2020 BASIC IMMUNOLOGY /a by Guido Forni

Piero Musiani and Guido Forni

2020 BASIC IMMUNOLOGY /b 15

Contents Foreword About the Authors 1 The immune system: An overview 2 Communication within the system: a. Cytokines 3 Immunity at surface 4 Sentinel cells 5 Cells of innate immunity 6 Communication within the immune system: b. The Major Histocompatibility Complex (MHC) 7 Peptide presentation by HLA glycoproteins 8 T cells 9 The thymic education of T cells 10 Activation of virgin T cells 11 T killer T cells 12 T helper cells 13 B cells 14 The B Cell Receptor and antibodies 15 Generation of B and T Cell Receptor repertoires 16 Binding site - antigen interaction 17 The antibodies 18 Activation of B cells 19 Secondary lymphoid organs 20 Direct and indirect antibody activities 21 The Complement system 22 Monoclonal antibodies (mAb) 23 The memory response 24 Vaccines 25 Negative controls of the immune response 26 Immune tolerance 27 Autoimmunity 28 Immunodeficiencies

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Foreword

This book intends providing an introduction to immunology for undergraduate and graduate students. As we were engaged for a few years in teaching in a Masterâ&#x20AC;&#x2122;s Degree the Medical University of HuĂ¨, Viet Nam, we (Piero Musiani and Guido Forni) debated at length what should be emphasized and, on the contrary, what could be left to an eventual personal investigation in a basic course of Immunology. Our first agreement was that the main emphasis should be on immunological strategies, while most of the molecular mechanisms could be left to student exploration. Molecular biology is now so pervasive that everyone easily swims in the sea of molecular mechanisms. Updated information on molecular pathways of the immune system can be obtained on internet without difficulty. By contrast, the peculiar logic of immune reactions is sometimes not obvious and not easy to grasp. A second shared view was that a schematic drawing could be more informative than a long text. Therefore, this book is mainly based on text illustrations, while only an explanatory comment is reported in legends. Finally, we wish to thank exquisite Mrs. Jehanne Marchesi for the numerous corrections and suggestions.

A 2018 printed version of this book is available directly from Piccin Nuova Libraria, Padua, Italy (https://www.piccin.it/it/immunology/2333-basicimmunology-9788829928828.html?search_query=forni&results=4) or through Amazon, also in a Kindle version.

About the Authors

Piero Musiani, MD (Right in the picture) was a semi-professional basketball player, but then he became a Surgical Pathologist working first at the Catholic University, Rome and later at the University of Chieti, Italy. Initially, his main experimental interest was on the thymus gland and secretory immunoglobulins. At the University of Chieti he played a pivotal role in the foundation of the Excellence Center on Aging (CeSI). The sophisticated equipment acquired by the CeSI allowed Pieroâ&#x20AC;&#x2122;s team to combine a state of art pathological analysis with experimental data. On many occasions, these sophisticated data led to a fresh interpretation of new immunological findings. In the 2014 Piero died in a motorbike accident while he was drawing the illustrations of this book. Guido Forni, MD (Left in the picture) is a National Member of the Accademia dei Lincei, Rome. From 1970 to 2011 he was Professor of Immunology at the University of Torino Italy. He has experience in basic and translational cancer immunotherapy and has a considerable expertise in transgenic mouse models of cancer and DNA vaccination. His work at the University of Torino, Italy on the role of cytokines in the immune recognition and vaccines to prevent tumors is recognized worldwide. His laboratory devoted considerable effort towards acquiring new knowledge concerning the direct role of vaccine-induced antibodies on tumor growth, their ability to trigger cell-mediated reactions and activate Complement cascade and to trigger a cell-mediated immune memory to tumor antigens.

CHAPTER 1. The immune system: An overview Fig.1.1. WHY WE DO HAVE THE IMMUNE SYSTEM? The progressive diffusion of mammalians on our planet prompted microbes to develop increasingly efficient invasion strategies. The incessant threat created by microbial attempts to invade has forced the mammalians to develop efficient defense mechanisms. Over the evolutionary history of mammalians, several primitive defense mechanisms have been conserved and re-adapted. Defense mechanisms progressively evolved as the second more complex system of our body, involving thousands of billions of cells. The efficacy by which immune mechanisms block microbial invasions modulates the ability of microbes to spread and cause a disease. The immune system restrains the pathogenicity of the microbes. Mutualistic, commensal, pathogenic opportunistic and truly pathogenic microbes may become capable of causing a disease as soon as a body barrier breach or a jam of an immune mechanism takes place. In addition, the immune system is becoming able to perceive anomalies of body molecules and to eliminate the cells expressing them. This ability to check the integrity of body cells has become increasingly important as the cellular and molecular complexity of the organism increased.

Fig. 1.2. NEVER ALONE! The flow of defense reactions passes from body barriers to the elicitation of a quick and furious innate immune reaction and to a later specific adaptive immunity reaction followed by a long-lasting immune memory of the past war. These four defense reactions follow different strategies. Nevertheless, they are highly integrated with each other. Indeed, a constant feature of

6 immune reactions is continuous cell-to-cell communication. An immune cell perceiving an intruder, or an anomalous body situation delivers signals that trigger the activation of several other immune cells. Body barriers (See Chapter 3) are always operative in hindering microbial invasions. Their peculiar barrier features and defense mechanisms make effective microbial incursions an infrequent event. When an invasion is sensed by the cells of body barriers, in a lapse of few minutes to a few hours a storm of alarm signals (See Fig. 4.2), chemokines and cytokines (See Chapter 2) recruits and activates several cell populations of innate immunity. Commonly, this fast and furious reaction effectively overcomes the intruder (See Fig. 5.35). In the rare occasions in which the invasion is not quickly repressed, innate immunity signals along with the diffusion of the intruder concur in triggering a different strategy of reaction, the adaptive immunity. This new defense strategy based on the precise (specific) reaction of T cells and antibodies against the molecular peculiarities of the intruder, requires about one or two weeks to be operative. Thanks to their ability to recognize the intruder with high precision, adaptive immunity mechanisms guide innate immunity cells towards a more efficient elimination of the invaders. Lastly, a successfully repressed invasion leaves a state of long-lasting immune memory (See Chapter 23). Memory epithelial cells and macrophages can trigger effective secondary reactions. Memory T and B cells are quickly activated by a second arrival of the same intruder and build up a reaction 10 to 1000 times stronger than their primary reaction. In most of the cases, the efficacy by which T and B cell memory mechanisms control subsequent invasions are so strong and the reaction is so quick as to pass almost unnoticed.

Fig. 1.3. WHAT DOES THE IMMUNE SYSTEM DO?

Fig. 1.4. HOW DO IMMUNE CELLS REALIZE THAT SOMETHING IS NOT GOING WELL? In the absolute darkness of the inside of the body, the immune system adopts different strategies to perceive cues of body abnormalities. Non-receptor dependent immune activation. The alterations of a few body molecular structures may directly trigger an immune reaction. For example, this happens when immune sensors recognize microbial toxins in the cytoplasm and lead to the assembly of a multiprotein complex called inflammasone (See Fig. 4.9) that triggers an inflammatory response, or when microbial molecu1les stabilize the C3b fragment of the lectinic pathway of Complement activation (See Fig. 21.4). Activation of the immune response through receptors shared among several immune cell populations. Pattern Recognition Receptors (PPR, See Figs. 4.6 and 4.7) expressed by several cell populations perceive the presence molecules commonly associated with the damage of body cells (See Fig. 4.3) and microbial invasion (See Fig. 4.4): By contrast, every T and B cell expresses a unique receptor interacting with a specific molecular target. Lastly, several immune functions are always active, ready to be implemented as soon as the brake given by inhibitory receptors is removed. A typical example is the inhibition of the activity of the NK cells by the normal body cells.

Fig. 1.5. THE NUMEROUS AND DIFFERENT CELLS OF THE IMMUNE SYSTEM. The various families of immune cells enclose several subsets of cells sharing common features but perceiving distinct targets and activating different reaction mechanisms.

Fig. 1.6. THE SIGNALS PERCEIVED BY THE VARIOUS IMMUNE CELL POPULATIONS ARE VERY DIFFERENT. In the total darkness of the inside the body, innate immune cells sense several traces of both microbial invasion and anomalies of the body cell behavior. The distinct receptors expressed by these cells transform environmental cues into intracellular signals that change cell behavior. For DAMP, PAMP and PRR see Chapter 4

Fig. 1.7. EACH VIRGIN LYMPHOCYTE RECOGNIZES A DISTINCT MOLECULAR TARGET. While innate immune cells sense the common traces of microbial invasion and body anomalies, each T and B cells interacts with a distinct and unique molecular target. Conventionally, the target recognized by T and B cells is called “antigen”, ANTIbody GENErator

Fig. 1.8. THE INFLUENCE OF THE IMMUNE SYSTEM OF THE HUMAN LIFE SHIFTS WITH THE CHANGES OF THE ENVIRONMENT. The aspects of human life that are affected by the immune system are almost countless. Nevertheless, the ability of the immune system to keep at bay microbial infections played a central role in its evolution. Without an efficient way to block microbes, human life was (and is) impossible. During the evolution, the increasing complexity of the body of living things and the prolonged close interaction of the fetus with the mother body typical of mammalians made mandatory to have also a sophisticated control of body cell mutations. Cancer could somewhat be considered a mistake made by these control mechanisms. Poor environmental conditions, high neonatal mortality and short life expectancy at birth emphasize the importance of the immune control of microbial infections (middle panel). By contrast, in the world areas denoted by good environmental conditions (lower panel), almost absent neonatal mortality and long-life expectancy at birth, are the alterations of the immune system and cancer that cause prominent health care problems. In these countries, hospitals are dealing mostly with patients with diseases due to alteration of the normal function of the immune system (See Chapter 26). In effect, the immune system evolved when the life expectancy of human beings was short. With the prolongation of human life, the immune system may often appear to be too complex to keep functioning properly (lower panel).

CHAPTER 2. COMMUNICATION WITHIN THE SYSTEM: a. CYTOKINES. Fig. 2.1. COMMUNICATION WITHIN THE CELL POPULATIONS OF THE IMMUNE SYSTEM. In the absolute dark of the inner of our body, thousands of billions of immune system cells incessantly communicate with each other. These interactions are necessary to coordinate the rapid activation and the appropriate modulation of effective defense reactions involving several millions of cells. The sophisticated communication system used by the immune system is based on the combination of different codes. Usually, the immune messages are captured by specific receptors expressed on the cell membrane. In some cases, the receptor expression is constitutive, i.e. the receptor is constantly expressed on the cell membrane during the life of the responder cells. By contrast, other receptors are inducible, i.e. are expressed only in response to other signals or only during a peculiar maturation stage of cell life. The communication code based on soluble molecules in circulation in the body fluids, the socalled cytokines, is the subject of this Chapter, while the communication code based on molecules expressed on the cell membrane, the MHC/HLA glycoproteins, is discussed in Chapter 6. In addition, all immune cells are subject to many and complex hormonal influences. In Fig: PPR, Pattern Recognition Receptors (See Figs 4.6 and 4.7), TCR, T cell receptor (See Figs. 8.11-8.15).

Fig. 2.2. CYTOKINES. I. MAIN FEATURES. Cytokines are proteins secreted by an immune cell that modulates the behavior of other cells.

11 Fig. 2.3. CYTOKINES. II. RELATED FAMILIES. Cytokines are molecules with many names. Commonly they are called Interleukins (IL), cytokines that interconnect (Inter: between) leukocytes (leuk) and activate (Greek, Kinos: movement) them. The progressive numbers of IL derive from their successive discovery. Somewhat arbitrarily, in other cases their name derives from the first biological activity attributed to the cytokine: Interferons (IFN), cytokines that were discovered for their ability to interfere with viral infections: an early step in the host response to viral infection involves a burst of synthesis of type I IFN that allow cells to quickly fight back against the offending viruses. IFN-ď § (type II IFN) is a pro-inflammatory cytokine that plays multiple roles in both innate and adaptive immunity. It is a primary activator of macrophages (See Fig. 12.10), Tk cells (See Fig. 12.9) and natural killer cells (See Figs. 2.22 and 12.10); Tumor Necrosis Factors (TNF), cytokines that were discovered for their ability to cause the necrosis of some tumors, are involved in the regulation of a wide spectrum of cell functions including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation (See Figs. 10.15 and 10.16); Colony Stimulating Factors (CSF), cytokines that were discovered for their ability to promote the proliferation and differentiation of bone marrow progenitor cells towards granulocytes and macrophages; Transforming Growth Factors (TGF, See Fig. 25.6) are cytokines inducing cell proliferation, differentiation and neoplastic transformation.

