIS Research Paper: Schneider A.,et al. In Active Relapsing-remitting Multiple Sclerosis, Effector T Cell Resistance to Adaptive Tregs Involves IL-6–mediated Signaling. Science Translational Medicine 5, 170ra15 (2013).

PMID: 23363979 Regulatory T cells (Tregs) are critical for suppressing harmful autoimmune reactions. In chronic inflammatory diseases such as multiple sclerosis, Tregs are often present but are incapable of suppressing the activity of pathogenic effector T cells. The authors studied the mechanism of Tregresistance in patients with multiple sclerosis and found that it was critically dependent on IL-6 signaling.

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Using assays of T cell suppression, the authors demonstrated that effector T cells in patients with ongoing flares of relapsing-remitting multiple sclerosis (RRMS) were resistant to Tregmediated suppression. Effector T cells from RRMS patients in remission and from normal individuals were suppressed normally by Tregs.

Previous studies have shown that IL-6 signaling and phosphorylation of STAT3 in effector T cells can contribute to effector T cell resistance to suppression.

Effector T cells in RRMS patients with active disease showed increased IL-6 receptor alpha expression and elevated IL-6 signaling as shown by increased levels of phosphorylated STAT3 (pSTAT3), as compared to patients with quiescent disease and healthy controls.

Increased levels of pSTAT3 and IL-6 receptor expression correlated with resistance to suppression in in vitro suppression assays.

Blockade of STAT3 phosphorylation in vitro in effector T cells restored the ability of these cells to be suppressed by Tregs.

Active and effective regulatory T cell function is critical for maintaining self tolerance. The authors find that effector T cells become resistant to Treg suppression during flares of MS and that this resistance is likely mediated by IL-6 signaling. These studies suggest that inhibiting IL-6 signaling in effector T cells may have the potential to enhance tolerance, suppress disease flares and slow disease progression in multiple sclerosis and possibly other autoimmune diseases as well.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital.

IMMUNE CROSSTALK AND OFF TARGET IMMUNITY Research Paper: Su, et al. Virus Specific CD4+ Memory-Phenotype T Cells are Abundant in Unexposed Adults.

Immunity (2013) S1074-7613(13). PMID: 23395677 In order to train the immune system to fend off pathogens, we design antigen specific vaccines to stimulate the activation of naïve B and T cells in order to create a pool of memory B and T cells that will remain at the ready to protect against any attempt by that pathogen to infiltrate our physical and biological defenses. However, there has been recent mouse model work showing that there can be memory-phenotype T cells in a host in the absence of exposure to the relevant antigens. In this paper, Su et al investigate the relationship between antigen exposure and memory cell populations in human T cell populations. Interestingly, they were able to delve deeper into the role of non-cognate antigen exposure by comparing rates of antigen specific memory T cells in the absence of viral exposure in adults and neonates (using cord blood). Potential mechanisms for the generation of antigen specific memory without evidence of viral infection are explored.

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Su et al. characterize antigen specific T cell frequencies for unexposed viral antigens and self antigens (implicated in oncologic and autoimmune processes) in 26 healthy adults using an enrichment technique with class II MHC tetramers bearing the selected peptide antigens. o They compared the frequency of anti-viral T cells in individuals who had and had not been exposed to a given antigen (for example, seropositive vs. negative for HSV) and found that antigen exposure did not always predict a larger population of antigen specific T cells. o Interestingly, there was also not a significant difference between the frequencies of antigen specific T cells that recognize self antigens versus non-exposed viral antigens. o In addition, they noted that anti-self antigen T cells were more frequent in females versus males, consistent with the known gender bias in autoimmunity incidence.

When they examined the nature of the antigen-specific T cells, they noted that many T cells that recognized unexposed viral antigens were in fact memory phenotype cells. o To show that these memory phenotype cells were functionally memory cells they assayed the ability of tetramer positive cells to produce cytokines more quickly than naïve and tested known gene expression profiles associated with naïve and memory T cells. Finally, there was evidence of increased clonality in the memory phenotype versus the naïve phenotype cells.

To examine the potential role of non-cognate activation, they compared adult samples to umbilical cord blood samples. While there were a similar frequency T cells specific for nonantigen exposed pathogen and self-antigen specific T cells they did not show similar frequency of memory phenotype cells at birth in comparison to the adults studied in the first part of the study.

The chemokine CCL22 was identified as a direct miR-34a target. Importantly, there was an inverse relationship between CCL22 and miR-34a expression in HBV+ HCC. CCL22 binds CCR4, a chemokine receptor expressed on regulatory T (Treg) cells. In an elegant cell culture system, inhibition of miR-34a in HCC cells resulted in increased CCL22

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and robust migration of Tregs. Importantly, a neutralizing CCL22 antibody blocked Treg migration.

To study the possible source of antigen specific memory phenotype T cells in the absence of relevant pathogen exposure in adults, they created 24 HIV specific T cell clones from three donors and then stimulated them with HIV-1 antigens and with similar peptide sequences from other microorganisms, finding that more than 20% of the clones produced cytokines in response to similar peptides from a wide range of bacteria, algae and plants. In other words, anti-HIV-1 T cells can be stimulated by activating the immune system with many, varied microorganisms in our environment.

Finally, they vaccinated two volunteers that had not had influenza vaccines in > 5 years, which resulted in influenza antibody titers and the development of cross-reactivity to other microorganisms.

This paper carefully investigates the nature of cross reactivity in human CD4 T cells. The demonstration that adults who have not been exposed to a given pathogen can have a significant population of antigen specific T cells with a memory phenotype, as well as the capacity for speedy functional responses and gene expression profiles consistent with memory cell populations is fascinating. The delineation of the mechanism of the generation of these cells is the most interesting question, coming down to the two most likely mechanisms—homeostatic proliferation versus exposure to environmental crossreactive antigens. With many groups focusing on the details of human immunology, with an upsurge in focus at the single cell level as well as on comparisons between mouse model data and human investigations, data is being accumulated at an incredible rate. This paper investigates the correlates in human T cells of intriguing results in mouse data. Here, while adults have antigen specific populations of memory phenotype T cells in the absence of exposure to the specific pathogens, the same antigen-specific T cells (in the absence of relevant antigen exposure) in newborns do not have memory phenotypes, which suggests a role of a lifetime of exposure to infectious and environmental antigens in the repertoire of both antigen-specific and crossreactive T cells. In addition, T cells enriched based on a specific viral antigen specificity are shown to have TCRs with the capacity to bind peptides from a wide variety of organisms. Finally, when healthy humans were vaccinated with influenza vaccine, they developed a population of T cells whose TCRs could bind peptides from other microorganisms. Of course, there are always questions that remain to be answered—does the ability of a TCR to bind to related peptides in an MHC complex prove that the hosts can effectively defend themselves against those pathogens? If so, is the ability to generate T cells that can respond to pathogens to which the host has not been exposed of benefit to humans (in that we are protected against pathogens we have not experienced) or is it dangerous (in that we are now likely in possession of T cells that recognize both pathogens we have conquered as well as possibly self antigens we’d rather remain tolerant towards) or does the balance of those considerations come out even, at least at the population level? Given the importance of these questions and the diversity of human repertoires, it will require large cohorts to investigate these questions, with a focus on the tantalizing possible “off target” effects of both vaccination and infectious disease with the possible long term correlations including protection from infectious disease, the development of autoimmunity and, possibly, even oncologic transformation.

Reviewed by Sarah Henrickson, MD, PhD Boston Children’s Hospital and Harvard Medical School.

OF MOUSE AND MAN Research Paper: Seok, et al. Genomic Responses in Mouse Models Poorly Mimic Human Inflammatory Diseases.

Proceedings of the National Academy Sciences. 2013. 110:3507-12. PMID: 23401516 While mice are arguably the key animal model in immunology, elucidating links between murine and human physiology and pathology remains a topic of active investigation, especially given the importance of animal model data in clinical trials and in choosing candidates and questions for logistically complex human studies. While many differences in the two systems have been noted, these could be argued to result from differences in both underlying biology and/or differences in the experimental paradigms (in many cases, likely both could be implicated). This paper takes on the task of comparing inflammatory conditions between murine models and human disease, finding many differences. •

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Seok et al aimed to compare murine and human responses to various systemic inflammatory conditions, including trauma, burn injury and endotoxin exposure by studying gene expression from PBMCs. They showed impressive similarity between human burn and trauma, with some similarity of both to human endotoxin exposure. In comparison, there were poor correlations between gene expression of orthologs in human and murine models of a given condition, and poor correlations among the three murine models.

Seok et al also compared the timing of transcriptional changes across human and murine clinical scenarios. Perhaps as expected, given the short duration of murine lifespans, while the initial post-inflammatory transcriptional changes occurred on similar timescales between human and murine systems, the duration of recovery was months in humans and hours to days in mice.

In addition, Seok et al investigated the pathways and individual genes underlying these inflammatory models (human and murine), comparing each to human burns. In each case, human injury more closely resembled human burns than any mouse model, including murine burns.

As a community, it is fundamental to our use of murine models in our pursuit of deeper understanding of human disease that we can find significant parallels in physiology, pathophysiology and response to therapeutic interventions between humans and the model system of choice. It is intriguing that they have investigated the details of complex, inflammatory conditions in this study, rather than simply focusing on cells at rest. However, while this study is fascinating, it is important to note, as the authors have, that in comparing disease states, there are not only differences in the inherent biology of the organisms (both at a species level and the amount of difference seen between individuals from inbred mouse strains and humans) but also fundamental differences between a critically ill human and an induced model of critical illness in a mouse. This study carefully focuses on some of the key differences, including the appropriate timepoints to choose for analysis, given the differences in lifespan of the two organisms. In addition, the samples studied are unsorted PBMCs, which does not allow consideration of differences between similar cell types in the two species. Overall, this study provides an important window into the key assumption made by many immunologists that mice and humans can be considered nearly interchangeable from an immunological perspective. The focus within the work on the differences between human disease and murine models of inflammation will hopefully stimulate a robust discussion around how to choose and validate models, both for basic research and clinical trials.

Of note, a related study came out last month as well, and in Shay et al (PNAS (2013) 110: 29462951), there is a demonstration of a highly detailed comparison of two compendia of transcriptional studies of sorted mouse and human cell lineages, during both resting and activated states with

evidence of both shared gene transcription programs as well as tantalizing differences between orthologs. Clearly, there will need to be further studies to follow up on this and prior work that will help us continue to improve our understanding of the benefits and limitations of mice as models for understanding the physiology and pathophysiology of the human immune system.

Reviewed by Sarah Henrickson, MD, PhD Boston Children’s Hospital and Harvard Medical School.

REGULATORY B CELLS MAY FACILITATE CHRONIC JOINT INFLAMMATION IN RHEUMATOID ARTHRITIS Research Paper: Flores-Borja F., Bosma A., Ng D., Reddy V., Ehrenstein M.R., Isenberg D.A., Mauri C. CD19+CD24HICD38HI B Cells Maintain Regulatory T Cells While Limiting TH1 and TH17 Differentiation.

Science Translational Medicine. 2013 Feb 20;5(173):173ra23. PMID: 23427243 Regulatory B cells (Bregs) are a small subset of CD19+CD24HICD38HI B cells that constitute approximately 1% of the peripheral blood leukocyte population. They are thought to regulate T cell function via the production of IL-10 and the engagement of costimulatory molecules, which results in inhibition of interferon-γ (IFNγ) and tumor necrosis factor-γ (TNFγ) by effector T cells. They may also promote the generation of induced regulatory T cells (Tregs). While they have been shown to inhibit autoimmune disease in mouse models, their role in human autoimmune disease is less clearly established. In the manuscript by Flores-Borja, Mauri and colleagues, the authors test the hypothesis that defective Breg function might mediate the defect in Tregs observed in rheumatoid arthritis (RA) patients while permitting the generation of proinflammatory T helper 1 (TH1) and TH17 cells. They compare the frequency and function of B-regs obtained from the peripheral blood of healthy controls and RA patients with active or quiescent disease. There are several interesting findings from this work: • • •

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B-regs from healthy individuals limited differentiation of naïve CD4 T cells into TH1 and TH17 cells. They also promoted the generation of CD4+FoxP3+ Tregs from naïve T cells.

B-regs obtained from RA patients with active disease were able to inhibit TH1 differentiation as measured by production of IFNγ and TNFα. However, they failed to prevent TH17 development and were not able to promote differentiation of effector CD4+ T cells into functional Tregs.

Importantly, co-culture of B-regs from healthy controls with RA patient T cells demonstrated that effector T cells from RA patients were not intrinsically refractory to differentiation into TH17 or Tregs with suppressor function. In contrast, RA patient Bregs could not induce the differentiation of Tregs from healthy control naïve CD4+ T cells.

The number of Bregs was significantly lower in the blood of patients with active RA relative to healthy controls and was inversely correlated with C reactive protein (CRP) levels, a marker of disease activity. Interestingly, serial monitoring of RA patients demonstrated that Breg numbers normalized when disease was under control and inactive in RA patients and decreased with disease flares.

Breg number in RA patients did not correlate with the age of the patient or treatment regimen.

Breg numbers in patients with other autoimmune diseases including psoriatic arthritis, Sjogren’s syndrome, and ankylosing spondylitis were similar to healthy controls, suggesting that dysregulation of B-regs is a feature of active RA.

Historically, T cell dysfunction has been thought to play a central role in the pathogenesis of RA. This article is interesting because it highlights a central role for Bregs in RA. The authors postulate that a decrease in Bregs during active disease results in an inability to induce Treg differentiation, thereby enabling the establishment of TH17 cell differentiation and a proinflammatory environment. This

raises an important question: What regulates the differentiation and maintenance of Breg numbers in RA patients? It is interesting that the decrease in Breg numbers paralleled disease activity and appeared to not be influenced by the treatment regimen (although the study may not have been appropriately powered to firmly establish this). Addressing this issue will be important as the data to date suggest that enhancing Breg numbers and function could improve the control of RA. Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco.

IDENTIFICATION OF ‘PSEUDOEXHAUSTED’ T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) Research Paper: Riches J.C., Davies J.K., McClanahan F., Fatah R., Iqbal S., Agrawal S., Ramsay A.G., Gribben J.G. T cells from CLL Patients Exhibit Features of T Cell Exhaustion but Retain Capacity for Cytokine Production. Blood. 2012 Dec 17. [Epub ahead of print] PMID: 23247726 T cell exhaustion is an acquired state of T-cell dysfunction characterized by increased expression of inhibitory receptors such as programmed death-1 (PD1), CD160, and CD244 and poor effector function including decreased proliferative capacity, impaired cytolytic function, and reduced cytokine production. T cell exhaustion is believed to play a critical role in the optimal immune control of chronic viral infections and tumor cells. It has been shown to be a poor prognostic indicator in several malignancies. Consistent with this, inhibiting PD1 in human melanoma patients can reverse T cell exhaustion resulting in significant tumor responses in a subset of patients. While it has long been appreciated that CLL patients often have profound defects in T-cell function and increased susceptibility to infections, the mechanisms underlying this dysfunction are poorly understood. In this report, Riches and colleagues explore the role of T cell exhaustion in chronic lymphocytic leukemia patients (CLL). They compare the phenotypic and functional characteristic of peripheral blood T cells in 39 CLL patients with early stage disease and 20 age-matched healthy donors. To control for the potential confounding impact of chronic viral infection, donors were also balanced for CMV seropositivity (56% of CLL patients and 65% of controls). There are several intriguing findings from this work.

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CD8+ cells were preferentially expanded in CLL patients irrespective of CMV serostatus.

CD4 and CD8 T cells from CLL patients had increased expression of inhibitory markers CD244, CD160, and PD1 relative to healthy controls, suggesting a T cell exhaustion phenotype. Inhibitory receptor expression was highest on CD8+ T cells.

CD8+ T cells from CLL patients demonstrated functional defects in proliferation and cytoxicity.

The cytotoxic defect was caused by impaired granzyme packaging into vesicles and poor immune synapse formation that resulted in nonpolarized degranulation.

A common feature of T cell exhaustion is failure to produce effector cytokines. Surprisingly, CD8 T cells from CLL patients had normal IL-2 production and increased production of INFγ and TNFα. The augmented cytokine production correlated with increased expression of Tbet, a key regulator of TH1 cytokine production.

Although CMV serostatus modulated the distribution of lymphocyte subsets in patients and controls, it did not impact the augmented expression of inhibitory receptors or the functional defects in T cells from CLL patients.

Taken together, these data indicate that T cells from patients with CLL exhibit some features of T cell exhaustion in regards to poor proliferative responses and defective cytotoxic activity. Intriguingly, unlike classically described T exhaustion, the ability to produce cytokine was retained and even augmented. This suggests T cell dysfunction in the context of malignancy may vary depending on the

underlying cancer subtype. These findings also raise the possibility that immunomodulatory approaches such as PD1 blockade may be efficacious in CLL. Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco.

OPPOSING EFFECTS OF GEMCITABINE AND 5-FLUOROURACIL ON ANTITUMOR IMMUNITY Research Paper: Chemotherapy-triggered Cathepsin B Release in Myeloid-derived Suppressor Cells Activates the Nlrp3 Inflammasome and PromotesTtumor Growth. Nature Medicine 2013; 19:57-64. PMID: 23202296 Chemotherapy is a major treatment modality for cancer that is primarily thought to suppress the immune system. However, recent evidence suggests that some chemotherapeutic agents promote antitumor immune responses through various mechanisms including increasing antigen presentation, sensitizing tumor cells to NK and T cell mediated lysis, and selectively killing immunosuppressive cell subsets. Gemcitabine (Gem) and 5-fluorouracil (5FU) are two chemotherapeutic agents used in the clinic that have been shown to promote antitumor immunity through the depletion of myeloid-derived suppressor cells (MDSC), a population of immature myeloid cells known to suppress antitumor T cell responses. In this study, Melanie Bruchard and colleagues demonstrate that the effects of Gem and 5FU on MDSC can also promote tumor growth. This effect occurs prior to the induction of MDSC cell death and is mediated through the activation of NOD-like receptor family, pyrin domain containing-3 protein (NLRP3) inflammasomes in MDSC, leading to IL-1β release and the polarization of protumorigenic TH17 T cell responses.

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The authors showed that in vitro treatment of MSC-2 cells, a murine MDSC cell line, with 5FU or Gem induced caspase-1 activation prior to, and independently from the induction of MSC-2 cell death. They confirmed their in vitro results in MDSC from EL4 lymphoma-challenged mice treated with 5FU or Gem, and showed that treatment-induced caspase-1 activation was similar in both granulocytic and monocytic MDSC subsets. They also showed that 5FU treatment induced caspase-1 activation in MDSC from metastatic colorectal patients.

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MDSC from EL4-bearing mice treated with 5FU and Gem were shown to secrete IL-1β, and treatment of EL4-bearing mice with 5FU resulted in elevated serum concentrations of IL-1β. IL1β serum levels were also found to be increased in 9 of 12 colorectal patients following 5FU treatment. Using caspase-1 and NLRP3 deficient mice, and NLRP3-specific shRNA transduced MDSC, the authors showed that 5FU and Gem induced IL-1β secretion from MDSC through caspase-1 mediated activation of the NLRP3 inflammasome.

Chemotherapy-induced MDSC inflammasome activation was shown to be mediated through the permeabilization of lysosomes and the release of mature cathepsin B, and could be inhibited by lysosomal acidification using bafilomycin A, cathepsin B inhibitors, and cathepsin B-specific knockdown using shRNA. 5FU-induced IL-1β expression by MDSCs markedly decreased the antitumor effect of 5FU in EL4 lymphoma, 4T1 mammary carcinoma, B16F10 melanoma and Lewis lung carcinoma animal models. To confirm the role of IL-1β, the authors demonstrated that the antitumor efficacy of 5FU was enhanced in these cancer models when co-administered with the soluble IL-1 receptor antagonist anak←inra.

The pro-tumorigenic effect of 5FU was shown to be mediated through the IL-1β-dependent polarization of TH17 T cell responses. To confirm this, the authors showed that the antitumor effect of 5FU was enhanced in IL17-deficient mice.

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The data in this paper demonstrate that 5FU and Gem have opposing effects on antitumor immune responses. On one hand, these chemotherapeutic agents promote antitumor immune responses through the selective depletion of immunosuppressive MDSC. On the other hand, 5FU and Gem abrogate their antitumor immune effect by activating the NLRP3 inflammasome in MDSC, resulting in the secretion of IL-1β and the polarization of pro-tumorigenic TH17 responses. These data help explain why previously reported studies have shown conflicting results for these agents and their effects on antitumor immunity. In addition, these data also suggest that the antitumor efficacy of 5FU and Gem may be enhanced by combining them with IL1 inhibition in the clinic. Importantly, this study provides an excellent example of how chemotherapy and immunotherapy can be appropriately combined to achieve improved antitumor efficacy.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

IMMUNIZATION ROUTE MATTERS FOR MUCOSAL TUMORS Research Paper: Sandoval F., et al. Mucosal Imprinting of Vaccine-induced CD8+ T Cells is Crucial to Inhibit the Growth of Mucosal Tumors. Science Translational Medicine. 2013; 172,172ra20. PMID: 23100628 Systemic lupus erythematosus (SLE) is associated with the production of autoantibodies reactive against host DNA. Although the role of DNA-specific antibodies in SLE is not clear, anti-DNA antibodies commonly have toxic effects on cells. 3E10 is an anti-DNA monoclonal antibody isolated from a mouse model of SLE that is not toxic to normal cells, but is capable of penetrating the nuclei of cells. Because entry is mediated by a ubiquitous nucleoside transporter, and is independent of the constant domain, nuclei of both normal and malignant cells can be penetrated by full-length 3E10, and its single-chain variable fragment (3E10 scFv). Pre-clinical studies suggest that 3E10 scFv can be used as a vehicle for delivering cargo molecules to cell nuclei. In this study, Peter Glazer and colleagues investigated the potential of using a 3E10 scFv-HSP70 fusion protein to protect cells from ionizing radiation. In the process, they serendipitously discovered that 3E10 by itself can sensitize DNA repair-deficient tumors to DNA-damaging therapy and have provided the rationale for testing 3E10 as a targeted cancer therapy.

As expected, the authors showed that in vitro treatment with 3E10 scFv-HSP70 protected the human MCF-7 breast cancer cell line from ionizing radiation. However, unexpectedly they noticed that treatment with 3E10 scFv alone significantly increased MCF-7 sensitivity to radiation. 3E10 scFv treatment also sensitized the U251 human glioma cell line to radiation and the U87 human glioma cell line to the DNA-damaging drug doxorubicin, but not to the microtubule-disrupting drug paclitaxel.

Furthermore, in vivo treatment with full 3E10 antibody sensitized U87 glioma xenografts to both radiation and low-dose doxorubicin. These data suggested that both 3E10 and 3E10 scFv specifically sensitizes tumor cells to DNA-damaging therapies.

The authors also showed that 3E10 preferentially binds single-strand DNA tails; and sensitizes tumor cells to DNA-damage by inhibiting critical steps involved in both DNA single-strand and double-strand break repair.

Given the known sensitivity of BRCA2-deficient tumor cells to DNA-damage, the authors tested 3E10 and 3E10 scFv against BRCA2-deficient and proficient tumor cell lines. As expected, 3E10 and 3E10 scFv exposure was synthetically lethal to BRCA2-deficient human ovarian and pancreatic cancer cell lines, but not BRCA2-proficient ovarian cancer cell lines in vitro.

Furthermore, 3E10 treatment dramatically and selectively sensitized BRCA2-deficient, but not BRCA2-proficient, human ovarian cancer cells to doxorubicin and pancreatic cancer cells to radiation.

These data thus demonstrate that the anti-DNA antibody 3E10, and its single chain variable fragment, sensitize DNA repair-deficient tumor cells to DNA-damaging therapies, such as ionizing radiation and doxorubicin, by interfering with DNA single-strand and double-strand break repair. 3E10 has been proven safe in phase I clinical studies testing the antibody as a potential vaccine for SLE. These data support testing 3E10 as a targeted therapy in patients with DNA-repair deficient cancers such as BRCA2-deficient malignancies. The authors also propose that DNA-repair inhibition resulting from anti-DNA autoantibodies could be responsible for lower incidences of breast, prostate and ovarian cancers in SLE patients, since these are cancers that are frequently associated with debilitated DNA repair mechanisms.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

Highlights from Clinical Immunology, Official Journal of FOCIS EPIGENETICS AND CHROMATIN REMODELING IN HENOCH-SCHÖNLEIN PURPURA Research Paper: Luo S., et al. Aberrant Histone Modifications in Peripheral Blood Mononuclear Cells from Patients with Henoch-Schönlein Purpura. Clinical Immunology (2013) 146:165-175. PMID: 23353785 Henoch-Schönlein purpura (HSP) is a systemic vasculitis that classically presents as palpable purpura with arthritis, abdominal pain, or kidney dysfunction in the weeks following an upper respiratory tract infection. The majority of cases of HSP occur in children, although it can affect adults as well, and the pathophysiology is thought to involve IgA-immune complex deposition, which leads to complement activation, neutrophil influx, and microvascular destruction. Despite much interest in HSP, its precise etiology remains unclear and a variety of host and environmental factors have been identified as potential contributors. One area that has not been previously explored in HSP is the role of epigenetics and histone remodeling, which can affect the transcriptional accessibility of specific genetic loci. This avenue of research is of particular interest in light of recent evidence supporting the notion that epigenetic regulation may influence T cell function and susceptibility to autoimmune diseases such as lupus, Sjögren’s syndrome, and rheumatoid arthritis. To test the hypothesis that epigenetic changes might contribute to HSP, Luo et al. analyzed histone modifications and gene expression in PBMCs collected from 24 adult patients with HSP and 22 healthy controls of similar age. The authors found that: •

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Histone modifying genes were differentially expressed in PBMCs of HSP patients relative to healthy controls. HSP patients had increased expression of histone acetyltransferases (e.g.

Creb-binding protein) and histone methyltransferases (e.g. SETD1A), as well as reduced expression of histone deacetylases (e.g. HDAC1) and histone demethylases (e.g. LSD1).

Acetylation and methylation of particular histones were increased in PBMCs of HSP patients.

Specifically, histone H3 acetylation was increased in all HSP patients, while H3K4 methylation was increased only in those HSP patients with concurrent signs of kidney dysfunction.

Th2 associated genes (e.g. IL-4, IL-13, GATA3, TIM1, CXCL4) were upregulated in PBMCs of patients with HSP. However, no differences were seen between groups in the expression of Th1 associated genes (e.g. IFN-γ, Tbet).

The IL-4 locus was enriched for hyperacetylated histone H3 and hypermethylated histone H3K4 in CD4+ PBMCs of HSP patients, as determined by chromatin immunoprecipitation.

•

However, no differences were seen between groups at the IFN-γ locus with regards to acetylated H3 or methylated H3K4.

Among patients with HSP, increased H3 acetylation and H3K4 methylation were positively correlated with increasing clinical disease severity and serum levels of inflammatory markers and cytokines (e.g. CRP, ESR, IL-4).

The study authors demonstrate the presence of specific histone modifications, namely hyperacetylated H3 and hypermethylated H3K4, in PBMCs of adult patients with HSP. In addition, the authors correlate these changes with an increase in the expression of Th2-associated genes, such as IL-4 and GATA-3, as well as with worsening clinical disease severity of HSP. These findings represent a novel observation of the role that epigenetics and chromatin remodeling may play in HSP and other vasculitides. In addition, they suggest that Th2-skewing and increased IL-4 production may be important consequences of histone modification in HSP. Further research would be necessary, however, to better demonstrate the extent to which the development of HSP is dependent on epigenetic dysregulation and Th2 activation. Given that HSP predominantly affects children, it would also be important for future work to validate the findings of Luo et al., which were obtained in adult HSP patients, in the pediatric population. By examining the sequelae and outcomes of HSP patients, future research could also provide valuable information as to the prognostic utility of testing for specific histone modifications.

Reviewed by Gabriel Griffin, B.A. Duke University School of Medicine, and Andrew H. Lichtman, MD, PhD, Brigham and Women's Hospital.

HUMAN IMMUNOPHENOTYPING UPDATE MINIATURIZING IMMUNE MONITORING ASSAYS By: Holden T. Maecker, PhD, Stanford University The most common immune monitoring assays are serological. This stems from the fact that antibodybased assays have a much longer history in immunology than do cellular assays, and because the technical demands for antibody assays tend to be lower. Because of their long history and widespread use, technological advances in antibody-based assays are many. Two of the main areas for advancement have been multiplexing (the ability to measure many analytes at once) and miniaturization (to allow use of smaller samples). Often, the two go hand in hand. Consider the following advances in multiplexing and/or miniaturization of antibody-based assays: Multiplexed ELISA’s. These include multiplexed assays with readouts other than the traditional colorimetric ELISA (or radiological RIA). A prime example is the electrochemiluminescence platform from MesoScale Discovery (1). By spotting capture reagents for up to 10 analytes per well, the sample volume required per readout is reduced up to 10-fold. Furthermore, the wide linear dynamic range of electrochemiluminescence allows for measurement of samples without multiple serial dilutions.

Bead-based immunoassays. These are available from many vendors, most commonly designed for the Luminex platform (2-4) (see also the previous FOCIS Immunophenotyping Update on this technology). Bead-based arrays allow for a much higher level of multiplexing, theoretically up to several hundred analytes per well with current systems. In practice, most protein-based assays top out at 30-50 analytes, due to considerations such as antibody cross-reactivity and target protein

abundance. Still, this considerable level of multiplexing results in a 25 microliter aliquot of serum or plasma being used to measure a large number of targets.

Protein microarrays. Similar to genomic microarrays, these usually have target proteins or peptides spotted on membranes or other substrates. The degree of multiplexing here is enormous. There are commercial arrays containing several thousand human proteins, or thousands of peptides derived from hundreds of microbes. While they are generally considered to be less quantitative than sandwich-type immunoassays, they can quickly read out specificity patterns of potential autoantibodies or exposure to pathogens, for example. A sample of just a few microliters is used to probe all of the targets.

In addition to these direct protein measurement tools, there are multiplexed platforms to perform qPCR for cytokines, and even hybrid methods that use nucleic acid tags as intermediates to detect proteins prior to signal amplification by PCR (Proseek, Olink Technologies).

So, where are the highly multiplexed, miniaturized cellular assays? To be sure, cellular immunology has undergone some technical revolutions, most notably with the development of flow cytometry. No single technology has done as much to further our understanding of the diversity and function of immune cells (5). And, flow cytometry has evolved from one- and two-color assays in the 1980’s to up to 17-color assays that are possible today (6)‌.with 40+ parameters available by the use of mass labels in the CyTOF (7-9) (See also the FOCIS Immunophenotyping Update, Fall 2011).

In other ways, cellular assays have evolved from messy, bulk radiological tests (3H-thymidine incorporation for proliferation, 51Cr release for cytotoxicity) to dye-labeled single-cell equivalents (including CFSE dilution (10) and CD107-based degranulation assays (11), to name two common ones).

But none of these assays are truly miniaturized. When performed on cryopreserved PBMC, the requirement in terms of starting blood volume is particularly high, owing to the loss that comes with cryopreservation. Often, a 10 cc tube of blood gets processed to a single cryovial of PBMC and devoted to one flow cytometry assay. The high cell requirement could be substantially reduced if whole blood were assayed directly. One way to facilitate this is through point-of-collection devices that can stimulate and fix blood for later functional assays (Smart Tube, Inc.). Although this commercial system is currently designed for 1 mL blood per stimulation condition, it could certainly be further miniaturized without loss of reproducibility. And presumably, plate-based versions could be developed that would allow for further multiplexing of samples and stimulators. If combined with a CyTOF readout, many hundreds of cell subsets could be enumerated and their functional capacities interrogated for each stimulation condition. Such an assay, currently in development in our laboratory, would read out more information from 1-2 mL of blood than current state-of-the art assays can read out from ten times that volume.

One of the ways that cells are lost in current assays is through washing steps. However, using acoustic waves to move and align cells in microfluidic chambers, it is possible to achieve efficient buffer exchange without loss of any cells (12, 13). Devices that perform such operations, even automating the staining and washing of cells in a microchip device, could revolutionize the front-end processing currently required for flow cytometry. In fact, these devices might be coupled to flow cytometers, or else placed in scanning cytometers for readout. The latter has the advantage that, in theory, the same cells could be repetitively analyzed in situ, to follow dynamic processes in real time.

At least one group has used microdevices to trap immune cells in nano-scale wells and perform multiple assays on them over time (14-16). Cell supernatants are printed onto slides for assay of secreted cytokines, while the cells themselves are phenotyped using fluorescent-labeled antibodies and detection by fluorescent microscopy. This system, while currently a bit labor-intensive, allows for the maximum information content to be derived from a relatively small starting population of cells.

Taking this idea a step further, the immunoassay of the future could possibly be something like the following. A few hundred microliters of blood are pipetted into a microchip, where plasma and cellular components are separated. The plasma is run over a series of biosensors to measure soluble cytokines and to probe antibody types and specificities. The cellular fraction is separated by acoustic or electrical means into erythrocytes and leukocytes. Since this is the future, there will probably be important assays to be performed on the erythrocytes as well as the leukcocytes, but these will likely not be done in a mixture. At any rate, the leukocytes might be directed into microchambers where they are stimulated with a battery of cytokines, antigens, etc. They might be stained in situ with the addition of antibodies, whose binding is read by laser-scanning cytometry. Meanwhile, their secreted products can be analyzed in real time using biosensors. Alternately, the cells could be sent after staining into a flow (or mass) cytometer.

A bioinformatics module would take the many readouts from such a chip and summarize them in ways such as: • Absolute numbers and percentages of hundreds of phenotypic cell subsets • Functional profiles of each of these cell types with regards to major signaling pathways • Antigen reactivity of B, T, and NK cells to major antigens of interest • Circulating levels of a number of cytokines • Levels of immunoglobulin isotypes and reactivity profile of antibodies One could envision that such a super-assay could be customized for particular purposes, by varying the stimuli, antibody probes, etc. But a somewhat standardized version of it would be a tremendously valuable (and easy to implement) biomarker discovery tool. With enough data from healthy and diseased individuals, normal ranges could be established and metrics of diseases identified. This could become the cellular immunologist’s version of a whole-genome gene expression array, but in many ways more powerful, because its information would be generated on a single-cell level.