Fig. 2.4. CYTOKINES. III: SELECTIVITY OF SIGNALS. The cell (a) secretes a cytokine in its microenvironment. The cytokine is perceived only by the surrounding cells expressing specific receptors for that cytokine (b). Following the capture of the cytokine, this cell changes its behavior as dictated by signal transduced by the receptor. The other cells (c), while in contact with the secreted cytokine, does not express the specific receptor for this cytokine and do not perceive the cytokine message. The expression (or not) of the specific receptor makes selective a non-selective cytokine message

Fig. 2.5. CYTOKINES. IV: PARACRINE SECRETION. The small amount of a cytokine secreted by a cell (as in the Fig) is perceived only by the cells that are in its close microenvironment and express the specific receptor for that cytokine (cell b). The cytokine does not diffuse as a hormone because it is secreted in a very small amount and is rapidly and physiologically inactivated by specific inhibitors and quickly degraded in the kidneys.

Fig. 2.6. CYTOKINES. V. POLARIZED SECRETION. A small amount of a cytokine is secreted only in the synaptic area where the membrane of the secreting cell (Cell a in the Fig) interacts with the membrane of the cell that will receive the signal (Cell b).

Fig. 2.7. CYTOKINES. VI. AUTOCRINE SECRETION. A cytokine can be secreted and utilized by the same cell. This happens when the cell secreting the cytokine also expresses on the cell membrane the specific receptor for that cytokine.

Fig. 2.8. CYTOKINES. VII. ENDOCRINE SECRETION. In a few cases only, a cytokine has an endocrine activity. For instance, IL1 and IL6 secreted in the area invaded by microbes can reach the anterior hypothalamus and change the temperature setting and induce fever.

Fig. 2.9. CYTOKINES. VIII. Cytokines can be view as the verbs of an immune language. The messages of this language can trigger or suppress various cell functions. Only the cells that express the specific cytokine receptor can capture the message and accordingly change their activity. The meaning of a cytokine message is markedly modulated by the concurrence of other cytokines. The new behavior assumed by the cell results from the combination of the various cytokine receptor transduction pathways.

Fig. 2.10. CYTOKINES. IX. IL1. IL1 is the prototype of the proinflammatory cytokines acting on cells of different kind. It is produced as an inactive form, then proteolytically cleaved in mature IL-1 (See Fig. 4.9). When secreted in large amounts, IL1 endocrinally activates a systemic inflammation. Smaller amounts of IL1 amplify1 local inflammation and adaptive responses

Fig. 2.11. COMMON FEATURES OF CYTOKINE RECEPTORS. The expression of cytokine receptors changes during the various stages of White Blood Cell (WBC) maturation and activation. Thus, the expression or not of a specific cytokine receptor confers selectivity to the nonselective cytokine message. In addition, several WBC modulate the expression of cytokine decoy receptors. These receptors capture the cytokine but are unable to transduce an activation signal. Decoy receptors play an important role in inhibiting cytokine activity and controlling cytokine diffusion in the micro-environment.

Fig. 2.12. CYTOKINE RECEPTORS: MAIN FAMILIES. Cytokine receptors interact with the cytokine and transduce the signal to the nucleus. Often these receptors are made by two (dimers) or three (trimers) chains. Receptors of the common  chain family share the same signal transducing chain (CD 123). Also, the receptors of the common  chain family share the same signal transducing chain (CD 131). Since the common  chain is a component of an important family of receptors, mutations of the common  chain gene cause a severe break down of the immune system (severe immunodeficiency) due to the failure of numerous distinct immune mechanisms. Since the gene for the common  chain is located on the X chromosome this immunodeficiency is called X-linked severe combined immunodeficiency (X-linked SCID, See Fig. 28.3).

Fig. 2.13. THE IL2 RECEPTOR. I. This important two-three chain receptor illustrates how a cytokine receptor varies during the progression of cell activation conferring selectivity to the cytokine message.

Fig. 2.14. THE IL2 RECEPTOR. II: THE POOR AFFINITY TWO CHAINS RECEPTOR. Resting lymphocytes express only the  and the  chains of the IL2 receptor. Following the interaction with IL2, both chains transduce signals to the cell nucleus. However, the  chain does not bind IL2, while the  chain binds IL2 with very low strength (low affinity). Therefore, only anomalous very high concentration of IL2 in the lymphocyte microenvironment allows the binding of IL2 to the  receptor chain. Once bound to IL2, the  chain assumes a new structural conformation.

Fig. 2.15. THE IL2 RECEPTOR. III: THE POOR AFFINITY TWO CHAINS RECEPTOR. Only the presence of an anomalous high concentration of IL2 in the lymphocyte micro-environment allows the binding of IL2 to the  chain. The new conformation acquired by the  chain bound to IL2 now permits its interaction with the  chain.

Fig. 2.16. THE IL2 RECEPTOR. IV: THE POOR AFFINITY TWO CHAINS RECEPTOR. Both the  and  chain of the new complex transduce signals to the nucleus. The intracytoplasmatic tails of both receptors are first phosphorylated (P in the Fig.) by the Janus kinases (JAK). Then the phosphorylated chains act as binding sites for the Signal Transducer and Activator of Transcription (STAT) factor (See Fig. 2.20). In this way high concentrations of IL2 directly activate T, B, and NK cells.

Fig. 2.17. THE IL2 RECEPTOR. V: THE  CHAIN. A lymphocyte pre-activated by a ligand bound to antigen receptor and by membrane signals starts to express the  chain (CD25), a new chain of the IL2 receptor. The  chain binds IL2 at very lowaffinity and does not transduce activating signals.

Fig. 2.18. THE IL2 RECEPTOR. VI: THE HIGH AFFINITY THREE CHAINS RECEPTOR. In the presence of IL2, the  chain interacts with the  chain forming a new - receptor that binds IL2 with high affinity. The - receptor joining to the  chain forms a three-chain high affinity IL2 receptor able to bind lL2 even when IL2 is present in low physiological concentrations. Then the  and  chains transduce the signals to the cell nucleus.

Fig. 2.19. THE IL2 RECEPTOR. VII: THE HIGH AFFINITY THREE CHAINS RECEPTOR. Following the interaction of the  and  chains with small, physiologic amounts of IL2, a trimeric -- receptor is assembled. The cytoplasmatic tails of  and  chains are phosphorylated (P in the Fig.) by the Janus kinases.

Fig. 2.20. THE IL2 RECEPTOR. VIII: THE HIGH AFFINITY THREE CHAINS RECEPTOR. Both in the two chain and three chain IL2 receptors, the phosphorylated tails of the  and  receptor chains become the docking sites for STAT transcription factors. Bound STAT molecules are then phosphorylated by the Janus kinases (JAK). Dimers of phosphorylated STAT migrate to the nucleus where they activate the promoter of particular genes and trigger the progression of the cell cycle, cell proliferation and the formation of a cell clone.

Fig. 2.21. CYTOKINE RECEPTORS SHARING THE COMMON GAMMA CHAIN. The transmembrane receptor  chain does not bind any cytokine while it acts as the main transducer chain for several distinct cytokine receptors. The  chain is encoded by a gene on the X chromosome. Since this chain is involved in the transduction of several important cytokine signals, the inactivation of the gene encoding the common  chain leads to the X-linked Severe Combined Immunodeficiency (Xlinked SCID) (See Fig. 28.3).

Fig. 2.22. THE INTERFERON-GAMMA (IFN-).

Fig. 2.23. THE IFN- RECEPTOR

Fig. 2.24. CHEMOKINES: GENERAL FEATURES. Chemokines (chemotactic cytokines) are classed in four families designated CC, CXC, CX3C, and XC on the basis of the spacing of disulfide bonds. Chemokines of the CC family have two intra-chain disulfide bonds (In red in the Fig.) and the first two cysteines (C in the Fig.) residues are adjacent. Also, chemokines of the CXC family have two intra-chain disulfide bonds but the first two cysteine residues are separated by a no conserved amino acid residue (X, red arrow). Chemokines of the CX3C family have two intra-chain disulfide bonds, and the two cysteines are separated by three spacer amino acids. Finally, those of the XC family have a single intra-chain disulfide bond. The chemokines are referred to as CC, CXC, CX3C and XC chemokine ligands (CCL1, 2,28; CXCL1, 2â&#x20AC;Ś17; CC3L1; XC1, 2). The same abbreviations are used for their receptors (CCR1R.., CXC1Râ&#x20AC;Ś, CX3C1Rand XC1R..).

Fig. 2.25. CONSTITUTIVE AND INFLAMMATORY CHEMOKINES. Constitutive chemokines play a central role in governing the homing of the various populations of White Blood Cells (WBC) and thus the general architecture of the immune system. Inflammatory chemokines released by sentinel cells in injured or infected tissues recruit locally reactive WBC. First inflammatory chemokines act on endothelial cells of blood capillaries and increase their expression of adhesion molecules (selectins, ICAM; See Fig. 5.35). Then chemokines up-modulate the expression of integrins on WBC. In this way, chemokines favor WBC adhesion to the activated endothelial cells and their exit from blood vessels. Once transmigrated out of the capillary, WBC are guided towards the highest chemokine concentration (chemotaxis; See Fig. 3.34).

20 Fig.2.26. CHEMOKINE RECEPTORS. Chemokine receptors (CCR, CXCRâ&#x20AC;Ś.) are expressed on the cell membrane of White Blood Cells (WBC). They are formed by seven transmembrane domain proteins coupled with G proteins. In this Fig., a prototypic chemokine receptor is drawn in blue. The gray arrow shows the chemokine binding site while the green arrow shows the G chain transduced signal.

Fig. 2.27. CHEMOKINE CIRCUITS. When macrophages, Dendritic Cells as well as other sentinel cells perceive a microbial infection, they secrete a series of chemokines. These pro-inflammatory chemokines are then selectively captured by specific receptors expressed on the membrane of several immune cells. The interaction between a chemokine and its receptor activates the gradientâ&#x20AC;&#x201C;dependent chemotaxis of the cell towards the area where the chemokine is secreted. In Fig. the main cell populations activated by various CC and CXC chemokines are shown.

21 Fig. 2.28. TWO DISTINCT CHEMOKINE RECEPTORS MAY ACT AS CO-RECEPTORS FOR (HIV) INFECTION. When the Human Immunodeficiency Virus (HIV) is bound to a CD4 cell membrane receptor, a domain of the gp120 on the HIV envelope changes conformation and binds the CCR5 and CXR4 chemokine receptors. This interaction is critical for the absorption of the HIV on the cell (See Figs. 28.6 and 28.7). Viruses that use CCR5 are generally responsible for viral transmission, while viruses that use both CCR5 and CXCR4 receptors (dual tropic viruses) emerge during infection progression.

Fig. 2.29. THE -32 DEFECTIVE CCR5 CHEMOKINE RECEPTOR (CCR532). A few persons inherit the -32 variant of the gene coding for the CCR5 receptor. The non-functioning CCR532 receptor encoded by this gene variant lacks 32 base pairs and therefore it does not bind the Human Immunodeficiency Virus1 (HIV-1) proteins. The HIV-1 inability to bind to CCR532 hampers (when the -32 gene is in heterozygosis) or impedes (when it is in homozygosis) the HIV infection (See also Fig. 28.9). The frequency of -32 variant of the CCR5 gene expression decreases from 14% in Northern Europe to 4% in Southern Europe, while its expression is rare or absent in Japan and Western and Central Africa.

CHAPTER 3. IMMUNITY AT SURFACE

Fig. 3.1.THE SOPHISTICATE ENVELOPES OF THE HUMAN BODY. A barrier tissue dysfunction is often a fundamental feature of microbial infections.