Because it would presumably be automated, largely self-contained, and performed with a small amount of blood, it could become a powerful clinical diagnostic tool as well. Of course, it would require a major engineering effort to create. But we cellular immunologists can dream.

References: 1. S. V. Sennikov et al., Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method. J Immunol Methods 275, 81 (Apr 1, 2003).

2. S. Pang et al., A comparability study of the emerging protein array platforms with established ELISA procedures. J Immunol Methods 302, 1 (Jul, 2005).

3. J. F. Djoba Siawaya et al., An evaluation of commercial fluorescent bead-based luminex cytokine assays. PloS one 3, e2535 (2008).

4. M. F. Elshal, J. P. McCoy, Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods 38, 317 (Apr, 2006).

5. L. A. Herzenberg, D. Parks, B. Sahaf, O. Perez, M. Roederer, The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48, 1819 (Oct, 2002).

6. S. P. Perfetto, P. K. Chattopadhyay, M. Roederer, Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4, 648 (Aug, 2004).

7. D. R. Bandura et al., Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry.

Analytical chemistry, (Jul 14, 2009).

8. O. Ornatsky et al., Highly multiparametric analysis by mass cytometry. J Immunol Methods, (Jul 21, 2010).

9. S. C. Bendall et al., Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687 (May 6, 2011).

10. P. K. Wallace et al., Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution. Cytometry. Part A : the journal of the International Society for Analytical Cytology 73, 1019 (Nov, 2008).

11. M. R. Betts et al., Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281, 65 (Oct 1, 2003).

12. J. Shi, X. Mao, D. Ahmed, A. Colletti, T. J. Huang, Focusing microparticles in a microfluidic channel with standing surface acoustic waves (SSAW). Lab on a chip 8, 221 (Feb, 2008).

13. M. E. Piyasena et al., Multinode acoustic focusing for parallel flow cytometry. Analytical chemistry 84, 1831 (Feb 21, 2012).

14. N. Varadarajan et al., A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis. J. Clin. Invest. 121, 4322 (Nov 01, 2011).

15. Q. Han et al., Polyfunctional responses by human T cells result from sequential release of cytokines. Proceedings of the National Academy of Sciences 109, 1607 (Feb 31, 2012).

16. N. Varadarajan et al., Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving. Proceedings of the National Academy of Sciences 109, 3885 (Apr 06, 2012).

CONCEPTS IN BASIC IMMUNOLOGY: PRINCIPLES AND THERAPEUTIC APPLICATIONS A NEW WAVE OF RATIONAL THERAPEUTICS FOR IMMUNOLOGICAL DISORDERS: SMALL MOLECULE SIGNALING INHIBITORS Shiv Pillai, MD, PhD, Massachusetts General Hospital History For over two millenia, compounds with therapeutic efficacy have largely been discovered by chance at best by a process of trial and error. In ancient Greece, India and China and later in the Islamic world and pre-Columbian America, arsenals of efficacious medicinal preparations were discovered centuries ago by physicians and herbalists and some of these preparations are still in use today. A little over a hundred years ago, Paul Ehrlich searched for drugs derived from dyes and related organic compounds and early in the 20th century gave us our first chemotherapeutic, Salvarsan. This opened the doors to more discoveries that helped bring synthetic chemists into the brave new world of pharmacology. Alexander Fleming's discovery in the 1940s of the first antibiotic derived from a fungal spore led to Ernst Boris Chain's chemical synthesis of penicillin. Organic chemists have since created large compendiums of compound libraries - these libraries as well as collections of molecules derived from natural products have long formed the core of large screens aimed at identifying random molecules that happen to possess some biological efficacy of relevance to the treatment of disease.

The first baby steps towards rational therapy coincided with the birth of modern immunology. Behring and Kitasato injected large animals with bacterial toxins that had been chemically modified using iodoform in order to generate anti-toxins. Passive serum therapy with anti-toxins was a huge success.

The discovery of monoclonal antibody technology by Kohler and Milstein in the 1970s paved the way for the first true rational therapeutic. This emanated from the discovery by Maini and Schulman that the synovium in rheumatoid arthritis subjects contains large amounts of TNF. Their pioneering experiments with monoclonal antibodies against TNF in the treatment of rheumatoid arthritis ushered in the era of biologics, largely monoclonal antibodies and some immunoglobulin-receptor ectodomain fusion proteins that typically block receptor function. Some of the most successful biologics used in immunological disorders have included TNF inhibitors, anti-CD20 antibodies for B cell depletion,

antibodies that block integrins and antibodies that negate inhibitory signaling by PD-1 - among many, many others.

Targeting Signaling Pathways Since the mid 1980s there has been an explosion in our understanding of signaling pathways in vertebrate cells - including immune cells and cancer cells. A highly touted example of rational therapy in cancer in the past decade was also something of an accident. In chronic myelogenous leukemia, a fusion protein containing a cytosolic protein called Bcr (not the antigen receptor in B cells!) and the cAbl tyrosine kinase is generated from the vicinity of the translocation breakpoint in the Philadelphia chromosome. The Bcr-Abl tyrosine kinase is therefore cancer specific - it is not found in normal cells.

Attempts to generate a kinase inhibitor that inhibited protein kinase C led to the identification of a molecule that was actually far more capable of inhibiting the Bcr-Abl kinase. This inhibitor, Gleevec or Imatinib, has emerged as a remarkable therapeutic and remains one of the poster children for rational therapy. It remains the first small molecule rational therapeutic.

A number of small molecules have of course been used as therapeutics in transplantation and autoimmunity for decades. Although the mechanism of action of drugs like cyclosporin A, FK506 and rapamycin have been worked out in detail they were identified from fungal extracts and their mechanisms of actions were discovered after they had already proven to be biologically efficacious. It is not an accident that these compounds, and indeed the majority of small molecule therapeutics, are enzyme inhibitors. Small molecules typically function by binding tightly to hydrophobic clefts in proteins. It has been estimated that only about 15% of all proteins possess a hydrophobic cleft.

Enzymes by nature of their ability to interact closely with and "re-organize" the structure of substrates invariably contain hydrophobic clefts amenable to small molecule attack while the overwhelming majority of non-catalytic proteins lack such a feature. The growing knowledge about the enzymes that are activated downstream of antigen receptors, cytokine receptors and other receptors in the immune system has generated a series of targets for rational therapy aimed at disorders involving the immune system.

There are a number of advantages to small molecule inhibitors. The first is “dosability “- achieving the right therapeutic dose is considerably easier with a molecule given a couple of times a day rather than once every few months. Side effects can be more easily reversed by altering doses of a small molecule compared with a biologic. In addition, there are large swaths of the world in which biologics will remain a pipe dream because of issues of delivery and cost. Small molecules therefore hold great promise for a very wide variety of reasons.

Small Molecule Inhibitors Under Development A number of therapeutic trials are currently in progress involving small molecules targeting enzymes that are crucial in immune cells. We will briefly discuss here inhibitors of three particular categories of protein tyrosine kinases that have already exhibited great promise in disorders affecting the immune system and in certain tumors. Not all the inhibitors currently in use are unique in their target specificity. One interesting example involves inhibitors for Btk (Bruton's tyrosine kinase). Btk is an enzyme activated downstream of the B cell receptor in B cells; it is also activated by immune receptors in myeloid cells. B cell receptor activation involves the sequential activation of members of three different families of tyrosine kinases. Src family kinases such as Lyn are first activated, followed by the recruitment and activation of Syk, a tyrosine kinase with tandem phosphotyrosine specific SH2 domains, and the consequent activation of Btk. Btk is a tyrosine kinase of the Tec family and Btk inhibitors currently in trials are fairly specific molecules which covalently bind to a cysteine residue in this kinase and thus irreversibly inactivate it. This particular residue of Btk, cysteine -481, lacks a counterpart in the vast majority of protein kinases, as a result of which these inhibitors are very specific. These covalent inhibitors are quite potent and have already shown to be of value in B lymphoid malignancies, particularly those in which B cell receptor signaling plays a presumed trophic role. These malignancies include chronic lymphocytic leukemia, small cell lymphoma and diffuse large

B cell lymphoma. Irreversible inhibitors might be more suited to malignancies that to chronic inflammatory diseases. Attempts are ongoing to generate non-covalent, reversible Btk inhibitors that may prove useful in autoimmune disorders such as systemic lupus erythematosus.

Syk functions downstream of the B cell receptor in B cells and plays a similar role in myeloid cells downstream typically of Fc receptors for IgG in many phagocytic cells and distal to the high affinity Fc receptor in eosinophils and mast cells. Inhibitors of the Syk tyrosine kinase have been tried successfully in B cell malignancies, rheumatoid arthritis and in allergic lung disease and the spectrum of diseases in which this category of small molecules may prove effective is likely to widen.

There are four Jak tyrosine kinases (sometimes called Janus kinases) that are activated downstream of a large number of cytokine receptors. They are known as Jak1, Jak2, Jak3, and Tyk2. Ligation of Type I and Type II cytokine receptors activates specific Jak kinases which in turn cause the phosphorylation, dimerization and nuclear transport of specific STAT transcription factors. Inhibitors that preferentially target Jak3 (specificity is not absolute) compromise signaling via common γ chain cytokines such as IL-2, IL-4, IL-7, IL-15, and IL-21. These inhibitors can therefore compromise allergic inflammation (Th2) and also attenuate Th17 type responses. They have shown promise in allergic diseases, psoriasis and rheumatoid arthritis. Jak1/Jak2 inhibitors can block signaling via IFN-γ and IL6. They can therefore compromise Th1 and Th17 responses and have been shown to be effective in psoriasis, rheumatoid arthritis, myelofibrosis, acute myelogenous leukemias and refractory lymphomas.

Numerous other small molecule inhibitors continue to be developed at an astonishing rate even as signaling pathways continue to be parsed and three-dimensional structures of more enzymes of interest determined. The small molecule therapeutic wave has hit the shore and the ripples will flow for a long time.

SELECTED RECENT CLINICAL TRIAL RESULTS Edited by Carla J. Greenbaum, MD, Benaroya Research Institute

DIMETHYL FUMARATE IN RELAPSING MULTIPLE SCLEROSIS: THE DEFINE AND CONFIRM STUDIES Clinical Trials: • Fox J. et al., Placebo-Controlled Phase 3 Study of Oral BG-12 or Glatiramer in Multiple Sclerosis. New England Journal of Medicine 2012; 367:1087-97.) PMID: 22992072 • Gold R et al., Placebo-Controlled Phase 3 Study of Oral BG-12 for Relapsing Multiple Sclerosis. New England Journal of Medicine 2012; 367:10107. PMID: 22992073 Disease: Multiple Sclerosis Study Design: • Two large Phase 3 studies of dimethyl fumarate (BG-12) in relapsing Multiple Sclerosis (MS) were recently reported in the same issue of the NEJM. • The DEFINE study was a double-blind, randomized, three-arm trial over 96 weeks evaluating BG-12 in two regimens (240mg twice daily and 240mg thrice daily) versus placebo. The primary endpoint was the proportion of patients who experienced a relapse of MS during the study. Secondary endpoints included the annualized relapse rate, the time to disability progression (defined as sustained, 1.0 point increase in the Expanded Disability Status Scale

•

[EDSS]), and several MRI measures of MS lesions. Eligible patients (n = 1,237) were adults with relapsing-remitting MS (RRMS) and 1 or more relapses within the last 12 months.

The CONFIRM study had a similar design, except it included an open-label arm of glatiramer acetate 20 mg SQ DAILY. The primary endpoint was the annual relapse rate at the end of study (96 weeks). Secondary endpoints included the proportion of patients with relapse within the study period, disability progression, and MRI measures. Eligible patients (n = 1430) had identical eligibility criteria.

Drug: • Dimethyl Fumarate (BG-12) is an orally-administered fumaric acid ester that has been used in the EU as a treatment for severe systemic psoriasis since the 1990s. Development in MS resulted from observation by German clinicians that oral fumarate stabilized MS in psoriatic patients with MS. Mechanism of action is not fully understood. It may have neuroprotective effects via activation of the E2-related transcription factor 2 (Nrf2) – which upregulates NAD(P)H;quinone reductase, increasing glutathione levels (an important antioxidant). Among other potentially beneficial effects, BG-12 may cause a shift from a TH1 to TH2 cytokine profile, downregulate ICAM-1 and VCAM, and downregulate differentiation antigens on (and induce apoptosis of) dendritic cells.

Results: • In DEFINE, the proportion of patients with relapse was 27%, 26%, and 46% for BG-12 twice daily, BG-12 thrice daily and placebo, respectively (P • Interestingly, the reduction of disease progression was not significant for BG-12 or glatiramer in CONFIRM, while it was significant for both BG-12 doses versus placebo in DEFINE (38% and 34&, P • The main adverse events with use of BG-12 were flushing, GI events such as diarrhea or upper abdominal pain, decreased WBC counts and elevated liver enzymes. There was no association with risk of serious infection, opportunistic infection, or neoplasm.

Why is the trial of interest to the broader FOCIS community?

BG-12 has features that make it a model for the kind of therapies that will be readily approved and widely adopted for use in chronic inflammatory diseases in the future. First, because it has been used to treat psoriasis in the EU since the 1990s, BG-12 has a two-decade safety record that will address any long-term safety concerns raised by regulatory agencies. Second, it is an orally-administered small molecule, which is appealing to both patients who will likely be very adherent on the drug. Of course, equally important to the future success of BG-12 is the unexpectedly robust efficacy data in these two Phase 3 studies as measured by MRI and relapse rates. The key question as to whether this therapy does or does not affect disability progression will require additional studies.

Reviewed by Carla Greenbaum, MD and Steven Lamola, MD Benaroya Research Institute

PHASE 1/2A OPEN-LABEL, DOSE-ESCALATION STUDY OF OVALBUMIN-SPECIFIC TREG CELLS IN REFRACTORY CROHN’S DISEASE (CROHN’S AND TREG CELLS STUDY [CATS1]) Clinical Trial: Desreumaux P et al., Gastroenterology 2012; 143:1207-1217. PMID: 22885333 Disease: Crohn's Disease Study Design: • 12-week, open-label, single-injection of escalating-dose of Treg cells. Phase 1/2a study in 20 patients with refractory Crohn’s Disease.

• Key inclusion criteria were age 18-65, diagnosis of Crohn’s Disease > 12 months, with active disease and prior inadequate response or intolerance to conventional treatment. Key exclusion criteria were active infection or malignancy, and immunosuppressant or anti-TNF-α therapy.

• Safety and efficacy were assessed at follow-up visits at weeks 1, 2, 3, 5, 8 and 12. Efficacy was primarily assessed by measure of Crohn’s Disease Activity Score (CDAI). Proliferation of PBMCs and serum cytokines were also evaluated, as were C-reactive protein (CRP).

Drug: • Treg cells isolated from patients’ PBMCs were exposed to ovalbumin. At day 0, patients received a single IV injection of 106 (n = 8), 107 (n = 3), 108 (n = 3) or 109 (n = 6) of these ovalbumin-specific type-1 Treg cells. Patients also ingested a meringue cake to enable activation of ovalbumin-specific Tregs in the inflamed gut.

Results: • Both pro-inflammatory CD14+ CD16+ monocytes and FoxP3-expressing Tregs cells decreased in responding patients – however serum cytokine levels did not. Ovalbumin-specific proliferative responses of PBMCs were significantly lower in responders vs. nonresponders (especially in the lowest dose group) at week 8. Mean change in CRP at week 1 was significantly lower in CDAI responders at week 5 (n = 8) vs. nonresponders.

• Overall the treatment was well-tolerated, with only 2 related AEs and 3 related SAEs (no deaths, and all recovered). Response to treatment, per standard definition of 100 point reduction in CDAI, was observed in 40% of patients at weeks 5 and 8, and in 75% of the lowest dose group (106 Treg cells). It was maximal at 5 weeks post-treatment, and subsequently declined.

Why is the trial is of interest to the broader FOCIS community?

This is the first-in-man study of antigen specific Treg therapy in human inflammatory bowel disease (Tregs previously been used in Type 1 diabetes and graft-versus-host disease). As in any small, open-label trial, results should be interpreted with caution.

Reviewed by Carla Greenbaum, MD and Steven Lamola, MD Benaroya Research Institute

PHASE 2A STUDY OF SECUKINUMAB IN CROHN’S DISEASE Clinical Trial: • Hueber, W., et al. Gut. 2012; 61:1693-1700. PMID: 22595313 Diseases: Crohn's Disease Study Design: • Double-blind, randomized, placebo-controlled Phase 2a trial of 59 adult patients with moderate-to-severe Crohn’s Disease assigned in a 2:1 ratio to either secukinumab ( IL-17A monoclonal antibody) or placebo • Safety and efficacy evaluated at weeks 1, 2, 3, 4, 5, 6, 10, 14 and 18.

• The primary endpoint was the probability of the study drug achieving a reduction in the Crohn’s Disease activity index (CDAI) at week 6 Drug: • Secukinumab, an IL-17A monoclonal antibody, given at a dose of 10 mg/kg and administered as 2 hour IV infusions on days 1 and 22 Results: • The trial was prematurely terminated when it was determined at a scheduled interim analysis of data from the first 41 patients that pre-specified futility criteria had been met. Secondary analysis showed statistically significant worsening of disease with secukinumab treatment during weeks 4-10.

• Worsening of disease in the active treatment arm was further reflected in a high rate of Crohn’s Disease related serious adverse effects compared with placebo. Additionally, 44% (17/39) of patients on secukinumab developed an infection (compared to none on placebo), including 4 fungal infections.

Why is the trial is of interest to the broader FOCIS community?

Although larger studies of secukinumab have demonstrated clinical efficacy and safety in psoriasis and rheumatoid arthritis, the drug has been shown in this Phase 2a study to lack efficacy in Crohn’s Disease -- and furthermore be harmful in at least a subset of patients. Brodalumab, a monoclonal antibody directed against IL-17RA, with efficacy in psoriasis has also failed in Crohn’s Disease. As pointed out in an accompanying editorial (1) this divergence has come to pass despite substantial shared genetic risk between psoriasis and Crohn’s Disease (including having the same risk haplotypes at IL23R) – highlighting the hazards of predicting pathophysiology from genetic risk alone.

These divergent results highlight the complexity of TH17 biology and emphasize the need for crossautoimmune disease evaluation of pathway similarity and differences.

References: 1. Raine, T and Kaser, A. Gut. 2012; 61:1653-4 Reviewed by Carla Greenbaum, MD and Steven Lamola, MD Benaroya Research Institute

TRANSLATIONAL IMMUNOLOGY UPDATE Volume 4, Issue 2 Editor: Andrew H. Lichtman Editorial Board: Abul K. Abbas, Carla J. Greenbaum

HIGHLIGHTS FROM RECENT LITERATURE TH1-LIKE INNATE LYMPHOID CELLS ACCUMULATE IN THE INTESTINAL LESIONS OF PATIENTS WITH CROHN’S DISEASE Research paper: Bernink, J. et al. Human Type 1 Innate Lymphoid Cells Accumulate in Inflamed Mucosal Tissues.

Nature Immunology 14, 221–229 (2013). PMID: 23334791 Innate lymphoid cells (ILC) are recently described lymphoid cells that lack rearranged T cell receptors and are thought to participate in innate immune reactions and tissue remodeling. Subsets of ILC have been described that have cytokine production profiles similar to those of helper T cell subsets. In this article, the authors describe a type of ILC that resembles Th1 cells and show they accumulate in the intestines of patients with Crohn’s disease.

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•

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Studying cells from human tonsils, the authors identified a novel population of T helper type 1 cell (Th1 cell)-like ILC distinct from NK cells that produced IFNγ in response to IL-12. The authors designated these cells ILC1 cells.

Crohn’s disease is a type I mediated inflammatory disease with increased production of IFNγ, IL12 and IL-18.

The authors compared ILC populations from healthy intestine and from the inflamed and noninflamed intestines of patients with Crohn’s disease. They observed increased numbers of ILC1 cells and decreased numbers of Th17-like ILC3 cells in the intestinal lesions of Crohn’s disease vs. healthy controls.

Irradiated immunodeficient mice reconstituted with human hematopoietic stem cells had an ILC pool similar to non-inflamed human gut. Treatment of these mice with dextran sodium sulfate (DSS) led to gut inflammation that was not dependent on human immune cells but nonetheless was associated with increased numbers of resident ILC1 cells. These studies suggested that ILC1 cells accumulated as a result of inflammation.

The authors observed that ILC populations were plastic, and that both immature ILCs and ILC3 cells could differentiate into ILC1 cells in vitro in the presence of IL-2 and IL-12.

The authors describe ILC that produce IFNγ and are distinct from previously identified ILC3 and ILC2 cells. These ILC1 cells accumulated in the mucosal lesions of human Crohn’s disease. Mouse studies showed these cells accumulated because of inflammation but did not necessarily cause inflammation.

The authors observed functional plasticity among ILC in that both immature ILC and mature ILC3 cells could differentiated into ILC1 cells in vitro. In summary, the authors describe a novel population of Th1-like ILC that accumulate in the inflamed lesions of human Crohn’s disease. The role of these cells in healthy immunity and their contribution to pathology remain to be determined.

Reviewed by Rachael A. Clark, MD, PhD, Department of Dermatology, Brigham and Women's Hospital.

T FOLLICULAR HELPER-LIKE CELLS APPEAR IN THE CIRCULATION AND LEAD TO A BURST OF ANTI-VIRAL ANTIBODY PRODUCTION BY MEMORY T CELLS FOLLOWING INFLUENZA VACCINATION Research Paper: Bentebibel, S., et al. Induction of ICOS+CXCR3+CXCR5+ TH Cells Correlates with Antibody Responses to Influenza Vaccination. Science Translational Medicine 5,176ra32 (2013).

PMID: 23486778 The immune events that lead to protective antibody production after influenza vaccination remain poorly characterized in humans. The authors studied circulating T cell subsets following influenza vaccination identified a particular type of T cell that correlates with the production of protective antiinfluenza antibody by memory T cells.

The authors observed an increased number of CD4+ T cells expressing ICOS after vaccination with split influenza vaccines. These cells resembled T follicular helper (TFH) cells but lacked the Bcl-6 expression observed in true TFH found in secondary lymphoid organs.These cells were induced in patients of different ages and in response to different split influenza vaccine formulations.

Emergence of these cells correlated with increased global protective anti-influenza antibody titers against previously recognized strains but not against novel strains, suggesting these cells contributed to boosting pre-existing antibody titers but did not induce primary antibody responses.

A subset of these circulating ICOS+ TFH-like T cells was specific for influenza antigens. These cells produced IFNγ, IL-2, IL-10 and IL-21, efficiently helped memory B cells differentiate into plasma cells, and specifically assisted influenza specific B cells to differentiate into plasma cells making influenza specific antibodies.

However, these cells did not provide help to naïve B cells and therefore did not support the formation of primary anti-influenza immune responses. The authors demonstrate that the appearance of circulating ICOS+ TFH-like CD4+ T cells is correlated with an increase in pre-existing anti-influenza antibodies but not with the generation of primary antibody responses following vaccination with split influenza vaccines. These T cells induced influenza-specific B cells to differentiate into plasma cells that produced protective anti-influenza antibodies but they were unable to provide help to naïve T cells. In addition to clarifying the role of ICOS+ TFH-like CD4+ T cells, these results suggest that split influenza vaccines primarily boost production of existing antibodies and fail to induce strong primary responses. Alternative vaccine strategies or inclusion of adjuvants may be necessary to induce protective primary immune responses against different, newly encountered strains of influenza.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital.

OPEN ACCESS SYSTEMS IMMUNOLOGY AND VACCINOLOGY Research Paper: Obermoser et al. Systems Scale Interactive Exploration Reveals Quantitative and Differences in Response to Influenza and Pneumococcal Vaccines. Immunity (2013) 38: 831-844.

PMID: 23601689 The application of high throughput profiling strategies to disease pathogenesis and progression has recently been extended to deepen our understanding of vaccines. We have been able to generate vaccines against many of the infectious diseases that stalked human populations (and still do in

unvaccinated populations). However, we have been most successful in crafting vaccines to prevent invariant pathogens (like measles, rubella, etc) it has remained a monumental challenge to develop effective vaccines against HIV, TB, Hepatitis C virus and malaria, among others. Influenza vaccination, while effective, requires costly, time consuming yearly re-design of the immunization.

There are many contributing factors to the challenges faced by vaccine researchers, from the ability of some pathogens to rapidly mutate their fundamental building blocks to escape detection and our defenses, to the humoral nature of the immunity elicited by our standard vaccine designs and the ability of many viruses to stealthily combat our immune system, yielding poor natural immunity to infection (let alone vaccination). In this context, many groups are trying to better understand the mechanism by which our best and our most commonly used vaccines function, from the mechanism of the limited set of clinically approved adjuvants to the implications of subunit component selection or vaccine site selection. In order to accomplish this goal, the application of an unbiased approach to gathering relevant data about the cell circuits that underlie protection (or lack thereof) in response to a vaccine, yield massive datasets, which can be impenetrable to researchers beyond the primary team.

This work assesses the immune response in a small group of healthy patients to two commonly used non-live vaccines, those for influenza and pneumococcus. In addition, they crafted an intriguing user friendly interface to allow full immersion and consideration of the wealth of data they have collected. •

They recruited 6 healthy volunteers who randomly received influenza vaccine, 23-valent pneumococcal vaccine or saline injections. These participants had a baseline blood draw preimmunization (day -7) and then had blood drawn for transcriptome analysis at 0, 1, 3, 7, 10, 14, 21 and 28 days. In addition, this was correlated with serum antibody titers in response to the relevant vaccine at days 0 and 28. o Throughout the study, the immunological findings are intriguing, with creative and clarifying use of novel static and interactive figures. The ability to link gene expression results to flow cytometry and cytokine results is a powerful model going forward, and raises the bar for dissemination of complex datasets in a fashion that aims to spur detailed analysis of the datasets in labs across the world. o They performed a modular analysis, with modules defined by previous investigations. This allowed a granular analysis (in addition to a fine analysis) of the major coordinated gene expression circuits that fundamentally underlie these responses. By comparing two vaccines, they were able to show both similarities and differences in the responses elicited.  While a number of gene expression changes were shared, each vaccine had modules that were uniquely stimulated or repressed by one of the two studied vaccines. They group the gene expression changes linked to PCV23 as inflammatory, while the modules associated with influenza were related to innate immunity and interferon signaling. o Detailed analysis of early transcriptional events is made possible by a parallel study of 6 independent patients who were assayed frequently after immunization via finger stick blood draws (at -168, 0, 1.5, 3, 6, 9, 12, 15, 24, 36 and 48h) which were assessed for transcriptional changes in response to vaccination and then compared to the transcriptional changes in cells exposed to a variety of pathogens (as well as the saline control patients). o They focus on the interferon module and assess transcription in sorted cell subsets (neutrophil, CD4 and CD8 T cells and monocytes), implicating neutrophils and monocytes as the predominant source of interferon early in the vaccine response. o Finally, they identified plasmablast responses by gene expression and flow cytometry at day 7, as seen in other studies.

This paper deeply investigates the response of the human immune system to two non-live vaccinations (influenza and pneumococcus), generating a deep and fascinating dataset.

However, this study embraces the spirit of collaboration and takes on (rather than simply acknowledging) the practical challenges of this type of work, by creating and sharing an interface

that allows both experts and novices to engage directly with the various high throughput profiling data sets in a coordinated and clear fashion. While the data set is drawn from two small samples of healthy adults, the assessment of both early and medium terms responses in a detailed fashion, with a saline control for comparison for both, provide an optimal assessment of those patients.

This is a fascinating paper, adding to the vaccinology, systems immunology and computational biology fields simultaneously.

Reviewed by Sarah Henrickson, MD, PhD Boston Children’s Hospital and Harvard Medical School.

EVALUATION B CELL TOLERANCE CHECKPOINTS OF MULTIPLE SCLEROSIS Research Paper: Kinnunen T., et al. Specific Peripheral B Cell Tolerance Defects in Patients with Multiple Sclerosis.

Journal of Clinical Investigation. 2013. 123(6):2737-41.

PMID: 23676463 Multiple sclerosis (MS) is an autoimmune disease that preferentially attacks myelin resulting in immune mediated demyelination and subsequent neurologic sequelae. Historically, MS had long thought to be a T cell mediated disease. It was thus surprising when MS patients improved clinically after B cell depletion with the anti-CD20 antibody Rituximab. While this highlights an important role for B cells in MS, how they contribute to disease pathogenesis has remained incompletely understood.

Meffre and co-workers address this knowledge gap by comparing the B cell repertoire of treatmentnaïve MS patients and healthy controls. Each day humans produce an excess of ~10 billion lymphocytes. Because generation of B and T cell receptors occurs through random recombination, the majority of these lymphocytes are autoreactive. In humans, autoreactive B cells are culled from the repertoire at two key checkpoints, one in the bone marrow (central tolerance) and one in the periphery (peripheral tolerance) prior to emergence of newly emigrant B cells into the mature naïve B cell compartment. To evaluate the reactivity of the B cell repertoire, the authors clone and produce recombinant antibodies from individual newly emigrant/transitional and mature naïve B cells and evaluate each B cell antibody’s reactivity to self antigens. Prior work by the Meffre laboratory utilizing similar techniques has demonstrated that both checkpoints are defective in patients with rheumatoid arthritis (RA) or type 1 diabetes (T1D). Using a similar approach, they analyze the autoreactivity of B cells at each of these checkpoints in MS patients. There are several interesting findings from this work.

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The proportion of newly emigrant/transitional B cells that were polyreactive with specificities to self antigens were comparable between most MS patients and healthy controls. This indicates that the central B cell tolerance checkpoint is intact in the majority of MS patients.

This is in stark contrast to RA and TID patients that show a high frequency of polyreactive new emigrant/transitional B cells due to a defective central B cell tolerance checkpoint.

Similar to RA and TID patients, evaluation of mature naïve B cells revealed an increased frequency of polyreactive B cells in MS patients relative to healthy controls, indicating that the peripheral B cell checkpoint is perturbed in MS. Importantly, these mature naïve B cells from MS patients were reactive to white matter extracts from the brain.

Loss of peripheral tolerance is accompanied by increased activation and homeostatic proliferation of mature naïve B cells but not newly emigrant/transitional B cells.

The authors note that the loss of peripheral but not central B cell tolerance in MS mirrored the patterns seen in IPEX patients. IPEX patients suffer from impaired Treg function due to mutations in the FOXp3 gene. While functional studies were unfortunately not performed, the authors do

demonstrate an abnormal accumulation of memory Tregs relative to healthy controls in MS patients similar to that seen in IPEX patients. Based on this observation, they postulate that loss of peripheral tolerance in MS could be due to defective B-T cell interactions.

The observation that defective peripheral B cell tolerance is common to MS, TID, and RA while the central tolerance defect is absent in MS is intriguing. The authors speculate that this could potentially explain why B cell depletion strategies tend to result in a more sustained remission in MS relative to other autoimmune disorders. They reason that once autoreactive B cells are eliminated in the periphery due to therapy, newly generated B cells exiting the bone marrow should be properly counterselected in MS patients but not patients whose autoimmune disease is characterized by defective central tolerance.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco.

ARMED AND WAITING: ROLE OF CDαα+T CELLS IN REGULATION OF HUMAN HERPES VIRUS INFECTION Research Paper: Zhu J., et al. Immune Surveillance by CD8αα+ Skin-resident T Cells in Human Herpes Virus Infection.

Nature. 2013 May 23;497(7450):494-7.

PMID:23657257 Clinical studies have shown that most HSV-2 reactivations are subclinical and of short duration. The mechanisms that regulate rapid asymptomatic HSV-2 containment upon reactivation had been incompletely elucidated. Prior studies had shown that CD8+ T cells persist at the dermal-epidermal junction (DEJ) at the sites of neural release of reactivating virus in genital skin and mucosa. However, the mechanistic basis by which these cells control viral reactivation were poorly understood due to the inherent challenges of studying primary immune cells during infection in humans. Zhu, Peng, Corey and colleagues use cell-type specific laser capture microdissection, transcriptional profiling, and T-cell antigen receptor β chain (TCRβ) sequencing on sequential genital skin biopsies from 10 patients to tackle this challenge. By comparing individual CD8+ T cells from the DEJ of infected areas (DEJ CD8) to control CD8+ T cells located near blood vessels (BV CD8) or at the DEJ of the contralateral HSV-2 unaffected genital tissue (control CD8), they provide important new insights into the CD8 T cell population responsible for containment of HSV-2 reactivation.

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A unique population of CD8αα+ T cells persists at the DEJ for at least 8 weeks post healing in human HSV-2 infection. In contrast, traditional CD8α+β + T cells, which are mostly absent in the previously infected DEJ regions, are present in biopsies obtained near the blood vessel and in uninfected DEJ regions.

In contrast to normal CD8α+β+ T cells, the DEJ-associated CD8αα+ T cells lack expression of the chemokine receptors CCR7, CCR8, CXCR4, CXCR6, CXCR7, and S1PR1 that are required for lymphocyte egress and trafficking. The authors hypothesize that this may aid in the long-term persistence of these cells at former sites of infection.

Transcriptional and protein expression analysis of the DEJ-associated CD8αα+ T cells demonstrated that these cells have an activated phenotype with upregulation of genes important for antiviral and cytolytic function, unlike the CD8α+β+ T cells in the control site biopsies.

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Sequencing of the TCR repertoire in the DEJ CD8αα+ T cells reveals that these cells are oligoclonal with a diverse usage of TCR variable-β genes and demonstrates that these cells are distinct from mucosa-associated invariant T cells and NK T cells.

Sequential analysis of the TCR repertoire in a subset of patients over 2.5 years revealed dominant clonotypes that overlapped among multiple recurrences, supporting the notion that the DEJ CD8αα+ T cells are a long-lasting tissue-resident population.

Taking their data together, the authors conclude that the DEJ CD8αα+ T cells may play a critical role in immune surveillance and initial containment of HSV-2 reactivation in human peripheral tissue. This work has important implications for vaccine strategies and other immunotherapeutic approaches focused on regulation of HSV2 infection and reactivation. This study also provides important precedence for dissecting the immune response in other types of mucosal infections.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco.

IDENTIFICATION OF T REGULATORY TYPE 1 CELL-SPECFIC SURFACE MARKERS Research Paper: Gagliani N. et al. Coexpression of CD49b and LAG-3 Identifies Human and Mouse T Regulatory Type 1 Cells. Nature Medicine 2013 Apr 28; doi: 10.1038/nm.3179 PMID: 23624599 Multiple types of T regulatory cells (Tregs) are involved in maintaining immune tolerance and preventing autoimmunity, including natural Tregs (nTregs) and inducible Tregs (iTregs). Unlike nTregs, which develop in the thymus, iTregs are induced in the periphery and consist of several subtypes that utilize different suppressive mechanisms and can be distinguished by the cytokines they produce. The different subsets of Tregs also express varying levels of Foxp3, the major transcription factor responsible for driving nTreg differentiation. T regulatory type 1 (Tr1) cells are a type of iTreg that express low levels of Foxp3 and produce high levels of the cytokine IL10. Tr1 cells have been shown to be highly immunosuppressive and capable of restoring tolerance in several immunemediated diseases. Elevated Tr1 cell levels have also been associated with improved outcomes following hematopoietic stem cell transplantation (HSCT). However, the study and clinical evaluation of Tr1 cells has been hindered by the lack of specific cell surface markers for isolating and tracking them. In this study, Nicola Gagliani and colleagues may have solved this problem. They demonstrate that the coexpression of two markers on CD4+ T cells, lymphocyte activation gene 3 (LAG-3) and CD49b, the α2 integrin subunit of very late activation antigen-2 (VLA-2), identifies Tr1 cells in both humans and mice.

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The authors first compared gene expression profiles between human Tr1 cell clones and undifferentiated CD4+ TH0 cells to identify T cell markers differentially expressed between the two subsets. They identified 17 differentially expressed genes, including the genes encoding LAG-3 and CD49b. Differential expression of the three markers was also confirmed at the protein level by FACS analysis.

CD49b and LAG-3 coexpressing CD4+ T cells isolated from human peripheral blood were shown to possess the key phenotypic features of Tr1 cells. Specifically, they were Foxp3 low, produced large amounts of IL10, and suppressed the proliferation of effector T cells in vitro.

In a previous study, the authors had shown that Tr1 cells accumulate in the small intestine of mice following treatment with CD3-specific monoclonal antibodies. In this study, the authors

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showed that CD4+CD49b+LAG-3+ T cells isolated from intestines and spleens from these mice produce high amounts of IL10, and suppress T cell proliferation in vitro. They also showed that the adoptive transfer of CD4+CD49b+LAG-3+ T cells from these mice could reduce colitis in a model of inflammatory bowel disease (IBD) indicating that CD49b and LAG-3 coexpression also identified mouse Tr1 cells.

The authors then showed that CD49b and LAG-3 were specifically coexpressed on Tr1 cells, and not TH1, TH2, TH17 or Foxp3+ Treg isolated from mice.

Human T cells cultured under Tr1-polarizing conditions were also shown to upregulate coexpression of CD49b and LAG-3. In addition, FACS sorted CD4+CD49b+LAG-3+ T cells were shown to be more suppressive and produce more IL10 than unsorted bulk Tr1 cultures indicating that CD49b and LAG-3 could be used to purify Tr1 cells following in vitro polarization.

Higher percentages of circulating CD4+CD49b+LAG-3+ T cells were measured in subjects with B-thalassemia who demonstrated persistent mixed chimerism following allogeneic HSCT, which has been shown to be associated with increased levels of circulating Tr1 cells.

This study identified two selective markers for Tr1 cells, in both humans and mice, that will facilitate the investigation of these cells in a variety of clinical and experimental settings. Ultimately, use of these markers should make it possible to isolate and track Tr1 cells, and could lead to novel therapeutic strategies for transplantation, cancer, and autoimmune diseases.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

POTENTIAL VACCINATION APPROACH FOR DIVERTING NATURAL IMMUNODOMINANCE AND TARGETING MORE DIVERSE T CELL EPITOPES Research Paper: Hansen S.G. et al. Cytomegalovirus Vectors Violate CD8+ T Cell Epitope Recognition Paradigms.

Science 2013; 340, 1237874.

PMID: 23704576 CD8+ T cells detect intracellular pathogens through T cell receptor (TCR)-mediated recognition of short 8-10 amino acid long pathogen-derived peptides (epitopes) loaded onto and presented by MHC class I (MHC-I) on the surface of infected cells. Although thousands of epitopes can potentially be generated by a pathogen, T cell responses are generally restricted to relatively few pathogen-derived epitopes (immunodominant epitopes). Despite this, most pathogens can be controlled by T cell responses directed against narrow pools of immunodominant epitopes. However, other pathogens, such as HIV and its simian relative SIV, can evade immunodominant T cell responses and are unable to be controlled by naturally induced immune responses. In this study, Scott Hansen and colleagues demonstrate that it may be possible to genetically manipulate vaccine vectors in order to direct T cell responses against a broader noncanonical repertoire of epitopes. They show that this vaccination strategy can induce protective immune responses against SIV. •

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In a prior study, the authors showed that vaccination of rhesus macaques with SIV proteinencoding rhesus cytomegalovirus (RhCMV) vectors resulted in protective immune responses in approximately 50% of treated animals. However, virologic control was not associated with enhanced T cell responses to canonical SIV epitopes.

In this study, the authors used intracellular cytokine analysis to compare SIV epitopes recognized in rhesus macaques with naturally controlled SIV infections, or after vaccination with conventional SIV vaccines or RhCMV vectors.

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As previously shown, RhCMV vectors failed to elicit CD8+ T cell responses against canonical epitopes associated with SIV infection and conventional SIV vaccination. Instead, RhCMV vaccination induced responses to a broader (> 3 times greater number of epitopes) repertoire of highly promiscuous unconventional epitopes, over half (63%) of which were restricted to MHC class II (MHC-II).

CD8+ T cells recognizing these unconventional SIV epitopes were capable of recognizing SIVinfected cells indicating that the processing and presentation of these unusual epitopes are CMV-independent. However, CMV gene expression was required for priming T cell responses directed against them.

To determine which RhCMV genes were responsible for redirecting T cell responses, vectors with different RhCMV gene deletions were tested. The RhCMV-encoded Rh189 gene (human CMV US11 ortholog) was shown to be responsible for suppressing canonical CD8+ T cell responses, and T cell responses against unconventional epitope were only induced in the absence of Rh157.5, Rh157.4 and Rh157.6 (human CMV UL128, UL130 and UL131 orthologs).

These data suggest that hierarchies of CD8+ T cell responses are more flexible than had been thought, and may primarily be controlled by immunoregulation during priming rather than by limitations in antigen processing and presentation or the composition of the T cell repertoire. These data also identified several CMV genes capable of regulating the profile of epitopes recognized by CD8+ T cells, providing a novel approach for selectively programming CD8+ T cell responses with CMV vector – based vaccines.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

HUMAN IMMUNOPHENOTYPING UPDATE DETECTION AND ANALYSIS OF RARE EVENTS BY FLOW CYTOMETRY By: J. Philip McCoy, Jr., National Heart, Lung and Blood Institute (NHLBI) and the Center for Human Immunology, National Institutes of Health There is an ever-increasing demand for those using flow cytometry to detect and quantify rare events, such as dendritic cell subsets, circulating tumor cells (CTCs), circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs), to name a few. Whereas the enumeration of relatively abundant T cell subsets such as CD4 and CD8 is straightforward, the accurate detection of rare events demands rigorous attention to elimination of potential artifacts. There is no frequency of events that defines what is ‘rare’, but it is generally accepted that cellular populations constituting less than 1% of the total population require special measures to ensure the accuracy of immunophenotyping. In this article, we will review sources of potential artifact in rare event analysis and strategies to cope with these pitfalls, regardless of the rare population to be detected. Specific marker combinations for various rare populations will not be addressed, as these are often controversial and would require more discussion than is permitted by current space.

Sources of artifacts and potential solutions: 1. Dead cells. Dead cells are a key source of non-specific staining as these often bind, or trap, various fluorochrome-conjugated antibodies. This can lead to dead cells being mis-identified as the rare cells of interest. Dead cells are seldom a problem when immunophenotyping abundant populations in fresh peripheral blood of healthy donors, as the overwhelming majority of leukocytes are live in these specimens if processed promptly. However, when attempting to detect rare events such as CTCs or CECs, which may have a frequency of 1 cells per million leukocytes (or less), even an occasional dead cell in the sample can be a problem. To address this problem one can use of a viability stain to identify the dead cells and exclude them from analysis. A number of stains are available for this purpose, ranging from propidium iodide and other nuclear dyes to the newer live/dead stains that are available with various emission spectra. By carefully selecting the appropriate viability stain, these can be incorporated into most antibody staining panels without a problem.

2. Cell doublets. Cytometers recognize events, or particles, passing through the laser beam, but do not recognize cells per se. Thus two cells (or more), or one cell and debris, stuck together will be recognized as only a singular event. If these cells have different phenotypic characteristics, the detected event will appear as a composite of these phenotypes, resulting in a misinterpretation of the actual staining of each cell. This can lead to a myriad of confusing phenotypes. Exclusion of cell doublets can be performed by pulse width analysis, and by simply acquiring both area and height measurements of forward and side scatter, plotting these parameters against one another, and excluding outliers from further analysis.

3. Nucleation. With the exception of mature erythrocytes and platelets, all cells in the human peripheral blood are nucleated. A least one group has reported that clumps of platelets can be misinterpreted as CECs (ref), and it is likely clumps of cellular debris could also be misinterpreted. The inclusion of nuclear stain such as Hoechst, which can stain nuclei in viable cells, will ensure that the events identified are nucleated and therefore cellular. It should be emphasized that this staining is distinct from the viability stain described above, which

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identifies dead cells, sometimes by staining nucleic acids. Those stains are not membrane permeant whereas dyes such as Hoechst are membrane permeant.

Collection of sufficient events. Rare events are often found at frequencies of 1 event per hundred thousand or per million leukocytes. It should be evident therefore that the collection of a 10,000 or 20,000 event file will not suffice. Even file sizes as large as 100,000 or 1 million events may not be sufficient to gather statistically significant, and reproducible, data (1). In planning these analyses one should look at the expected frequencies of the rare events and plan to acquire files large enough to contain an appropriate number of rare events that will satisfy a consulting statistician. These files may contain millions of events and could require a lengthy period of time to acquire (perhaps even an hour per file).

Carry-over in the cytometer. An often overlooked source of artifacts in flow cytometry is the carry-over of cells from previous experiments. The small diameter tubing in cytometers can harbor cells after analysis, even though virtually all modern cytometers have systems to backflush these events out. Nonetheless there can be a small number of residual events that are not flushed out, but can be carried over into the next experiment. In most routine analysis, this contamination is of such low magnitude that it is doesn’t interfere with analysis of the data.

In rare events analysis, where acquisition may take a long time, perhaps even an hour for one file, these rare contaminants can be problem. An easy test is to run a particle-free solution as a sample on the cytometer for 30 minutes and note the number of events collected. Removal of the contaminants can generally be accomplished by a thorough cleaning of the instrument (30 minutes or more). Efficacy of cleaning can be monitored by repeating the test above and looking for a decrease in the number of events collected.

Interruption in the fluidics. Flow cytometry is dependent upon a stable fluidic system delivering the cells in a focused stream though points of laser excitation. While these fluidic systems are generally stable, minor fluctuations can occur, particularly when large data files are acquired over a long period of time. Such fluctuations have been demonstrated to directly affect to fluorescence detected on the cells (2). Brief changes in fluorescence could lead to false positive events. Causes of these fluctuations can include small bubbles, vibrations, and air supply interruptions, among others. Perturbations in the fluidics can easily be monitored in each experiment by including “time” as a parameter. This simply lets one display any parameter (usually a fluorescence channel) against the relative time at which it was acquired.

By plotting this, any fluctuations in the fluidics become readily apparent and can be gated out from further analysis.

Non-specific binding of antibodies. A well-known source of artifact in flow immunophenotyping is the non-specific binding of antibodies to irrelevant cells. The most common solution for this is the use of heat-inactivated normal serum to pretreat the cells and take up these binding sites, although FcBlock (a cocktail of CD16+CD32) is also used for this purpose. When staining human cells with mouse monoclonal antibodies there is some debate whether it is best to use mouse serum or human serum. Strong arguments can be made for either. Our laboratory uses mouse serum because of easy availability and reduced biohazard concerns.

Exclusion of cells not of interest. (“Eliminate all other factors, and the one which remains must be the truth.” – Sherlock Holmes). For extremely rare events it is often quite helpful to gate out more common events that express markers not found on the rare cells. For CTCs and CECs, this is usually CD45, as it is present on all leukocytes but neither CTCs nor CECs. In other situations, a lineage mixture (such as CD19+CD3+CD33+CD14 all in the same fluorophore) might be used if it is suspected that the target cells might have dim CD45 expression. By

gating out the predominant population of cells, it now becomes an easier task to use specific markers to identify the rare populations.

In addition to the principles above, investigators undertaking rare event analysis should also consider how they will report their findings. The unit of measure should be stated (events per ml of blood, per 1 million mononuclear cells?) and it should be clearly stated how these calculations were made. The investigator should also be cautious about stating sensitivity levels of their assays based on spiking experiments with cell lines, as cell lines often have different antigen intensities, cell size, and autofluorescence, than do the actual rare events. Consideration should also be given to the precision of the assay – if the same sample is run ten times, what is the variance of the results? Finally, there should be strong consideration to validation of the assays. How does one know that the rare cell identified is actually the cell type of interest and not an artifact? Can that cell be sorted and used for single cell PCR to validate the phenotype?

Rare event analysis by flow cytometry is tedious and filled with potential artifact, but it is hoped that the principles outlined above will be of use for laboratories attempting these assays, and ultimately result in more reproducible data. Listed below are suggested references for further reading.

References and Suggested Reading: 1.

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Roederer M: How Many Events Is Enough? Are You Positive? Cytometry Part A. 73A (2008) 384385.

Seamer L, Sklar LA: Time as a flow cytometric parameter. Methods Cell Biol. (2001) 63:16983.

Khan et al: Detection of Circulating Endothelial Cells and Endothelial Progenitor Cells by Flow Cytometry. Cytometry Part B (Clinical Cytometry) (2005) 64B:1–8.

Donnenberg & Donnenberg. Rare-Event Analysis in Flow Cytometry. Clin Lab Med 27 (2007) 627–652.

Tibbe et al: Statistical Considerations for Enumeration of Circulating Tumor Cells. Cytometry Part A (2007) 71A 154-162.

Coumans et al: Challenges in the Enumeration and Phenotyping of CTC. Clin Cancer Res (2012) 18:5711-5718 Hedley and Keeney: Technical issues: flow cytometry and rare event analysis. Int J Lab Hematol (2013) 35:344-350

CONCEPTS IN BASIC IMMUNOLOGY: PRINCIPLES AND THERAPEUTIC APPLICATIONS INNATE LYMPHOID CELLS: NEW AWARENESS OF SOME OLD PLAYERS IN IMMUNITY AND DISEASE Andrew H. Lichtman, MD, PhD, Brigham and Women's Hospital Introduction Progress in understanding the innate immune system, which is phylogenetically older and arguably more essential for day to day survival than the adaptive immune system, had been shadowed for several decades by the brisk pace of advances in the molecular and cellular basis of adaptive immunity beginning in the 1980’s. The discovery of immune function of a fly Toll like receptor (TLR) in 1996 and characterization of mammalian TLRs soon after heralded the beginning of a new phase in innate immunity research, in which the molecular basis of pathogen associated pattern recognition and responses has been elucidated in great detail. Nonetheless, it appears that we remain in a catchup phase in which fundamental knowledge of the cellular effectors of innate immune responses are still being sorted out. A prime example of this is the emerging knowledge of subsets of innate lymphoid cells (ILCs) that secrete different cytokines typically attributed to helper T cell subsets.

These ILC subsets, some of which were not even known to exist a few years ago, appear to be of central importance in constitutive barrier immunity, innate responses to infection, and the pathogenesis of inflammatory diseases. The majority of research on ILCs has been done in murine models, which allow fate mapping studies and elimination of adaptive immune sources of cytokines, both of which have been essential for discovery in this field. Human ILCs comparable to the newly identified murine cells have also been found, and their roles in human disease are now being actively studied.

Distinguishing Features of Innate Lymphoid Cells ILCs are bone marrow derived, and have the morphology of lymphocytes, but they do not rearrange or express TCR or Ig genes, nor do they express cell surface markers typical of myeloid cells. ILCs do arise from the common lymphoid progenitor and require expression of the IL-7 receptor or, in the case of NK cells, the 1L-15 receptor. In contrast to B and T cell lineages, ILC development also requires the transcriptional regulator called inhibitor of DNA-binding 2 (ID2). Two types of ILCs that have been thoroughly investigated for many years include Natural Killer (NK) cells and lymphoid tissue-inducer (LTi) cells. NK cells were first described in 1975, and have similar anti-viral functions as CD8+ cytotoxic T lymphocytes. LTis, which were first described in 1997, produce cytokines required for the formation of lymphoid tissues during embryonic development. In adults they are needed for regeneration of lymphoid organ structure after viral infections. More recently, additional members of the ILC family of cells distinguished by the cytokines they produce have been identified, and the grouping of ILCs into three subsets, all arising from a common ID2+ precursor, has been proposed(1) .

These three subsets have distinct transcription factor and cytokine expression profiles which are respectively similar to TH1, TH2 and TH17 cells.

Innate Lymphoid Cell Subsets Group 1 ILCs include IFNγ and TNF expressing cells, some of which require the T-bet transcription for development and function, all features shared by TH1 cells. The prototype of this subset is NK cells, mentioned above. There is some controversy about the T-bet dependency of most NK cells, and some investigators in this field do not agree that NK cells should be grouped with other ILCs. In addition, T-bet-dependent IFNγ secreting ILCs with minimal cytotoxic function are also put in Group 1, even though they may arise from plastic Group 3 ILCs, described below. The major functions of Group 1 ILCs are immunity against viruses and other intracellular pathogens, via perforin and granzyme mediated cytotoxicity and by enhancement of inflammatory responses. Group 2 ILCs

(previously called natural helper cells or nuocytes) mainly produce one or more of the “type 2” cytokines IL-5, IL-9, IL-13 as well as an epidermal growth factor-related cytokine called amphiregulin, and they require the transcription factors GATA3 and RORα for their development and function. In mice, group 2 ILCs are required for protection against helminths and also for homeostasis and amphiregulin mediated repair of bronchial epithelium. Group 3 ILCs produce either or both IL17A and IL-22, and depend on the transcription factor RORγt as well as the aryl hydrocarbon receptor (AHR) for development and function, features shared by TH17 cells. Within this Group 3 subset are LTi cells, mentioned earlier, which produce LTα, LTβm, IL-17A and IL-22. Two additional types of Group 3 ILCs have been identified, which exist mainly in intestinal mucosa. These include NCR+ ILC3 cells, which produce IL-22 and express the NK activating receptor NCR1, and NCR-ILC3 cells which produce IFNγ, IL-17 and IL-22 but not NCR1. While LTi cells are uniquely involved in formation of lymphoid tissues, all three identified types of Group 3 ILCs cells contribute to immunity to certain extracellular bacteria. In addition, the IL-22 secreting ILC3 subtypes maintain mucosal epithelial barrier homeostasis, and in the gut ILC3 cells expressing class II MHC and IL-22 present antigen to and suppress CD4+ T cell responses to commensal bacteria (2).

Human ILCs have been described that have cytokine expression patterns similar to those of Group 1, 2 and 3 subsets. The different human ILCs are identifiable as LIN- cells (lineage-negative referring to the absence of markers of T and B cells) that express the same transcription factors seen in the mouse subsets, but have different sets of surface markers than the mouse cells (3). Group 1 human ILCs include the classic NK cells, as well as CD56+ NCR1,2,3+IL-7Rα- poorly cytotoxic ILC1s. Human Group 2 ILCs express ST2 (IL-1RL1 or IL-33 receptor), IL-17RB, and CRTH2 (prostaglandin D2 receptor 2). Group 3 human ILCs include ILC3 cells that are CD56+ NCR1,2,3+IL‑7Rα+, and LTi cells that are IL‑7RαhiCD45intRORγt+.

Activation and Stability of ILC Subsets The signals that simulate cytokine production by ILCs are only partially understood, but a common theme across the subsets is that cytokines from other cell sources are the major signals that induce ILC cytokine expression (1). For Group 1 ILCs, it has been known for many years that NK cells can be activated to secrete IFNγ by other cytokines, including IL-12, IL-15, IL-18, and IL-2, independent of signals from the NK activating receptors that bind to membrane bound ligand on other cells. IL-5 and IL-13 secretion by ILC2 cells is stimulated by IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) produced by epithelial cells. The homeostatic role of ILC3 cells in the intestine is mediated by continual steady state IL-22 secretion, i.e in the absence of infection, and this is dependent on IL-1 from mononuclear phagocytes exposed to commensal bacteria, while IL-22 secretion by ILC3 cells in response to infection depends on mononuclear IL-23.

Some plasticity has been documented in ILCs, and cytokines may drive conversion of one phenotype to another. In particular, IL-12 and IL-18 may act on IL-22 secreting ILC3 cells and convert them to IFNγ secreting ILC1 cells, and this switch may be accompanied by loss of RORγT and gain of T-bet expression. This is a similar story to that described for TH17 cells changing into TH1 cell in some mouse models of autoimmunity.

The Role of ILC Subsets in Disease Pathogenic roles of ILCs have been uncovered in several mouse models of inflammatory diseases.

Many of these roles are mediated by cytokines that helper T cell subsets can produce, but the importance of the ILCs as a source of the cytokines in vivo is supported by studies in Rag knockout mice, which lack T (and B) cells. ILC2 cells contribute to airway disease, including virus induced airway hyperactivity and allergy and allergic asthma, both dependent on IL-13, and papain-induced airway inflammation, a purely experimental model dependent on IL-9. Rag-/- mice treated with antiCD40 or infected with Helicobacter hepaticus develop colitis, and IL-17 and /or IFNγ producing ILCs are required for the intestinal inflammation to develop. It is likely that the pace of work on human

disease associations of different ILC subsets will increase rapidly. Recent examples include reports of increased blood ILC1 cells in severe asthmatic patients (4), and high frequency of intestinal IFNγ producing ILC1 cells in patients with Crohn’s disease (5). A major barrier to assessing the contribution of ILCs to disease, both in experimental models and in humans, is a paucity of tools to selectively purify and delete these subsets from otherwise immunologically intact individuals.

References 1. Spits, H., D. Artis, M. Colonna, A. Diefenbach, J. P. Di Santo, G. Eberl, S. Koyasu, R. M. Locksley, A. N. McKenzie, R. E. Mebius, F. Powrie, and E. Vivier. 2013. Innate Lymphoid Cells--A Proposal for Uniform Nomenclature. Nat Rev Immunol 13: 145-149.

2. Hepworth, M. R., L. A. Monticelli, T. C. Fung, C. G. Ziegler, S. Grunberg, R. Sinha, A. R. Mantegazza, H. L. Ma, A. Crawford, J. M. Angelosanto, E. J. Wherry, P. A. Koni, F. D. Bushman, C. O. Elson, G. Eberl, D. Artis, and G. F. Sonnenberg. 2013. Innate Lymphoid Cells Regulate CD4 T Cell Responses to Intestinal Commensal Bacteria. Nature.

3. Walker, J. A., J. L. Barlow, and A. N. McKenzie. 2013. Innate lymphoid Cells--How Did We Miss Them? Nat Rev Immunol 13: 75-87.

4. Barnig, C., M. Cernadas, S. Dutile, X. Liu, M. A. Perrella, S. Kazani, M. E. Wechsler, E. Israel, and B. D. Levy. 2013. Lipoxin A4 Regulates Natural Killer Cell and Type 2 Innate Lymphoid Cell Activation in Asthma. Science of Translational Medicine 5: 174ra126.

5. Bernink, J. H., C. P. Peters, M. Munneke, A. A. te Velde, S. L. Meijer, K. Weijer, H. S. Hreggvidsdottir, S. E. Heinsbroek, N. Legrand, C. J. Buskens, W. A. Bemelman, J. M. Mjosberg, and H. Spits. 2013. Human Type 1 Innate Lymphoid Cells Accumulate in Inflamed Mucosal Tissues. Nat Immunol 14: 221-229.

Figure. Subsets of Innate Lymphoid Cells ( ILCs). The common lymphoid precursor ( CLP) gives rise to an ILC precusor that expresses the inhibitor of DNA-binding 2 (ID2) transcription factor, which in turn gives to all the different types of ILCs. IL-7 is required for all ILC differnataton except NK cells, which require IL-15. The transcription factors required for each pathway of differnataton are shown in the boxes overlying the solid blue arrows. The cytokines produced by each ILC type are shown in the pink boxes. Three groups of ILCs, indicated at the left, are distinguished by the types of cytokine they produce, analagous to Th1, Th2 and Th17 cells.The cytokines that activate the ILCs to produce their signture cytokines are shown over the dotted arrows.

SELECTED RECENT CLINICAL TRIAL RESULTS Edited by Carla J. Greenbaum, MD, Benaroya Research Institute

PHASE 2A TRIALS OF DUPILUMAB IN PERSISTENT ASTHMA WITH ELEVATED EOSINOPHIL LEVELS Clinical Trial: Wenzel S. et al., Dupilumab in Persistent Asthma with Elevated Eosinophil Levels. New England Journal of Medicine. Published online May 21, 2013 PMID: 23688323 Disease: Asthma Drug: Dupilumab is a fully human monoclonal antibody to the IL-4 receptor α subunit that inhibits both IL-4 and IL-13 signaling. In this trial, 300mg of dupilumab (or matching placebo) were administered as SQ weekly injections for 12 weeks.

Study Design: • Phase 2a randomized, placebo-controlled trial assessing whether dupilumab is safe and effective in patients with persistent, moderate-to-severe asthma and elevated eosinophil levels.

• Patients were randomly assigned to receive once weekly subcutaneous (SQ) injections of dupilumab (n=52) or matching placebo (n=52) for 12 weeks. Patients also initially received standard therapy of fluticasone, an inhaled glucocorticoid, and salmeterol, a long-acting beta agonist. The salmeterol was discontinued at week 4, and the fluticasone was tapered and discontinued from weeks 6 through 9. Thus, in the early part of the study the effects of dupilumab were evaluated in addition to two or one drug standard-of-care therapy and later as monotherapy.

• Patients (age 18-65) were those not well-controlled on standard therapy with moderate-tosevere asthma (with lung function measured as FEV1 ≥ 50%) and elevated blood eosinophil count (≥300 cells per µL) or elevated sputum eosinophil level (≥3%).

• The primary efficacy endpoint was occurrence of an asthma exacerbation. A number of Th2associated biomarkers were also measured including the eosinophil-attracting eotaxins (CCL26) and thymus and activation-regulated chemokine (TARC, or CCL17). These are C-C chemokines produced by structural cells (such as fibroblasts and epithelial cells) that are known to be upregulated by Th2 cytokines.

Results: • A total of 491 patients were screened in order to identify 104 eligible patients for randomization. Two-hundred sixty (260) of these screen failures were due to low eosinophil count.

• The primary endpoint was met: 23 patients on dupilumab had an exacerbation, while only 3 patients on dupilumab did (p<0.001), an 87% reduction in exacerbations with dupilumab.

The clinical relevance of the definition of exacerbations in this study is uncertain. Moreover, in the time to exacerbation analysis, the survival curves only start to favor the dupilumab group after withdrawal of standard therapy.

• Patients on dupilumab had significantly higher absolute FEV1 and asthma symptom questionnaire scores starting at week 2 and week 3, respectively. These differences were both statistically and clinically significant (e.g. absolute FEV1 improvement of 200ml). TARC,

eotaxin-3, and IgE levels were significantly lower by week 4, and were sustained or lower through the duration of the study. While the majority of patients had little or change in peripheral blood eosinophil levels in either group, 4 patients in the dupilumab group had increased blood eosinophilia suggesting a reduction in the migration of eosinophils from the blood to the lung compartment.

There were so significant safety issues or serious adverse events related to study drug. More patients on dupilumab experienced injection-site reactions, nasopharyngitis, nausea, and headache.

Among the adverse events (AE) leading to discontinuation of study drug was one related-AE of angioedema in the dupilumab group.

Why the trial is of interest to the broader FOCIS community: With increased understanding of mechanisms broadly underlying inflammatory and immune mediated disease, targeted therapies such this one inhibiting IL-4 and IL-13 therapy have been developed.

Previous trials of the monoclonal antibodies lebrikizumab and tralokinumab did not demonstrate improvements in asthma symptoms and quality of life; this may be attributed to the fact that these therapies only target IL-13 or because of patient selection.

Thus equally important to identifying targets for disease pathogenesis is identifying the right subjects out of the heterogenous population with clinical evidence of disease. In this study, investigators built on the observed relationship between hypereosinophilia and therapeutic outcome in trials with other agents targeting these pathways. The investigators enrolled only those individuals with a hypereosinophilia as well as those with poorly controlled disease on standard therapy. While this selection targeted only ~20% of otherwise eligible subjects, it enhanced the probability that the drug would prove effective for the endpoints tested. Though the clinical importance of some of these endpoints is debatable, the marked effect on the primary endpoint as well as the clear demonstration of changes in targeted biomarkers make this an important proof of concept study. The concept being proved is not only that a correct target was selected; but that selectively enrolling subjects may limit the non-responders and enable a therapy to continue towards clinical development that otherwise might have been discounted. The down side of this approach from a clinical or public health point of view is that a therapy that is effective in only a selected group of subjects may still be demanded by clinicians and patients with the same disease without the same characteristics, thus resulting in increased use (and therefore costs) of a therapy without real benefit. Ultimately, results of this Phase 2 study showing clinical efficacy with withdrawal of background therapy will need to be replicated in long-term, Phase 3 studies before it is known whether it will be employed in treating those with asthma someday. These issues in no way detract from the importance of this trial demonstrating that targeting therapeutics to the right population can give you the results you expect.

Reviewed by Steven Lamola, MD Benaroya Research Institute

PHASE 2 TRIAL OF ANTI-IP-10 ANTIBODY FOR ULCERATIVE COLITIS Clinical Trial: Mayer L. et al., Anti-IP-10 antibody (BMS-936557) for ulcerative colitis: a phase II randomised study.

Gut 2013;0:1-9. PMID: 23461895 Disease: Ulcerative Colitis

Drug: BMS-936557 (formerly MDX-1100) is a fully human monoclonal antibody that targets the chemokine interferon-γ-inducible protein-10 (IP-10), also referred to as CXCL10.

Study Design: • •

•

8-week, Phase II, double-blind, multicenter, randomized study to evaluate safety and efficacy of BMS-936557 in patients with moderately-to-severe active ulcerative colitis (UC).

Patients were randomly assigned to receive 10 mg/kg to 1000 mg of BMS-936557 (n=55) or matching placebo (n=54) intravenously every other week for 8 weeks. Eligible patients were ≥18 years old with an active UC flare within 2 weeks of enrollment while on stable treatment with standard, non-biologic therapy.

The primary efficacy endpoint was the rate of clinical response 2 weeks after the last study treatment. Clinical response was defined on the basis of standardized scores which include patient reported symptoms such as stool frequency and rectal bleeding.

Results: •

•

•

The primary and secondary efficacy endpoints were not met in this study. The proportion of patients who achieved clinical response was not significantly different in the BMS-936557treated group (52.7%) versus placebo-treated group (35.2%; p=0.083). However, patients who failed to adequately record symptoms were considered non-responders. That means that all such patients (n=7) were analyzed as if the drug was not helpful. This “imputation model” is an accepted statistical approach to deal with missing data points. If instead of imputing these subjects’ results as being negative the trial was analyzed (post-hoc) using the data that actually was available, the clinical response rates were significantly higher in the active treatment group.

The investigators also explored (post-hoc) the relationship of the drug levels [(trough concentrations (Cmin)] with clinical response rate. Sixteen patients with the highest Cmin values were observed to have a significantly higher clinical response rate (87.5%) versus the placebo group (p<0.001). Also, a significantly greater proportion of patients with BMS-936557 Cmin ≥ 100 µg/ml were determined to be in histological remission compared with placebo (73% vs.

41%, p=0.004), exhibiting a marked reduction in inflammatory infiltrates and decrease in erosion, ulceration and crypt destruction.

There were no related significant adverse events or deaths during this study. The BMS936557-treated group had a numerically higher frequency of infections compared with placebo. This was not the case in a previous Phase 2 study of BMS-936557 in patients with rheumatoid arthritis. The safety profile was the same for patients in the Cmin ≥ 100 µg/ml subgroup.

Why the trial is of interest to the broader FOCIS community: Chemokines and chemokine receptors are central to the inflammatory process and therefore attractive therapeutic targets. This trial illustrates a potential advantage of targeting chemokines as opposed to their receptors in specific autoimmune diseases. Binding of the chemokine IL-10 to its receptor, CXCR3, induces differentiation and recruitment of Th1 and Th17 cells responsible for inflammation and tissue destruction. In addition, IL-10 has been shown to operate independently of its receptor, as a direct inhibitor of epithelial and endothelial cell proliferation,1 and as a contributor to bone destruction (by upregulating RANKL)2 and apoptosis of pancreatic β-cells.3 Why then did this trial have a negative outcome? This trial illustrates how significantly study design can impact efficacy outcomes. Failure to meet the primary endpoint might be attributed to insufficient compliance on the part of participants to complete the electronic diary of symptoms. Thus, this trial not only had a negative outcome, it is a failed trial in that we are still unclear as to whether or not this therapy is worth pursuing. Though use of imputation attributing missing data as negative is an appropriate pre-specified analysis plan; this trial also illustrates that the choice of analytic approach may often dramatically affect the “results” of a study.