Fig. 3.2. SKIN AND MUCOSAE. Mechanisms of isolation and protection.

Fig. 3.3. THE SKIN: A COMPLEX AND DYNAMIC BARRIER. The isolation and protection from the environment provided by the skin is due to the combination of several distinct mechanisms. Physical barrier: The skin is constituted by three cell layers: the upper epidermis, the deeper dermis, and the subcutaneous tissue or hypodermis. A thin layer of extracellular proteins, called the basal membrane divides epidermis from dermis. The epidermis is made by a multi-layered keratinized stratified epithelium, the outermost layer of which (Stratum corneum) is composed of a-nucleated keratinized epithelial cells (Horny cells) and epidermal lipids, both produced in the granular layer and secreted by the sebaceous glands. The external surface of the Stratum corneum is dry, acidic lipidic rich and with a high salt concentration. Epidermal lipids and the thigh junctions among epithelial cells form a waterproof barrier. The proliferation of epithelial stem cells in the basal layer of epidermis provides a steady renewal of epithelial cells that accompanies the continual desquamation of horny cells. Complete skin cell renewal takes place every 2-4 weeks. Desquamation is an effective way to remove microbes and substances adhering to the skin. Physical stress leads to an enhanced proliferation of epithelial cells and the formation of a ticker Stratum corneum (Callous). Fibroblasts produce extracellular matrix (ECM) molecules that regulate skin strength and resilience. In the few areas where the skin is particularly thick a stratum lucidum made by dead cells containing eleidin, a transparent protein, is evident between the

24 stratum corneum and stratum granulosus. Skin responds to the damage due to ultraviolet (UV) radiations with the proliferation of melanocytes and melanin secretion. This forms a protective barrier against ultraviolet-light-induced DNA damage. This process is associated with an enhanced proliferation of epithelial cells replacing dead and damaged cells. Several microbes produce cytolytic proteins and peptide (short protein) toxins in order to disrupt epithelial barriers and initiate a mucosal infection. The fatty cells on the subcutaneous tissue provide an effective thermal insulation. The contraction of pili muscles leading to goosebumps and porous restriction reduces the dispersion of body heat. By contrast the sweat secretion and the subsequent water evaporation lower body temperature. Signals provided by cells damaged by wounds trigger dermal fibroblasts and epithelial cell proliferation leading to wound healing. Biochemical barrier: Epithelial cells secrete several antimicrobial peptides (See Fig. 3.4). The acidic pH of the skin (pH 4-4.5) impairs microbial proliferation. Acidic pH is due to salts, lactic acid in sweat and the resident microbial flora. In addition, fatty acids secreted by sebaceous glands and sweat components (Lysozyme, see Fig. 3.5 and Lactoferrin) are endowed with marked antimicrobial activity. Direct immune activity: Epithelial cells express a series of Pattern Recognition Receptors (PPR, See Fig. 4.4) sensing microbial invasion and penetration. Their activation induces the release of alarm signals (danger signals; See Fig. 4.2; chemokines, and cytokines, see Chapter 2) that elicit innate immune reactions and play a key role in promoting and polarizing the following adaptive immunity. Intraepithelial Langherhans cells (epidermis immature Dendritic Cells, DC), dermal Dendritic Cells, macrophages and blood-derived neutrophils (See Chapter 5) activated by factors released by epithelial cells ingest cell debris and kill microbes. These cells along with lymphocytes and epithelial cells secrete a large array of anti-microbial molecules. Sentinel activity: Mast cells, DC, neutrophils, macrophage, lymphocytes, Innate Lymphoid Cells (ILC) and endothelial cells of blood vessels (Chapter 5) are the further outpost of the innate and adaptive immunity. The large array of cytokines and pro-inflammatory factors released by these cells when an intruder is perceived trigger and bias both a local and a systemic innate and adaptive immune response. In effect, the peculiar state of differentiation acquired by antigen-presenting cells (APC) and their migration to lymphoid organs trigger and modulates both antibody and cell-mediated adaptive immune responses (See Chapters 10 - 12). Skin microbes can fall anywhere along the continuum between mutualism and pathogenicity. To control microbes that normally colonize the barrier surfaces such as those of the skin and gut innate NK cells (iNK cells) induce a form of immunity that not directly retaliate against the microbes, but instead aids tissue repair and microbe containment. Microbial antibiosis: Numerous and diverse microbes (>1012, viruses, fungi and bacteria) referred to as skin microbiome reside on the epidermis and the hair follicles. These microbes metabolize skin proteins and lipids and produce bioactive molecules that inhibit skin infection by invading microbes and are involved in the skin pH acidification. The body odor is mostly produced by skin microbiome. Memory response: Epithelial stem cells respond more rapidly to a secondary microbial assault since they remember the primary assault by acquiring epigenetic changes, i.e. changes that regulate gene expression without changing DNA sequences (See Chapter 23). A loss of methylation and different chromatin accessibility allow a quicker transcription of the genes activated by the primary assault. REFERENCES: H Hammad & BN Lambrecht, Immunity, 2015,43:29; YE Chen, MA Fischbach & Y Belkaid, Nature 2018,553:427.

Fig. 3.4. MOLECULES SECRETED BY EPITHELIAL CELLS. Barrier epithelial cells are the very first line of defense. They secrete both molecules with direct antimicrobial activity and danger signals alarming innate and adaptive immune responses. Often combinations of molecules released by epithelial cells program antigen-presenting cells (APC) to induce a Th2 cell-mediated response (See Chapter 12).

Fig. 3.5. THE LYSOZYME. An effective anti-microbial enzyme

Fig. 3.6. THE BRONCHIAL MUCOSA: A BALANCE BETWEEN FUNCTION AND PROTECTION. The respiratory tract is incessantly exposed to microbes and substances from the inhaled air. The upper respiratory tract and trachea, bronchi and bronchioles are lined by a delicate mono-stratified ciliated epithelium shielded by mucus. Physical barrier: Ciliated epithelial cells of the respiratory tract are much thinner than the skin and therefore respiratory infections are the most common in the human population. The continuous beats of cilia move mucus forming the Phlegm and the microbes trapped on it towards the throat where it is swallowed. Mast cell degranulation causes constriction of the muscular layer (bronchial constriction). Biochemical barrier: The mucus is a dense and sticky substance covering the respiratory tract mucosa. It is made by water and mucins, substances secreted by goblet cells and submucosal serous mucus glands. Goblet cells are scattered among the respiratory tract ciliated epithelial cells. They are present also in the mucosa of the digestive tract. Mucus protects against airborne microbes. It contains lysozyme (See Fig. 3.5), antibodies, inorganic salts, lactoferrin, and mucins. Innate Lymphoid Cells (ILC) activated by IL23, IL33 and TSLP secrete IL13, a cytokine that enhances mucus production by goblet cells (See Fig. 12.13). Direct immune activity: Dendritic Cells (DC), granulocytes and macrophages ingest microbes and foreign substances and migrate to draining lymph nodes. Moreover, these cells along with lymphocytes and epithelial cells secrete a large array of anti-microbial molecules. However, an organized lymphoid tissue in lungs (BALT, Bronchus-associated lymphoid tissue) is rarely found in humans. Sentinel activity: Here too DC, macrophages, lymphocytes and Innate Lymphoid Cells (ILC) are the further outpost of adaptive immunity.

Fig. 3.7. THE MUCOSA OF THE LIPS AND MOUTH: A DELICATE PHYSICAL BARRIER. The lips and the oral cavity are lined by a mucous membrane consisting of a stratified squamous epithelium similar to that of the skin. However here the keratinized stratum corneum is scarce or absent. The underlying lamina propria is a thin connective tissue layer containing blood and lymphatic vessels, many nerve fibers and elements of the immune system. This kind of mucosa is an effective physical barrier even if less protective than skin. Biochemical barrier: Several minor salivary glands are located in the submucosa Direct immune activity, Sentinel activity, and Microbial antibiosis are similar to those of the skin and the bronchial mucosa, furthermore the saliva produced by the salivary glands is endowed with digestive and antimicrobial action. The antimicrobial immune activity depends on lysozyme, lactoferrin, myeloperoxidase and antibodies.

Fig. 3.8. THE MUCOSA OF THE DIGESTIVE TRACT. The intestinal mucosa functions as a barrier interacting up dynamically with the external environment since it has to perform a complex management of food absorption, microbiota containment and inflammation due to microbial incursion. In fact, the human intestine houses a vast consortium of symbiotic bacteria (up to 100 trillion microbial cells, >90% anaerobic bacteria, the gut microbiota) which are a potential risk to the host. In addition, gut mucosa plays a crucial role in food absorption and the maintenance of immune tolerance to food compounds. Gut mucosa is constituted by intestinal glands lined by a monostratified epithelium formed by columnar absorbing cells. The inner portion of the glands contains mucus-secreting goblet cells (See Fig. 12.13), endocrine cells and Paneth cells. All these cells emerge from stem cells at the base of intestinal gland pits. The intestinal epithelium is the most rapidly self-renewing tissue of the body: Cells have a life cycle of 3-4 days. Lymphoid cells and lymphatic nodules are in the submucosa. Physical barrier: The glandular monostratified epithelium is much thinner than in the skin, and therefore intestinal infections are common. Peristaltic movements, forcing mucus mixed with digested food and fecal contents toward the anal end control bacterial growth by mechanical cleansing which dislodges and removes potential colonies of microbes. Biochemical barrier: The intestinal mucosa is protected from microbes by mucus layers produced by goblet cells. Abundant numbers of commensal microbiota are present in the outer mucus layer, while a firm inner mucus layer acts as a physical barrier to microbial contact maintaining the segregation of microbes and epithelial cells, thus reducing the risk of intestinal invasion (See Figs. 3.9 and 27.11). Paneth cells release into the lumen granules containing antimicrobial proteins, including lysozyme and alpha-defensins and other antimicrobial peptides. Direct immune activity: The Gut Associated Lymphoid Tissue (GALT). An organized lymphoid tissue is often associated with mucosal barriers, a very large and fragile area to be protected against invaders

29 microbes and symbiotic microbiota. The Mucosal-Associated Lymphoid Tissue(MALT) is composed by numerous immune cells specialized to combat against the invaders most commonly encountered at the different mucosal surfaces: the BALT, Bronchus-Associated Lymphoid Tissue (poorly evident in humans); the pharyngeal Waldeyer’s tonsillar ring at the entrance to digestive and respiratory tract, and the GALT. The GALT is made by thousand submucosal lymphoid nodules, hundred oval lymphoid aggregates similar to lymph nodes called Peyer’s patches, the lymphoid appendix and very numerous scattered lymphoid cells. B cells (See Chapter 13), CD8+ killer T cells (See Chapter 11) and CD4+ helper T cells (Th17, Th1, and Th2; See Chapter 12) and iNKT cells (See Figs. 8.19 and 9.16) are present along with the Mucosal-Associated Invariant T cells (MAIT) cells. MAIT cells are a T cell subpopulation with both innate and adaptive immune properties that plays an important role in controlling bacterial infections. Paneth cells along with other lymphoid cells secrete a large array of anti-microbial molecules. Sentinel Innate Lymphoid Cells (ILC) activated by IL1 and IL23 release IL17, IL22 and IL23 (See Figs. 4.11 and 4.12) play an important role in direct the containment of microbiota incursions. In addition, IL22 protects intestinal stem cells against genotoxic damages caused by microbial products. The combination of IL17 and IL22 enhances the production of anti-microbial molecules by Paneth cells. IL22 produced by intestinal ILC that are sensing a genotoxic compound in the food activates the DNA repair machinery of epithelial stem cells. REFERENCE: K Gronke et al, Nature,2018., XX Dendritic Cells (DC), macrophages and neutrophils in the lamina propria ingest microbes and foreign substances and migrate to submucosal lymphatic nodules and Peyer’s patches where they trigger the T cell response. The peculiar cytokine combinations secreted by the cells at submucosal surfaces switch the antibody production by locally activated plasma cells towards IgA (See Figs. 17.11 - 17.15). Danger signals of various nature perceived by intestinal epithelial induce the activation of B1 B cells which differentiate in plasma cells producing poly-reactive IgA that are protective against enteric inflammation. Incessantly, the combination of multiple innate and adaptive immune mechanisms tempers the expansion of symbiotic microbiota and protects gut epithelium against new invading microbes. Sentinel activity: Here too DC, macrophages, lymphocytes, and ILC are a further outpost of adaptive immunity (See Figs. 8.1). The sentinel activity of oddball tuft (brush) cells relies on their sensory capabilities to detect pathogens and allergens. Signals delivered by microbes of gut microbiota create complex interaction between epithelial and tuft cells, DC, macrophages, and ILC able to influence the functions of innate immunity, the local differentiation and polarization of T cells (See Chapter 12) and promoting local IgA class-switch and production (See Fig. 18.13). Microbial antibiosis: Besides being a constant potential danger, our enormous gut microbiota regulates various aspects of our body physiology including immune response, tolerance to food compounds, metabolic functions and behavior. This vast consortium of symbiotic bacteria ferments carbohydrates, synthesizes vitamins, prevents the growth of pathogenic species by competing for nutrition and attachment and producing toxins (bacteriocidins) and quorum sensing factors that inhibit the growth of other microbial species. The balance between inflammatory, adaptive and tolerogenic reactions to foreign nutrients, microbiota and invading microbes is discussed in Fig. 27.11.