In addition, the relationship between drugs levels and outcome suggests that a higher dose might have resulted in a different outcome. Because this was a post-hoc analysis, even this premise would need additional testing. There is still reason to believe that, at the right dose, an anti-IP-10 antibody may be effective in UC because of both inhibition of Th1 cell differentiation and trafficking, and promotion of crypt epithelia cell proliferation and regeneration. Based on both this study in UC and the previously published Phase 2 study in rheumatoid arthritis – which showed modest but significant response –,4 additional dose-ranging studies of BMS-936557may be informative.

References: 1.

2.

3.

4.

Campanella et al., PLoS ONE 2010: 5(9): e12700.

Kwak et al., Arthritis Rheum 2008:58:1332-42.

Schulthess FT et al., Cell Metab 2009:9:125-39.

Yellin M et al., Arthritis Rheum 2012:64(6): 1730-1739.

Reviewed by Steven Lamola, MD Benaroya Research Institute

INTERLEUKIN-1 ANTAGONISM IN TYPE 1 DIABETES OF RECENT ONSET Clinical Trial: Moran A. et al., Interleukin-1 Antagonism in Type 1 Diabetes of Recent Onset: Two Multicenter, Randomized, Double-blind, Placebo-controlled Trials. Lancet. Published online April 5, 2013 PMID: 23562090 Disease: Type 1 Diabetes Drugs: Canakinumab is a human monoclonal anti-IL-1 antibody, given in this trial as a monthly subcutaneous (SQ) injection of 2mg/kg (maximum 300 mg). Anakinra is a recombinant human IL-1 receptor antagonist, given as a 100mg SQ dose every morning.

Study Design: • •

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Results of two randomized, placebo-controlled Phase 2a trials assessing whether interleukin-1 blockade improves β-cell function in recent-onset type 1 diabetes (T1D) have been published in the same Lancet paper.

In one study, 69 patients were randomly assigned to receive 12 monthly injections of either canakinumab (n=47) or placebo (n=22). Eligible patients were 6-45 years old at onset of T1D diagnosed within 100 days of enrollment, were positive for at least one diabetes-associated autoantibody, and had a stimulated peak C-peptide during mixed meal tolerance test (MMTT) ≥ 0.2 nmol/L. In the other study (AIDA Study) 69 patients were randomly assigned to receive daily injections of anakinra (n=35) or placebo (n=34) for 9 months. Eligible patients were 18-35 years old, had their first reported symptoms of T1D within 12 weeks of enrollment, were positive for GAD-65 autoantibodies, and had a stimulated peak C-peptide during MMTT ≥ 0.2 nmol/L. All subjects in both trials received standard intensive diabetes treatment.

The primary endpoint of both studies was comparison of the area under the curve (AUC) stimulated C-peptide response over a 2-hour MMTT, at the end of the treatment period.

Results: •

• •

The primary endpoint was not met in either study: stimulated C-peptide concentrations did not differ between the active treatment and placebo arms. The difference in AUC C-peptide in the canakinumab study at 12 months was 0.01 nmol/L (95% CI -0.11 to 0.14; p=0.86); in the anakinra study at 9 months it was 0.02 nmol/L (95% CI -0.09 to 0.15; p=0.71).

Secondary endpoints also did not demonstrate significant difference between active and placebo groups: e.g. in neither trial was HgA1c or insulin dose over time different between treatment arms.

There were no significant safety signals in either study, with the exception of a higher rate of moderately severe injection site reactions in the anakinra group versus placebo. Despite the possible anti-inflammatory effects of these drugs, neither active treatment group experienced more frequent or severe infections. Neutropenia also did not differ significantly between active and placebo groups.

Why the trial is of interest to the broader FOCIS community: The failure of these trials to demonstrate efficacy of IL-1 blockade in T1D in no way refutes the decades of preclinical research implicating this master cytokine as an important player in the pathogenesis of T1D. There is substantial evidence that IL-1 (or specifically IL-1β) has both direct toxic effects on β-cells and promotes expansion and differentiation of pathologic T-cells, including Th1

and Th17 cells. Instead, these trials illustrate that it is not enough to pick the right target. Equally critical is intervening on that target at the stage of disease development at which research suggests it plays its major role. As pointed out in this paper, IL-1β gene expression in peripheral blood monocytes is elevated at diagnosis of T1D, but levels are back to normal within 1 month of diagnosis.

It is also known that diabetes autoantibody-positive first-degree relatives of individuals with T1D – who are at higher risk of developing T1D – have higher levels of IL-1β expression by monocytes and dendritic cells. It is therefore reasonable to expect that it may be too late to neutralize IL-1 after development of T1D. It should also be pointed out that blockade of IL-1β signaling has only slowed progression to diabetes in NOD mouse, not prevented or reversed disease. Therefore, in addition to intervening at the right time, use of a therapy like IL-1β blockade may only be effective in rational, synergic combination with other agents.

Reviewed by Steven Lamola, MD Benaroya Research Institute

TRIAL OF SIROLIMUS CONVERSION TO REDUCE RISK OF SCC RECURRENCE IN RENAL TRANSPLANT RECIPIENTS Clinical Trial: Hoogendijk-van den Akker JM et al. Two-Year Randomized Controlled Prospective Trial Converting Treatment of Stable Renal Transplant Recipients With Cutaneous Invasive Squamous Cell Carcinomas to Sirolimus. Journal of Clinical Oncology 2013; 31(10):1317-23. PMID: 23358973 Disease: Squamous cell carcinoma (SCC) in setting of renal transplantation Drug: Sirolimus is an inhibitor of mammalian target of rapamycin (mTOR), a key serine-threonine kinase involved in regulation of cell growth and proliferation.

Study Design: • Randomized trial to evaluate recurrence of cutaneous invasive squamous cell carcinoma (SCC) in renal transplant recipients two years after assignment to either changing their immunosuppression regimen to include sirolimus (n = 74) or maintaining current immunosuppression (n = 81). Patients and transplant physicians were aware of trial assignment, however, evaluating dermatologists and pathologists were not (i.e. they were masked).

• Participants were adults who were more than 12 months post-kidney transplant and had at least one biopsy-confirmed cutaneous invasive SCC. Subjects were all receiving maintenance calcineurin inhibitor, azathioprine, mycophenolate, and/or steroids for at least 12 weeks before randomization.

Exclusion criteria included metastatic cutaneous SCC and internal malignancies. Randomization was stratified by transplantation center, number of SCCs, and recipient age.

• Evaluation of invasive SCC recurrence was conducted by a dermatologist every three months (suspect lesions were biopsied for interpretation by a dermatopathologist). Laboratory data were obtained every three months, and adverse events evaluated through the two years of the study.

Results: •

At the primary analysis timepoint of 2 years, subjects who were converted to sirolimus experienced a nonsignificant reduction in risk of SCC recurrence (HR of 0.76, p=0.255) compared with subjects who remained on conventional immunosuppression. However, there

•

was a significant 50% risk reduction in development of new lesions after one year (p=0.021).

Similarly, the subgroup of subjects who entered the trial with only one previous SCC were significantly less likely to develop a new lesion after one year (p=0.044).

42% of subjects discontinued sirolimus because of adverse events.

Why the trial is of interest to the broader FOCIS community: The risk of immunosuppressive drugs causing lymphoma – specifically lymphomas positive for Epstein-Barr virus (EBV) – has been recognized for some time. Less appreciated is the fact that cutaneous squamous cell carcinomas (SCCs) are the most common post-transplantation cancers.

The risk of SCC in an organ transplant recipient is reportedly 65 to 250 times that of the general population, an effect usually attributed to inhibition of T cell-mediated immune surveillance. However, other mechanisms have also been proposed. There is experimental evidence as well as previous clinical studies that suggest use of mTOR inhibition would reduce the risk. For example, mouse studies suggest that calcineurin inhibitors (cyclosporine and tacrolimus) directly promote cancer development via a TGF-β mechanism and that mTOR inhibitors may block tumor progression in various cancer models. Understanding the mechanism for this markedly increased risk and what strategies may reduce this risk is important for immunotherapeutic approaches in other inflammatory and autoimmune diseases.

Why then, was the primary measure in this trial (reduction of new SCC two years after randomization to sirolimus) negative – i.e. why didn’t the change in immunosuppression regimen significantly reduce new cancers? The answer is that we don’t know because almost half the subjects assigned to sirolimus stopped taking the drug. This is an example therefore of a failed clinical trial – not because the primary outcome was negative, but because the results leave us without an answer to the question posed.

The demonstration that the exploratory endpoints at 1 year suggested a benefit of sirolimus only indicates that a new study is needed. As noted by the authors, additional testing of this concept will require strategies to reduce the adverse effects of sirolimus.

Reviewed by the European Society of Organ Transplantation (ESOT) and the Centre for Evidence in Transplantation (CET)

TRANSLATIONAL IMMUNOLOGY UPDATE Volume 4, Issue 3 Editor: Andrew H. Lichtman Editorial Board: Abul K. Abbas, Carla J. Greenbaum

HIGHLIGHTS FROM RECENT LITERATURE TUMOR INFLATING CD8+ T CELLS: THE SOLUTION OR ANOTHER PART OF THE PROBLEM? Research paper: Spranger, et al. Up-Regulation of PD-L1, IDO and Tregs in the Melanoma Tumor Microenvironment is Driven by CD8+ T Cells. Science Translational Medicine, 5, 200ra116 (2013). PMID:23986400 Despite recent advances in targeted and immune therapies, treatment options for patients with metastatic melanoma are limited and long-term survival is poor. In some patients, melanoma tumors are infiltrated by CD8+ T cells and yet the tumor is tolerated by the immune system. To address why this might occur, the authors studied metastatic melanoma lesions in both humans and mice.

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Suppression of T cell responses within tumors can result from a number of inhibitory pathways in the tumor microenvironment, including suppression by FoxP3+ regulatory T cells (Tregs), metabolic dysregulation via tryptophan catabolism by indoleamine-2,3-dioxygenase (IDO) and engagement of the inhibitory receptor PD1 by its ligand PD-L1. (PD-L1).

The authors studied human metastatic melanoma specimens by transcriptional profiling and immunohistochemical studies. They found that tumors that contained activated T cells also had the highest expression of genes associated with all three of these distinct immunosuppressive pathways (Tregs, IDO and PD-L1).

Using a mouse model, the authors demonstrated that up-regulation of both IDO and PD-L1 within the tumor microenvironment was dependent on the presence of CD8+ T cells and the production of IFNγ.

Recruitment of Tregs to the tumor and the induction of proliferation of Tregs within the tumor also required the presence of CD8+ T cells, but was not dependent on IFNγ. Instead, Tregs recruitment was found to be dependent on the production of CCL22 by CD8+ T cells, which supported the recruitment of CCR4+ Tregs. The authors also demonstrated that human CD8+ T cells recruited human Tregs in vitro and in a xenograft melanoma model in mice.

In summary, the authors found that immunosuppressive conditions only developed after T cell infiltration of tumors, suggesting infiltrating immune cells may themselves induce immunosuppressive mechanisms. In mice, all three major mechanisms of immune suppression were induced or worsened by the presence of CD8+ T cells infiltrating the tumor. This study demonstrates that tumor infiltrating CD8+ T cells contribute to the development of an immunosuppressive environment that in turn blocks their function. Combined therapy with multiple agents to combat the separate mechanisms of immunosuppression may therefore be needed to overcome local immune suppression and enhance antitumor T cell responses.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital

AN ABNORMAL POPULATION OF GUT BACTERIA IS ASSOCIATED WITH HIV PROGRESSION Research Paper: Vujkovic-Cvijin I., et. al. Dysbiosis of the Gut Microbiota is Associated with HIV Disease Progression and Tryptophan Catabolism. Science Translational Medicine, 5, 193ra91 (2013). PMID:23843452.

Evidence from both animal and human studies suggests that the progression of HIV disease is driven by chronically activated T cells and systemic inflammation. The source of this inflammation is not known, but breakdown of the gastrointestinal mucosal barrier and increased transfer of immunostimulatory microbial products from the gut lumen into this circulation have been suggested to participate. Increased tryptophan catabolism through interferon-inducible enzyme indoleamine 2,3dioxygenase 1 (IDO1) is associated with disease progression, but how it contributes is unknown. In this study, authors examined the gastrointestinal microbiota of patients with HIV to determine the role that microflora may play in disease progression.

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Using high resolution bacterial community profiling, the authors found that a particular abnormal microflora composition existed in some HIV-infected people and that this microflora was associated with evidence of mucosal immune disruption, T cell activation and chronic inflammation.

This abnormal gut microbiome was characterized by increased numbers of Proteobacteria and decreased numbers of Bacteroidia members.

This abnormal microbiota was observed even in patients on highly active anti-retro viral therapy (HAART) and its presence correlated with levels of tryptophan catabolism and plasma concentrations of IL-6, two markers that have been previously associated with HIV disease progression.

Bacteria with the ability to break down tryptophan into kynurenine were increased in the gastrointestinal tracts of patients with HIV, and the levels of these bacteria correlated with kynurenine levels in the blood.

The authors have demonstrated the first link between mucosal-adherent colonic bacteria and the chronic inflammatory state that is thought to weaken the immune system and lead to HIV disease progression. Moreover, they found that this abnormal gut microbiota can be present and contribute to pathology even in patients in whom HIV viral production is suppressed with HAART. These exciting results suggest that modulation of the gastrointestinal microbiota may be a useful approach for preventing or reversing the chronic inflammation associated with HIV disease progression Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital

UNCOVERING KEY T CELL EPITOPES IN HUMAN DISEASE Research Paper: Newell E.W., et. al. Combinatorial Tetramer Staining and Mass Cytometry Analysis Facilitate T Cell Epitope Mapping and Characterization.Nature Biotech, 31. 623-631 (2013). PMID:23748502.

For T cells to become activated, their T cell receptors (TCRs) must recognize and bind peptide antigens on major histocompatability complex molecules (MHC; in humans referred to as Human Leukocyte Antigen or HLA). In natural responses, the peptide-MHC (or HLA) are displayed on antigen presenting cells. To study the antigenic specificity of human T cells, it is possible to use peptide-MHC multimer reagents in traditional flow cytometry, but this has been fairly inefficient to

understand the depth and breadth of a natural immune response (since it is limited by the number of flurophors that can be distinguished from each other, even when used combinatorially). Previous studies have been able to study up to 64 TCR specificities simultaneously using multiple flurophor tagged pMHCs. The application of cytometry by time of flight (CyTOF), also known as single cell mass spectrometry, allows the assessment of a given T cell using up to 40 easily distinguished single metal labeled reagents (including pMHC multimers), but was used in this study in a combinatorial fashion to allow simulataneous assessment of 120 reagents labeled with unique three metal combinations as tags (in this case, each pMHC tetramer is conjugated to three different metal-tagged streptavidins, mixed and used as a single reagent).

This study applied this technique to uncover T cell epitopes for rotavirus, as a model system for the application of their technique to any infectious disease (or, arguably, to many forms of human disease).

Methods: Leukocyte reduction system cones from platelet apheresis donors were obtained (and for some experiments, selected HLA-A*0201+). In addition, deidentified proximal jejunum tissues from bariatric surgery were collected. HLA-A*0201 molecules were generated and refolded with various peptides and biotinylated and mixed with metal labeled streptavidins.

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They began by predicting epitopes that would bind to the relevant HLA molecule from viral peptides and selected candidate peptides conserved across rotavirus strains (132 candidate peptides).

Each candidate peptide was assessed for binding to soluble HLA-A*201 (in competition with a peptide known to bind to the HLA molecule). Only those that met a binding threshold were kept, leaving 77 candidate peptides.

They generated a set of 109 peptide-MHC tetramers, including 77 with rotavirus epitopes and 32 with either negative or positive control peptides (including epitopes from influenza, CMV, EBV).

Magnetically separated CD8+ T cells (and whole) patient samples (from 17 healthy blood donors and nine bariatric surgery patient jejunal tissue samples; chosen since rotavirus is a gastrointestinal virus) were stained with the panel of MHC tetramers and 23-27 additional surface markers (to assist in determining the cell subtype and activation status). Of note, patients with known CMV, influenza or EBV negative status were used to verify that the analysis techniques used showed that those samples had appropriately low levels of T cells that could bind epitopes they had not been exposed to. Of note, they verified these results with two different mixtures of three-metal labeled tetramers.

They compared the cell surface marker phenotypes (i.e. naïve versus effector memory versus central memory CD8+ T cells) of the T cells responding to the positive control epitopes and found them to be consistent with prior studies.

They focused on the rotavirus specific T cells and found that only T cells that recognized epitopes from VP3 (a viral peptide) had an effector phenotype and that only those cells were found in the jejunal biopsies. They also identified non-VP3 epitope specific T cells.

Applying the powerful single cell mass spectrometry technique, this paper describes a method for starting from a known pathogen (here an infectious disease that has been targeted by multiple vaccine programs which led to a vaccine given to infants at 2, 4 and 6 months of age), predicting and validating candidate pathogen epitopes and then assessing the contribution of those epitopes to the immune responses of a healthy human population, in both PBMC and gut, the target tissue of this infection. Harnessing combinatorial tagging of peptide-MHC complexes, in tandem with deep characterization of surface markers, has the potential to allow to further rationalize our approach to

both preventative and therapeutic vaccine design, as well as augmenting our understanding of the factors that determine key epitopes underlying immune responses in human diseases.

Reviewed by Sarah Henrickson, MD, PhD Boston Children’s Hospital and Harvard Medical School

THE EARLY BIRD GETS THE WORM: ANTIGENIC SENIORITY AND INFLUENZA A VIRUS Research Paper: Miller M.M., et. al. Neutralizing Antibodies Against Previously Encountered Influenza Virus Strains Increase Over Time: A Longitudinal Analysis. Science Translational Medicine, 5, 198ra107 (2013). PMID:23946196 Over time, influenza A viruses (IAVs) undergo both antigenic shift and drift, challenging both vaccine design and our immune systems. To truly understand how our cumulative lifetime exposure to slightly (or significantly) different IAVs each year affects our ability to respond to the next strain we encounter, a large-scale study across a significant time span was necessary. While cross-sectional studies can yield a great deal of information, there is dramatic power in watching the evolution of the immune response over time in multiple individuals. This study considers the role of antigenic seniority, a theory wherein the amplitude of the neutralizing antibody response to the first strain of a given subtype is the greatest (via an as-yet unknown mechanism). In addition, the current focus on developing broadly universal influenza vaccines (that would not have to be given yearly) based on generating neutralizing antibodies to the conserved hemagglutinin (HA) stalk domain, requires a more detailed understanding of the optimal antigen exposures to yield desired humoral immune responses.

Methods: Samples were collected approximately every five years over two decades from 40 patients in the Framingham Heart Study who were born between 1917 and 1952 (to confirm that they had been exposed to both seasonal and three pandemic influenza A virus strains, 1957 H2N2, 1968 H3N2 and 1977 H1N1). Tests on these samples included HA inhibition (HAI) titers for both pandemic viruses (as above) and seasonal strains. Both IAV and HCMV titers were measured, at each time point.

• Interestingly, HAI titers against a pandemic strain that disappeared more than 40 years ago have increased over the four study samples, a consistent pattern seen with other pandemic strains. They hypothesize that increases in titers well after exposure to the pandemic strains may be secondary to exposures to descendent viruses with conserved epitopes. This would then support the model of antigenic superiority, because the first strain in a given subtype would have its response boosted by each subsequent exposure to a similar virus, retaining a superior response for the patient’s lifetime.

• How does this boosting of antibodies against historical pandemic IAVs affect responses to novel IAVs? By examining responses to drifted viruses, it is possible to assess the effect of boosting responses to historical exposures. While pandemic strains had an advantage in the absolute titers, when compared to drifted variants, the IAVs that had undergone antigenic drift still showed increases in titers across the four study time points.

• How do periods of low antigenic drift affect development (and change) of neutralizing responses to the antigenically stable HA stalk of IAVs? This was examined in both group 1 HA (where periods with low antigenic drift still led to increases in HA stalk antibody titers) and group 2 HA (a more restricted group with a single dominant lineage in circulation; here they also demonstrate increase in titers over the course of the study). In agreement with the model that significant amplitudes of neutralizing anti-HA stalk antibodies require exposure to a set of viruses with antigenic diversity, the group 1 viruses, with greater antigenic drift, yielded greater amplitude HA stalk antibody titers.

• Interestingly, they contrasted the rapidly evolving IAVs with the stable human cytomegalovirus.

This herpesvirus sets up lifelong latency with episodic reactivation. The viral antigens do not

change over time and titers of HCMV neutralizing antibodies also remained stable throughout the study.

This study quantitates the changes in human immune responses to IAVs across adult lifetimes with likely exposures to both well studied pandemic IAVs and seasonal IAVs. With the breadth and depth of data gathered, this study has been able to assess the effects of sequential exposures to drifted and shifted IAVs, with implications for both the theories of antigenic superiority and the importance of exposure to antigenically diverse IAVs for generation of neutralizing antibodies to the HA stalk. The value of a human study that focuses on these complex and subtle issues (fundamental to human health) is clear. However, the challenges of uncontrolled natural infection of healthy humans versus controlled infection of animal models does dramatically increase complexity. In addition, while influenza vaccine development is a key issue, extending this study to infant vaccination or therapeutic vaccination would be fascinating.

Reviewed by Sarah Henrickson, MD, PhD Boston Children’s Hospital and Harvard Medical School

HOW DO HLA CLASS II ALLELES AFFECT HIV VIREMIA? Research Paper: Ranasinghe S., et al. Association of HLA-DRB1-Restricted CD4+ T Cell Responses with HIV Immune Control. Nature Medicine, 19: 930-933 (2013). PMID:23793098.

A challenge in the field of HIV research is understanding the mechanisms that dictate why some HIVinfected individuals are able to control their disease with minimal intervention while other progress rapidly. While a genetic association of HLA class I alleles with HIV control is well established, the role of HLA class II-restricted responses in control of HIV infection is more poorly defined. Previously published genetic studies have suggested that HLA class II alleles of the HLA-DRB1 locus may influence HIV viremia; however, the mechanistic basis by which they may do so remains unclear.

Since HIV preferentially infects CD4 T cells, understanding the role of the HLA class II-restricted response in viremia control is of interest. To address this knowledge gap, Ranasinghe and colleagues explored the breadth of HIV peptide-specific responses for each of HLA-DRB1 alleles and characterized the avidity of the responses in HIV controllers and progessors. There are several intriguing observations from this work.

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Using a panel of 201 DRB1-restricted HIV specific CD4 T cells recognizing 67 peptide specificities generated from 42 HIV-infected patients, they find that HLA-DR variants show a high degree of promiscuity with multiple peptides restricted by two or more HLA-DR variants.

Moreover, detailed analysis of the OLP-41 peptide in Gag p24 demonstrated that promiscuous HLA-DRB1 variants share overlapping epitope-binding registers. The authors postulate that this promiscuity is mediated by the open confirmation of HLA class II. Despite the high degree of overall HLA-DRBI binding promiscuity, there were marked differences in the number of peptides restricted by each DRB1 variant. DRB1*01:01 and 04:01 had only three peptide restrictions, while DRB1*13:01, 13:02 and 13:03 had 18, 11 and 8 HIV peptide restrictions respectively. Interestingly, the DRB1*13 variants were also associated with lower HIV viral load.

Analysis of the HLA-DRB1 allele expression and viral load in a cohort of 1,085 treatment-naïve HIV-infected patients of European ancestry revealed a hierarchy of more protective to more hazardous HLA-DRB1 alleles. HLA-DRB1*15:02 most strongly associated with low viremia, while HLA-DRB1*03:01 was significantly associated with high viremia.

Functional analyses of an independent cohort of 26 patients (17 controllers and nine progressors) revealed that HLA-DRB1 variants linked to low viremia (i.e., controllers) were

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more promiscuous, particularly in their ability to present a variety of Gag peptides. Moreover, Nef proteins were exclusively restricted by variants linked with low viremia.

CD4+ T cells from individuals with HLA-DRB1 alleles linked with low viremia had greater breadth of HIV-specific CD4+ T cell responses ex vivo. Surprisingly, despite the increased promiscuity of protective HLA-DRB1 alleles, these CD4+ T cells had functionally lower peptide binding avidity than those from individuals expressing hazardous alleles.

These data suggest that HLA-DRB1 alleles regulate control of HIV infection through their peptidebinding promiscuity and avidity. The authors propose that the increased promiscuity, but decreased avidity of protective HLA-DRB1 alleles may lower viral titers by reducing T cell activation and proliferation, thereby minimizing susceptibility to HIV infection or activation-induced cell death. While additional studies are required to validate this hypothesis, these data suggest a new approach for stratifying HIV-infected patients and a novel mechanism of CD4+ T cell-mediated viral control.

Reviewed by Si-Han V. Hai and Michelle L. Hermiston, MD, PhD, University of California San Francisco

WHAT MEDIATES VACCINE NON-RESPONSIVENESS? Research Paper: Garner-Spitzer, et al. Tick-Borne Encephalitis (TBE) and Hepatitis B Non-Responders Feature Different Immunologic Mechanisms in Response to TBE and Influenza Vaccination with Involvement of Regulatory T and B Cells and IL-10. Journal of Immunology, 191:2426-36 (2013). PMID:23872054.

Depending upon the vaccine, 1-10% of individuals will fail to mount a sufficient protective antibody response after immunization. Mechanisms mediating vaccine non-responsiveness have been incompletely elucidated. Most studies have focused on the hepatitis B vaccine where risk factors for non-responsiveness include obesity, smoking, renal disease, HLA-DRB1 and HLA-DQB1 subtypes, and increased TGF-β and IL-10. A central question in this field is whether non-responsiveness is a generalizable phenomenon, suggesting a more global immunologic defect, or whether it reflects an antigen/vaccine specific mechanism. To address this issue, Garner-Spitzer, Wiedermann, and colleagues compared tick-borne encephalitis (TBE) and hepatitis B non-responders that were otherwise healthy. Individuals with high antibody responses served as a control group. All participants received a TBE booster and an influenza vaccine. Humoral and cellular immune responses were analyzed pre-vaccine and at one week, eight weeks, and six months post-immunization. There are several interesting findings from this work.

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While TBE titers upon TBE booster remained low in TBE non-responders, both TBE high responders and hepatitis B responders had appropriate increases in their TBE titers.

The antibody response to influenza vaccine was similar among all three groups.

IL-2 and IFN−γ production by PBMC’s stimulated with TBE ex vivo were significantly higher in TBE responders than TBE non-responders. However, both groups had equivalent cytokine production in response to stimulation with influenza antigens. Interestingly, hepatitis B nonresponders had low IL-2 and IFN−γ production in response to both TBE and influenza antigens despite producing adequate antibody titers. Hepatitis B non-responders also had high IL-10 levels at baseline.

Flow cytometric quantification of T cell subsets post booster revealed increased frequencies of CD4 T effector memory cells and FoxP3+ regulatory T cells in both TBE and hepatitis B nonresponders relative to controls. TBE, but not hepatitis B, non-responders also had elevated frequencies of CD8 T effector memory cells.

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Only hepatitis B non-responders showed expansion of CD19+CD24highCD38high transitional B cells, which are thought to be a precursor of regulatory B cells and a potential source of IL-10.

Functional studies to evaluate regulatory B cell function in this subgroup were not performed.

Confirming published reports, hepatitis B non-responsiveness was associated with the HLADRB1 and HLA-DQB1 alleles. There was no correlation with TBE non-responsiveness to these or any other DR-B1, B3/4/5 or DQ-B1 subtypes.

Taken together, these data suggest that vaccine non-responsiveness is antigen specific and not reflective of overall immune dysfunction. The data also suggests that pathways leading to vaccine non-responsiveness can be distinct. While TBE non-responsiveness appears strictly antigen specific, the associations of HLA alleles, increased IL-10 production, and poor T cell proliferation to multiple antigens in hepatitis B non-responders suggest that defective antigen presentation and cellular immunity plays a more prominent role in the response to hepatitis B. Further studies addressing the functional aspects of vaccine non-responsiveness are warranted.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California San Francisco

LETHAL CROSS-REACTIVITY FOLLOWING ADOPTIVE THERAPY WITH AFFINITY ENHANCED T CELLS: SUBTLE CHANGES IN T CELL RECEPTORS CAN HAVE UNPREDICTABLE ADVERSE CONSEQUENCES Research paper: Cameron B.J., et al. Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells. Science Translational Medicine (2013); 5(197):197ra103. PMID:23926201.

Adoptive transfer of autologous T cells engineered to target and kill tumors is a promising strategy being developed for cancer therapy. However, there are major challenges facing this approach. First, the success of this approach relies on the identification of T cell receptors (TCRs) with high affinity for antigens that are specifically expressed by tumor cells. The problem is that TCRs like this are rarely isolated from natural T cell repertoires because the majority of antigens expressed by tumors are selfantigens also expressed by normal tissues; and therefore, most high affinity TCRs capable of recognizing them are eliminated during thymic development. Fortunately, lower affinity TCRs can be isolated, and their affinity for antigen can be enhanced through the mutation of specific residues within the complementarily-determining regions (CDRs) of the TCR, which are the regions that interact with and control antigen specificity. Although affinity enhancement of TCRs can overcome the first challenge, it does not overcome serious toxicity risks associated with this therapeutic approach, including both on-target and off-target toxicity. Two recent clinical studies attempted to minimize ontarget off-tissue toxicity risks by targeting MAGE A3, a cancer-testis antigen that is expressed by several tumor types, but is only normally expressed in adult testes with affinity enhanced TCRengineered T cells. Although extensive preclinical evaluation suggested that there were no off-target antigen recognition concerns; in each study, the first patient treated died due to treatment-related cardiac toxicity. In this study, Brian Cameron and colleagues show that the lethal toxicity caused by transferring affinity enhanced MAGE A3-specific T cells was not a result of on-target off-tissue toxicity as was observed in a prior study using a TCR targeting a different MAGE antigen, but rather potent cross-reactivity against titin, a self antigen expressed in cardiomyocytes. Titin cross-reactivity was a direct effect of the CDR substitutions introduced to enhance the affinity for MAGE A3, and was not observed in T cells expressing the original non-mutated TCR. •

Examination of cardiac tissue from each of the two subjects treated revealed marked T cell infiltration, and indicated the underlying cause of death was T cell-mediated acute cardiac

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injury. However, the authors showed that this could not have been the result of on-target offtissue toxicity because MAGE antigen expression was not detected in cardiac tissue from either of the subjects.

Comparison of the frequency of T cells expressing the a3a MAGE A3 affinity-enhanced TCR to T cells expressing only the V 5.1 β chain used by the a3a TCR, suggested that TCR mispairing was not responsible for the toxicity.

Alloreactivity was ruled out because T cells expressing the a3a TCR were not activated by a panel of cells covering 95% of the HLA serotype population. In vitro activation only occurred in response to HLA-A1+ targets that expressed MAGE A3.

T cells expressing the a3a TCR were found not to recognize 38 cardiac-derived primary cell lines. However, they did recognize and kill iCell cardiomyocytes, which are derived from induced pluripotent stem cells and more closely resemble heart tissue. iCell cardiomyocytes were shown not to express MAGE antigens, which suggested that a different peptide was recognized by the a3a TCR. The authors could not identify the cross-reactive epitope being recognized by the a3a TCR by blasting the human genome for similar sequences. Instead, they used alanine and glycine substitution to determine the amino acid motif recognized by the a3a TCR. Using the ScanProsite tool, they were able to identify three candidate proteins containing the a3a TCR recognition motif.

They then screened each of the three candidate peptides and showed that the cross-reactive epitope being recognized by the a3a TCR was derived from the muscle protein titin and presented by HLA-A1. This peptide was not recognized by the parental non-mutated MAGE A3-specific TCR. Titin was shown to be expressed in iCell cardiomyocytes and normal cardiac tissue, but not primary cardiac cell lines. Furthermore, the titin peptide could be stripped from HLA-A1 molecules present on the surface of a B cell lymphoma line shown to express titin that could be recognized by the a3a TCR, indicating that the epitope could be naturally processed and presented.

Interestingly, the murine homologue peptide was not recognized by T cells expressing the a3a TCR, which suggests that evaluation of a3a TCR-expressing T cells in HLA-A1 transgenic mice would not have predicted the lethal cross-reactive toxicity observed in patients.

This study highlights the danger associated with transferring affinity-enhanced T cells as an approach for antitumor immunotherapy. Although the transferred cells have the potential to induce robust antitumor responses, they also have the potential to induce lethal toxicity. This study demonstrates that even in cases where on-target off-tumor toxicity are non-existent, which was reported to be the cause of fatal toxicity in a separate study, the possible presence of non-obvious peptide homologues must also be considered. The estimate is that a single TCR can recognize more than a million different peptides. As this study illustrates, modifying CDRs alters the affinity of the TCR for more than just the single epitope of interest, and can even lead to the recognition of new epitopes. Most importantly, this study has shown that we need improved methods for assessing off-target toxicity risks so similar adverse events can be avoided in future studies and this approach can be developed more safely.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

MICROBIAL METABOLITES IN THE GUT SEND MESSAGES TO THE IMMUNE SYSTEM Research Paper: Smith P.M., et al. The Microbial Metabolites, Short-chain Fatty Acids, Regulate Colonic Treg Cell Homeostasis. Science (2013); 341:569-73. PMID:23828891.

Immune homeostasis in the gut requires a proper balance between pro- and anti-inflammatory pathways. The disruption of this balance leads to intestinal inflammatory diseases, and can lead to increased cancer occurrence. Maintenance of immune homeostasis is influenced by over 500 different species of bacteria residing in the human intestine. Whereas some bacteria in the gut microbiota, like Clostridia strains belonging to cluster IV and XIVa, suppress inflammation in the gut, other strains, like the closely related Clostridium cluster XI, promote inflammation. One way that particular strains of intestinal bacteria influence immune homeostasis is by affecting the recruitment, development and function of FoxP3+ regulatory T cells (Tregs), cells that are known to have a critical immunoregulatory role in the gut. Current efforts are aimed at determining how bacteria communicate with Tregs, and why the effects are unique to particular strains of bacteria. In this study, Patrick Smith and colleagues demonstrate that one of the signals between bacteria and Tregs is mediated through short-chain fatty acids (SCFAs), metabolites generated during the fermentation of complex carbohydrates, such as dietary fiber.