30 Fig. 3.9. THE COLONIC EPITHELIUM. Absorptive colonocytes and tight-joined epithelial cells coordinate food absorption and anti-microbial immunity. The barrier formed by mucus secreted by goblet cells cannot be overcome by most of the microbes, which are thus unable to come into contact with the cells of the gut mucosa. The mucus is a a slippery aqueous and viscous gel mostly composed by mucin glycoproteins. By acting as a reservoir of salts, anti-microbial enzymes (lysozyme), peptides, proteins (lactoferrin) and secretory IgA (See Figs. 17.15 and 17.16), mucus immobilizes, agglutinates and kills the entrapped microbes. Its production is regulated by immune cells through IL13 and IL22 secretion. IL22 is produced by Th17 cells. Fig. 3.10. THE RECTAL MUCOSA: AN EFFECTIVE PROTECTION PRONE TO VIRAL INFECTIONS. Its internal portion is constituted by a glandular mucosa similar to that of the intestine while the more external portion consists of a pluristratified epithelium. Physical barrier: Mechanical stress easily damages the thin internal portion of the mucosa which is often made more vulnerable by the enlargement of the hemorrhoidal plexus. As mechanical stress, cuts and micro-abrasions increase the risk of Human Immunodeficiency Virus (HIV) infection (See below and Fig. 28.5). Indeed, the vast majority of new HIV infections are acquired via the rectal and vaginal mucosa. Biochemical barrier: As for the mucosa of the intestinal tract. Direct immune activity: As for the mucosa of the intestinal tract. Sentinel activity: The HIV infection as well by other viruses is made easier by the fragility of the mucosa to mechanical stress. The HIV virions are captured by Dendritic Cells and macrophages that pass the virus to CD4+ T lymphocytes. Then, infected mucosal CD4+ T lymphocytes display a massive production of HIV virions. Microbial antibiosis: As for the mucosa of the intestinal tract.

Fig. 3.11. THE MUCOSA OF THE GLANS PENIS AND VAGINA: A SIMILAR FRAGILE PROTECTION IN DIFFERENT ANATOMICAL SITES. These thin and fragile mucosae are the site of common infections including sexually transmitted infections. Physical barrier: In both the glans penis and vagina the mucosa is constituted by a pluristratified squamous epithelium similar to that of the skin. However here the stratum corneum is very scarce or absent. Here too the infection by the lymphotropic Human Immunodeficiency Virus (HIV, See Figs. 28.5) as well by numerous other viruses and microbes is made easier by the micro-abrasions and cuts due to the fragility of the mucosa. Biochemical barrier: No sebaceous or serous-mucus glands are present in the glans penis and vagina mucosa. The mucus covering the vaginal mucosa is produced by glands of the vulva and uterine cervix. Direct immune activity: The glans penis and vaginal mucosa produce lysozyme, lactoferrin, mannosebinding protein, and small antimicrobial proteins (peptides) endowed with broad antimicrobial activity. A rapid influx of neutrophils takes place during infections. Sentinel activity: The epithelium lies over a loose connective tissue with fibroblasts and a few Dendritic and lymphoid cells. As described for the rectal mucosa, HIV virions are captured by Dendritic Cells and macrophages that pass the virus to CD4+ T lymphocytes. Microbial antibiosis: Whereas microbial antibiosis virtually does not take place on the glans penis, it has a major importance in the vagina which is colonized by commensal microbiota whose composition changes under the influence of hormones from the neonatal to the reproductive period and menopause. Vaginal lactobacilli, the most important constituent of vaginal microbiota, produce lactic acid and hydrogen peroxide which maintain the low vaginal pH which protects against infections.

Fig. 3.13. THE WAR ON BODY SURFACE. On our skin and mucosae, an everlasting war is taking place against chemicals, microbes, parasites, and foreign cells. Defense mechanisms outlined in the previous figures have to continuously face ingenious and sophisticated invasion strategies that are incessantly evolved by microbes. Sometimes these may seem made-up, like a science fiction tale, while they hide a dramatic medical problem. For example, the Malaria parasite exploits the female Anopheles mosquitos to fly from one person to another and to overcome, with the sting, the thick and otherwise impenetrable skin barrier. The female mosquito alights on the skin and penetrates the epidermis with its proboscis to pierce a small vessel in the dermis (Blue arrows). The mosquito's saliva contains anticoagulants to keep the blood from clotting. If the mosquito is infested by the malaria parasite, the bite may be followed by a malaria infection. Currently, this imaginative and apparently unlikely strategy allows Malariaâ&#x20AC;&#x2122;s parasite to infect 250 million and kill one million persons every year.

CHAPTER 4. SENTINEL CELLS.

Fig. 4.1. SENTINEL CELLS. Frequently, micro-lesions as well as larger wounds of body barriers, allow a massive arrival of microbes. Immune cells should recognize and respond to invaders at the earlier stage of incursion, before the microbes adapt to the new environment and expand. Various cues of invasion (alarm or danger signals; see Fig. 4.2) delivered by stressed and dying cells and damaged tissues are perceived by distinct sentinel cells strategically located in the districts of the body where the invasions are more common. Alerted sentinel cells rapidly signal the invasion and recruit and activate various leukocyte populations of innate immunity which concur to counteract the invasion in a quick and effective way. In Fig. 1. Epithelial cells (See Fig. 3.3); 2. Mast cells (See Fig.5.7); 3. Macrophages (See Figs. 5.17-5.20) and Dendritic Cells (See Fig. 5.23); 4. Granulocytes: Neutrophils (See. Fig. 5.10) and Eosinophils (See Fig. 5.15); 5. Large granular lymphocytes/Natural Killer (NK Cells, see Fig. 5.26); 6. Lymphocytes and Innate Lymphoid Cells (ILC, see Figs. 4.11 and 4.12); 7. Fibroblasts; 8. Endothelial cells (See Figs. 5.33.

Fig. 4.2. A FEW ALARM SIGNALS RELEASED FOLLOWING A BODY BARRIER LESION. The release of a few of these alarm signals quickly activates an inflammatory response. Cell death by necrosis and necroapoptosis triggers a natural immunity reaction by releasing alarm signals belonging to the Damage Associated Molecular Patterns (DAMP) (See Fig. 4.3). The key role played by the Complement system in innate immunity is illustrated in Chapter 21; Cytokines are illustrated in Chapter 2; For Reactive Oxygen Intermediates (ROI) see Figs. 5.10, 5.20 and 5.23.

Fig. 4.3. DAMP: WHAT ARE THEY? DAMPs are alarm signals, molecules that are released by the damaged cells and tissues of the body.

Fig. 4.4. M/PAMP: WHAT ARE THEY? While DAMP are body molecules, M/PAMP are molecules of microbial origin, expressed microbes of many types, not only pathogenic.

Fig. 4.5. PATROLLING THE MICRO-ENVIRONMENT. In the dark of the interior of our body cells of innate immunity sense cues of microbial invasion, tissue damage, cellular stress, and alarm signals through a series of receptors called Pattern Recognition Receptors (PRR, See Figs. 4.6 and 4.7) that activate a reactive cell behavior. PPR provide a continuous surveillance for the presence of microbes and anomalous body conditions. Other receptors on the membrane of innate immunity cells guide their homing inside our body (Chemokine receptors, See Fig. 2.26), their activation (Chemokine and cytokine receptors, See Chapter 2), their interaction with other cells (Adhesion molecules), their metabolism (Hormone receptors), their involvement in reactions triggered by Complement activation (Complement receptors, See Fig. 21.8) or by antibodies (Receptors for the Fc fragment of antibodies, See Figs. 20.7). The expression of the various membrane receptors may change during the various stages of the life of the cells of the innate immunity. The conformational modification of the receptor caused by its interaction with ligands triggers signal transduction pathways that modify the cell activation state. A continuous integration of the myriad of signals perceived by numerous membrane receptors modulates gene activation and guides differentiation, behavior, and fate of immune cells. In a few cases (See Chapter 2), ligands interacting with cell membrane receptors can be compared to language messages. They are meaningful only if the cell is able to perceive them, i.e. to express a receptor able to capture the signaling molecule (the ligand) and transduce the messages to the nucleus. Every population of immune cells expresses distinct repertoires of receptors, and therefore perceives and reacts to different environmental signals. There are also receptors able to capture the message but unable to transduce the signal to the nucleus. These decoy receptors remove or attenuate a specific message. Only a few families of the numerous membrane receptors expressed by natural immunity cells are shown here.

Fig. 4.6. PRR: WHAT ARE THEY. The ability of cells of innate immunity to perceive traces of microbial invasion and damage to body tissues depends on the PRR repertoire that the cell expresses

Fig. 4.7. PRR: SITE OF EXPRESSION. The PPR expression on the cell membrane allows the detection of traces of invasion and alarm signals in the cell microenvironment (Upper panel). The expression of PPR on endosomes (Middle panel) or in the cytoplasm (Lower panel) permits to sense the presence of microbes that have entered the cell or live inside the cell (intracellular microbes: viruses, bacteria, protozoa…). Indeed, the recognition and destruction of incoming DNA from microbes is a fundamental mechanism of defense. RIG-like receptors recognize non-methylated viral RNA inside the cell (See Fig. 28.8). The cGAS-STIN DNA sensing pathway elicits a variety of inflammatory effector responses. LPS (lipopolysaccharide) is a bacterial endotoxin. Not methylated CpG (CpG, Cytosine—phosphate—Guanine) /dinucleotide: a base sequence typical of microbial DNA.

Fig. 4.8. REACTIVE CELL ACTIVITIES TRIGGERED BY PATTERN RECOGNITION RECEPTORS (PPR). PPR are sensors of very different kind who perceive various cues of microbial incursion or cell damage. After interacting with his ligand, every kind of PRR activates a peculiar defense mechanism. The RIG-like receptors recognize non-methylated viral RNA inside the cell and counteract viral invasion by producing interferons and triggering innate immunity. These receptors play a critical role in Human Immunodeficiency Virus (HIV) infection. See Fig. 28.8). Inflammasomes are cytoplasmatic multimeric complexes that assemble in response to microbial invasion and endogenous damage signals. Once assembled, inflammasomes activate caspases which permit the production of high amounts of important pro-inflammatory cytokines (IL1, IL18â&#x20AC;Ś) (See Fig. 4.9 and 2.10). cGAS (cyclic GMP-AMP synthase) enzyme perceives the abnormal presence in the cytoplasm of microbial DNA from infectious microbes or self-DNA from the nucleus or mitochondria. On binding to DNA, cGAS activates STING (Stimulator of Interferon Genes), an endoplasmic reticulum membrane protein to form dimers and tetramers, move to the Golgi apparatus and triggers the production of type I Interferon (IFN) and other pro-inflammatory cytokines.