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The authors compared levels of SCFAs in specific pathogen-free (SPF) mice, gnotobiotic altered Schaedler flora (ASF)-colonized mice, and germ-free (GF) mice and found that GF mice had lower concentrations of acetic acid, propionic acid and butyric acid.

The authors hypothesized that lower concentrations of these SCFAs were responsible for the decreased frequencies of Tregs seen in colons of GF mice compared to SPF and ASFcolonized mice.

They supplemented drinking water with SCFAs for three weeks and found that the SCFAs individually, or in combination, increased Treg frequencies and numbers in the colons, but not the spleens, mesenteric lymph nodes or thymuses of GF mice. The SCFAs did not significantly alter CD4+ TH1 or TH17 cells in the colon.

Microbiota-induced Treg development has been shown to be associated with increases in inducible Tregs (iTregs). However, the majority of the Tregs that were expanded by SCFAs expressed the transcription factor Helios, suggesting that SCFAs specifically induce natural Tregs (nTregs) in the colon rather than iTregs.

SCFAs were shown to induce FoxP3 and IL-10 expression in Tregs, but not TGFβ, suggesting that SCFAs can selectively alter Treg effector gene expression and function. SCFAs were also shown to enhance the proliferation and suppressive capacity of Tregs.

B. fragilis and Clostridium cluster XIV members known to affect Tregs in the gut were shown to produce higher levels of SCFAs than Clostidium clusters XI and XVII members that do not affect Tregs.

The authors showed that the GPCR43 surface receptor that binds SCFAs was specifically upregulated on Tregs in the colon, and that the upregulation of GPCR43 on Tregs was dependent on signals provided by the gut microbiota.

Using Ffar2 (the gene encoding GPCR43) knock-out mice, the authors demonstrated that the effects of SCFAs on Treg proliferation and function were mediated directly on Tregs through the GPCR43 receptor.

The authors showed that SCFAs specifically reduced the expression of HDAC6 and HDAC9, two histone deacetylases that inhibit nTreg function, suggesting that the effects of SCFAs on Tregs is mediated through the inhibition of HDACs.

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The authors demonstrated the therapeutic potential of their findings by showing that SCFAs could reduce the severity of colitis in a T cell adoptive transfer model of inflammatory bowel disease in a GPCR43-dependent manner.

The human gut harbors up to 1011 bacteria per gram consisting of over 500 different species. Recent studies have shown that different species in the gut microbiota have varying effects on immune homeostasis, and suggest that altering the composition of the gut microbiota through the introduction of particular strains may provide a therapeutic strategy for manipulating the gut immune environment.

However, whether this approach can permanently change the repertoire of bacteria in the gut and result in long-term restoration of immune homeostasis remains unknown. This study demonstrates that SCFAs generated during the fermentation of complex carbohydrates, such as dietary fiber, provide a signal that enhances the proliferation and function of nTregs and suppresses inflammation in the gut; and may explain the benefits of dietary fiber and particular bacterial strains. Furthermore, this study shows that the identification of the specific molecules mediating signaling between bacteria and the immune system may provide novel therapeutic approaches for specifically controlling immune balance and treating or preventing inflammatory diseases in the gut without tampering with the composition of the gut micriobiota.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

HIGHLIGHTS FROM CLINICAL IMMUNOLOGY, THE OFFICIAL JOURNAL OF FOCIS NEW INSIGHTS INTO THE PATHOGENESIS OF CIRRHOSIS Research Paper: Mchedlidze T., et al. Interleukin-33-Dependent Innate Lymphoid Cells Mediate Hepatic Fibrosis.

Immunity, 39: 357–371 (2013). PMID: 23954132.

Hepatic fibrosis is a common complication of many different infectious and metabolic diseases of the liver and often leads to end stage cirrhosis with hepatic failure. Chronic inflammation is implicated in liver fibrosis, but the specific mediators that drive extracellular matrix deposition and tissue remodeling are not well understood.TH2 cells and the cytokines they produce, including IL-5, IL-9 and IL-13, have been implicated in fibrotic diseases of the lung and other tissues. Emerging data has recently implicated a subset of innate lymphoid cells, called ILC2, as important source of “TH2” cytokines that play roles in protection against infections and contribute to diseases (see review in Translational Immunology Update, 6/13). In this study, Mchedlidze and colleagues provide compelling evidence for a pathogenic pathway of liver fibrosis that depends on IL-33 secreted by hepatic cells and stimulates ILC2 cells in the liver to secrete IL-13, which in turn drives fibrosis. The major findings of this study are: •

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Patients with hepatic fibrosis have increased IL-33 in serum and liver compared to patients without hepatic fibrosis. Two murine models hepatic fibrosis were used in this paper, one induced by CCL4 administration and one by common bile duct ligation. IL-33 was increased in both of these models as well.

Within the context of the murine models, forced expression of IL-33 in the liver, by a vector or transgene increased liver fibrosis, and IL-33 deficiency caused significantly reduced liver fibrosis and reduced expression of extracellular matrix remodeling genes (collagen and TIMP genes).

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IL-33 treatment prominently upregulated IL-13 gene expression in the liver, but IL-13 deficient mice, or mice deficient in either of the two chains of the IL-13 receptor (IL-4Rα and IL-13Rα1) were resistant to IL-33 induced fibrosis. Both these receptor chains were highly expressed in fibrotic areas of human cirrhotic liver. Bone marrow chimeric mice deficient IL-4Rα only in cells of hematopoietic origin remained susceptible to IL-33 induced liver fibrosis, showing that the target of profibrotic effect of IL-13 was an intrinsic tissue cell in the liver.

Hepatic stellate cells (perisinusoidal cells or Ito cells) are known to be key players in hepatic fibrosis. The authors isolated these cells from mouse liver, and showed they respond to IL-13 by STAT6 phosphorylation, proliferation and induction of profibrotic and chemokine genes.

Bone marrow chimeric mice deficient in the IL-33 receptor (Il1rl1-/-) only in cells of hematopoietic origin did not develop liver fibrosis, showing the IL-33 responsive cells were of hematopoietic origin.

Various data are presented supporting the conclusion that the source of IL-13 in the fibrotic livers was ILC2 cells. These cells were identified and shown to be increased in the affected livers, based on cell surface markers, dependence on RORα, and lack of T and B cell markers. In fact, hepatic fibrosis was maintained or worse in Rag1-/- mice but was diminished in Rag1-/--ɣc-/- mice, which lack ILC2 development, because it depends on IL-7, which signals though a ɣc receptor.

ILC2 cells were shown to mediate the profibrotic response to IL-13 by depletion studies, which diminished the response, and by adoptive transfer into IL-33 receptor deficient mice, which restored the fibrotic responses.

This paper contributes to the rapidly emerging knowledge base about the role of ILCs in disease, and provides new information about how liver fibrosis develops. The study raises the possibility that targeting ILC2 cells, IL-13 or IL-33 may be helpful to treat this very significant clinical problem. Some parts of this story require further work. The actual cellular source of the IL-33, and the precise signals that induce it are not defined in this study, nor is it proven that the Ito cells are the key responders to IL-13 in vivo. There is also some data in the recent literature that IL-33 may be protective in acute liver injury.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital

GAMMA DELTA DEPENDENT HAIR Research Paper: Gay, et al. Fgf9 from dermal ɣδ T Cells Induces Hair Follicle Neogenesis After Wounding. Nature Medicine, 19, 916–923 (2013). PMID: 23727932.

T cells are implicated in skin wound repair in both mice and humans. In particular, epidermal ɣδ T cells produce a variety of growth factors, including insulin growth factor-1 (IGF-1) and fibroblast growth factor ( Fgf)7 and Fgf10, which promote keratinocyte proliferation and migration that contributes to wound healing. However, cutaneous wound repair in humans usually results in scars without hair follicle regeneration, while hair follicles are regenerated in wounded mouse skin. In this study, the authors show that hair follicle regeneration in mice depends on Fgf9 initially produced by dermal ɣδ T cells. The main experimental findings are: • Using a full thickness excisional wound model of hair neogenesis, Fgf9 was found to be upregulated in the dermis before the detection of early stages of new hair follicle development.

• The importance of Fgf9 in hair follicle neogenesis after wounding was demonstrated by showing that dermal injection of anti-Fgf9 reduced new hair follicle formation and

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overexpression of Fgf9 in the epidermis using an inducible keratin-promoter Fgf9 transgenic mice caused an increase in the number of neogenic hair follicles. qRPC analysis of sorted populations of cells taken from mouse skin post wound day 12 showed that the primary source of Fgf9 was dermal Vɣ4+ ɣδ T cells.

The importance of Fgf9 expressed by ɣδ T cells in induced hair follicle neogenesis was shown using Lck-Cre; Fgf9fl/fl mice. These mice lacking Fgf9 in T cells showed markedly fewer new hair follicles compared to single transgene controls, and qRT-PCR analyses showed that only ɣδ T cells, and not other T cells expressed Fgf9.

Uinsg Wnt-reporter mice (Axin2-LacZ heterozygotes), the authors showed that Fgf9 from γδ T cells induces Wnt expression and subsequent Wnt activation in wound fibroblasts, and this induction is markedly attenuated in Axin2-LacZ;Tcrd−/− that lack ɣδ T cells.

The authors confirmed previous works showing that there are very few dermal γδ T cells, in human skin, potentially explaining their inability to regenerate hair after wounding.

This paper is of interest because it shows how the immune system can contribute to different aspects of tissue regeneration in a highly specific manner. In this case, one sub-subset of the γδ subset of T cells is activated by wounding to express a single growth factor that is the major inducer of only one aspect of skin wound, namely hair follicle regeneration. The lack of Fgf9-producing γδ T cells in human dermis may explain why wounded human skin usually does not regenerate hair, and this may be of relevance to alopecia therapy.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital

GUT BUGS AND FOOD ALLERGY Research Paper: Rivas N., et. al. A Microbiota Signature Associated with Experimental Food Allergy Promotes Allergic Sensitization and Anaphylaxis. Journal of Allergy and Clinical Immunology. (2013) PMID: 23201093.

In Rivas, et. al, the researchers were interested in profiling microbiome changes to observe whether oral sensitization to a food allergen resulted in a characteristic microbiota signature. Prior to this study, it was hypothesized that the microbiome played a role in food allergy from studies that documented alterations in the signature before and after treatment. However, this study utilized oligonucleotide microarrays for 16S RNA to probe the complete microbiome profile, before and after therapy, as well as rescue experiments using regulatory T cells.

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A mouse strain carrying a gain-of-function mutation for the IL-4 receptor that was susceptible to sensitization to the chicken egg protein, OVA, was used as a model for food allergy.

Compared to wild-type mice, the mutant mice that were sensitized to OVA had decreased levels in Firmicutes family Erysipelotrichi, Erysipelotrichales, Erysipelotrichaceae and significant increase in the the Proteobacteria family Gammaproteobacteria, Enterobacteriales and Enterobacteriaceae.

Furthermore, upon treating these sensitized mice with allergen-specific regulatory T cells, the microbiota signature was different from sensitized mice without regulatory T cell treatment, but also different from sham-sensitized wild-type mice, suggesting a distinctive microbial profile for induced tolerance from regulatory T cells.

The researchers also performed experiments demonstrating that germ-free wild-type mice with flora derived from the OVA-sensitized mutant mice also experienced anaphylaxis and IgEmediated allergic reactions to OVA.

In conclusion, these findings demonstrate that specific microbiota signatures play a role in the pathogenesis of food allergy in mice, suggesting potential therapeutic approaches targeting microbes.

The experimental system may prove useful for research into the mechanisms through which gut flora influence the immune system.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

BACH KEEPS THE IMMUNE SYSTEM COMPOSED Research Paper: Roychoudhuri, R., et. al. BACH2 Represses Effector Programs to Stabilize Treg-Mediated Immune Homeostasis. Nature, 498:506-10. (2013). PMID:23728300.

BACH2 is a x zinc-finger transcription factor previously shown to be required for B cell somatic hypermutation and class switching. Human genetic studies demonstrated polymorphisms in BACH2 are associated with several immune/inflammatory diseases, such as asthma, Crohn's disease, vitiligo, multiple sclerosis and Type 1 diabetes. Roychoudhuri, et. al. hypothesized that BACH2 plays a broad role in immune homeostasis and preventing inflammation. Using Bach2 knockout mice, these investigators provided evidence in support of that hypothesis.

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By three months of age, Bach2 knockout mice developed diffuse inflammatory pathology, autoantibodies and activated CD4+ T cell infiltrates in various tissues, including lung and gut.

Bone marrow chimeric mice with Bach2 KO marrow into recipient mice lacking recombination activating genes (RAG1) to prevent endogenous B and T cell differentiation, developed inflammatory disease, while co-transfer of Bach2 wild type bone marrow prevented disease.

The protective effect of Bach2-wild type bone marrow was dependent upon FoxP3.

Thymic Treg development was impaired in Bach2 KO mice.

Additionally, far less adoptively transferred naïve Bach2 KO T cells differentiate into iTreg compared to transferred wild-type naïve T cells, but retroviral-mediated expression of BACH2 rescued iTreg differentiation in the Bach2 KO cells.

RNA sequencing of BACH2 deficient CD4+ T cells showed upregulation of many effectorlineage-associated genes, including genes known to represss Treg differentiation. Chromatin immunoprecipitation of wild-type T cells showed that that BACH2 bound to many of these genes that were derepressed in the BACH2 deficient CD4+ T cells.,

In conclusion, these findings show that BACH2 functions to repress effector CD4+ T cell differentiation programs and to stabilize Tregs, thereby limiting inappropriate immune activation. Overall, from human genetic studies and this murine study, BACH2 appears to be of central importance in maintaining immune homeostasis and preventing autoimmune diseases.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine and Andrew Lichtman, Brigham and Women’s Hospital

HUMAN IMMUNOPHENOTYPING UPDATE PROFICIENCY TESTING SERVICES: WHATâ&#x20AC;&#x2122;S AVAILABLE AND FOR WHOM By: Holden T. Maecker, Stanford University School of Medicine The idea of standardized services that allow a laboratory to test its proficiency at a particular assay is common in the clinical diagnostics world, but is a relatively new concept for research labs. For flow cytometry assays, the use of CD4+ T cell enumeration in HIV clinical monitoring drove the creation of several proficiency testing programs in the U.S. and U.K. These mostly use a stabilized blood sample that can be sent to participating labs, which perform the CD4 counts and return data for evaluation.

Initial experiments of this kind showed the reproducibility between labs could be kept very tight.1 The ELISPOT assay, a test for enumerating cells secreting particular protein(s) such as cytokines, was one of the first to be subject to standardization among research labs. Because of its use in evaluating experimental HIV vaccines, a group of 11 vaccine testing labs led by Josephine Cox, then at the U.S. Military HIV Research Program, showed that qualitative concordance was possible, although precise quantitation varied between labs.2 Other groups have since claimed reproducible quantitation among labs3,4 by careful harmonization or standardization of protocols.

While CD4 counting has the longest history of flow cytometry proficiency testing programs, there are other assays for which such programs have also been developed. Notably, the Division of AIDS at the National Institute of Allergy and Infectious Diseases (NIAID) began a program in 2005 to test laboratories evaluating vaccine clinical trials.5,6 The target assay for this effort was intracellular cytokine staining (ICS), also used in the evaluation of experimental HIV vaccines. This Quality Assurance Program eventually became an ongoing effort, and currently tests laboratories for proficiency in ICS and ELISPOT assays. It is overseen by a central laboratory, termed EQAPOL, for External Quality Assurance Program Oversight Laboratory (http://eqapol.dhvi.duke.edu). The EQAPOL lab is based at Duke University, under the direction of Thomas Denny. EQAPOL has also begun to administer a Luminex proficiency panel, which was developed in collaboration with Michael Kalos and the Cancer Immunotherapy Consortium (CIC).

The CIC and the Association for Cancer Immunotherapy (CIMT) are the other major players in the development of quality assurance and proficiency testing programs.7 They have published the results of these programs together with major leaders in the field, including Pedro Romero and Cedrik Britten for T cell MHC multimer staining8 and Sylvia Janetzki for ELISPOT3. A current CIC effort is also underway to standardize gating of ICS assays, led by Lisa McNeil. This has produced a recent publication describing results from 110 participating labs, along with recommendations for best practices.9 An interesting recent development has been the shift of proficiency testing for CIC panels to a commercial vendor, Immudex (http://www.immudex.com/proficiency-panels.aspx). The CIC ELISPOT and MHC multimer panels were developed to promote assay harmonization, by finding and controlling the major sources of variability. A natural evolution of such harmonization is the provision of on-going proficiency testing, which will be the role of Immudex.

So where should labs go to test their proficiency at flow cytometry assays, provided they are not already part of a required program such as EQAPOL? For MHC multimer and ELISPOT assays, the obvious source is the CIC-Immudex collaboration. While the EQAPOL program is intended for laboratories in the NIAID DAIDS and CIC consortia, interested laboratories can contact the EQAPOL program to determine if their laboratory is eligible for participation. And, for those looking to harmonize

assays other than those listed here, the CIC/CIMT would seem a natural group with which to work, given their history in this area.

Finally, what are the benefits of ongoing proficiency testing in a research lab setting? Obviously, an initial external comparison is desirable to determine whether a lab has truly optimized a particular assay. Thereafter, a minimum of annual (and possibly even quarterly) testing is desirable to show consistency and to validate the performance of new personnel or other changes that can drift into the lab environment over time. Proficiency testing participation is among the criteria called out by the MIATA initiative, as a reportable part of the lab environment.10-12 Along with the development of consensus protocols and panels, proficiency testing can perform a vital role in the standardization/harmonization of flow cytometry assays.

References and Suggested Reading: 1.

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Reimann K.A., O'Gorman M.R., Spritzler J., Wilkening C.L., Sabath D.E., Helm K., Campbell D.E.: Multisite comparison of CD4 and CD8 T-lymphocyte counting by single- versus multipleplatform methodologies: evaluation of Beckman Coulter flow-count fluorospheres and the tetraONE system.The NIAID DAIDS New Technologies Evaluation Group, Clin Diagn Lab Immunol (2000), 7:344-351.

Cox J.H., Ferrari G., Kalams S.A., Lopaczynski W., Oden N., D'Souza M.P.: Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus Type 1 vaccine trials, AIDS Res Hum Retroviruses (2005) 21:68-81.

Janetzki S., Panageas K.S., Ben-Porat L., Boyer J., Britten C.M., Clay T.M., Kalos M., Maecker H.T., Romero P., Yuan J., Martin Kast W., Hoos A.: Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI), Cancer Immunol Immunother (2008) 57:303-315.

Boaz M.J., Hayes P., Tarragona T., Seamons L., Cooper A., Birungi J., Kitandwe P., Semaganda A., Kaleebu P., Stevens G., Anzala O., Farah B., Ogola S., Indangasi J., Mhlanga P., Van Eeden M., Thakar M., Pujari A., Mishra S., Goonetilleke N., Moore S., Mahmoud A., Sathyamoorthy P., Mahalingam J., Narayanan P.R., Ramanathan V.D., Cox J.H., Dally L., Gill D.K., Gilmour J.: Concordant proficiency in measurement of T-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents, Clinical and vaccine immunology: CVI (2009) 16:147-155. 2643552.

Maecker H.T., Rinfret A., D'Souza P., Darden J., Roig E., Landry C., Hayes P., Birungi J., Anzala O., Garcia M., Harari A., Frank I., Baydo R., Baker M., Holbrook J., Ottinger J., Lamoreaux L., Epling C.L., Sinclair E., Suni M.A., Punt K., Calarota S., El-Bahi S., Alter G., Maila H., Kuta E., Cox J., Gray C., Altfeld M., Nougarede N., Boyer J., Tussey L., Tobery T., Bredt B., Roederer M., Koup R., Maino V.C., Weinhold K., Pantaleo G., Gilmour J., Horton H., Sekaly R.P.: Standardization of cytokine flow cytometry assays, BMC Immunol (2005) 6:13.

Jaimes M.C., Maecker H.T., Yan M, Maino V.C., Hanley M.B., Greer A., Darden J.M., D'Souza M.P.: Quality assurance of intracellular cytokine staining assays: Analysis of multiple rounds of proficiency testing, J Immunol Methods (2010). van der Burg S.H., Kalos M., Gouttefangeas C., Janetzki S., Ottensmeier C., Welters M.J., Romero P., Britten C.M., Hoos A.: Harmonization of immune biomarker assays for clinical studies, Sci Transl Med (2011) 3:108ps144.

Britten C.M., Janetzki S., Ben-Porat L., Clay T.M., Kalos M., Maecker H., Odunsi K., Pride M., Old L., Hoos A., Romero P.: Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium, Cancer Immunol Immunother (2009) 58:1710-1713.

McNeil L.K., Price L., Britten C.M., Jaimes M., Maecker H., Odunsi K., Matsuzaki J., Staats J.S., Thorpe J., Yuan J., Janetzki S.: A harmonized approach to intracellular cytokine staining gating: Results from an international multiconsortia proficiency panel conducted by the Cancer

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Immunotherapy Consortium (CIC/CRI), Cytometry Part A : the journal of the International Society for Analytical Cytology (2013) 83:728-738.

Janetzki S., Britten C.M., Kalos M., Levitsky H.I., Maecker H.T., Melief C.J., Old L.J., Romero P., Hoos A., Davis M.M.: "MIATA"-Minimal Information about T Cell Assays, Immunity (2009) 31:527-528.

Britten C.M., Janetzki S., Butterfield L.H., Ferrari G., Gouttefangeas C., Huber C., Kalos M., Levitsky H.I., Maecker H.T., Melief C.J., O'Donnell-Tormey J., Odunsi K., Old L.J., Ottenhoff T.H., Ottensmeier C., Pawelec G., Roederer M., Roep B.O., Romero P., van der Burg S.H., Walter S., Hoos A., Davis M.M.: T Cell Assays and MIATA: The Essential Minimum for Maximum Impact, Immunity (2012) 37:1-2.

Janetzki S., Hoos A., Melief C.J., Odunsi K., Romero P., Britten C.M.: Structured reporting of T cell assay results, Cancer immunity (2013) 13:13. 3718734.

DEVELOPMENTS IN BASIC IMMUNOLOGY AND NOVEL THERAPIES EPIGENETIC CONTROL OF HELPER T CELL PLASTICITY Lai Wei and Robert B. Nussenblatt, National Eye Institute, National Institute of Health Introduction Na誰ve CD4+ T cells differentiate into various subsets of effector helper or regulatory T cells when facing pathogenic attack, including T helper (Th) 1, Th2, Th17, Th9, Th22, T follicular help (Tfh) and regulatory T (Treg) cells. Appropriately activated effector and regulatory T cells subsets are critical for host defense, while disregulation of them leads to autoimmunity and inflammatory diseases. The differentiation process of helper T cells is tightly controlled by distinct cytokine milieus, the products of innate immune responses to diverse microbial pathogens. Importantly, the phenotypic and functional changes acquired by na誰ve CD4+ T cells during differentiation is heritable to daughter cells and do not involve DNA sequence changes. Therefore, the epigenetic mechanisms underlying the cell fate decision become a key area of interest in studying the regulation of the adaptive immune response.

Basic concept of epigenetics Epigenetics studies the changes in gene expression or cellular phenotype, caused by mechanisms other than changes in the underlying DNA sequence. In another word, epigenetics studies how the body adjusts to the environment without changing its own DNA code. Currently, the main focus of epigenetic studies, however, is on the alteration of chromatin structure through adjusting the patterns of DNA methylation and histone modifications.

One of the first well studied epigenetic phenomenon is the 5-methylcytosine of DNA methylation, which is generally associated with gene silencing. It occurs predominantly in the symmetric CG context, called CpG. Most of CG dinucleotides of the genome are normally methylated and tend to cluster together and form CpG islands. Recent studies revealed multiple forms of DNA methylation that may be the derivative of the DNA demethylation process, including 5-hydroxymethylcytosine, 5formylcytosine and 5-carboxylcytosine. In addition to DNA methylation, the histone code, referring to over 100 types of post-translational covalent modifications of histones, plays a major role in epigenetic regulation of various cellular processes. The abundant forms of modifications on histone tails are acetylation, methylation, phosphorylation, ubiquitination, ADP-ribosylation, and sumolation. Recent studies also strongly suggest a crosstalk between DNA methylation and histone modifications in coordinating the epigenetic regulation of cellular functions. Therefore, cooperation of both DNA methylation and histone modifications critically decides the physiological and pathological outcomes of the organism.

Epigenetic regulation of signature cytokines Traditionally, effector T helper cell subsets are defined according to the signature cytokines they secrete. IFN-ɣ, IL-4, IL-17, IL-22, and IL-9 are broadly used as the markers of Th1, Th2, Th17, Th22, and Th9, respectively. In addition, another important character of Th subsets is the silencing of lineage-inappropriate cytokines. For example, fully polarized Th1 cells produce IFN-ɣ, while the expression of IL-4 and IL17 is effectively suppressed. Previous studies mapping the genome-wide histone modifications H3K4me3 (active marker) and H3K27me3 (silent marker) demonstrate that in fully polarized murine Th subsets, signature cytokine loci are selectively associated with H3K4me3 alone which marks the chromosome structure permitting active gene transcription in their featured subsets, while they are associated with H3K27me3 alone which marks the suppressive chromosome structure preventing gene transcription in all other Th subsets in which they are not expressed.

However, recent reports have suggested that helper T cells are more plastic than the simple scenario (one signature cytokine in one subset) previously defined and have challenged the traditional view of exclusive expression of signature cytokines in their Th subsets. Expression of IFN-ɣ can be detected in a subset of fully polarized Th17 and Th2 cells in both in vitro cultures of human T cells as well as in vivo murine disease model systems. Therefore, the partially heritable epigenetic control of signature cytokine loci is also dynamically regulated by the microenvironment helper T cells reside in.

Epigenetic regulation of transcription factors The successful differentiation of helper T cells requires induction of multiple lineage-specific transcription factors that drive the programing of cell fate decision, such as Tbet in Th1, Gata3 in Th2, Rorc in Th17, Bcl6 in Tfh and FoxP3 in Treg. It is believed that the dynamic epigenetic regulation of these key transcription factors during the differentiation process promotes the plasticity of effector T cells. Genome-wide H3K4me3 and H3K27me3 data indicate that a bivalent domain is present on the promoters of several master regulators, including Tbx21 (encoded Tbet), Gata3 and Bcl6 in the T cell subsets in which these transcription factors are not highly induced. The bivalent domain represents genomic regions with both accessible H3K4me3 and repressive H3K27me3 marks and poises the downstream genes for flexible induction, which provides a mechanism for plasticity of gene regulation.

Importantly, cells highly expressing Tbet and Gata3 as well as IFN-ɣ and IL-4 can be fully functional in host defense in vivo. Thus, helper T cell plasticity under epigenetic control of the key transcription factors may be critical for the generation of diverse cell mediated immune responses.

Epigenetic therapies A growing body of evidence indicates critical role of epigenetic regulation in the immune system.

Although the majority of DNA methylation and histone modification patterns remain unchanged and heritable during T cell differentiation and proliferation, the key cytokines and transcription factors defining the helper T cell subsets are all epigenetically regulated in response to environmental stimuli, providing a potential application of epigenetic therapies in controlling helper T cell mediated inflammatory diseases, for example psoriasis, inflammatory bowel disease, uveitis, age-related macular degeneration et al. Recently, DNA methyltransferase inhibitors including 5-azanucleoside, azacitidine and decitabine, as well as histone deacetylase inhibitors such as Vorinostat and Romidepsin have been approved by the FDA for treatment of specific types of cancers. Many of these drugs can be tested for controlling inflammation through modulating the helper T cell differentiation/proliferation process.

Conclusions and prospects Newly developed technologies have significantly advanced our ability to understand physiological and pathological responses in the immune systems. The comprehensive understanding of the interaction between genetic and epigenetic regulation of helper T cell differentiation have expanded our potential approaches for distinguishing and controlling both health and disease. Importantly, targeting the epigenetic regulators may serve as a powerful new therapy for treating autoimmune and inflammatory diseases.

References: 1. Kanno Y., Vahedi G., Hirahara K., Singleton K., O'Shea J.J. Transcriptional and epigenetic control of T helper cell specification: molecular mechanisms underlying commitment and plasticity. Annu Rev Immunol. (2012) 30:707-31.

2. http://en.wikipedia.org/wiki/Epigenetics 3. Wu H., Zhang Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev (2011) 25:2436-52.

4. Cedar H., Bergman Y. Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet (2009) 10:295-304.

5. Wei G., Wei L., Zhu J., Zang C., Hu-Li J., Yao Z., Cui K., Kanno Y., Roh T.Y., Watford W.T., Schones D.E., Peng W., Sun H.W., Paul W.E., O'Shea J.J., Zhao K. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity 30:155-67.

6. Wei L., Vahedi G., Sun H.W., Watford W.T., Takatori H., Ramos H.L., Takahashi H., Liang J., Gutierrez-Cruz G., Zang C., Peng W., O'Shea J.J., Kanno Y. Discrete roles of STAT4 and STAT6 transcription factors in tuning epigenetic modifications and transcription during T helper cell differentiation. Immunity 32:840-51.

7. Lee Y.K., Turner H., Maynard C.L., Oliver J.R., Chen D., Elson C.O., Weaver C.T. Late developmental plasticity in the T helper 17 lineage. Immunity. (2009) Jan 16;30(1):92-107.

8. Baylin S.B., Jones P.A. A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer (2011) 11:726-34.

9. Wei L., Liu B., Tuo J., et al. Hypomethylation of the IL-17RC promoter associates with agerelated macular degeneration. Cell Rep (2012) 2:1151-8.

10. Liu B., Wei L., Meyerle C., et al. Complement component C5a promotes expression of IL-22 and IL-17 from human T cells and its implication in age-related macular degeneration. J Transl Med (2011) 9:1-12.

11. Kelly T.K., De Carvalho D.D., Jones P.A. Epigenetic modifications as therapeutic targets. Nat Biotechnol (2010) 28:1069-78.

TRANSLATIONAL IMMUNOLOGY UPDATE Volume 4, Issue 4 Editor: Andrew H. Lichtman Editorial Board: Abul K. Abbas, Carla J. Greenbaum

HIGHLIGHTS FROM RECENT LITERATURE HIF SIGNALING COMBATS CD8+ T CELL EXHAUSTION Research paper: Doedens, A.L., et al., Hypoxia-Inducible Factors Enhance the effector responses of CD8+ T Cells to Persistent Antigen. Nature Immunolgy. 14, 1173-1182 (2013). PMID: 24076634 Under conditions of chronic antigen exposure, CD8 T cells often enter a state of exhaustion, in which effector functions are markedly decreased. Exhausted T cells are observed in chronic infections such as HIV and hepatitis and contribute to impaired antitumor immunity in both mice and humans.

Pharmacologic interventions that reverse T cell exhaustion are of great interest in the treatment of both chronic infections and cancer. In this study, the authors discovered an important role for hypoxiainduced factor (HIF) pathway signaling in preventing CD8 T cell exhaustion.

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The authors studied a mouse model in which expression of VHL, the gene encoding the negative regulator of HIF signaling, was turned on or off. The authors found that VHL-deficient (HIF active) mice died after infection with lymphocytic choriomeningitis virus (LCMV) but wild type mice survived.

Death of infected mice also occurred in wild type mice adoptively transferred with VHLdeficient virus specific CD8+ T cells, suggesting that death after infection was caused by overactive virus specific CTL responses.

These pathogenic CD8+ T cells did not express the terminal effector marker KLRG1 during chronic viral infection. Transcriptional profiling showed that VHL deficient CD8+ T cells had increased HIF signaling, increased cytotoxic functions, and did not enter an exhausted state.

Hypoxia alone also enhanced and prolonged CD8 T cell effector functions in normal T cells, and this response was dependent on HIF signaling. HIF-activated T cells also had decreased expression of Eomes, a transcription factor upregulated in exhausted T cells.

VHL deficient (HIF activated) T cells also provided superior antitumor immune protection in a mouse B16 melanoma model.

Immunomodulatory therapies that enhance T cell activity and reverse T cell exhaustion, including PD1 and CTLA-4 blockade, are proving to be effective in enhancing antitumor responses in cancer patients. These studies suggest that agents that increase HIF signaling could be promising as new therapies for patients with cancer and chronic infections.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital

SCIENCE COMES TO THE AID OF EMERGENCY ROOM PHYSICIANS Research Paper: Zaas, A.K., et al. A Host-Based RT-PCR Gene Expression Signature to Identify Acute Respiratory Viral Infection. Science Translational Medicine 5, 203ra126 (2013). PMID: 24048524 Billions of dollars of NIH research funds are spent each year investigating the effects of various infections on the immune system, most commonly in mouse models. Despite this remarkable outlay of resources, today’s emergency room and hospital physicians have only the crudest tools, such as fever, elevation of the white counts, and tests for specific pathogens to discriminate patients with dangerous bacterial infections from those with viral illnesses. In this study, the authors studied immune profiles of patients with viral vs. bacterial infections in order to determine a rapid and effective test to discriminate between these patients.

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The authors experimentally infected healthy patients with H3N2 or H1N1 influenza and carried out RT-PCR studies of blood to develop a reproducible signature of viral infection.

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They validated this signature by comparing these results to those obtained from healthy patients and “real world” patients seen in the emergency room with fever arising from bacterial vs. viral illnesses.

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The authors identified a set of 48 genes that, when expression levels were measured by RTPCR from blood samples, reliably distinguished patients with viral respiratory illnesses from those with bacterial infections.

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The test had an excellent sensitivity (87%-100%) for detecting influenza infection that was superior to commercially available viral PCR studies and provided superior ability to both rule in and rule out viral respiratory infections. The assay for viral infection also detected patients with unrelated viral infections, including the common cold rhinovirus.

The authors have demonstrated that profiling of a patient’s immune response is as good or better at detecting viral infection than more specific PCR’s for the individual infectious organisms. This rapid test could be used to effectively discriminate patients with viral infections from those with bacterial infections, allowing the appropriate use of antiviral versus antibiotics therapies. If adopted clinically, this test could be a major advance in rapidly diagnosing and appropriately treating patients with fever.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital

HIDE AND SEEK: IDENTIFYING TCRS TO ATTACK PATIENT SPECIFIC CANCER ANTIGENS Research Paper: Linnemann, C. et al. High Throughput Identification of Antigen-specific TCRs by TCR Gene Capture.

National Medicine. (2013) 19: 1534-1543. PMID: 24121928 T cells require multiple signals for activation, carefully protecting us from autoimmunity (a self antigen is not sufficient for activation) and preventing reactions against irrelevant antigens (a foreign antigen is not sufficient for activation). These signals include antigen, processed and then presented by antigen presenting cells to be recognized by specific T cell receptors (TCRs), as well as costimulation and cytokine signals. In this study, the group takes as a starting point the exciting recent advances in oncologic therapy that make use of techniques to engineer TCRs against specific tumor antigens.

They argue for the importance of generating libraries of TCRs that recognize diverse tumor antigens

in the setting of a wide variety of HLA alleles. One can also argue for the value of understanding the population of TCRs in many other clinical scenarios, from autoimmunity to infectious disease.

SUMMARY •

In order to capture the extremely diverse population of TCRs, the team devised a ‘bait’ system which uses conserved components of the TCRs to ‘fish’ for the variable components of the TCRs. It is important to remember the underlying recombination events that yield TCRs, which include the random selection of inherited ‘V’, ‘D’ and ‘J’ segments for VDJ rearrangement and the addition of random bases between the segments. Thus, there are elements of the TCR that are conserved and these can be used to find the novel regions. In this study, each ‘V’ and ‘J’ element in the section of the genome encoding the TCR was targeted.

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In practice, genomic DNA is sheared into very short pieces (~500bp) and the above ‘bait’ strategy is used. Once the regions of interest are ‘fished’ out of the massive quantity of short pieces of DNA, they perform deep sequencing on these regions and then use analysis techniques to reconstruct TCRs.

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To facilitate the process, individual T cells are ‘indexed’ so that they can be sequenced in parallel.

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Once the DNA is sequenced, it is possible to decode the VDJ rearrangements that took place (i.e. this TCR includes V1 and J4). Beyond that, they could also identify the highly variable complementarity determining region 3 (CDR3) component of the TCRs in silico.

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To initially validate the process, they examined CMV and HA-2 specific T cell samples and studied the limits of how few cells they need to successfully determine the VDJ rearrangement.

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To test the clinical efficacy of the technique, they collected blood and tumor infiltrating lymphocyte samples from melanoma patients, isolated T cells with responsiveness to known melanoma antigens and created TCR gene libraries using the TCR capture approach (identifying 147 T cell responses from 61 patients). The genes for 21 TCRs against 11 tumor antigens were identified were used to transduce human peripheral blood lymphocytes, (n = 14), which were then tested for antigen specific cytokine production. Using cell lines that expressed the relevant tumor antigens to test the transduced cells, they found that the TCRs could recognize the tumor antigens, and only one had non-specific activation.

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Interestingly, each tumor antigen yielded only a few TCRs.

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The group then moved on to show that they could identify novel TCRs from bulk tumor infiltrating lymphocytes or short-term expansions of single CD8+ T cells in melanomas (i.e. without preselecting T cells for tumor antigen specificity)

In this study, TCR gene capture demonstrates the potential to identify the TCRs responding to a wide range of antigens, from tumor antigens (including an individual’s tumor neoantigens) to self-antigens underlying autoimmunity and foreign antigens contained within infections. While the ability to identify the potentially novel protein antigens recognized by these TCRs remains a challenge, this work raises many new questions and the potential to assess the T cell repertoire of a host in rapid fashion in a number of fascinating and clinically important contexts Reviewed by Sarah Henrickson, MD, PhD Boston Children’s Hospital and Harvard Medical School

HOW DOES IL-21R/STAT3 SIGNALING REGULATE HUMAN B CELL FUNCTION? A review of Deenick E.K., et al. Naïve and Memory Human B Cells have Distinct Requirements for STAT3 Activation to Differentiate into Antibody-secreting Plasma Cells. Journal of Experimental Medicine., 210:2739-53, (2013). PMID: 24218138 The role of IL-21R/STAT3 signaling in regulating plasma cell generation from naïve and memory B cells is poorly understood. Previously published studies have revealed that STAT3 but not STAT1 is necessary for IL-10- and IL-21-mediated differentiation of human naïve B cells into plasmablasts.

Autosomal-dominant hyper-IgE syndrome (AD-HIES) is caused by a heterozygous mutation in STAT3 that acts in a dominant negative manner, reducing the pool of function STAT3. By comparing ADHIES patients to healthy controls, Deenich and colleagues reveal the distinct requirements for STAT3 activation to induce plasma cell differentiation in naïve and memory human B cells. The authors examine responses naïve and memory B-cell responses to STAT3-activating cytokine IL-21 and the molecular machinery necessary for plasma cell differentiation in this paper, which provides several intriguing observations: •

STAT3 mutations impair the response of naïve but not memory B cells to IL-21. The authors cultured naïve and memory B cells isolated from normal donors or STAT3-mutant patients with CD40L and IL-21 and measured Ig secretion. After 10-12 days in stimulating culture, the authors found that STAT3-deficient naïve B cells failed to secrete Ig while their memory B cell counterparts secreted similar levels of IgM, IgG and IgG as healthy controls. In addition, STAT3-mutant naïve B cells fail to proliferate upon stimulation with IL-21.

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Memory B cell pools in STAT3-deficient patients contain antigen-specific B cells. The authors examined the relative amounts of anti-tetanus IgG produced by B cells from STAT3-mutant donors vs. healthy controls. Naïve B cells from both STAT3-mutants and healthy controls produced very low to undetectable levels of anti-tetanus IgG. Surprisingly, STAT3-mutant memory B cells produced anti-tetanus IgG, albeit at lower levels than healthy controls indicating that STAT3deficient individuals are capable of generating Ag-specific antibody responses and a stable pool of memory B cells.

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Plasma cell lineage commitment is unaffected by mutations in STAT3. During differentiation of human B cells into plasma cells, PAX5 is down regulated, which increases Blimp-1 and XBP-1 expression. The authors examined expression of PAX5, PRDM1 (Blimp-1), and XBP-1 in naïve and memory B cells in STAT3-deficient and healthy donors. They found that STAT3-deficient naïve B cells fail to up regulate Blimp-1 and XBP-1 upon stimulation with IL-21. In contrast, memory B cells do not have this defect.

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Memory cells exhibit greater sensitivity to differentiation-effects of IL-21 and IL-10. To determine differences between naïve and memory B cell responses to IL-10 and IL-21, the authors cultured naïve and memory B cells with CD40L and increasing concentrations of IL-10 or IL-21 and measured expression of XBP-1 and Blimp-1. The authors found that memory B cells have greater sensitivity to both IL-10 and IL-21.

These data suggest that naïve and memory B cells have distinct requirements for STAT3 activation during differentiation into plasma cells. While STAT3 is not required for generation of antigen-specific

memory, naïve B cells fail to differentiate into plasma cells upon stimulation with IL-21. By targeting the STAT3-deficient memory populations, it may be possible to improve antibody-responses in ADHIES patients.

Reviewed by Si-Han V. Hai and Michelle L. Hermiston, MD, PhD, University of California San Francisco

ROLE OF MYELOID SUPPRESSOR CELLS IN DEVELOPMENT OF MYELODYSPLASTIC SYNDROME Research Paper: A review of Chen X et al., Induction of Myelodysplasia by Myeloid-derived Suppressor Cells. Journal of Clinical Investigation. 123:4595-4611 (2013). PMID: 24216507 Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders resulting in disordered hematopoiesis and progressive pancytopenia with irreversible loss of blood-forming cells. The mechanistic basis for MDS is incompletely understood. Direct genetic and epigenetic mutations in hematopoietic stem cells as well as an altered bone marrow immune microenvironment are thought to both contribute to disease pathogenesis. Indeed, although not curative, broad immunosuppression is one therapeutic approach. The paper by Chen, Wei, and colleagues provides new insights into the role of immune dysregulation in MDS pathogenesis. Focusing on the innate immune system, they evaluate the composition and function of the myeloid compartment in primary bone marrow cells obtained from patients with MDS, non-MDS cancer patients (4 breast and 4 lymphoma), and agematched healthy controls. There are several intriguing findings from this work.

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LIN-HLA-DR-CD33+ myeloid derived suppressor cells (MDSC) are expanded in primary bone marrow specimens of MDS patients (median 35% of bone marrow mononuclear cells versus <5% in controls). Mutation analysis demonstrated that the MDSC were not derived from the malignant MDS cells.

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LIN-HLA-DR-CD33+ produced excessive levels of the immunosuppressive cytokines IL10 and TGF-β as well as high levels of nitric oxide and arginase relative to LIN-HLA-DR-CD33+ from healthy controls or patients with other types of cancer.

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Functional studies demonstrated that MDSC cells from MDS patients had increased suppressive function relative to controls. Co-culture experiments demonstrated they could inhibit proliferation of autologous T cells. T cell proliferation and cytokine production could be restored with depletion of MDSC prior to co-culture. Burst-forming unit-erythroid (BFU-E) and GM-CFU colony formation was also lower in co-cultures with MDSC-supplemented versus MDSC-depleted samples.

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MDS-MDSC cells also had increased cytotoxic effector function with increased levels of granzyme B polarization and granule release when cultured with autologous erythroid precursors. This resulted in increased apoptosis of target erythroid precursors, revealing a potential direct mechanism contributing to progressive loss of these cells in MDS.

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Using a series of elegant transfection cell models, the authors demonstrate that the Siglec 3 receptor CD33, which is elevated in MDS-MDSC, can directly promote MDSC suppressive function.

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Using a high-throughput mass-spectrometric approach, the authors identified the S100A9 as a putative ligand for CD33. Functional studies confirmed a direct interaction between CD33 and S100A9. Interestingly, S100A9 levels were increased in MDS bone marrow relative to controls.

S1009 treatment of MDS-MDSC resulted in phosphorylation of CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), recruitment of the inhibitory phosphatse SHP-1, and production of IL10 and TGF-β. Depletion of S100A9 attenuated IL-10 and TGF-β and restored BFU-E and GM-CFU colony formation. In addition, overexpression of S100A9 in transgenic mice resulted in bone marrow dysplasia reminiscent of human MDS.

Taken together, this work highlights a potential role for MDSC in MDS that is mediated, at least in part, by the S100A9/CD33 pathway. Several important questions remain: What triggers the initial expansion of MDSC in MDS patients? Are they necessary for disease initiation, maintenance/progression, or both? Can this pathway be targeted therapeutically? While the authors postulate that sustained activation of innate immune signaling in the bone marrow microenvironment creates a permissive inflammatory milieu that allows for development of MDS, additional studies will be required.

Reviewed by Si-Han V. Hai and Michelle L. Hermiston, MD, PhD, University of California, San Francisco

LOCALIZED IMMUNOMODULATION WITH CTLA-4 BLOCKING ANTIBODY: SAME ANTITUMOR EFFECT WITHOUT THE SIDE EFFECTS Research Paper: Fransen M.F. et al. Controlled Local Delivery of CTLA-4 Blocking Antibody Induces CD8+ T Cell– dependent Tumor Eradication and Decreases Risk of Toxic Side Effects. Clinical Cancer Research.

2013;19(19):5381-5389. PMID: 23788581 Systemic delivery of CTLA-4 blocking antibodies induces antitumor immune responses in some preclinical models and patients, and has been approved for the treatment of advanced melanoma by the FDA. However, because systemic treatment with CTLA-4 blocking antibodies lowers the activation threshold for all T cells, and not just the tumor-reactive T cells, clinical use of CTLA-4 blocking antibodies is hampered by dose-limiting autoimmune and inflammatory side effects. In this study, Marieke Fransen and colleagues demonstrate that it may be possible to achieve the antitumor effect without causing adverse side effects by administering CTLA-4 blocking antibodies locally rather than systemically.

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The authors first compared the antitumor efficacy of systemic versus local administration of CTLA-4 blocking antibody against subcutaneously implanted murine colon carcinoma MC-38 tumors expressing Ovalbumin (MC-38-OVA). They demonstrate that a single local injection of 50 µg of antibody in Montanide ISA-51 was just as effective as treatment with two systemic doses of 200 µg of antibody. They also showed that localized treatment with CTLA-4 blocking antibodies induced antitumor responses in mice bearing MC-38 tumors that do not express OVA, and mice bearing more aggressive OVA-expressing EG7 thymoma tumors.

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Low dose treatment with 50 µg of antibody was shown to only be effective when delivered in the tumor-draining area, and not when given systemically, or in the contralateral flank of tumor-bearing mice.

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Similar to systemic treatment, localized treatment with CTLA-4 blocking antibody was shown to enhance systemic tumor-specific T cell responses, and as a result, was capable of controlling distant tumors.

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By depleting CD4+ and CD8+ T cells in treated mice, the authors demonstrated that CD8+ T cells, and not CD4+ T cells, were responsible for the antitumor effect in their model, indicating that CTLA-4 blocking antibodies can operate directly on CD8+ T cells.

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Analysis of serum from treated mice showed that antibody levels were more than 1,000-fold lower in mice treated with local doses of antibody compared to mice treated with systemic doses. Levels of the liver enzymes ALT and AST were also lower in locally-treated mice suggesting that local slow-release administration of the antibody decreases the induction of adverse effects.

Although systemic treatment with CTLA-4 blocking antibodies can induce significant antitumor effects in some patients, and has been FDA approved for the treatment of advanced melanoma, this treatment can also cause severe autoimmune and inflammatory side effects. This study provides a simple, yet novel delivery system for distributing CTLA-4 blocking antibody in the tumor-draining area.

In preclinical models, use of this delivery approach resulted in antitumor responses that were comparable to systemic antibody treatment, but were associated with 1,000-fold lower serum levels of antibody. These data indicate that use of this delivery approach may make it possible to achieve the same antitumor effects in patients without inducing unwanted side effects. This strategy could also be useful for other immunomodulatory agents being developed for cancer treatment.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

DIFFERENT ADJUVANTS HAVE CONTRASTING EFFECTS ON ANTIGEN-SPECIFIC T EFFECTOR AND T REGULATORY CELLS Research Paper: Perret, R.,. et al. Adjuvants That Improve the Ratio of Antigen-specific Effector to Regulatory T Cells Enhance Tumor Immunity. Cancer Research. 2013;73(22):6597-608. PMID: 24048821 The balance between tumor-antigen-specific effector T cells (Teffs) and regulatory T cells (Tregs) plays an important role in regulating antitumor immunity. Tumor-specific vaccines aim to selectively activate and expand Teffs to skew this balance in order to promote the induction of protective antitumor responses. Immunization with tumor antigen peptides is one vaccination approach that can stimulate tumor antigen-specific T cells. Because vaccination with peptide alone is not effective, and results in expansion of both Teffs and Tregs, numerous adjuvants are being developed to enhance the antitumor activity of peptide-based vaccines. However, prior to this study by Rachel Perret and colleagues, few studies have directly compared the impact of various adjuvants on the balance between antigen-specific Teffs and Tregs.

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The authors first established a model to evaluate the effects of vaccination on antigen-specific CD4+ and CD8+ Teffs and CD4+ Tregs by transferring Ovalbumin (OVA) specific CD8+ OT-1 T cells and CD4+ FOXP3-GFP OT-II T cells into immuncompetent mice.

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Using this model, they compared immunization with OVA257-264 and OVA323-339 peptides with and without various adjuvants. Adjuvants tested included the TLR-2 agonist Pam3Cys, TLR3 agonist Poly(I:C), TLR-4 agonist LPS, TLR-5 agonist Flagellin, TLR-7/8 agonist imiquimod, TLR-9 agonist CpG, and Quil A saponin.

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Analysis of OVA-specific Teffs and Tregs in inguinal vaccine-draining lymph nodes and spleens 7 days following immunization showed that Poly(I:C) and CpG induced significantly more expansion of OT-1 CD8+ Teffs compared to the other adjuvants. OT-II CD4+ Teffs were expanded similarly by all adjuvants, but expanded significantly less to imiquimod and Quil A. All adjuvants, except for Quil A, reduced the proportion of Tregs among OT-II cells compared to immunization with peptide alone.

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Comparison of ratios of OVA-specific Teffs to Tregs in spleens and draining lymph nodes demonstrated that Poly(I:C) and CpG significantly enhanced CD8+ Teff:Treg ratios compared to immunization with OVA peptides alone, whereas CpG, Poly(I:C) and Pam3Cys significantly enhanced OVA-specific CD4+ Teff:Treg ratios.

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Due to their contrasting immunologic effects, the authors chose to further analyze the antitumor effects of Poly(I:C), CpG, imiquimod and Quil A in mice bearing B16 melanoma tumors expressing OVA (B16-OVA). Analysis of tumor infiltrating lymphocytes 7 days following immunization demonstrated that significantly more CD8+ OT-1 Teffs infiltrated tumors following vaccination with Poly(I:C) and CpG compared to the other adjuvants tested.

Because too few OT-II cells were found infiltrating tumors, OVA-specific Teff:Treg ratios in the tumor could not be evaluated.

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OVA-specific Teff:Treg numbers and ratios in tumor-draining lymph nodes in tumor-bearing mice resembled those observed in vaccine-draining lymph nodes in tumor-free mice, and correlated with the degree of CD8+ OT-1 Teff tumor infiltration and tumor protection.

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When mice were vaccinated one week prior to B16-OVA challenge, and boosted 10 days after tumor challenge, mice treated with peptides plus CpG were completely protected, and Poly(I:C) resulted in a delay in tumor growth, whereas the other adjuvants had no effect on tumor growth.

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Cytotoxic analysis of CD8+ OT-1 Teffs 7 days following immunization showed that CpG and Poly(I:C) induced the highest level of OVA-specific cytotoxic function.

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Therapeutic vaccination with OVA peptide plus Poly(I:C) and CpG were also shown to induce enhanced tumor-protection in mice bearing 10-day old EG7 thymomas, whereas use of imiquimod or Quil A had no tumor-protective effect.

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The authors also confirmed their results in a self-antigen melanoma system by quantitating GP100-specific Pmel CD8+ Teffs and Trp-1-specific Trp-1 CD4+ Teffs and Tregs following immunization with peptides plus the various adjuvants. In addition, they showed that immunization with Poly(I:C) and CpG induced significantly more polyfunctional CD8+ and CD4+ Teffs than imiquimod or Quil A.

•

Immunization with peptides plus Poly(I:C) or CpG were also shown to induce the greatest cytotoxic and antitumor effects in the self-antigen melanoma model.

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Analysis of cytokine production in vaccine-draining lymph nodes 24 hrs following vaccination showed that Poly(I:C) and CpG induced significantly more production of the proinflammatory type I cytokines IFN-β, IL-12, IFN-γ and IL-6 compared to imiquimod and Quil A.

This study provides a comprehensive analysis of the effects of a broad panel of adjuvants on tumor antigen-specific Teffs and Tregs, and the induction of antitumor immunity. The authors demonstrated that, compared to imiquimod and Quil A, Poly(I:C) and CpG induce more potent antitumor immunity that is associated with the preferential expansion of antigen-specific Teffs over Tregs resulting in increased Teff:Treg ratios, and the induction of polyfunctional antigen-specific Teff responses, both of which have been shown in previous studies to correlate with improved antitumor immunity. Moreover, this study shows that not all adjuvants are equal, and suggests that Poly(I:C) and CpG are the most ideal adjuvants currently available for peptide vaccines for cancer.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

TSC1 REGULATES THE BALANCE BETWEEN EFFECTOR AND REGULATORY T CELLS Research Paper: [Journal of Clinical Investigation] Yoon Park, Hyung-Seung Jin, Justine Lopez, Chris Elly, Gisen Kim, Masako Murai,Mitchell Kronenberg and Yun-Cai Liu.. Published 25 November, 2013. PMID: 24270422 Investigators found that T cell-specific deletion of the gene encoding tuberous sclerosis 1 (TSC1), an upstream negative regulator of mammalian target of rapamycin, resulted in augmented Th1 and Th17 differentiation and led to severe intestinal inflammation in a colitis model.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

SKIN THYMIC STROMAL LYMPHOPOIETIN INITIATES TH2 RESPONSES THROUGH AN ORCHESTRATED IMMUNE CASCADE Research Paper: [National Community]. Pierre Hener, Paula Michea, Hajime Karasuyama, Susan Chan, Vassili Soumelis & Mei Li. Published 28 November 2013 PMID: 24284909 By dissecting the cellular network triggered by mouse skin thymic stromal lymphopoietin (TSLP) in vivo, researchers uncovered that TSLP-promoted IL-4 induction in CD4+ T cells in skin-draining lymph nodes is driven by an orchestrated ‘DC-T-Baso-T’ cascade, which represents a sequential cooperation of dendritic cells (DCs), CD4+ T cells and basophils.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

LEPTIN DEFICIENCY IMPAIRS MATURATION OF DENDRITIC CELLS AND ENHANCES INDUCTION OF REGULATORY T AND TH17 CELLS Research Paper: A review of Moraes-Vieira, P.M.M., et al. Leptin Deficiency Impairs Maturation of Dendritic Cells and Enhances Induction of Regulatory T and Th17 Cells. European Journal of Immunology. DOI: 10.1002/eji.201343592 PMID: 24271843 The authors investigated whether leptin mediates its activity on T cells by influencing dendritic cells (DCs) to promote Th17 and regulatory T (Treg) cell immune responses in mice. They observed that leptin deficiency reduced the expression of DC maturation markers, decreased DC production of IL12, TNF-α and IL-6, increased DC production of TGF-β, and limited the capacity of DCs to induce syngeneic CD4+ T-cell proliferation.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

TSP1-PRODUCING B CELLS SHOW IMMUNE REGULATORY PROPERTY AND SUPPRESS ALLERGY-RELATED MUCOSAL INFLAMMATION Research Paper: A review of Zhang, H.P., et al. TSP1-Producing B Cells Show Immune Regulatory Property and Suppress Allergy-Related Mucosal Inflammation. Scientific Reports. 2013; (3): 3345 PMCID: PMC2096769 Researchers aimed to elucidate the role of the thrombospondin (TSP)1-producing B cells in the immune regulatory role of specific immunotherapy (SIT). The results showed that after SIT, the frequency of CD35+ B cells was increased in the intestine of mice with food allergy.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

CONTROLLING IMMUNE RESPONSES BY TARGETING ANTIGENS TO DENDRITIC CELL SUBSETS AND B CELLS Research Paper: A review of Chappell, C.P., International Immunology. November 2013. PMID:24285828 Delivering antigens in vivo by coupling them to mAbs specific for unique receptors on antigenpresenting cells (APCs) is a promising approach for modulating immune responses. The authors focus on the literature surrounding the control of B-cell responses when antigen is delivered to various APC subsets.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

CD28 AND ITK SIGNALS REGULATE AUTOREACTIVE T CELL TRAFFICKING Research Paper: A review of Jain, N., et al. CD28 and ITK Signals Regulate Autoreactive T Cell Trafficking. Nature Medicine. 2013; (19) 1632-37.PMID: 24270545 The authors demonstrated that the CD28 co-stimulatory pathway regulates the trafficking of selfreactive cytotoxic T lymphocyte antigen-4 (Ctla4)−/− T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4−/− T cells to accumulate in secondary lymphoid organs.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

HIGHLIGHTS FROM CLINICAL IMMUNOLOGY, THE OFFICIAL JOURNAL OF FOCIS HISTOPATHOLOGY IN RELAPSING AND REMITTING EAE A review of: Soellner IA. et al. Differential Aspects of Immune Cell Infiltration and Neurodegeneration in Acute and Relapse Experimental Autoimmune Encephalomyelitis Clinical Immunology 149, 519– 529, 2013 PMID: 24239839 Relapsing–remitting multiple sclerosis is the most common clinical subtype of human MS, and is modeled by proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. The authors of this paper have previously shown that myelin oligodendrocyte glycoprotein (MOG)-induced EAE, which displays a chronic disease phenotype, is characterized by early acute inflammation leading to chronic neuro-degeneration, and that axonal damage was present early and remained constant but myelin damage increased chronically. In this paper, the authors characterized and quantified CNS inflammation, extent of demyelination, and axon damage in acute and relapse phase of PLP-induced EAE, with the goal of better understanding mechanisms by which relapsingremitting MS transitions to chronic-progressive disease. The major finding were: •

• • • •

The predominant cell type found in acute and relapse EAE within the CNS infiltrates were CD4+-cells, however there were differences in other cell types between the two phases, namely, a reduction of CD8+ T cells and macrophages and an increase in B in the relapse phase.

There was significantly less edema in the relapse phase. decreased in relapse EAE. In acute EAE, there was extensive demyelination taking place, while in the relapse phase, there was less myelin pathology, with both demyelinated nerve fibers and axons in the process of demyelination.

The number of dead axons also increased significantly in the relapsed phase, and there were more damaged axons without inflammation of myelin sheaths, than in the acute phase.

Damaged axons without myelin sheath pathology were increased significantly in relapse phase compared to initial acute phase.

Overall, the results are consistent with previous studies in showing that clinical severity of acute EAE correlates with the degree of spinal inflammation while late neurological disease correlates with loss of axons. The authors argue that their findings support the hypothesis that nerve fiber destruction in late EAE reflects neuro-degeneration independent from ongoing inflammation and myelin damage.

Based on EM findings in this study, they suggest early mitochondrial damage is one factor that leads to neuro-degeneration.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital

IMMUNOTHERAPY WITH HUMAN ALLOGENEIC CD8REG A review of: Horwitz, DA., et al. Therapeutic Polyclonal Human CD8+ CD25+Fox3+ TNFR2+ PD-L1+ regulatory cells induced ex-vivo. Clinical Immunology 149, 450–463, 2013. PMID: 24211847 Most of the effort on therapeutic use of Treg has focused on isolating CD4+ CD25+ Foxp3+ Tregs from blood of patients with autoimmune diseases or allografts, expanding them in vitro, and transferring them back into the patient. Horwitz and colleagues cite problems with expansion of these classical Treg from limited numbers isolated from blood and limited efficacy on pathogenic memory CD4+ effector cells as the rationale for characterizing the therapeutic potential of CD8+ regulatory T cells (CD8reg). In this data-rich paper, the authors describe the in vitro generation and in vivo suppressive activity of human CD8+ T regs that phenotypically resemble exhausted CD8+ CTL or mouse donorderived CD8+ Foxp3+ Tregs that are found in recipient mice after transplantation.

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Human CD8reg were generated from blood derived naïve CD8+ cells by stimulation with antiCD3/28 coated beads, IL-2, with or without TGF-β.

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The in vivo suppressive activity of these T cells was tested in a xenogeneic graft vs. host model in which human PBMC were transferred in into lightly irradiated NOD SCID IL-2Rγc deficient mice, which results in rapid engraftment of human T cells and death by GVHD in two weeks.

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Transfer of allogeneic CDr8eg generated with or without TGFβ quadrupled survival time in the GVHD model. When the PBMC and the CD8reg were from the same donor (autologous) there was no protection.

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The CD8reg expressed IL-2Rαβ chains, TGF-β dependent Foxp3, TNFR2, PD-L1, PD-1, and Tim3. In vitro, FOXP3 expression was enhanced by TGF-β, and sustained by IL-2.

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The in vivo protective activity of the CDreg was dependent on IL-10.

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The CD8regs lack cytotoxic activity against allogeneic T cells.

Based on their findings, the authors propose that polyclonal CD8regs generated with anti-CD3/28 beads will preferentially target allogeneic T cells when transferred into hosts with autoimmune disease, GVHD or allograft rejection. They suggest that the transferred allogeneic cells CD8regs would not be rejected because they would suppress the lymphocytes they come into contact with. The

findings on this paper are similar in several ways to another recent report that showed transfer of human CD8+ regulatory inhibit GVHD when transferred into humanized mice without causing generalized immunosuppression (Zheng J,Sci. Transl. Med. 5,2013). A limitation described by the authors of the CD4+ dependent xeno-GVHD model is that it does not test efficacy of the CD8reg on pathogenic class I MHC restricted pathologies. The precise mechanisms of in vivo suppression remain unclear, and testing the efficacy of human allogeneic CD8reg in models of autoimmunity will be challenging.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Womenâ&#x20AC;&#x2122;s Hospital

HUMAN IMMUNOPHENOTYPING UPDATE Imaging Flow Cytometry: Picture this!

J Philip McCoy, Jr., PhD NHLBI and the Center for Human Immunology, NIH A number of variants of traditional flow cytometry, such as CyTOF and Luminex, have recently emerged as exciting new technologies that extend the utility of cytometry for clinical research. Imaging flow cytometry, a technology in which immunophenotyping data can be acquired in concert with capturing images of every cell analyzed, should also be recognized as one of these revolutionary new cytometric endeavors. Since separate images are captured for every fluorochrome on each cell, which can be merged into a multicolor image, it is possible to a myriad of analyses with these data. The main advantage of imaging flow cytometry is that it is now possible to visualize the pattern of staining of every marker examined on every cell in a sample. Such visualization permits one to examine features with relative ease as one is obtaining routine immunophenotyping data. Furthermore, as one can obtain these data on hundreds of cells per second, it is practical to avoid the pitfalls that one may encounter using confocal microscopy such as insufficient cell numbers for rigorous statistical calculations or the subjectivity of selecting a particular field of view for study. Using imaging flow cytometry data from thousands of cells can be collected in only a few minutes without any potential bias in how these cells were selected for analysis.

So other than satisfying your curiosity about how your cells look, how can you apply this technology to your research? Adding morphology to your flow cytometry permits answering some rather obvious questions, such as is the staining capped or polarized? Thaunat and colleagues (1) demonstrated asymmetric segregation of polarized antigens on B lymphocyte division that correlated with the ability of progeny to present antigen and activate T cells. Visualization of cellular protrusions or podopods, for example, may permit one to ask which antigens are present on these structures.

Nobile and colleagues used this approach to visualize cell protrusions in lymphocytes induced by HIV1 Nef (2). It is also possible to discriminate nuclear from cytoplasmic staining, hence nuclear translocation of transcription factors such as NF-ÎşB is readily detectable (3). This technology is also capable to performing polychromatic immunophenotyping with roughly 6 to 8 different fluorochromes.

Furthermore, as images are captured of each color separately; it is possible to determine the extent of antigen co-localization by staining cells simultaneously with two fluorochrome-labeled antibodies to those antigens. One can also examine cellular functions such as apoptosis and autophagy on cell subsets defined by multiple markers, and a recent publication has demonstrated an imaging flow cytometry techniques for measuring average telomere length using labels for repetitive DNA sequences in the human telomeric repeat (4).

Even more applications can be devised which take even more advantage of being able to capture images of the immunophenotyping. For example, phagocytosis of fluorescent particles can

not only be detected, but also the number of phagocytosed particles can be counted (5). Imaging flow cytometry can also be used to directly visualize immunological synapses between T cells and antigen presenting cells (APCs) and the redistributions of antigens as these synapses form (6). Similarly, one can directly image target-effector conjugates as exemplified by Pascal et al (7). Erythrocyte morphology has been studied using imaging flow cytometry in studies of sickle cell anemia (8) and in Plasmodium faciparum (9).

Naturally, new technologies are not perfect in every way. Some shortcomings of the current generation of imaging flow cytometers include the lack of ability to sort – thus any cells identified by this technology cannot be purified for downstream analyses such as genomic studies. Imaging glow cytometry also has a much lower throughput and number of colors that can be run simultaneously than traditional flow cytometry, and the highest magnification is 60x. Finally, the sheer size of the data files limits the number of events that can be acquired in a single file. This is due to the fact that images are captured for every cell in every color, hence thousands of images are present in a single data file, yielding data files that can require 500MB for storage. Even with these caveats, imaging flow cytometry is an interesting technique that presents new opportunities for addressing difficult research questions.

References 1. Thaunat O, Granja AG, Barral P, Filby A, Montaner B, Collinson L, Martinez-Martin N, Harwood NE, Bruckbauer A, Batista FD. Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity. Science. 2012 Jan 27;335(6067):475-9. doi: 10.1126/science.1214100.

2. Nobile C, Rudnicka D, Hasan M, Aulner N, Porrot F, Machu C, Renaud O, Prévost MC, Hivroz C, Schwartz O, Sol-Foulon N. HIV-1 Nef inhibits ruffles, induces filopodia, and modulates migration of infected lymphocytes. J Virol. 2010 Mar;84(5):2282-93. doi: 10.1128/JVI.0223009.

3. George TC, Fanning SL, Fitzgerald-Bocarsly P, Medeiros RB, Highfill S, Shimizu Y, Hall BE, Frost K, Basiji D, Ortyn WE, Morrissey PJ, Lynch DH.Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. J Immunol Methods. 2006 Apr 20;311(1-2):117-29.

4. Ma H, Reyes-Gutierrez P, Pederson T. Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors. Proc Natl Acad Sci U S A. 2013 Dec 9.

[Epub ahead of print].

5. Ackerman ME, Moldt B, Wyatt RT, Dugast AS, McAndrew E, Tsoukas S, Jost S, Berger CT, Sciaranghella G, Liu Q, Irvine DJ, Burton DR, Alter G. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J Immunol Methods. 2011 Mar 7;366(1-2):8-19. doi: 10.1016/j.jim.2010.12.016.

6. Hoffmann S, Hosseini BH, Hecker M, Louban I, Bulbuc N, Garbi N, Wabnitz GH, Samstag Y, Spatz JP, Hämmerling GJ. Single cell force spectroscopy of T cells recognizing a myelinderived peptide on antigen presenting cells. Immunol Lett. 2011 Apr 30;136(1):13-20. doi: 10.1016/j.imlet.2010.11.005.

7. Pascal V, Laffleur B, Debin A, Cuvillier A, van Egmond M, Drocourt D, Imbertie L, Pangault C, Tarte K, Tiraby G, Cogné M. Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells.

Haematologica. 2012 Nov;97(11):1686-94. doi: 10.3324/haematol.2011.061408.

8. Fertrin KY, van Beers EJ, Samsel L, Mendelsohn LG, Saiyed R, Nichols JS, Hepp DA, Brantner CA, Daniels MP, McCoy JP, Kato GJ. Imaging Flow Cytometry Documents Incomplete Resistance of F-Cells To Hypoxia-Induced Sickling In Blood Samples From Patients With Sickle Cell Anemia. ASH, 2013.