Fig. 4.9. TOLL-LIKE RECEPTORS, INFLAMMASOMES AND PROINFLAMMATORY CYTOKINES. Toll-LikeReceptors (TLR, in blue in Fig.) are a family (from TLR1 to TLR 13) of Pattern Recognition Receptors (PPR, See Fig. 4.6) expressed by immune and non-immune cells. Every TLR senses a different cluster of Pattern or Microbial Associated molecules (M/PAMP, See Fig. 4,4). The signal they transduce triggers the synthesis of Reactive Oxygen Intermediates (ROI, see Figs. 5.10) and the release of oxidized mitochondrial DNA. The combination of TLR transduced signals triggers the assembly of disc-shaped multi protein complexes (the inflammasomes) that initiate innate immunity by recruitment and activation of caspase-1. Once assembled and activated, the inflammatory caspase 1 proteolytical activity cleaves the inactive form of IL1ď ˘ (See Fig. 2.10) and of IL18, two important proinflammary cytokines. The secretion of the mature form of these cytokines promotes the induction of an innate immunity inflammatory response. Moreover, inflammatory caspase 1 cleaves gasdermin D to cause cytokine release and pyroptotic cell death, a form of programmed cell death that occurs most frequently upon infection with intracellular pathogens. Toxic products of microbial origin can also be recognized independently of PRR. Their toxic action may directly trigger the assembly of inflammasomes and directly activate a caspase dependent /protective immune response. REFERENCE: A Sandstrom et al, Science 2019,364:43

41 Fig. 4.10. TUFT CELLS are brush kind of epithelial cells present in the epithelium of the respiratory tract, intestines and thymus (See Fig. 9.2). Tuft cell express chemical-sensing surface receptor that perceive the presence of allergens and microbes. These chemical environmental signals are translated into effector functions that promote immune type 2 (accomodatory) immune responses (See Fig 27.11) in the underlying tissue. For the sentinel role of Dendritic Cells see Fig. 10.4 and 10.5.

Fig. 4.11. INNATE LYMPHOID CELLS (ILC). ILC are a family of lymphoid cells with important sentinel and effector roles. All members of the ILC family have lymphoid cell morphology while lack the expression of peculiar surface molecules that identify other immune cells. In addition, despite having the typical morphology of lymphocytes, ILC dos express neither T cell receptor (TCR) nor the B cell receptor (BCR). Their activation is modulated by the peculiar combination of alarm signals and cytokines triggered by the invader. By quickly inducing and regulating an appropriate acute inflammatory response, ILC play a fundamental sentinel role.

Fig. 4.12. ILC: SUBSETS AND FUNCTIONS. As for T helper cells (See Chapter 12), the differentiation of ILC towards one subset or the other (transdifferentiation) is governed by the molecular features of the invader and by the combination of danger signals and cytokines released by the sentinel cells that first perceived the signs of the invasion. In response to these signals, ILC transcription factors bind to specific sequence motifs within the gene promoters. In this way, one genetic program dominates while alternative programs are silenced. Thus, in response to different stimuli, ILC differentiate in three major subsets (ILC1, ILC2, and ILC3) that provide a potent and early source of distinct cytokine sequences that tune up an appropriate innate immune reaction against different invaders. ILC1: The acute inflammation triggered by the cytokines released by ILC1 is effective against intracellular microbes (viruses, bacteria, protozoa). ILC2: The reaction triggered by cytokines released by ILC2 is effective against worms, contribute to multiple homeostatic processes, dampen inflammation and tissue repair. Neuropeptides modulate the activity of ILC2. ILC3: play an important protective role against tuberculosis: The CXCL13 chemokine acts on the CXCR5 receptor expressed on ILC3 cell membrane and recruit ILC3 in the lungs infected by the Mycobacterium tuberculosis. REFERENCES: A Ardain et al, Nature June 5, 2019. IL7 and IL23 cytokines released by ILC3 induce a rapid and effective reaction against extracellular bacteria and fungi. Moreover, ILC3-derived IL-2 supports intestinal T reg cells, immunological homeostasis and oral tolerance (See Fig. 27.11). A lightentrained and brain tuned circadian clock shapes the activity of enteric ILC3 REFERENCES: C Godinho et al, Nature 2019,574:254. For acute inflammation, see Fig. 5.36; For M1 and M2 macrophages, see Fig. 5.20; For Th1 and Th2 see Chapter 12; For goblet cells see Figs. 3.6, 3.8, 3.9 and 12.13; For Paneth cells see Figs. 3.8 and 3.9; For neutrophils see Fig. 5.10; TSLP is the Thymus Stromal Lymphopoietin, a cytokine known to play an important role in the maturation of T cell populations through activation of antigenpresenting cells (See Chapter 12).

Fig. 4.13. DEFENSE REACTIONS. Here, listed, are the most relevant effector reactions activated by sentinel cells of ate immunity. Several of these activities are illustrated in more detail in Chapter 5. ILC, Innate Lymphoid Cells.

CHAPTER 5. CELLS OF INNATE IMMUNITY. Fig. 5.1. ALL WHITE BLOOD CELLS (WBC) ORIGINATE FROM BONE MARROW. Hematopoiesis is the process of generating mature WBC. The bone marrow microenvironment has a key role in regulating the progressive differentiation of selfrenewing hematopoietic stem cells (HSC in the Fig.) into the various WBC. Panel A: The bone marrow is contained in flat bones and long bones such as tibias and femurs. There are two types of bone marrow: Red marrow, which consists mainly of hematopoietic tissue, and Yellow marrow, which is mainly made up of fat cells. Panel B: The differentiation of the rare population of long-lived and self-renewing HSC occurs in a specialized bone marrow extravascular spaces (HSC niches) between the sinusoids. In the nicest, the fluid differentiation of HSC is driven by multiple interactions. Bone lining osteoclasts (Oc) and osteoblasts (Ob) support HSC survival. Sympathetic nerve fibers, leptin-receptor-positive mesenchymal stromal cells (MSC), macrophages (M), adipocytes (Ad), megakaryocytes and Schwann cells and vascular endothelial cells expressing (or not) Notch delta ligands provide additional differentiation signals. HSC located on the endosteal niche are more quiescent, whereas those located at the perivascular side display a more active self-renewal and differentiation. Panel C: The main blood source to the red bone marrow is provided by the nutrient artery which crosses the bone cortex and gives rise to numerous capillaries. These capillaries enter the medullary vascular sinuses forming a dense network of sinusoids through the medullary cavity. Osteoblasts (Ob), macrophages (M), reticular cells producing the CXCL12 chemokine (Cell Abundant of CXCL12 Reticular, CAR), Nestin rich Mesenchymal Stem Cells (MSC), endothelial cells (See Fig. 5.32), and adipocytes (Ad) form the specialized nice microenvironment that regulates HSC self-renewal and differentiation. Red marrow extravascular spaces are filled by HSC-derived myeloid and lymphoid cell precursors together with their cell progeny. Only mature leukocytes, erythrocytes and platelets are released into the sinusoids and enter in the circulatory blood. The bone marrow trabeculae provide a network of supportive frameworks. Adipocytes are dispersed throughout the marrow.

Fig. 5.2. FROM HEMATOPOIETIC STEM CELL TO MATURE BLOOD CELL. Diagram showing the developmental stages of hematopoiesis of lymphocytes, erythrocytes, other leukocytes, and platelets. Erythrocytes and all white blood cells (WBC) derive from the myeloid progenitor.

Fig. 5.3 CELL FATE DECISIONS DURING DIVERSIFICATION OF BLOOD CELLS. Our body produces about 300 billion blood cells/day. This enormous production needs to have the fluid flexibility to adapt to changes of the demand caused by different pathological situations. Stem cells possess a broad range of differentiation programs. Their diversification rests on a series of nodal cell-fate decisions, i.e. choices between alternative gene expression programs in response to extracellular signals: one program is winning out, while alternative programs are extinguished. The concurrence of various signals triggers the expression of a particular transcription factor that binds to specific sequence motifs within gene promoter, enhancer and silencer regions, and recruits epigenetic regulators to modulate the activation status of a gene. Cells upregulate one program and down-regulate the others. The induced gene activation pattern is then transmitted subsequent cell generations as a permanent epigenetic modification. REFERENCE: E Laurent & B Gottgens, Nature 2018,553:418

Fig. 5.4. LEUKOCYTES PRESENT IN HUMAN BLOOD: THE WHITE BLOOD CELLS (WBC). The great majority of innate immunity cells (neutrophils, eosinophils, basophils and monocytes) rests on a major proliferative activity of Hematopoietic Stem Cells differentiating towards these cell lineages.

Fig. 5.5. INNATE IMMUNITY. I: THE PROTECTIVE POTENTIAL OF INFLAMMATORY REACTIONS. The

reaction made by innate immunity cells is rapidly upregulated. It acts to inhibit microbial invasions and control body damage.

Fig. 5.6. INNATE IMMUNITY. II: WHAT IS PERCEIVED BY THE CELLS OF INNATE IMMUNITY. Though immune memory is considered the hallmark of adaptive immunity, even the cells of innate immunity can acquire a form of immune memory that allows them to respond better against subsequent invasion.

Fig. 5.7. BASOPHILS AND MAST CELLS: ULTRASTRUCTURAL (panels A and C) AND HISTOLOGICAL (panels B and D) ASPECTS. Basophils, along with eosinophils and neutrophils constitute a group of White Blood Cells (WBC) known as granulocytes, i.e. cells with many cytoplasmatic granules. Basophils: The name basophils derives from their ability to be stained by basic dyes. Basophils are smaller than other white blood cells; their nucleus is usually bi-lobed (two lobes) and the cytoplasm is often obscured by granules containing heparin and histamine. Basophils and mast cells derive from hematopoietic bone marrow precursors and share several functions while they differ in size, shape of the nucleus and number of cytoplasmic granules. Mast cells: Mast cells are larger than basophils with irregular, elongated shapes and cytoplasmic extensions. The nucleus is round, and the cytoplasm is packed with basophilic granules that may obscure the nuclear margin. Basophils mature in the bone marrow and stay in the blood whereas immature mast cells migrate in the lamina propria beneath the basal membrane of skin, conjunctiva, mouth, digestive tract and lung mucosa where they undergo final differentiation. Activity: Through Pattern Recognition Receptors (PRR, see Fig. 4.4), basophils and mast cells recognize invading microbes. Within seconds of activation, they release the contents of pre-formed mediators present within cytoplasmic granules, an activity defined as degranulation. After that, these cells start to synthesize newly formed mediators. The released mediators increase local blood flow and vascular permeability, induce the recruitment of eosinophils, neutrophils and NK cells, increase mucus (See Fig. 3.6) production by goblet cells (See Figs. 3.6; 3.8; 3.9, 12.12, and 12.13) and intestinal mobility and act on smooth muscles to increase the expulsion of mucosal parasites. Basophils and mast cells are also involved in allergic reactions since their degranulation can be activated through the cross-linking of IgE bound on their membrane (See Figs. 17.26 and 20.10), and by constituents of Complement cascade adhering to Complement Receptors (See Fig. 21.8), immunocomplexes (See Fig. 20.4) and by direct injury. A massive degranulation of mast cells induces the often-life-threatening phenomenon of systemic anaphylaxis (from Greek, lack of protection) or the anaphylactic shock (see Fig. 20.10).

Fig. 5.8. BASOPHILS (right) AND MAST CELLS (left): THE CONTENT OF THEIR GRANULES

Fig. 5.9. BASOPHILS AND MAST CELLS: MAIN EFFECTS OF GRANULE SECRETION

Fig. 5.10. THE NEUTROPHIL: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Neutrophils are phagocytes, capable of ingesting microorganisms or small particles. The name derives from their ability to be stained by neutral dyes. These cells derive from hematopoietic bone marrow stem cells (See Figs. 5.2 and 5.3), with a production of 1011 neutrophils/day (!): therefore, they are the larger population of White Blood Cells. Neutrophil lifespan in the circulation is about 5.4 days while they survive for 1â&#x20AC;&#x201C;2 days after migration into tissues. IL5 is the major cytokine responsible for the growth, differentiation, recruitment, activation and survival of neutrophils. Neutrophils have a multilobed nucleus, and the cytoplasm contains small Golgi apparatus, sparse mitochondria and ribosomes and about 200 granules of three kinds: azurophilic granules (or primary granules), specific granules (or secondary granules) and gelatinase granules (or tertiary granules). The molecules contained in the granules are endowed with a strong antimicrobial activity, but they can also damage normal and neoplastic cells. The massive release of granules outside the cell, with the death of the neutrophils, is called degranulation. Dead neutrophils are the major component of pus. Alternatively, neutrophils phagocyte and kill microbes. Upon phagocytosis they produce nitric oxide (NO), activate NADH oxidase and generate O2- and other Reactive Oxygen Intermediates (ROI, See Figs. 5.20 and 5.22). ROI production, as well as the release of the toxic content of granules, kills the ingested microbes. By the degranulation of granules neutrophils release an assortment of anti-microbial substances, enzymes, Properdin (See Fig. 21.4) and pro-inflammatory molecules. A further antimicrobial activity of neutrophils is the release of DNA nets that trap microbes in the micro-environment (See Fig. 5.14). Neutrophils are one of the first responders of inflammatory cells to migrate towards the site of inflammation.

Fig. 5.11. NEUTROPHILS: THE CONTENT OF CYTOPLASMATIC GRANULES. The enzymes of neutropil granules kill invading bacteria but also damage normal body cells and extracellular matrix. The release of Properdin, a Complement constituent stored in secondary granules, enhances the lytic activity of Complement activated by the Alternative pathway (See Fig. 21.4).

Fig. 5.12. NEUTROPHILS: CONSEQUENCES OF DEGRANULATION.

52 Fig. 5.13. NEUTROPHILS: PHAGOCYTOSIS. A neutrophil develops cytoplasmic projections, called pseudopods (or pseudopodia) that engulf the microbe. After the formation of a phagosome containing the microbe, there is the fusion of the phagosome with a lysosome to form a phagolysosome. Lysosomes are membrane-bound spherical organelles containing acid hydrolases, which are able to break down virtually all kinds of biomolecules in an acidic environment. A microbe in a phagolysosome is killed and digested by lysosomal enzymes. Indigestible and residual material is discharged by exocytosis.

Fig. 5.14. NEUTROPHILS: EXTRACELLULAR TRAPS. Once the invading microbes is perceived through several Pattern Recognition Receptors (PPR, see Fig. 4.4), neutrophils release Neutrophil Extracellular Traps (NET) which are webs of DNA covered with antimicrobial molecules (such as myeloperoxidase, neutrophil elastase, lactoferrin, gelatinase, cathepsin G, etc.) derived from primary azurophilic, secondary and gelatinase granules. After stimulation, the neutrophil chromatin undergoes decondensation, a process mediated by enzymes stored in the primary azurophilic granules which are relocated in the nucleus. The nuclear chromatin expands inside the cell and is mixed with anti-microbial factors of the granules. Finally, the cell membrane breaks releasing NET. Microbes entrapped in NET are killed by oxidative and non-oxidative mechanisms.

53 Fig. 5.15. EOSINOPHILS: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Eosinophils derive from hematopoietic bone marrow precursors but form only a minor population among White Blood Cells. Their nucleus is often bilobed (two lobes) and the cytoplasm displays several distinct granules stained with eosin dye. Specific or crystalloid granules: These granules are very numerous (more than 200/cell). They contain a core of crystallized Major Basic Protein, peroxidase, cationic proteins, and a neurotoxin. All these proteins are toxic for helminths (See Fig. 11.13), parasites, microbes, and tumor cells. Moreover, these molecules are powerful promoters of allergic inflammation since they trigger histamine release by mast cells (See Figs 5.8-5.9.). Primary granules: These granules are less numerous and contain Charcot-Leyden crystal protein, a phospholipase. Small granules: These granules contain acid phosphatase, arylsulfatase B, catalase, and cytochrome lipid bodies (about 5/cell) contain arachidonic acid esterified into glycerophospholipids. Eosinophils bind antibodies on the surface of parasites and cancer cells and then degranulate to release proteins and enzymes which disrupt the plasma membrane of parasites and tumor cells. Eosinophils also phagocytose and destroy bacteria but are less efficient than neutrophils.

Fig. 5.16. NEUTROPHILS AND EOSINOPHILS: MAIN FEATURES. Aged neutrophils and eosinophils modulate the expression of their chemokine receptors and home in the bone marrow where they die.

Fig. 5.17. MONOCYTES: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Monocytes are the largest of all White Blood Cells (WBC) and have a large, single bean-shaped nucleus which gives them their name which means mononuclear leukocyte. The cytoplasm contains many lysosomes and lipid bodies. These cells derive from hematopoietic bone marrow precursors (See Fig. 5.5 and 5.6) and form a significant population among WBC. They stay for a few hours in the blood and then migrate into the tissues where they differentiate into macrophages.

Fig. 5.18. THE MONOCYTE: A LONG-LIVING CELL WHICH DIFFERENTIATES INTO DISTINCT SPECIALIZED CELLS. Monocytes and macrophages comprise a variety of subsets with diverse functions. Following the migration from blood vessels to tissues, monocytes differentiate into cells with a peculiar morphology and very specialized functions. In the central nervous system, microglia cells phagocytize protein aggregates and cellular debris. During brain development, microglia cells play a key role in stimulating or eliminating neuronal synapses. Monocytes may remember previous exposure to certain stimuli and generate an enhanced response to subsequent immune insult (See Chapter 23), REFERENCE: S Saeed et al, Science 2014,45:1250684.

55 Fig. 5.19. THE TISSUE MACROPHAGE: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. Once monocytes exit from blood vessels and marginate into a tissue they differentiate in macrophages. These cells display an irregularly shaped nucleus. The cytoplasm with a few mitochondria contains lysosomes, phagosomes, and phagolysosomes with residual bodies, which are undigested material. Macrophages often present numerous cytoplasmic projections, called pseudopods, indicating that these cells are actively engaged in phagocytosis.

Fig. 5.20. MACROPHAGE DIFFERENTIATION. A macrophage is a terminally differentiated cell. When activated by microbes, interferons (IFN), tumor necrosis factor (TNF) and other proinflammatory Th1 cytokines (See Fig. 12.8) macrophages are termed M1 macrophage. By contrast, when a macrophage is activated by Th2 cytokines (IL4, IL13â&#x20AC;Ś) and parasite products it is termed M2 macrophage (See Figs. 12.12 and 12.13). The names M1/M2 were chosen because; in their turn, M1 and M2 promote Th1 and Th2 responses (See Chapter 12). However, M1/M2 subdivision is a limited attempt to define the remarkable plasticity of macrophages.

Fig. 5.21. M1/M2 MACROPHAGES. Pattern Recognition Receptors (PPR) of macrophages (and Dendritic Cells) cells perceive the presence of Microbe Associated Molecular Patterns (M/PAM, See Fig. 4.4) and Damage Associated Molecular Pattern (DAMP, See Fig. 4.3). Moreover, macrophages sense their physical microenvironment through PIEZO-1 ion channel (REFERENCE AG Solis et al, NATURE 2019,573:69). Integration of the information provided by the various sensors and receptors simultaneously, triggers distinct differentiation programs. The most extreme points of this gradual differentiation are schematized in the concept of M1 and M2 macrophages. M1 MACROPHAGES acquire a high microbicidal activity, secreting pro-inflammatory cytokines and Reactive Oxygen Intermediates (ROI, See Fig. 5.23). Activated M1 macrophages display a respiratory burst and an increased glycolysis. M2 MACROPHAGES display an anti-parasitic activity (See Figs. 12.12 and 12.13). Moreover, M2 macrophages release molecules that work toward resolution of inflammation, promote cell proliferation, tissue repair, wound healing and angiogenesis. In addition, M2 macrophages are implicated in allergy and asthma. M2 macrophages have low glycolysis rates but a marked fatty acid oxidation activity.

Fig. 5.22. PHAGOCYTOSIS. Various forms of endocytosis are performed by all the body cells. However, macro phagocytosis is done in a professional way by tissue macrophages, Langerhans cells, immature Dendritic Cells, and -in a less intense way- by neutrophils and eosinophils. Microbes and foreign particles are perceived by distinct Pattern Recognition Receptors that trigger the emission of pseudopods that engulf the particle in a phagosome. In addition, macrophages digest their own parts (autophagy, See Fig. 7.5). Protein fragments from digested microbes, endocytosed proteins as well as

57 protein fragments generated by autophagy can be presented as peptides on glycoproteins of the Major Histocompatibility Complex (HLA in humans, see Chapter 6) and trigger a T cell response (See Chapter 7).

Fig. 5.23. M1 MACROPHAGES: MICROBE TRAPPING AND KILLING. Microbes are sensed by macrophages through a large array of Pattern Recognition Receptors (PPR, See Figs. 4.4) displayed on the membrane of long pseudopods stretched by the activated macrophage. Moreover, MAC 1 receptors on macrophage membrane interact with the C3b constituent of Complement cascade adhering to the membrane of a microbe (See Chapter 21). A lysosome containing various hydrolytic enzymes in an acidic matrix able to destroy proteins and nucleic acids fuses with the phagosome giving rise to a phagolysosome. On M1 activated macrophage NADPH oxidase assembled on the wall of phagolysosomes produces various Reactive Oxygen Intermediates (ROI). The combination of hydrolytic enzymes and ROI kills the majority of microbes picked up. Their debris are expelled from the cells or are presented as protein fragments (peptides) on Class II HLA glycoproteins and trigger a T cell response (See Chapter 7). Activated M1 macrophages display an enhanced phagocytic activity, ROI production, killer activity and expression of the glycoproteins of the Major Histocompatibility Complex (HLA in humans).

FIG. 5.24. DENDRITIC CELLS (DC): ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. DC orchestrate the crosstalk between innate and adaptive immunity. Through the Pattern Recognition Receptors (PRR) DC sense and capture antigens. Then, they process the endocyted molecules and migrate to lymph nodes where they present the antigen to T cells (See Figs. 10.4-10.6). Immature DC differentiate from hematopoietic progenitors as well from monocytes. Immature DC are located in tissues in contact with the external environment such as the dermis and the lamina propria beneath the basal membrane of skin, conjunctiva, mouth, digestive tract and lung mucosa. In the skin, immature DC are known as Langerhans cells. DC have an oval or lobulated nucleus. The cytoplasm contains a small Golgi apparatus, a moderate endoplasmic reticulum, some mitochondria, several lysosomes and a variable number of postGolgi vesicles that correspond to Class II HLA proteins containing organelles. When differentiated into mature DC, these cells become particularly efficient in presenting protein fragments (peptides) of phagocytized proteins on Class II HLA glycoproteins and trigger a T cell response (See Chapter 10). DC play a critical role in T helper cell differentiation (See Figs.12.2-12.3), allergen capture (See Fig. 20.11) and sensing intestinal antigens (See Figs. 3.4 and 27.11).

Fig. 5.25. DENDRITIC CELLS (DC): GENERAL FEATURES. The critical role played by DC in T cell activation is presented in Chapter 10.

59 Fig. 5.26. NATURAL KILLER (NK) CELLS: ULTRASTRUCTURAL (panel A) AND HISTOLOGICAL (panel B) ASPECTS. NK cells are a minor cell population present in the blood (10-15 % of circulating lymphocytes, not all White Blood Cells!), lymphoid organs and tissues. They differentiate from bone marrow progenitors under the influence of IL7 and IL15. As Innate Lymphoid Cells (ILC) NK cells are lymphocytes with a nucleus with the typical chromatin appearance of a mature lymphoid cell similar to that of T and B lymphocytes. However, NK cells are a little bit larger than T and B cells and display small cytoplasm with distinct azurophilic granules containing mainly perforin and granzymes (See Figs. 11.6, 11.7 and 11.8), so these cells are also defined as Large Granular Lymphocytes (LGL). As ILC, NK cells do not express RAG-mediated recombined T cell receptor (TCR) nor the B cell receptor (BCR). Since NK cells do not express distinctive markers, they are recognized through their peculiar combination of markers co-expressed by other cells. Besides a killer activity, NK cells release several cytokines that have a major role in driving the inflammatory response and the normal formation of the trophoblast during pregnancy. Both killer activity and cytokine secretion are modulated by several cytokines released by the cells in the microenvironment (See Fig. Chapter 2). Fig. 5.27. NK CELLS: CYTOKINES MODULATING KILLER ACTIVITY AND CYTOKINE RELEASE. The killer activity of NK cells (also known as cytotoxicity) rests on the release of perforin and granzymes and other granule contents on the membrane of the target cell (See Figs. 11.6 and 11.7). For cytokine release see Chapter 2.