9. Safeukui I, Buffet PA, Perrot S, Sauvanet A, Aussilhou B, Dokmak S, Couvelard A, Hatem DC, Mohandas N, David PH, Mercereau-Puijalon O, Milon G. Surface area loss and increased sphericity account for the splenic entrapment of subpopulations of Plasmodium falciparum ring-infected erythrocytes. PLoS One. 2013;8(3):e60150. doi: 10.1371/journal.pone.0060150.

DEVELOPMENTS IN BASIC IMMUNOLOGY AND NOVEL THERAPIES THE YIN AND YANG OF IL-2 By: Abul K. Abbas, University of California San Francisco The historical perspective Interleukin-2 (IL-2) has a storied history in the annals of Immunology. It is the first cytokine to be purified and characterized, and shown to stimulate the proliferation of T cells, the activity that gave rise to its early name T cell growth factor. It is also produced mainly by T cells, and thus functions as an autocrine growth factor. The discovery of IL-2 launched the concept that the immune system was regulated by soluble, secreted factors; this concept was revolutionary when first proposed and is, of course, now considered self-evident. IL-2 was also the first cytokine whose gene was cloned, and the first whose cellular receptor was identified and characterized. The IL-2 receptor (IL-2R) is considered the founding member of the “type 1 cytokine receptor” family. It consists of three chains, all of which are required for high-affinity ligand binding. The α chain is unique to IL-2 and the β and γ chains are shared with other receptors. The receptor signals by a Jak-Stat pathway as well as pathways involving Ras-MAP kinase and Akt. In addition to serving as a proliferative signal for T cells, IL-2 also stimulates the differentiation of naïve T cells into effector and memory cells. There is great interest in defining the actions of IL-2 on follicular helper T cells and, therefore, on germinal center B-cell reactions, and on innate lymphoid cells.

The established T-cell stimulating activity of IL-2 provided the impetus for the clinical applications of this cytokine for boosting immune responses in patients with advanced cancers. This led to another first – IL-2 was the first cytokine, and, in fact, probably the first immunologically active biological agent, given to patients. The high incidence of serious adverse events, likely related to the production of other cytokines induced by high doses of IL-2, has seriously limited the use of this agent in cancer patients. The immune stimulatory activity of IL-2 was the basis of a much later clinical trial combining low doses of the cytokine with anti-retroviral drugs to boost immune responses in HIVinfected patients. Although IL-2 significantly increased CD4 T-cell counts, it showed no clinical benefit over anti-retroviral drugs alone. These disappointing clinical trial results have led to uncertainty about systemic IL-2 therapy to enhance immune responses in patients.

Revisions to the history: the unexpected actions of IL-2 Dogmas about the functions of IL-2 were shaken by the unexpected finding that germline deletion of the IL-2 gene led not to immune deficiency, as might have been predicted, but to severe lymphoproliferation, dominated by T cells, and systemic autoimmunity. The autoimmunity is manifested primarily by colitis or hemolytic anemia in different mouse strains. This was soon followed by observations that knockout of the IL-2Rα or β chain led to a similar lymphoproliferative and autoimmune disorder. (Knockout or mutations of the IL-2Rγ chain result in a severe combined immunodeficiency [SCID] phenotype because this chain, also called the common γ [γc] chain, is a component of the receptor for the essential lymphopoietic cytokine IL-7.) The excessive proliferation and expansion of T cells in the absence of the prototypic T cell growth factor was an obvious paradox.

The solution to this conundrum came with the finding that IL-2 is necessary for the development and maintenance of Foxp3+ regulatory T cells (Treg). It was known from the earliest discovery of Treg that these cells express high levels of CD25, the IL-2Rα chain, so it was not a surprise that they also respond to the cytokine. Mice lacking IL-2 have a profound deficiency of Treg and, as a result, uncontrolled activation of conventional (non-regulatory) T cells. It is still not known which cytokines or other stimuli that drive this massive T-cell proliferation in the absence of IL-2 or IL-2R signaling.

Nevertheless, the conclusion of the knockout mouse studies is that the essential, or non-redundant, function of IL-2 is to limit immune responses by acting on Treg, and its role in stimulating immune responses is redundant, and can be replaced by other stimuli.

Retrospective analyses of patients treated with IL-2 have shown a predicted increase in the numbers and suppressive activity of blood Foxp3+ T cells. Very few patients have been described with mutations in CD25. These patients typically present with immune deficiency and infections, as well as features of autoimmunity. In two of the patients who have been studied, Foxp3+ T cells are present but appear to be functionally defective.

The role of IL-2 in the life of Treg The realization that IL-2 is essential for Treg has led to a vast number of studies asking how the cytokine generates and maintains these cells. Despite some minor contradictions in the literature, the bulk of the evidence, mostly from mouse models, suggests that IL-2 has multiple actions that collectively enhance Treg numbers and functions.

• IL-2 is a survival factor for Treg in the periphery, and in its absence Treg lose their competitive fitness and undergo death by apoptosis.

• IL-2 promotes the function of Treg by maximizing expression of key effector molecules such as CTLA-4.

• IL-2 maintains the stability of Treg, in part by inducing demethylation of the Treg-specific demethylated region in the Foxp3 gene locus.

• IL-2 may promote the development of Treg in the thymus, in response to recognition of self antigens.

One of the puzzles in this area is that IL-2 functions mainly as an autocrine growth factor, so the same T cells produce and respond to the cytokine, but Treg do not produce IL-2. It is believed that small amounts of IL-2 produced by conventional T cells during normal responses to environmental antigens may serve to maintain the peripheral pool of functional Treg. Thus, one under-appreciated role of immune responses to foreign antigens may be to indirectly maintain tolerance (unresponsiveness) to self antigens!

The therapeutic potential of IL-2: prospects and challenges As the ability of IL-2 to maintain Treg has become increasingly established and accepted, the possibility of using the cytokine to suppress pathologic immune responses has been raised. Two

proof-of-concept studies were published in 2011 showing, in small numbers of patients, that treatment with low-doses of recombinant IL-2 had a beneficial effect in steroid-resistant graft-vs-host disease (GVHD) and in hepatitis virus-associated vasculitis. Based on these encouraging results, many more studies are now in the planning and execution stages, in type 1 diabetes, transplant rejection, and GVHD. The continuing challenge in the therapeutic application of IL-2 is that it does serve dual opposing roles – it stimulates and inhibits immune responses, by acting on different T cell populations. A central question then becomes how to selectively manipulate its activity so it does what is desired. So far many of the clinical studies have relied on dosing, based on the idea that activation of effector and memory T cells requires transient exposure to high concentrations of IL-2 whereas maintenance of Treg requires prolonged exposure to low concentrations. It is, however, difficult to develop precise quantitative criteria for the appropriate concentration or dose of any cytokine needed for diverse actions. Several other approaches are being tried.

• A very interesting possibility was raised by the finding that IL-2 could be targeted to Treg or effector T cells by making complexes with different anti-IL-2 antibodies. This phenomenon has been demonstrated in mice and humans. The antibodies may conceal different receptor-binding sites on the cytokine, thus leaving it available to bind to CD25 (and thus preferentially to Treg) or to the IL-2Rβγ receptors (thus focusing the cytokine preferentially on CD8+ T cells and NK cells).

• Based on these results, attempts have been made to structurally modify IL-2 to target it to different receptors. A version of the cytokine has been synthesized that targets it to CD8+ T cells (and, probably, NK cells). So far, there is no modified IL-2 that preserves its activity on Treg only.

• Another approach is to combine IL-2 with drugs that block different immune responses. The first clinical trial of IL-2 with rapamycin, predicted to inhibit effector T cells and preserve Treg, was done in type 1 diabetes but was terminated because the effector response was not adequately inhibited. As newer kinase inhibitors are developed to inhibit different lymphocyte populations, this type of combination therapy clearly has potential, especially if it becomes possible to identify differences in the signaling pathways used by effector T cells and Treg.

Summary and conclusions The first cytokine to be characterized continues to reveal surprises. Perhaps the most impressive aspects of the story of IL-2 are how basic research in the biology of the cytokine has overturned dogma and provided unexpected new insights, and how these insights are being translated into therapies. IL-2 remains the paradigm for an immune modulator with often opposing actions, raising great possibilities as well as substantial challenges.

SELECTED RECENT CLINICAL TRIAL RESULTS Edited by Elisa Boden, MD, Benaroya Research Institute

PHASE 1/2 STUDY OF TRANSDERMAL DELIVERY OF MYELIN PEPTIDES IN RELAPSINGREMITTING MULTIPLE SCLEROSIS Clinical Trial: Walczak, A. et al. JAMA Neurol. 2013; 70(9): 1105-1109 PMID: 23817921 Disease: relapsing-remitting multiple sclerosis Drug: • Myelin peptides (MBP85-99, PLP139-151, MOG35-55) applied transdermally via skin patch Study: • Phase 1/2, single center, randomized, double-blind, placebo-controlled study of transdermal delivery of myelin peptides in subjects with relapsing-remitting multiple sclerosis.

•

30 patients were randomized to placebo or 2 doses of myelin peptides delivered transdermally for one year; placebo (n=10), 1 mg myelin peptides (n=16), 10 mg myelin peptides (n=4).

•

MRI was performed at screening and every 3 months.

•

The primary endpoints were safety and efficacy measured as the number of active gadolinium positive lesions on MRI per patient per scan.

•

Secondary endpoint measures were mean volume of gadolinium positive lesions and cumulative number of new T2 lesions at months 3, 6, 9, and 12 and T2 lesion and T1 lesion volume change from baseline to 12 months.

•

Secondary clinical endpoints included annual relapse rate, proportion of relapse-free subjects, proportion of subjects with 3 months of confirmed disability worsening on the Expanded Disability Status Scale (EDSS) at month 12.

Results: • There were some differences in baseline characteristics of subjects randomized to placebo versus myelin patches with a higher proportion of female subjects, shorter duration of disease and slightly higher baseline EDSS scores among subjects receiving placebo.

•

There were no serious adverse events documented in the study. The most common side effect of myelin peptide patches was a local reaction in the area of the skin patch.

•

The primary efficacy endpoint was met for the 1 mg dose with a 66.5% reduction in the number of gadolinium positive lesions per patient per scan (p= .02).

•

In terms of secondary endpoints, there was a trend toward reduction in the volume of gadolinium positive lesions per patient and the cumulative number of new T2 lesions per patient among patients receiving the 1 mg dose. There was a statistically significant reduction

in the mean T1 and T2 lesion volume at 12 months among patients receiving the 1 mg dose of myelin peptides. â&#x20AC;˘

There were no differences in primary or secondary outcome measures for MRI between placebo and the 10 mg myelin peptide dose, although numbers in this group were small.

â&#x20AC;˘

Subjects receiving both the 1 mg and 10 mg myelin peptide patch had statistically significant reductions annual relapse rates compared to placebo. In the 1 mg myelin patch group, there was also significant increase in the number of relapse-free subjects compared to placebo.

There was a trend toward fewer patients with disability progression measured by EDSS in subjects receiving myelin peptide patches.

Why this trial is of interest to the broader FOCIS community: The majority of therapeutic agents for the treatment of autoimmune diseases are globally immunosuppressive and leave patients vulnerable to serious infections and lymphoproliferative disorders. Therapies capable of inducing antigen-specific tolerance hold great promise as safer alternatives to current therapeutics for autoimmune disease. Specific immunotherapy (SIT) is such a therapeutic strategy that has been used clinically for many years to induce antigen-specific tolerance in allergic diseases. This method delivers repeated subcutaneous doses of antigens or peptides which ultimately induces antigenic tolerance that lasts beyond the duration of therapy. Although the mechanisms that lead to tolerance have not fully been elucidated, evidence suggests induction of regulatory T cells and/or immune deviation from Th2 to Th1 response play an important role1.

In the current study, SIT has been adapted for use in the treatment of multiple sclerosis by dermal delivery of peptide epitopes implicated in the immune response that drives multiple sclerosis.

Although the study included a small number of patients, significant reductions in new brain lesions were identified in subjects on therapy which correlated with a reduction in symptomatic relapses. In a previously published study, the authors demonstrated that subjects receiving transdermal peptide therapy had reduced myelin-specific proliferative responses and increased IL-10 production in peripheral blood lymphocytes, providing evidence of immunoregulatory modulation by dermal peptide therapy2.

Interestingly, while the 1 mg peptide dose had statistically significant improvements in primary and secondary outcomes, the higher 10 mg peptide dose did not appear to be as effective. This may simply have been a result of the very small sample size in the 10 mg dose group. However, this may be reflective of a dose-dependency to the tolerogenic response, which has been described in tolerance to orally delivered antigens3.

While the results from this study are encouraging and dermal peptide therapy appears safe, the study population is very small and differences in the baseline characteristics subjects may have favored a positive outcome for the treatment group. Notably, while oral delivery of myelin has been demonstrated to prevent and treat animal models of multiple sclerosis, a phase III study of orally administered bovine myelin did not show clinical benefit in patients with multiple sclerosis3. Should significant clinical benefits of dermal myelin peptides hold up in a larger cohort, it may be instructive to study how dermal and oral delivery of antigens result in differential immune responses.

References Moldaver D, et al. Allergy. 2011; 66 (784-91) 2 Jurynczyk M, et al. Ann Neurol. 2010; 68 (5): 593-601.

3 Faria AM et al. Immunol. Rev., 206 (2005): 232-259.

1

PHASE 3 STUDIES OF VEDOLIZUMAB IN ULCERATIVE COLITIS AND CROHN’S DISEASE Edited by Elisa Boden, MD, Benaroya Research Institute

Clinical Trials: Feagan, B., et al. NEJM. 2013; 369; 8: 699-710 PMID: 23964932 Sandborn, W., et al. NEJM. 2013; 369; 8: 711-721.

PMID: 23964933 Diseases: Ulcerative colitis, Crohn’s disease Drug: • Vedolizumab, a humanized IgG1 monoclonal antibody that specifically recognizes he α4β7 intigrin given at a dose of 300 mg intravenously (1) Vedolizumab in ulcerative colitis: •

Phase 3, multicenter, randomized, double-blind, placebo-controlled study of Vedolizumab in subjects with moderate to severely active ulcerative colitis who failed or were intolerant to standard therapies.

•

Two integrated studies to evaluate induction and maintenance therapy.

•

The induction arm included 374 patients who were randomized 3:2 to receive induction doses of Vedolizumab or placebo at weeks 0 and 2.

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A second cohort of 521 subjects received open-label induction with Vedolizumab.

•

The maintenance arm of the study randomized responders from both induction cohorts to Vedolizumab every 4 or 8 weeks or placebo (1:1:1) for 52 weeks.

•

The primary endpoint for induction was clinical response defined by reduction in the Mayo clinic activity score including rectal bleeding at 6 weeks. The primary endpoint for maintenance was remission at week 52 (Mayo score <2).

•

Safety and efficacy were evaluated at weeks 2, 4, 6 and every 4 weeks thereafter.

Results: • The primary efficacy endpoint for the induction study was met with clinical response observed in 47.1% of subjects who received Vedolizumab versus 25.5% of subjects who received placebo at 6 weeks (p<0.001). Secondary analysis also showed a statistically significant increase in the percentage of patients achieving remission and mucosal healing in the Vedolizumab-treated group.

•

The primary efficacy endpoint for the maintenance study was also met with remission achieved in 41.8% of subjects receiving Vedolizumab every 8 weeks, 44.8% of patients receiving Vedolizumab every 4 weeks and 15.9% of patients in the placebo group (p<.001).

Secondary endpoints of mucosal healing and steroid-free remission were more frequent

among subjects receiving Vedolizumab. There were no differences in treatment outcomes among subjects with prior anti-TNF failure.

•

No significant differences in adverse events were observed in subjects exposed to Vedolizumab.

(2) Vedolizumab in Crohn’s disease: •

Phase 3, multicenter, randomized, double-blind, placebo-controlled study of Vedolizumab in subjects with moderate to severely active Crohn’s disease who failed or were intolerant to standard therapies.

•

Two integrated studies to evaluate induction and maintenance therapy.

•

The induction arm consisted of 368 patients randomized 3:2 to receive doses of Vedolizumab or placebo at weeks 0 and 2.

•

A second cohort of 747 subjects received open-label induction Vedolizumab.

•

The maintenance arm of the study randomized responders from both induction cohorts to Vedolizumab every 4 or 8 weeks or placebo (1:1:1) for 52 weeks.

•

Primary endpoints for induction were clinical remission (Crohn’s disease acivity index (CDAI) <150) and response (decrease of more than 100 points in CDAI) at week 6.

•

The primary endpoint for maintenance was clinical remission at week 52.

•

Safety and efficacy were evaluated at weeks 2, 4, 6 and every 4 weeks thereafter.

Results: • One of two primary efficacy endpoints for the induction study was met. 14.5% of subjects who received Vedolizumab were in clinical remission at week 6 versus 6.8% of subjects who received placebo (p = 0.02). There was no difference in response rates at week 6 (31.4 % in Vedolizumab-treated versus 25.7% in placebo-treated, p=0.23).

•

The primary efficacy endpoint for the maintenance study was met with 39% of subjects who received Vedolizumab every 8 weeks, 36.4% of subjects who received Vedolizumab every 4 weeks and 21.6% of patients who received placebo in clinical remission at 52 weeks (p=.004).

Secondary endpoints of response and steroid-free remission at 52 weeks were more frequent in subjects who received Vedolizumab. There were no differences in treatment outcomes among subjects with prior anti-TNF failure.

•

Infections and serious infections were more common in patients receiving Vedolizumab than placebo. Serious adverse events including deaths were more frequent among subjects who received Vedolizumab (24.4% versus 15.3%).

Why these trials are of interest to the broader FOCIS community: Inhibition of leukocyte migration has been an attractive and successful approach for the treatment of organ-specific immune-mediated diseases including multiple sclerosis and Crohn’s disease. Integrins are cell surface transmembrane receptors, expressed as heterodimers, that mediate adhesion to the extracellular matrix and are critical for a diverse set of functions including immunologic surveillance and migration. The α4β7 integrin binds to Mucosal Addressin Cellular Adhesion Molecule 1

(MAdCAM-1) expressed in the vascular endothelium of the gastrointestinal tract and mediates migration of memory T cells to the intestines. Natalizumab, a monoclonal antibody that inhibits α4 integrin, is effective for the treatment of Crohn’s disease and multiple sclerosis1-3. In addition to affecting lymphocyte migration to the gut via α4β7, Natalizumab blocks α4β1 integrin that binds to VCAM-1 and facilitates migration of memory T cells to the central nervous system, bone marrow and skin. Unfortunately, the use of Natalizumab has been limited in clinical practice, because it significantly increases the risk of progressive multifocal leukoencephalopathy (PML), an opportunistic brain infection that typically results in severe neurologic disability or death4.

Vedolizumab provides increased specificity of integrin blockade by targeting the α4β7 complex.

Animal models have suggested that Vedolizumab does not affect leukocyte trafficking to the CNS5.

None of the 1713 patients treated with Vedolizumab in these studies developed PML. The efficacy of Vedolizumab for induction of remission in Crohn’s disease was modest and it failed to meet one of its primary endpoints. Vedolizumab appeared to be more effective for induction in subjects with ulcerative colitis. The reason for this discrepancy is not clear. The authors cite two potential factors that may explain the mediocre induction rates in Crohn’s disease. First is that the subjects in this study with Crohn’s disease had very high baseline CDAI and may be a more difficult group of patients to treat. Second, they hypothesize that for Crohn’s disease the 6week assessment point may have been too early, as Natalizumab induction effects continue to increase up to 12 weeks23 . In an accompanying review6, it is also suggested that Natalizumab, by blocking of both α4β1 and α4β7 may provide global leukocyte defects that impact the more systemic nature of Crohn’s disease.

These landmark studies demonstrate that highly specific integrin blockade can be a successful treatment strategy for organ-specific immune-mediated diseases. Key knowledge also gained from this study is that Vedolizumab is effective in patients who have failed currently available therapies, particularly TNFα inhibitors.

This underscores importance of developing new classes of therapeutics that address the heterogeneity of patient responses. While the safety profile is similar to other biologics used to treat IBD, there is a suggestion of increased risk of serious infection in patients Crohn’s disease. While no cases of PML have been reported in patients treated with Vedolizumab, larger numbers of patient exposures will be required to fully assess this risk.

References Polman C, et al. NEJM. 2006; 354:899-910 2 Sandborn, W et al. NEJM. 2005; 353: 1912-25 3 Targan, S et al. Gastro. 2007; 132: 1672-83 4 Bloomgren G, et al. NEJM. 2012; 366: 1870-80 5 Haanstra, K et al. J Immunol. 2013; 190: 1961-73 6 Cominelli, F. NEJM. 2013; 369; 8: 775-6 1

TRANSLATIONAL IMMUNOLOGY UPDATE Volume 5, Issue 1 Editor: Andrew H. Lichtman Editorial Board: Abul K. Abbas, Carla J. Greenbaum

HIGHLIGHTS FROM RECENT LITERATURE A SURPRISING IMMUNE ETIOLOGY FOR NARCOLEPSY Research paper: De la Herrán-Arita, et al., CD4+ T Cell Autoimmunity to Hypocretin/Orexin and Cross-Reactivity to a 2009 H1N1 Influenza A Epitope in Narcolepsy. Science Translational Medicine 5, 216ra176 (2013).

PMID: 24353159 Type I narcolepsy is a lifelong disorder characterized by sleepiness, aplexy, and rapid transition from wakefulness to REM sleep. Type I narcolepsy is strongly associated with HLA DQ0602 and results from the loss of the approximately 70,000 posterior hypothalamus neurons that produce the wake promoting neuropeptide hypocretin.

• • • • •

The authors identified two immune epitopes of the hypocretin neuropeptide that bound to DQ0602. These two epitopes of hypocretin activated a population of CD4+ T cells in patients with narcolepsy but not in DQ0602-positive healthy controls.

The development of narcolepsy has been previously associated with infection by the 2009 H1N1 influenza A strain (pH1N1).

The authors administered a seasonal influenza vaccine containing pH1N1 to patients with narcolepsy and found an increased frequency of circulating T cells specific for the two immunogenic epitopes of hypocretin.

In addition, the authors also identified a hemagglutinin epitope of the influenza virus specific to the pH1N1 influenza strain that had homology to the two immunogenic hypocretin peptides.

In vitro stimulation of CD4 + T cells from patients with narcolepsy with the influenza pH1N1 proteins or the hemagglutinin epitope alone increased the frequency of T cells responding to the two hypocretin peptide epitopes.

Narcolepsy, a lifelong disease with devastating impact on quality of life has long been associated with a particular HLA molecule but an immune etiology has been elusive. The authors have demonstrated that T cells responding to the H1N1 influenza A strain pH1N1 and possibly to a single hemagglutinin epitope of this virus, likely cross-react with two immune epitopes for the critical neuropeptide hypocretin. These results suggest that cross-reactivity induced by antiviral immunity may be responsible for the destruction of hypocretin producing posterior hypothalamus neurons and therefore for some cases of narcolepsy developing after 2009. This elegant study suggests a role for molecular mimicry in the development of narcolepsy and sets the stage for further evaluation of immune epitopes in this neurologic disease.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital

PUTTING NEUTROPHILS INTO REVERSE: FISHING FOR NEW ANTI-INFLAMMATORY MOLECULES Research Paper: Robertson, et al. A Zebrafish Compound Screen Reveals Modulation of Neutrophil Reverse Migration as an Anti-flammatory Mechanism. Science Translational Medicine 5, 225ra29 (2014). PMID: 24554340 New therapies are needed for inflammatory disorders that can aid in the resolution of inflammation.

Targeting molecules that help resolve inflammation has been difficult because limited knowledge exists about the various molecules that contribute to resolution of inflammation. In order to identify new targets and new drugs, the authors developed and utilized a novel transgenic zebrafish model screen.

• • • • •

The authors developed an in vivo screen for molecules that helped resolve inflammation in using transgenic zebrafish. In this model, sterile tissue injury was induced and a panel of known compounds was screened for their ability to accelerate resolution of inflammation.

The screen found that a substance derived from a Chinese medicinal herb, tanshinone IIA, strongly accelerated resolution of inflammation.

This molecule blocked proinflammatory signals, induced neutrophil apoptosis and also had a surprising effect of inducing reverse migration of neutrophils in vivo in zebrafish.

Tanshinone IIA promoted neutrophil reverse migration by inducing nondirectional redistribution of the cells, rather than by inducing active fugetaxis.

The authors extended their studies to human neutrophils and demonstrated that tanshinone IIA overrode human neutrophil survival signals and induced apoptosis of human neutrophils.

Resolution of inflammation is a process that is poorly understood and novel therapeutics that enhance inflammatory resolution are needed for the treatment of autoimmune and inflammatory disorders. The authors identified a novel enhancer of immune resolution using a new in vivo zebrafish assay. The identified compound, tanshinone IIA, promoted neutrophil death in both humans and zebrafish and had the interesting effect of inducing increased random migration of neutrophils, thereby dispersing them away from the site of inflammation. These studies are significant because they identify a novel therapeutic that can now be further tested in humans and they demonstrate the utility of using a zebrafish screen to identify novel agents that enhance inflammatory resolution.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital

THE WHOLE 9 YARDS: A CAREFUL CHARACTERIZATION OF HUMAN TH9 CELLS Research Paper: Schlapach, C., et al. Human Th9 Cells are Skin-Tropic and have Autocrine and Paracrine Proinflammatory Capacity. Science Translational Medicine 6, 219ra8 (2014). PMID: 24431112 The immune system contains a panoply of cells and soluble factors to recognize and assess antigens and then determine an appropriate set of responses. Within the adaptive cell-mediated immune response, T cells have been subdivided into numerous subsets, with increasing appreciation of the existence of many intermediate states and functions in previously defined subsets, as well as entirely new subsets. Th9 cells are one of the newer subsets of CD4+ T cells, and they have led to extensive study in mouse models as well as studies on differentiating them from naïve or memory T cell populations or in a few human diseases. In this study, Schlapbach et al provide a detailed analysis of Th9 cells in healthy human donor PBMCs, including their frequency, their tissue tropism and their response to common pathogens and polyclonal stimulation. This is taken deeper, with investigations into Th9 cells from tissues and in samples from patients with varied skin disorders, and provides the most comprehensive picture yet of this cell subset.

SUMMARY •

• •

•

• •

•

The study begins by assessing responses of skin tropic (CLA+), gut tropic (α4β7+) and nonskin tropic, non-gut tropic (CLA-, α4 β7-) memory CD4+ T cells to autologous monocytes pulsed with peptides from a variety of pathogens . As one might expect, T cells with tropism for a given tissue responded more strongly to pathogens that are generally found at that site (i.e. Candida albicans and CLA+ CD4+ T cells).

Interestingly, IL-9 production was found almost exclusively in the skin tropic (CLA+) subset and only when stimulated with a skin-flora pathogen (C. albicans). In addition, the production of IL9 was transient (here, peaked at day 6).

Next, they examined the effects of polyclonal stimulation of human healthy donor PBMCs. Again, they isolated three populations: skin tropic (CLA+), gut tropic (α4β7+) and non-skin tropic, non-gut tropic (CLA-, α4β7-) CD4+ T cells and assessed cytokine production and proliferation post-stimulation. Most IL-9 was still produced by skin tropic CD4+ T cells, and most of those cells did not co-produce other cytokines commonly associated with major helper T cells subsets (i.e. IFNγ, IL-13, IL-17). Of note, skin homing cells also produced IL-13 (most after IL-9 production had halted), gut homing cells primarily produced IFNγ and non-skin, nongut homing T cells produced more IL-17. In this context, IL-9 production was again transient, though it peaked at day 2.

Beyond studies of PBMCs, the study also characterized IL-9 cells in skin, lung and gut from healthy human tissue samples by isolating resident memory T cells (TRM) from those tissues and assessing IL-9 production after stimulation. Skin resident memory T cells were the major source of IL-9 and were also found to co-produce TNFα and stain positive for granzyme B. Given reports that IL-9 may come from ILCs in mice, they assessed the effect of IL-33 and IL-2 (which stimulate IL-9 production from human ILCs) on skin TRM with no production of IL-9.

With respect to how IL-9 producing helper T cells affect the other subsets of helper T cells, blockade of the early, transient production of IL-9 reduced subsequent production of both additional IL-9 (autocrine) as well as IL-17, IL-13 and IFNγ (paracrine) from CLA+ skin tropic CD4+ memory T cells, in addition to reducing proliferation.

Finally, they characterized Th9 cells in skin samples from a Th2 skewed disease (atopic dermatitis) and a Th1/Th17 skewed disease (psoriasis) and found Th9 cells in both types of skin lesions, with a significant increase in psoriatic lesions versus healthy skin.

In this study, Th9 cells are identified in healthy human skin and PBMCs and the paper demonstrates that the secretion of IL-9 is from skin tropic CD4+ memory cells in PBMCs or skin resident memory CD4+ T cells and can be stimulated using tissue relevant pathogens or polyclonal stimulation techniques. They demonstrated that the production of IL-9 is co-incident with TNFa and granzyme B and comes from a CD3+ population (i.e. does not seem to be exclusively from ILCs in humans). In addition, the early and transient secretion of IL-9 by Th9 cells was shown to enhance the secretion by other core helper T cell subsets of their own signature cytokines (i.e. IL-17 from Th17 cells). Finally, psoriatic lesions from human patients showed an increase in Th9 cells versus healthy skin. Much work remains to be done to link these cells to the pathogenesis of disease, and starting with inflammatory skin disorders seems like a perfect first step. The ways in which this cell type are tied into the complex network of inhibitory and activating factors that drive helper T cell differentiation and function will also likely fuel efforts for years to come. This study provides many new avenues for investigation and a clear set of questions to pursue next.

Reviewed by Sarah Henrickson, MD, PhD, Childrenâ&#x20AC;&#x2122;s Hospital of Philadelphia

DRINKING FROM A FIRE HOSE: COLLECTING AND ANALYZING MASSIVE DATA SETS A review of Newel, E.W. and Davis, M.M., et al. Beyond Model Antigens: High Dimensional Methods for the Analysis of Antigen-Specific T Cells. Nature Biotechnology. 32, 147-149, (2014). PMID: 24441473 The complexity of the human immune system has dramatic variability among individuals based on genetic diversity as well as unique exposures to pathogens, commensal organisms and myriad environmental factors. Recent reviews in Nature Immunology and Nature Biotechnology (Feb. 2014) highlight the many challenges and tools available and under development for analysis of the increasingly deep and massive data sets that are being generated in this field.

Newell et al focuses on the challenges and strategies in deriving a complete understanding of the antigen specific T cell response to immunological challenge. Kidd et al focuses on massively multidimensional datasets and the best strategies for getting our hands on the full implications of these datasets, making use of the best algorithms and packages that are currently available.

SUMMARY â&#x20AC;˘

â&#x20AC;˘

Newell et al begins by discussing the frontiers of techniques available for phenotyping the states present in T cells, from traditional flow cytometry (with both user defined and non-linear strategies for data analysis) to mass cytometry (which allows simultaneous assessment of greater numbers of characteristics per cell, with much lower throughput). In addition, they discuss the use of microwells for single cell phenotyping of T cells and single cell transcriptomics.

Next, they focus on advanced techniques and recent papers that focus on the assessment of antigen specific T cells. They begin by discussing the history and recent advances in the use of peptide-MHC tetramers for antigen specific T cell enumeration, including methods for generating tetramers with full coverage or targeted coverage of epitopes from pathogens with small or large genomes. This includes a clear discussion of merging mass cytometry with the use of reagents to characterize antigen specific T cells.

• •

• •

Finally, they discuss the use of TCR sequencing and how to possibly derive antigen specificity from TCR sequences (with a focus on library based strategies versus structural prediction models).

Kidd et al take a more broad focus and discuss strategies to describe the entire immune response from many vantages, and provide a set of tools to use from each vantage point.

These techniques include transcriptional analysis (including DNA microarrays, DNA sequencing and RNASeq) with specific recommendations for the merging of simultaneous analysis of DNA sequencing and mRNA profiling to allow expression quantitative trait locus (eQTL) analysis. Mentioned also is how to deconvolute datasets containing profiles of a mixed (e.g PBMC) population from a computational perspective into cell specific datasets. They also discuss methods for assessing mass cytometry data.

From there, they discuss the complex methods for constructing networks from varied data sets, including a number of recent fascinating publications.

In addition to a wonderful list of packages for each type of analysis, they also provide a discussion of the many datasets and repositories of human data available for investigators to study.

Both reviews provide overviews for the generalist and the specialist of both the available tools, the most recent articles developing and exploiting these tools and the challenges that remain. Among these challenges remains the need for resources to allow systematic collection of appropriate samples (both in healthy donors from pediatric, young adult and elderly populations across races and ethnicities as well as in human disease) as well the development of analysis packages that can be used by non-computational biology researchers with deep domain knowledge in the samples under study.

Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia

DOES CANCER TRIGGER AUTOIMMUNITY? Research Paper: Joseph, C.G., et al. Association of the Autoimmune Disease Scleroderma with an Immunologic Response to Cancer. Science. 2014; 343(6167):152-157. PMID: 24310608 Systemic delivery of CTLA-4 blocking antibodies induces antitumor immune responses in some preclinical models and patients, and has been approved for the treatment of advanced melanoma by the FDA. However, because systemic treatment with CTLA-4 blocking antibodies lowers the activation threshold for all T cells, and not just the tumor-reactive T cells, clinical use of CTLA-4 blocking antibodies is hampered by dose-limiting autoimmune and inflammatory side effects. In this study, Marieke Fransen and colleagues demonstrate that it may be possible to achieve the antitumor effect without causing adverse side effects by administering CTLA-4 blocking antibodies locally rather than systemically.

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The authors first compared the antitumor efficacy of systemic versus local administration of CTLA-4 blocking antibody against subcutaneously implanted murine colon carcinoma MC-38 tumors expressing Ovalbumin (MC-38-OVA). They demonstrate that a single local injection of 50 µg of antibody in Montanide ISA-51 was just as effective as treatment with two systemic doses of 200 µg of antibody. They also showed that localized treatment with CTLA-4 blocking antibodies induced antitumor responses in mice bearing MC-38 tumors that do not express OVA, and mice bearing more aggressive OVA-expressing EG7 thymoma tumors.