60 Fig.5.28. WHY IS THE KILLER STRATEGY OF NK CELLS IS SO SPECIAL? Commonly immune cells are in a resting state. Signals of various kinds activate the cell and trigger the cell killer activity. By contrast, the killer program of NK cells is always active. Their killer activity is called natural precisely because it does not require activating signals to be turned on. Rather, this natural killer activity is usually blocked by various inhibitory receptors (See Figs. 25.3 and 25.4) perceiving signals delivered by the body normal cells. Fig. 5.29. THE BLOCKADE OF NATURAL KILLER ACTIVITY. The NK cell (Left in the Fig.) expresses both activating receptors (In red) and Killer Inhibitory Receptors (KIR) (In blue) whereas the target cell (Right in the Fig) expresses both normal inhibitory Class I HLA glycoproteins and other molecules that may trigger NK killer activity (In green). The integration of signals that the NK cell receives through KIR and activating receptors regulates the natural cytotoxicity of NK cells. KIR recognizing normal HLA Class I glycoproteins on the membrane of the target cells transduce signals that stop the killer program. Signals delivered by KIR are dominant and block the killer program even when activating receptors are triggering it. The most important inhibitory signals are delivered by KIR recognizing normal Class I glycoproteins of the HLA complex expressed on the membrane of the target cell (For HLA see Chapter 6). A human NK cell expresses on the cell membrane KIR of a different kind that recognize various allelic glycoproteins coded by HLA-A, HLA-B, HLA-C and HLA-E genes (See Fig. 6.5)

Fig. 5.30. THE RELEASE OF THE NATURAL CYTOTOXICITY. The NK cell (Left in the Fig) expresses both activating receptors (In red) and Killer Inhibitory Receptors (KIR) (in blue) whereas the target cell (Right in the Fig) does not express normal inhibitory Class I HLA glycoproteins but instead anomalous HLA glycoproteins and other activating molecules (In green and blue). The lack of inhibitory signals due to the poor expression or absence of normal Class I HLA glycoproteins on the cell membrane of the target cell allows activating receptors to unleash the NK lytic activity and cytokine release. On the cell membrane of NK cells, a set of distinct activating receptors recognize Class I HLA glycoproteins that are not normally expressed by healthy cells and other membrane glycoproteins whose expression increases during neoplastic transformation, cell stress, senescence or microbial infection (See Fig. 6.19)

Fig. 5.31. NK CELLS: WHAT ACTIVATING RECEPTORS RECOGNIZE. A human NK cell expresses on the cell membrane different kind of activating receptors that recognize various molecules that are not expressed by happy and healthy cells. Therefore, their killer activity plays a critical role in the control of infections by intracellular microbes, anomalous cells, tumor progression, and metastasis spread. For ADCC see Fig. 20.8.

Fig. 5.32. NK CELLS: ACTIVITIES MODULATED BY THE MULTIPLE CYTOKINE RELEASED. The roles played by NK cells through cytokine release are not less important than those of their killer activity. For the remodeling of decidual vessel see Fig. 26.14. The multiple activities of blood circulating NK cells play a critical protective role against Mycobacterium tuberculosis infection. REFERENCE: RR Chowdhuery et al, Nature2018,560:644.

Fig. 5.33. NK CELLS: ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC). The killer activity of NK cells can be guided by antibodies recognizing their target with high specificity. For details see Figs. 20.8 and 20.9.

63 Fig. 5.34. ENDOTHELIAL CELLS OF POST CAPILLARY VENUE. Blood vessels distribute oxygen, nutrients, hormones and immune cells through the body. Endothelial cells (EC) that line post-capillary venules are not just passive conduits for delivering blood. On the contrary EC guide White Blood Cell (WBC) migration to inflammatory sites. Post-capillary venules are minute vessels intermediate in structure and location between venules and capillaries. Their narrow lumen is lined with a single layer of EC surrounded by pericytes. The EC cells and pericytes are enveloped in a basal membrane. EC adhere to one another through junctional structures (tight junctions and adherent junctions) formed by transmembrane adhesive proteins. These junctions together with intercellular clefts create a barrier controlling the permeability of fluids and small molecules. Caveolae present as invaginations in the endothelial plasma membrane or as vesicles in the cytoplasm are involved in flow sensation and endocytosis. Weibelâ&#x20AC;&#x201C;Palade bodies contain the von Willibrand factor which is involved in blood coagulation and P-selectin, an adhesion molecule which plays a central role in the ability of inflamed EC to recruit passing leukocytes allowing them to extravasate.

Fig. 5.35. ACTIVATED ENDOTHELIAL CELLS: THE CONTROL OF WHITE BLOOD CELL (WBC) EXIT FROM BLOOD CAPILLARIES. Prostaglandins and histamine released by sentinel leukocytes, mast cells and platelets increase the permeability of postcapillary venules by inducing endothelial cells and pericytes to widen inter-cellular spaces. In addition, once activated by inflammatory stimuli, cytokines, and chemokines, the endothelial cells start to up-modulate the expression of adhesion molecules [Selectins; IntraCellular Adhesion Molecules (ICAM)]. The binding of these adhesion molecules to integrins expressed on the membrane of activated leukocytes (Rolling neutrophils in the Fig.) initiates a rolling adhesion of neutrophils to the vessel's wall. Chemokines and cytokines also up-modulate the neutrophil expression of membrane integrins which bind to ICAM on endothelial cells creating a firmer adhesion.

64 The exit of WBC from blood vessels (called diapedesis, transendothelial migration or margination) is guided by further adhesion interactions involving molecules such as PECAM-1 which is expressed by endothelial cells at intercellular junctions. Neutrophils pass between endothelial cells by disrupting various cell-cell junctions. Alternatively, they exit passing through pores generated within activated endothelial cells. Thus, WBC recruitment and extravasation can be separated into four steps: a) Initial attachment and low velocity rolling of WBC; b) Their arrest; c) The activation of leukocyte integrin expression; d) WBC transmigration and margination in the tissue.

Fig. 5.36. INNATE IMMUNITY: THE FAST AND FURIOUS RECRUITMENT OF WHITE BLOOD (WBC) CELLS TO THE BATTLE ZONE. In the cartoon, the broken body barrier lined by a fibrin clot and platelets allows the microbe invasion. Alarm signals (See Fig. 4.2) and proinflammatory chemokines (See Fig. 2.22) produced by damaged epithelial cells, dermal mast cells and sentinel lymphocytes lead to a fast and massive local recruitment of with Blood Cells (WBC) followed by their activation by pro-inflammatory cytokines. Vessel dilatation combined with the thickening of the blood due to the leaking out of plasma causes a slowing-down of blood flow rate. This leads leukocytes to stick to the vessel walls, especially in postcapillary venules. Neutrophils are the first and the more numerous WBC that exit from blood vessels (extravasate) and enter the invaded tissues (marginate) moving along chemotactic gradients. Numerous neutrophils present in acute and suppurative inflammation massively release cell-damaging

65 enzymes (degranulate) leading to microbes, cells and tissues necrosis and apoptotic death. Dead cells, secreted molecules, and released nucleic acids give rise to pus. This fast and furious acute inflammation is an early and effective protective response against the invaders that triggers both the adaptive immune response and the regeneration of the damaged tissues. Acute inflammation must be potent but carefully controlled to limit self-tissue damage. When the intruder is eliminated, negative receptors expressed on the inflammatory WBC sensing Damage Associated Molecular Patterns (DAMP) released by dead cells restrict neutrophil recruitment into the diseased organ and limits immunopathology (See Fig. 25.4; REFERENCE: F Salazar and GD Brown, Science 2018,362:292). Wound healing and injury repair facilitate the resolution of inflammation by restoring barrier function, followed by tissue formation and remodeling. Nevertheless, the storm of alarm signals and cytokines leading to the quick recruitment and activation of WBC along with a massive release of self-antigens by the damaged tissues may trigger the induction of late autoimmune reactions (See Chapter 27). Nowadays, people take anti-inflammatory drugs to avoid pain and dampen the intensity of acute inflammation.

Fig. 5.37. MICROBES AT MUCOSAL SURFACES REMOTELY CONTROL THE THYMIC MATURATION OF MUCOSAL-ASSOCIATED INVARIANT T CELLS (MAITC). The diffusion of microbiota metabolites (specifically Vitamin B2 precursor derivatives) induces the thymic maturation of a specialized population of T cells, all expressing an identical (invariant) antigen receptor. Once activated by antigen presenting cells (see Chapter 7) these MAITC recognize and respond to microbial metabolites and play an important role in tissue repair, wound healing and anti-microbial defense. REFERENCE: J Oh and D Unutmaz, Science, 2019,366:429.

Fig. 5.38. INNATE IMMUNITY: SOLUBLE ANTI-MICROBIAL MOLECULES. The presence in all body fluids of several molecules endowed with a direct anti-microbial activity makes difficult the survival of microbes inside the body. The concentration of anti-microbial molecules is especially high in inflammatory edema due to the local influx of plasma from activated endothelia. CDCC, Complement Dependent Cellular Cytotoxicity: Complement components adhering on the surface of target cells or microbes interact with Complement receptors (See Fig. 21.8) displayed on the cell membrane of granulocytes, macrophages, and NK cells. The interaction between Complement components and Complement receptors activates the cell reaction.

CHAPTER 6. COMMUNICATION CODES OF THE IMMUNE SYSTEM: b. THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC). Fig. 6.1. THE MHC/HLA GLYCOPROTEINS: A CLUSTER OF MOLECULES GUIDING ADAPTIVE IMMUNITY. The HLA/MHC glycoproteins are expressed on the cell membrane of almost all cells of the body. They guide the differentiation and the activity of − T cells and NK cells. By contrast, the cells of innate immunity do not express receptors able to recognize the peculiarities of MHC/HLA polymorphism.

Fig. 6.2. THE MHC/HLA GENES. I: A VERY PECULIAR CLUSTER OF GENES. The genes encoding the MHC/HLA glycoproteins are highly polymorphic. It means that for each of these genes there are numerous alleles. Alleles are the various forms that a gene can take. It is remarkable that this gene cluster is maintained substantially identical in all vertebrates. The reason why these genes did not undergo gene diaspora, a common genetic feature of other genes, is not yet clear.

Fig. 6.3. THE MHC/HLA GENES. II: MAJOR FEATURES. Some HLA alleles are relatively common in the human population while others are very rare.

Fig. 6.4. THE MHC/HLA GENES. III: A SIMPLIFIED MAP

Fig. 6.5. THE MHC/HLA GENES IV: PRINCIPAL CLASS I AND CLASS II GENES.

Fig. 6.6. FEATURES OF HLA GLYCOPROTEINS CODED FOR BY CLASS I GENES. I: This schematic drawing of the HLA Class I glycoprotein should be integrated with the scheme of the same glycoprotein in Fig. 6.7.

Fig. 6.7. FEATURES OF HLA GLYCOPROTEINS CODED FOR BY CLASS I GENES. II: This schematic drawing of the HLA Class I glycoprotein should be integrated with the scheme of the same glycoprotein on previous Fig.

Fig. 6.8. THE BETA (ď ˘)-2 MICROGLOBULIN. This non-HLA coded protein is an essential component of Class I HLA glycoproteins.

Fig. 6.9. THE HLA CLASS I GLYCOPROTEINS ARE POLYGENIC AND POLYMORPHIC. To be polygenic means that each person possesses several loci coding for slightly different HLA Class I glycoproteins. To be polymorphic means that in the human population for each of these HLA Class I loci there are several alleles.