•

Low dose treatment with 50 µg of antibody was shown to only be effective when delivered in the tumor-draining area, and not when given systemically, or in the contralateral flank of tumor-bearing mice.

•

Similar to systemic treatment, localized treatment with CTLA-4 blocking antibody was shown to enhance systemic tumor-specific T cell responses, and as a result, was capable of controlling distant tumors.

•

By depleting CD4+ and CD8+ T cells in treated mice, the authors demonstrated that CD8+ T cells, and not CD4+ T cells, were responsible for the antitumor effect in their model, indicating that CTLA-4 blocking antibodies can operate directly on CD8+ T cells.

•

Analysis of serum from treated mice showed that antibody levels were more than 1,000-fold lower in mice treated with local doses of antibody compared to mice treated with systemic doses. Levels of the liver enzymes ALT and AST were also lower in locally-treated mice suggesting that local slow-release administration of the antibody decreases the induction of adverse effects.

Although systemic treatment with CTLA-4 blocking antibodies can induce significant antitumor effects in some patients, and has been FDA approved for the treatment of advanced melanoma, this treatment can also cause severe autoimmune and inflammatory side effects. This study provides a simple, yet novel delivery system for distributing CTLA-4 blocking antibody in the tumor-draining area.

In preclinical models, use of this delivery approach resulted in antitumor responses that were comparable to systemic antibody treatment, but were associated with 1,000-fold lower serum levels of antibody. These data indicate that use of this delivery approach may make it possible to achieve the same antitumor effects in patients without inducing unwanted side effects. This strategy could also be useful for other immunomodulatory agents being developed for cancer treatment.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

TEST-DRIVING CARS (CHIMERIC ANTIGEN RECEPTORS) FOR SOLID TUMOR IMMUNOTHERAPY Research Paper: Beatty, G.L., et al. Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies. Cancer Immunol. Res. 2014; 2(2):112-120. PMID: 24579088 The adoptive transfer of CAR-expressing T cells has shown promising results as a treatment for CD19+ hematologic malignancies. However, efforts to extend this approach to solid tumors have been less successful and hindered with high toxicity. A major obstacle has been on-target off-tumor toxicity directed against normal tissue expressing the antigenic target of the CAR. In this study, Beatty and colleagues provide a potential approach to circumvent this issue.

• •

The authors established a platform for engineering transient CAR-expressing T cells using in vitro transcribed mRNA. In this article, they present case reports for the first two patients (one with mesothelioma and one with metastatic pancreatic adenocarcinoma) receiving mRNA-engineered CAR T cells specific for mesothelin, a cell-surface antigen expressed by several different solid tumors including mesothelioma and pancreatic adenocarcinoma, but also at low levels on normal

• •

• •

peritoneal, pericardial, and pleural mesothelial surfaces. mRNA mesothelin CAR T cells were infused intravenously in both patients, and intratumorally only in the pancreatic cancer patient.

Using quantitative PCR assays specific for the CAR transgene, the authors demonstrated that mRNA-engineered mesothelin CAR T cells persisted transiently in peripheral blood following intravenous infusion, but also trafficked to primary and metastatic tumor sites.

Infusion of mRNA-engineered mesothelin CAR T cells was associated with anti-tumor activity in both patients as monitored by CT and PET imaging, and stabilization of serum tumor markers. In addition, in vitro cytolytic assays were used to demonstrate that mRNA engineered mesothelin CAR T cells specifically lysed mesothelin-expressing tumor targets.

CAR T cell infusion was also associated with signs of immune induction including the development of novel antigen specific serum antibody responses, supporting the induction of epitope-spreading.

Importantly, no overt signs of on-target off-tumor toxicity (e.q. peritonitis, pleuritis and pericarditis) were observed in either of the two patients. This is interesting because treatment with antibody-toxin drug conjugates targeting mesothelin has been limited by pleuritis.

This study demonstrates the feasibility of using mRNA-engineered T cells to evaluate the antitumor activity, and off-tumor toxicity potential of CARs targeting tumor-associated antigens. This strategy provides a transient and controlled approach for more safely evaluating CAR-expressing T cells for the treatment of solid tumors.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

A LOOK AT THE FULL SPECTRUM! Research Paper: Xue, et al. Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation. Immunity. 40, 2014; 274-288. PMID: 24590056 According to the current conceptual framework, macrophages can be polarized into classically (M1) or alternatively (M2) activated cells with a role in promoting Th1 and Th2 responses respectively.

Insights gained into activation, polarization, and function of macrophages based on this framework have indeed been helpful in understanding the immune responses in health versus disease.

Nonetheless, transcriptional programs regulating these processes remain poorly characterized. Also, observations obtained from macrophages involved in chronic inflammation, chronic infection, or cancer are strongly suggestive of a rather broader transcriptional repertoire extending beyond the current bipolar model. In order to study the macrophage transcriptional programs better, the authors generated a data set of 299 macrophage transcriptomes by in vitro stimulation of human macrophages with 28 diverse activation signals.

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Thorough analysis of this data set through powerful analytical tools such as coregulation analysis (CRA), self-organizing-map (SOM) clustering, and correlation coefficient matrices (CCM) revealed a more extensive ‘spectrum’ model of macrophage activation that indeed goes beyond the existing M1 versus M2 model. The spectrum model was endorsed by in phenotypic and functional characterization studies.

Through this analysis, the authors could also identify certain genes that were selectively elevated in only one of the stimulation conditions in their data set. At the same time, necessity of multi-gene combinations to distinguish complex input signals at transcriptional level was perceived.

Using weighted gene coexpression network analysis (WGCNA), the authors could identify 49 distinct coexpression modules displaying divergent patterns corresponding to individual

• • •

stimuli. Gene ontology enrichment analysis (GOEA) and prediction of transcription factor binding based on the module genes revealed novel associations between stimulus-induced gene expression patterns and in vivo immune response.

Most interestingly, applying these transcriptional programs to human alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease (COPD) revealed an unexpected loss of inflammatory signatures in COPD patients.

Through reverse network engineering (RNE), the authors could define common denominators of macrophage activation, confirming the involvement of unknown transcriptional regulators, and identifying unexpected yet unexplored candidate regulators.

Finally, by integrating murine data from the ImmGen project, the authors put forth a refined, activation-independent core signature for human and murine macrophages.

With immunology entering into the era of high dimensional data generation and analysis, this paper primarily serves as an excellent example to demonstrate how capitalizing on the advanced analytical tools helps reveal erstwhile obscured cellular heterogeneity. The spectrum model thus uncovered indeed opens new avenues for future research in macrophage responses. The demonstrated application of this model to predict macrophage programs in vivo certainly holds tremendous potential in translational immunology. Similar studies employing clinically relevant stimuli (for e.g. those derived from pathogens, tumor cells, or allergens) to generate a transcriptional data set are needed in this context, and this study will most definitively be helpful in guiding the analytical strategy for such future experiments.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

BITTER END FOR MICROBES IN THE AIRWAY Research Paper: Lee, et al. Bitter and Sweet Taste Receptors Regulate human Upper Respiratory Innate Immunity.

Journal of Clinical Investigation. 2014; 124(2): 1393-1405. PMID: 24531552 Mucociliary action is a primary clearance method employed by upper airway epithelium to defend against encroaching toxins, particulates, and microbial populations. An alternative and novel role for bitter taste receptors, T2Rs, in bacterial clearance has recently emerged with the discovery that T2R38 enhances ciliary beat frequency and increases α-microbial peptides secretion and nitric oxide release. Lee et al. define an alternative method for bactericidal killing in which activated bitter receptors secrete α-microbial peptides upon bitter ligand administration. The authors further demonstrate that bitter receptors are in turn regulated by sugar and sugar substitutes through activated sweet receptors of upper airway chemosensory cells, thereby preventing α-microbial secretions.

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Using surgically removed human explants, the author set-up an air-liquid interface (ALI) to mimic the polarized upper airway epithelium. Administration of the bitter compound denatonium benzoate to apical nonciliated human sinonasal chemosensory cells in ALI cultures induces a calcium response that propagates to adjacent cells via gap junctions.

Apical co-expression and activation of sweet receptors T1R2/3 by glucose and sugar substitutes overrides denatonium-induced calcium responses, thereby regulating T2R calciumdependent signaling pathways.

Administration of denatonium benzoate to the apical side of ALI cultures results in calciumdependent bactericidal effects on numerous bacterial populations. Co-administration of glucose in a dose-dependent manner reverses α-microbial responses of denatonium-induce

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T2R activation. Preliminary findings suggest that cAMP and protein kinase signaling regulate α-microbial secretion.

Denatonium-stimulated secretions contain low-molecular weight proteins corresponding to αmicrobial peptides, specifically β-defensin-1 and -2. Glucose-stimulated T1R2/3 sweet receptors inhibit β-defensin secretion.

Patients with Chronic Rhinosinusitis (CRS) have recurrent upper respiratory infections and have elevated glucose levels in the nasal cavity similar to levels found to inhibit T2Rs by in vitro analysis. These data suggest that increases in nasal glucose in CRS patients exacerbates microbial infections by activing sweet receptors and subsequently inhibiting calcium-dependent T2R β-defensin secretion.

In summary, the authors postulate that in a healthy individual normal glucose levels repress bitter receptor expression of α-microbial peptides. Upon upper airway infections, microbial glucose consumption and shedding of bitter-derived microbial compounds relieve inhibition of T2R-dependent α-microbial peptide secretion.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

FOXP3 GENE TRANSFER-A NEW THERAPY FOR IPEX SYNDROME Research Paper: Passeerini, L., et al. CD4+ T Cells from IPEX Patients Convert into Functional and Stable Regulatory T Cells by FOXP3 Gene Transfer. Science Translational Medicine 5, 215ra174 (2013). PMID: 24337481 Immune dysregulation, polyendocrinopathy, enteropahty, X-linked (IPEX) syndrome is a primary immunodeficiency disorder that is characterized by loss of function of thymus-derived CD4+CD25+ regulatory T cells (Treg). Hematopoietic stem cell transplantation (HSCT) is the only curative available for IPEX syndrome patients. Data from animal models and recent successful gene therapy trials in graft-versus-host-disease patients undergoing allogeneic HSCT show promise for the use of Treg cells as therapeutic agents in diseases. In this study, the authors explore the possibility to restore immune regulatory function in FOXP3 mutated T cells by wild-type FOXP3 gene transfer thereby converting these conventional T cells in fully functional Treg like cells with suppressor function.

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They successfully generated CD4 FOXP3 Treg like cells using lentiviral vector system from healthy donors. These cells were stable and showed low levels of cytokine secretion (IL-2, IFN-g, IL-5, and IL-22) even under inflammatory conditions when stimulated with IL-6 and IL-1b.

Using a humanized murine model of xeno-GVHD (graft-versus host disease) in NSG mice they show in-vivo functionality of these CD4FOXP3 Treg cells. They show increased survival and decreased weight loss in NSG mice after transfer of CD4FOXP3 Treg cells.

Transfer of these cells resulted in development of substantial chimerism in peripheral blood and these cells did not hamper the engraftment of Teff cells but instead controlled their effector function and proliferation.

They show that CD4FOXP3 Treg cells generated using naïve CD4 cells are more stable and less proliferative compared to CD4FOXP3 Treg cells generated using antigen

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experiences memory CD4 T cells indicating that FOXP3 mediated Treg conversion of T cells is more efficient in naïve CD4 T cells compared to memory T cells.

To next evaluate the possibility of using these cells in a clinical setting, they transduced FOXP3 expression in FOXP3 mutant conventional CD4 T cells from 5 IPEX patients with different FOXP3 mutations. They successfully generated transduced T cells with stable FOXP3 expression that acquired Treg like phenotype- CD25hi, CTLA4hi and CD127low and upregulation of Helios. These transduced FOXP3 cells also showed reduced cytokine secretion similar to that observed in cells generated in healthy donors. These FOXP3 Treg cells also acquired functional properties similar to those of conventional Treg cells- cell suppression of allogenic CD4 effector cells and low self proliferation upon CD3 stimulation.

In vivo studies using the lethal GVHD humanized mouse model also demonstrated in vivo functionality of these cells generated from IPEX patients by protecting these animals from lethal GVHD and loss of weight.

Overall these data demonstrate that CD4FOXP3 T cells generated from FOXP3-mutated CD4+ T cells from IPEX patients are fully functional, both in vitro and in vivo. These de novo generated cells are highly stable if generated from naïve T cells and perform regulatory functions even in inflammatory conditions in vitro and in vivo. Taken together these data suggest that gene transfer of FOXP3 can be a potential therapeutic approach for IPEX syndrome patients.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine

HIGHLIGHTS FROM CLINICAL IMMUNOLOGY, THE OFFICIAL JOURNAL OF FOCIS A SLAP IN THE FACE OF AUTOIMMUNITY A review of: Peterson L. et al. SLAP Deficiency Decreases DsDNA Autoantibody Production. Clinical Immunology 150, 201–209, 2014 PMID: 24440645 Src-like adaptor protein (SLAP) down-regulates BCR signaling by promoting c-Cbl-mediated ubiquitin ligation of BCR complex proteins, leading to their degradation. This SLAP function reduces BCR levels in developing B cells. The authors investigated if SLAP deficiency could enhance central B cell tolerance by increasing BCR signaling in developing autoreactive B cells leading to deletion or receptor editing. They tested the effects of SLAP-deficiency using two models of anti-dsDNA autoimmunity. The first model uses vaccination with a peptide mimeotope, which induces an antidsDNA response in mice, and glomerular deposition of Ig. The second model is a Ig heavy chain transgenic mouse (56R), in which the transgene was cloned from a B cell derived from autoimmune MRL/lpr mouse. The 56R mouse spontaneously produces anti-dsDNA IgG autoantibodies with various light chains. The major findings were: • •

dsDNA peptide mimetope immunization induced an anti-dsDNA responses in wild type BALB/c but not SLAP-/- BALB/c mice. There were equivalent amounts of anti-peptide antibodies in both groups.

SLAP−/− 56R mice showed reduced amounts of anti-dsDNA antibodies compared to 56R control mice. A subset of the SLAP−/− 56R mice did not show reduced levels of anti-dsDNA antibodies, but the anti-dsDNA abs in the SLAP-deficient mice showed significantly less class switching to IgG2a, which is the major anti-dsDNA isotype in 56R mice

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Analysis of B-cell subsets and Vκ and Vλ light chain usage showed no differences between 56R and SLAP−/− 56R mice, indicating that the decreased production of autoantibodies in SLAP−/− 56R mice was not due to receptor editing.

However, SLAP deficiency did result in shift of the light chain usage in 56R mice from mainly Vκ38 to the complete editor Vκ21. The author argue this reflects repertorie selection due to biased light chain pairing and not based on receptor editing.

Overall, findings in this study are consistent with the hypothesis in the setting of SLA deficiency, increased BCR signal strength in developing B cells reduces autoantibody production. The authors suggest that targeting the ubiquitination of the BCR complex in developing B cells, is a potential strategy to decrease autoreactive B cell development.

Reviewed by: Andrew Lichtman, Brigham and Women’s Hospital

THERAPEUTIC PEPTIDES COME FULL CIRCLE A review of: Ali, M. et al. Cyclization Enhances Function of Linear Anti-arthritic Peptides. Clinical Immunology 150, 121–133, 2014 PMID: 24207019 In this paper, the authors document the enhanced biological activities of a cyclic peptide (C1) derived from a previously characterized linear peptide (CP), the later based on the TCR-α transmembrane region, including two polar amino acids (K, R) that are essential for TCR complex assembly. CP was previously shown to inhibit T cell activation in vitro, and when given subcutaneously or intraperitoneally, reduced the severity of adjuvant induced arthritis. C1, the cyclic variant of CP described in this paper, consists of alternating D-, L-amino acids, which may permit stacking into nanotubes within membranes, and thereby impart greater biological activity than linear peptides. The major finding of the study include the following: •

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In comparison to a control linear peptide, the C1 cyclic peptide was very effective in inhibiting T cell hybridoma IL-2 production in response to SEA plus APCs (an MHC dependent mechanisms), and was less albeit somewhat effective in inhibiting hybridoma IL-2 production in response to antiCD3, anti-TCRβ, and PMA and ionomycin. The C1 was not toxic to the cells in these assays.

NMR studies of C1 were consistent with C1 entering membranes and adopting a transmembrane orientation, without forming lethal pores.

Surface plasmon resonance was used to show that C1 bound very strongly to membrane, essentially irreversibly.

C1 also showed enhance stability and overall permeation through skin after 2 and 6 days, using an in vitro assay.

C1 delivered subcutaneously was as effective as cyclosporine in blocking inflammation in a rat adjuvant induced arthritis model.

In a mouse Ova-induced asthma model, C1 was effective in reducing eosinophil accumulation in BAL, reducing IL-5 and IL-13 in blood, and C1 suppressed airway hypersensitivity as effectively as methylprednisone.

Overall, this study shows that cyclization of a previously characterized linear immunomodulatory peptide greatly enhanced its effectiveness in binding to membranes, blocking T cell activation in vitro,

and reducing inflammatory parameters of T cell mediated disease process in rodent models. The authors results suggest that cyclization may be promising approach to enhancing the delivery and therapeutic effectiveness peptide inhibitors of immune mediated diseases.

Reviewed by: Andrew Lichtman, Brigham and Women’s Hospital

HUMAN IMMUNOPHENOTYPING UPDATE DATA NORMALIZATION: HOW TO HANDLE BATCH EFFECTS IN LARGE STUDIES Holden T. Maecker, PhD, Stanford University Janet Siebert, MS, MEd, CytoAnalytics, Inc. There is a theme among those doing translational immunology that “mice are easy, humans are hard”.

One of the more subtle ways that this truism plays out is in the greater need to deal with batch effects in human studies. Partly because of genetic heterogeneity, human studies often involve more subjects than a typical animal study. Also, while animal studies can be manipulated so that all the animals reach a particular endpoint at the same time, human studies often cannot. Thus, in situations requiring fresh samples, one must perform the human assays longitudinally. For both of these reasons (number of subjects and the occasional need for real-time assays), one often faces the prospect of comparing human data across multiple “runs” or batches. Given the variability of many immune monitoring assays, this generally results in some run-to-run variability, or “batch effect”, and the consequent need for normalization of data across batches (1). Here, we will discuss some highlevel considerations that should be given when designing studies for downstream normalization, and what normalization options are available for various study designs.

Standardization. The first consideration, of course, is to minimize the batch effects in the first place.

It is always advisable to secure a single lot of reagents for a study, and to ensure that those reagents have a shelf life that exceeds the duration of the study. Lyophilized antibody staining cocktails can be helpful in this regard (2, 3).

It is also advisable to adhere strictly to a validated SOP, as the validation will have established the level of inter-assay variability to be expected (4, 5). As many variables as can be controlled from run to run, should be controlled.

Sample distribution. Crucial comparisons should be made within a batch, if at all possible. For example, in a longitudinal study, all time points for a given individual should be run together. In this way, each subject serves as their own control, and normalization across batches generally becomes irrelevant.

Intra-assay concerns. For some assays, there are potential concerns about the ordering of samples within a run, or the placement of samples within a plate. For example, a fluorescence signal may decrease over the time of data acquisition, or there could be differences between edge and center wells of a plate over long-term culture. In such cases, one should try to avoid a systematic bias, i.e., don’t always run the treatment group last, etc. It may also be useful to place replicate controls at two distinct locations (e.g., the beginning and end of the run) to assess the within-batch variation. Even

without such concerns, replicates (as many as practical) of the same control sample should be run in each batch. This allows the degree of batch-to-batch variability to be assessed, and compared to the intra-assay variability.

Balancing batches. It is also wise, whenever possible, to design “balanced” batches with regard to the types of samples they contain. For example, if comparisons are to be made between three treatment regimens, each batch should contain a similar proportion of samples from all three treatment regimens. If it is a case-control study, cases and controls should be evenly divided among all batches. Obviously, this is not possible when assaying fresh samples in real time. But even in such “batch of one” scenarios, it is still worth trying to devise a control (such as replicates of a fixed or frozen specimen) that can be run with each new patient sample as a control.

While simple, standardization and batch design are factors that cannot be over-emphasized, since flaws in these factors may not be possible to overcome once the data is collected. After data collection, one needs to ask a series of questions before proceeding with normalization or other analysis.

To normalize or not? First, we can ask, what is the level of batch variability in a given study? By having the same control sample(s) in each plate, one can calculate a measure of variability (e.g., the coefficient of variation, CV) for each analyte across batches. If this is not obviously higher than the intra-assay CV, then there is no need to normalize. The intra-assay CV can be ascertained either from previous validation studies, or ideally, by calculating the CV of replicate controls within each batch. Alternatively, assuming batches are balanced and of a reasonable size, batch can be included as a categorical covariate in standard statistical analyses. This, or a more sophisticated conditional likelihood approach (6), provides a mechanism to test for statistical significance of batch effects, and a possible alternative to normalization.

There are some cases where assays are essentially self-normalizing. For example, immunophenotyping by flow cytometry requires individualized positivity gates to be set for each marker, specific to the batch or even to each donor. For the most part, this accounts for batch variability in the assay, and further normalization is usually not needed. Stimulated assays with functional readouts are a different situation, and normalization is more likely to be needed. However, some assays are always analyzed using an internal control, i.e., comparing longitudinal samples to baseline for each subject. If the baseline sample is always in the same batch as the compared samples, this is largely self-normalizing, as mentioned above.

Outlier batches. Whether the analysis approach can be considered “self-normalizing” or not, there are reasons to check the consistency of repeated control samples across batches. Outlier batches, which look clearly different from the others (and/or that meet statistical criteria as outliers), can then be discarded or targeted for repeat if possible.

Choice of normalization strategy. Of course, it’s often the case that, with or without outlier batches, variability between batches significantly exceeds intra-assay variability, and no self-normalizing analysis approach is possible. In these cases, normalization is indicated. The next question to ask is what kind of normalization should be used? Here, the batch design becomes important. If it is wellbalanced (as described above), then one can choose between global normalization or normalization to a control sample(s). In global normalization, sample values are divided by a group value such as the median of that batch. By contrast, normalization to a control means that sample values are all

divided by the corresponding control value (or mean of replicate control values) for that batch.

Clearly, global normalization generally has more stability, since it is not dependent upon a single or a small number of controls. On the other hand, global normalization is highly dependent upon the balance of samples within each batch; it thus generally works best when batches are large.

Z-scores. A variation of global normalization is the calculation of a z-score. In this form of normalization, the variance of each measurement is equalized, so that all values in the batch are centered on zero. A value of 1 indicates a sample that is one standard deviation (SD) above the mean; -1 corresponds to one SD below the mean, etc. Z-scores work best with data that is normally distributed, so it may be best used after log transformation (base 10 or base 2), which tends to bring high outliers closer to the mean, and thus force typical biological data to more closely approximate a normal distribution.

For downstream analysis of multiple readouts, z scores have the advantage of creating equal dynamic range for all analytes. All the readouts will also be of comparable magnitude, whether they originally had values that were all very low or very high.

Summary. Biological assays have inherent batch effects. Batch design and assay standardization are the most important variables to consider before beginning a study. Batches should be thoughtfully balanced prior to data collection. Once data is collected, one needs to first evaluate the degree of batch variability to determine if there are outlier batches, and/or if normalization is warranted.

Second, one needs to choose a normalization strategy, global or control-based. This decision should be driven by the size and balance of the batches, versus the number and reliability of control samples in each batch. See the flow chart summarizing the sequential decisions involved. Careful attention to these considerations should result in improved analysis of data collected over time.

References 1. Rosner, B. 2011. Fundamentals of Biostatistics, 7 ed. (M. Taylor, and D. Seibert, eds).

Brooks/Cole, Boston, MA. 2. Maecker, H. T., A. Rinfret, P. D'Souza, J. Darden, E. Roig, C. Landry, P. Hayes, J. Birungi, O. Anzala, M. Garcia, A. Harari, I. Frank, R. Baydo, M. Baker, J. Holbrook, J. Ottinger, L. Lamoreaux, C. L. Epling, E. Sinclair, M. A. Suni, K. Punt, S. Calarota, S. El-Bahi, G. Alter, H. Maila, E. Kuta, J. Cox, C. Gray, M. Altfeld, N. Nougarede, J. Boyer, L. Tussey, T. Tobery, B. Bredt, M. Roederer, R. Koup, V. C. Maino, K. Weinhold, G. Pantaleo, J. Gilmour, H. Horton, and R. P. Sekaly. 2005. Standardization of cytokine flow cytometry assays. BMC Immunol. 6: 13.

3. Maecker, H. T., J. P. McCoy, and R. Nussenblatt. 2012. Standardizing immunophenotyping for the Human Immunology Project. Nat. Rev. Immunol. 1–10.

4. Horton, H., E. P. Thomas, J. A. Stucky, I. Frank, Z. Moodie, Y. Huang, Y.-L. Chiu, M. J. McElrath, and S. C. De Rosa. 2007. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J. Immunol. Methods 323: 39– 54.

5. Maecker, H. T., J. Hassler, J. K. Payne, A. Summers, K. Comatas, M. Ghanayem, M. A. Morse, T. M. Clay, H. K. Lyerly, S. Bhatia, S. A. Ghanekar, V. C. Maino, C. Delarosa, and M. L. Disis. 2008.

Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides. BMC Immunol. 9: 9.

6. Wang, M., W. D. Flanders, R. M. Bostick, and Q. Long. 2012. A conditional likelihood approach for regression analysis using biomarkers measured with batch-specific error. Statistics in medicine 31: 3896–3906.

DEVELOPMENTS IN BASIC IMMUNOLOGY AND NOVEL THERAPIES Old Diseases in a New Bottle: IgG4-related disease Shiv Pillai, MD, PhD, Massachusetts General Hospital, Harvard Medical School Over the past decade a new disease entity has attracted a lot of interest. Patients presenting with tumescent, often painful, lesions affecting a variety of anatomic sites, initially often thought to have a malignant disease, have had biopsies analyzed that reveal a constellation of features that lead to a diagnosis of an entity now called IgG4-related disease or IgG4-RD. The histological features include storiform fibrosis, obliterative phlebitis, and the presence of a lymphoid and plasmacytic cell infiltrate in which forty percent or more of the IgG expressing plasma cells stain for the IgG4 isotype. Plasma levels of IgG4 may also be elevated.

This concept of IgG4-related disease was established in Japan by Hamano, Kamisawa and others at the beginning of this century (1,2). Virtually any organ in the body may be affected in IgG4-RD. This diagnosis now encompasses patients with salivary gland enlargements once thought to have Mikulicz’s disease, patients who would previously have been described as having Reidel’s thyroiditis, as well as subjects with retroperitoneal fibrosis, autoimmune pancreatitis, hypertrophic pachymeningitis, interstitial lung disease, tubulointerstitial nephritis and a host of other disorders (reviewed in 3, 4).

Why does an inflammatory disease in which fibrosis is a prominent component present with an increase in IgG4 secreting cells? What is the biological role of IgG4? Are disorders that fit the definition of IgG4-RD actually a small subset of a broader group of related fibro-inflammatory conditions? What broader lessons about immunology and disease might be learnt by studying IgG4RD? IgG4 is an unusual and relatively poorly understood immunoglobulin isotype. IgG4 antibodies bind poorly to activating Fc receptors and fix complement poorly. By conventional thinking this isotype should be unable to drive inflammation in the context of Fc-dependent effector mechanisms. In addition, when IgG4 is internalized into endosomes by FcRn, it can split down the middle between the two heavy chains and hybrid IgG4 molecules can form in a process called "Fab-arm exchange" (5-7).

As a result many IgG4 molecules are functionally monovalent in vivo. IgG4 is therefore not suited for immune complex formation, since bivalency is required for antigen-antibody lattice formation. IgG4 can associate with the inhibitory Fc receptor, FcÎłRIIb, so it may function primarily as an inhibitory immunoglobulin. However, it has NOT been established beyond any doubt that IgG4 is not inflammatory. IgG4 containing immune complexes have been described in certain renal disorders. It is unclear if these complexes contain both IgG4 and other IgG isotypes and thus fix complement, or if NGlycans on IgG4 recruits the mannose binding lectin and thus fix complement. The jury is still out on what IgG4 actually does in terms of disease pathogenesis, but it appears likely that IgG4 antibodies do not drive the inflammation and fibrosis seen in patients with IgG4-RD. It is possible that the increase in IgG4 in this disease reflects an attempt to dampen inflammation, albeit an attempt that has not succeeded.

Disease lesions in IgG4-RD share many histological features. CD4+ T cells are abundant at tissue sites of disease and they most likely drive the disease process. Apart from plasmablasts, plasma cells, and activated myofibroblasts, as well as organized tertiary lymphoid organs, essentially follicular structures with germinal centers, are also found in disease lesions. These germinal centers as well as those in draining lymph nodes are presumably major sites of IgG4 class switching. The promoters (so called I regions) adjacent to the IgG4 and IgE switch regions are structurally similar but detailed molecular studies on IgG4 class switching have not been performed. It is likely that T follicular helper cells that secrete IL-4 may participate in the IgG4 class switch, but it is also possible that unique yet to be defined T follicular helper cells are key to this class switching event. IgG4 remains a mysterious isotype and the study of IgG4 related disease may start to provide insights about this immunoglobulin sub-class.

The nature of the T cells that drive the disease is unclear. Fibrosis can be driven by a range of helper T cell subsets depending on the disease context. Th2 cells drive allergic fibrosis as seen in schistosomiasis and in this context interferon-Îł may be inhibitory (8). When one considers the fibrotic nature of lesions in diseases like tuberculosis and Crohn's disease, it is clear that Th1 and Th17 cells may also drive fibrosis in certain contexts. In IgG4-RD the case has sometimes been made that the disease is driven by Th2 cells although a role for Th1 cells has also been suggested (9-11). However recent studies suggest that Th2 cells are only expanded in the circulation of patients with IgG4-RD who have concomitant atopic disease but not in subject with active disease who do not have atopic manifestations (12).

It is unclear whether IgG4-RD is an autoimmune disease and the driving antigens or antigens in this disease remain to be identified. Unlike most autoimmune diseases, IgG4-RD is largely a disease of older males. Studies examining the potential role in disease pathogenesis of genetics, the microbiome and epigenetics remain to be performed.

Is there a role for IgG4 in pathogenesis? Do IgG4 producing B cells collaborate with CD4+ T cells, perhaps by presenting antigen at disease sites? About sixty to seventy percent of subjects respond to steroids, but the most effective therapy is B cell depletion using Rituximab (13). Since B cells help maintain CD4+ effector/memory T cells (reviewed in 14), it is possible that Rituximab, by depleting

antigen presenting B cells, indirectly depletes disease-causing rogue CD4+ effector/memory T cells. If IgG4 is not involved in pathogenesis, is it possible that similar mechanisms involving T cells cause disease in patients with IgG4-RD as well as in other fibrotic inflammatory diseases such as systemic sclerosis, lupus nephritis and possible idiopathic pulmonary fibrosis? The advent of immune repertoire analysis by Next Gen Sequencing and the development of many novel tools with which to interrogate the human immune system all suggest that we may soon have answers to a number of these longstanding puzzles.

References 1. Hamano H, Kawa S, Horiuchi A, Unno H, Furuya N, Akamatsu T, Fukushima M, Nikaido T, Nakayama K, Usuda N, Kiyosawa K. 2001. High serum IgG4 concentrations in patients with sclerosing pancreatitis. N Engl J Med 344: 732-8 2. Kamisawa T, Funata N, Hayashi Y, Eishi Y, Koike M, Tsuruta K, Okamoto A, Egawa N, Nakajima H. 2003. A new clinicopathological entity of IgG4-related autoimmune disease. J Gastroenterol 38: 982-4 3. Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med. 2012 Feb 9;366(6):539-51.

4. Mahajan VS, Mattoo H, Deshpande V, Pillai S, Stone JH. IgG4-Related Disease. Annu. Rev. Pathol. 2014, 9, 315-47.

5. Aalberse RC, Stapel SO, Schuurman J, Rispens T. 2009. Immunoglobulin G4: an odd antibody.

Clin Exp Allergy 39: 469-77 6. Rispens T, Ooijevaar-de Heer P, Bende O, Aalberse RC. 2011. Mechanism of immunoglobulin G4 Fab-arm exchange. J Am Chem Soc 133: 10302-11 7. van der Neut Kolfschoten M, Schuurman J, Losen M, Bleeker WK, Martinez-Martinez P, Vermeulen E, den Bleker TH, Wiegman L, Vink T, Aarden LA, De Baets MH, van de Winkel JG, Aalberse RC, Parren PW. 2007. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science 317: 1554-7.

8. Wynn TA. 2004. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol 4: 583-94 9. Tanaka A, Moriyama M, Nakashima H, Miyake K, Hayashida JN, Maehara T, et al. Th2 and regulatory immune reactions contribute to IgG4 production and the initiation of Mikulicz disease.

Arthritis Rheum 2012; 64:254e63.

10. Zen Y, Fujii T, Harada K, Kawano M, Yamada K, Takahira M and Nakanuma Y. Th2 and regulatory immune reactions are increased in immunoglobin G4-related sclerosing pancreatitis and cholangitis. Hepatology 2007; 45(6): 1538-46.

11. Okazaki K, Uchida K, Ohana M, Nakase H, Uose S, Inai M, Matsushima Y, Katamura K, Ohmori K, Chiba T. Autoimmune-related pancreatitis is associated with autoantibodies and a Th1/Th2-type cellular immune response. Gastroenterology. 2000 Mar;118(3):573-81.

12. Mattoo H, Della-Torre E, Mahajan VS, Stone JH, Pillai S. Circulating Th2 memory cells in IgG4 Related Disease are restricted to a defined subset of subjects with atopy. Allergy,2014 69, 399-402.

13. Khosroshahi A, Bloch DB, Deshpande V, Stone JH. 2010. Rituximab therapy leads to rapid decline of serum IgG4 levels and prompt clinical improvement in IgG4-related systemic disease.

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