Fig. 6.10. THE EXTENSIVE POLYMORPHISM OF PRINCIPAL HLA CLASS I GLYCOPROTEINS. The high number of alleles (polymorphism) present in the human population for HLA Class I gene (A, B, and C) and Class II genes (DP, DQ, and DR genes, see Fig. 6.15) makes it improbable to find two human beings expressing all the same HLA Class I and Class II glycoproteins. Of each of these numerous HLA-A, HLA-B and HLA-C alleles, every person expresses only two alleles, the one inherited from the mother and the one inherited from the father.

Fig. 6.11. CO-DOMINANCE OF THE HLA ALLELES INHERITED FROM THE MOTHER AND THE FATHER.

FIG. 6.12. WHICH ARE THE CELLS THAT EXPRESS HLA CLASS I GLYCOPROTEINS AND WHICH ARE THE CYTOKINES THAT INCREASE THEIR EXPRESSION?

Fig. 6.13. FEATURES OF HLA GLYCOPROTEINS ENCODED BY CLASS II GENES. I: This schematic drawing of the Class II HLA glycoprotein should be integrated with the scheme of the same glycoprotein in Fig. 6.14

72 Fig. 6.14. FEATURES OF THE HLA HETERODIMER ENCODED BY CLASS II GENES. I: The  and  chains are made by two extracellular domains (alpha 1, alpha2; beta 1 and beta2), a transmembrane domain (S in the Fig.) and a short intra-cytoplasmatic tail (l in the Fig.).This schematic drawing of the Class II HLA glycoprotein should be integrated with the scheme of the same glycoprotein in Fig. 6.13.

Fig. 6.15. POLYMORPHISM OF PRINCIPAL HLA CLASS II GLYCOPROTEINS. The high number of alleles (polymorphism) present in the human population for DR-A and DR-B, DQ-A and DQ-B, and DP-A and DP-B genes makes it improbable to find two human beings expressing all the same HLA Class I and Class II glycoproteins. Of each of these numerous DR-A and DR-B, DQ-A and DQ-B, and DP-A and DP-B alleles, every person expresses two alleles only, one inherited from the mother and one inherited from the father.

Fig. 6.16. MEMBRANE EXPRESSION AND MODULATION OF HLA CLASS II GLYCOPROTEINS.

Fig. 6.17. CO-DOMINANCE OF HLA ALLELES INHERITED FROM THE MOTHER AND THE FATHER: The creation of our molecular individuality.

Fig. 6.18. MAIN FUNCTIONS OF HLA CLASS I AND CLASS II GLYCOPROTEINS

Fig. 6.19. MULTIPLE FUNCTIONS OF “ADDITIONAL” CLASS I (CLASS Ib) GLYCOPROTEINS.

Fig. 6.20. MULTIPLE ROLES OF ADDITIONAL CLASS II GLYCOPROTEINS INVOLVED IN PEPTIDE PRESENTATION. For Peptide presentation and Proteasome see Chapter 7.

Fig. 6.21. MAIN FEATURES OF CLASS III HLA GLYCOPROTEINS. In addition, several olfactory receptors are coded by Class II genes.

Fig. 6.22. A FEW CRITICAL OUTCOMES OF THE POLYMORPHISM OF HLA GENES. The variant of HLA genes we inherited by our mother and father affects several aspects of our life, some of which not yet fully defined. The kind of Class I and Class II grooves we have inherited allows (or not) the binding and presentation of certain protein fragments (peptides), a critical issue in the activation of T cells. To have inherited certain HLA variants allows (or not) to mount an efficient T cell reaction. Thus, some combinations of HLA genes could be especially protective against a particular microbial disease, and therefore propagate in the population of the world area where the disease is endemic. The expression (or not) of HLA glycoproteins inhibits (or activates) NK activity (See Fig. 5.29). Moreover, the intensity of our reaction to microbes depends not only by the grooves of Class I and Class II glycoproteins we have inherited but also by the levels of the soluble anti-microbial molecules coded by Class III genes. The intensity of these reactions also shapes our microbial flora (or microbiome; Sees Fig. 3.3 and 3.8). In this way, HLA polymorphism affects our individual smell. The individual smell and the polymorphism of olfactory receptors coded by Class III genes appear to bias the selection of sexual partners in mice and perhaps unconsciously contribute to this selection also in humans.

CHAPTER 7. PEPTIDE GLYCOPROTEINS.

PRESENTATION

HLA

Fig. 7.1. HLA GLYCOPROTEINS ARE FLOATING ON THE CELL MEMBRANE OF HUMAN NUCLEATED CELLS. On the cell membrane, both Class I and Class II HLA glycoproteins display a protein fragment (a peptide) bound to them (In red in the Fig.).

Fig. 7.2. HLA GLYCOPROTEINS DISPLAY BOUND PEPTIDES. A. The majority of Class I HLA glycoproteins present on the cell membrane display an 8-10 amino acid long protein fragment (a peptide) entangled in the groove formed of alpha 1 and alpha 2 domains of the  chain. Moreover, the majority of Class II HLA glycoproteins display a peptide entangled in the more flexible groove made by alpha 1 and beta1 domains of the  and  chains. Peptides in the groove of Class II HLA glycoproteins are longer (13 amino acids or more). Peptides associated with Class I HLA glycoproteins derive from proteins broken down in the cytosol whereas those associated with Class II HLA glycoproteins arise from proteins broken down by acidic hydrolysis in endocytic vesicles. B. Ribbon diagram of the structure of the HLA Class I groove. The floor is made by eight antiparallel beta strands while two alpha helices form the lips of the peptide binding groove.

Fig. 7.3. THE UBIQUITIN-PROTEASOME SYSTEM. Cell proteins are continuously renewed: old, surplus or altered proteins are destroyed and substituted by new synthesized proteins. The 10 x 106 ribosomes associated with the endoplasmic reticulum of a c//ell produce about 10 x 106 protein/minute. Old, incorrect or surplus proteins are bound to the 100 x 106 ubiquitin molecules present in the cytoplasm and eliminated. Proteins bound by ubiquitin lose their conformational structure and interact with the lid of the proteasome. Ubiquitin is then removed and re-utilized while the protein introduced inside the proteasome is chopped into 8-9 amino acid long peptides.

Fig. 7.4. THE PROTEASOME. The 26S proteasome structure as determined by electron microscopy (Left in the Fig.) and its schematic drawing (Right). The 26S proteasome is a cylinder-shaped particle consisting of a multi-catalytic core, the 20S proteasome, and two regulatory complexes known as the 19S lid or regulatory complexes. The 20S proteasome core consists of two outer rings, made up of seven different subunits with chymotryptic, tryptic and postglutamyl peptide hydrolytic activity.

7.5. THE AUTOPHAGY. Autophagy is another dynamic process of cellular housekeeping in which disfunctional cytoplasmic organelles, large proteins aggregates, and endocellular microbes are removed and degraded. First, these structures are aggregated, then engulfed in a cytoplasmic vesicle, the autophagosome. When the autophagosome fuses with a lysosome, a membrane-bound spherical organelles containing acid hydrolases, engulfed structures are digested in short peptides and amino acids that are discharged by exocytosis or are recycled in the cytoplasm (REFERENCE: Pohl et al., Science 2019,366, 8182.

Fig. 7.6. WHERE CYTOPLASMATIC PEPTIDES MET CLASS I HLA GLYCOPROTEINS? Cytoplasmatic peptides emerging from the proteasome or autophagy are rapidly digested in amino acids by the aminopeptidases in the cytosol. However, a few lucky peptides are able to bind to particular transporters (Transporters associated with Antigen Processing, TAP1, and TAP2) on the membrane of the endoplasmic reticulum. TAP1 and TAP2 transporters are coded by Class II genes of the HLA complex. Lucky peptides reaching the endoplasmic reticulum through TAP1, and TAP2 transporters survive longer and may bind to the groove of nascent HLA Class I glycoproteins. In the endoplasmic reticulum the recently synthesized  chain of the HLA Class I glycoprotein binds a first chaperone protein, Calnexin. Subsequently, the  chain gets associated with the 2microglobulin, dissociates from Calnexin and binds Calreticulin another chaperone protein that senses nascent glycoproteins and Tapasin, a chaperone protein that interacts with TAP transporters. The interaction of Tapasin with TAP1 and TAP2 transporter helps the interaction between the nascent glycoprotein and peptides. The loading of the peptide in the groove induces structure rearrangements in the nascent glycoprotein resulting in its dissociation from chaperone proteins. Other proteins scan the quality of newly formed peptide-Class I glycoprotein complex. By interacting with the structural elements that anchor the peptide in the groove (See Fig. 7.6) they displace the peptides that have a modest affinity for the groove. Finally, Class I glycoproteins with a peptide in the groove leave the endoplasmic reticulum and move towards the cell surface. Also, a small fraction of glycoproteins without the peptide in the groove reach the cell surface. These Class I glycoproteins can be loaded by peptides present in the body fluids. REFERENCE: A Bleeps et al., Nature 2017,551:526.

80 Fig. 7.7. HOW THE LUCKY PEPTIDES BIND THE GROOVE OF CLASS I HLA GLYCOPROTEINS. The lucky peptides surviving a quick digestion to amino acids may bind the groove of recently formed Class I HLA glycoproteins. To remain associated with a Class I HLA, a peptide has to establish multiple interactions (ionic interactions, hydrogen bonds) with the amino acids of the floor and the walls of the groove. The ability to bind or not and the strength of the binding (the affinity) depends on both the structure of the groove and the amino acid sequence of the peptide. The groove of each Class I HLA allelic variant has a distinct amino acid sequence. Therefore, grooves with different shape and electric charges can (or cannot) interact with the anchor amino acids of the peptide. Therefore, some Class I allelic variants bind certain peptides very well while other alleles are not able to do so. This genetic control of the ability to present a peptide is an issue of crucial importance since it is possible that a person with certain Class I HLA alleles presents a particular peptide very well whereas another person does not present or poorly presents this particular peptide, having inherited a different Class I HLA allele. Moreover, the same peptide may bind the various grooves differently assuming a different conformation in order to adapt to the grove of distinct Class I HLA glycoproteins. The different conformations assumed by the Class I HLA glycoprotein and the bound peptide (HLA-p) are recognized by different receptors on the surface of T cells. Every groove of a Class I HLA glycoprotein can bind about 1000 different peptides. Moreover, every person displays multiple Class I HLA grooves (six at least considering only the HLA-A, -B, and â&#x20AC;&#x201C;C glycoproteins inherited from the mother and the father). This promiscuity of the peptide-groove interaction and the multiplicity of distinct Class I HLA glycoproteins increase the individual probability to bind and present a particular peptide at least with one of the groves. In addition, the polymorphism of Class I HLA glycoproteins present in a population increases the probability that at least a few persons have a groove able to efficiently present a given peptide. A few variable portions of the variable (V) domain of the T Cell Receptor (TCR) interact with the peptide (Complementarity Determining Region 1, CDR1); while other portions interact with the HLA glycoprotein (CDR2 and CDR3) (See Figs. 8.10, 8.12, 8.13 and 10.8).

Fig. 7.8. PEPTIDES PRESENTED BY CLASS I HLA SHOW WHAT IS GOING ON INSIDE THE CELL. On the cell membrane, the half-life of HLA glycoproteins bound to a peptide is about 6 hours. Newly made Class I HLA glycoproteins transit from the endoplasmic reticulum to the post-Golgi vesicles moving toward the cell membrane. This means that new HLA glycoproteins continuously display peptides derived from proteins recently destroyed in the cytoplasm. In the absence of a microbial incursion and antigenic stimulation, peptides expressed by Class I HLA glycoproteins derive from proteins digested either by the ubiquitin-proteasome system or autophagy.

Fig. 7.9. FROM WHERE ARE COMING THE PEPTIDES WHO END UP IN THE GROOVE OF CLASS II HLA GLYCOPROTEINS? While the synthesis of Class II HLA glycoproteins goes in parallel with that of HLA Class I, the peptides bound to their grooves have a different origin. In the endoplasmic reticulum, the  and  chains of HLA Class II glycoproteins are synthesized independently. The chaperone Invariant chain binds them while the Clip fragment seals their peptide groove. Therefore, cytosolic peptides transported by TAP 1 and TAP2 into the endoplasmic reticulum cannot bind to the groove of Class II HLA glycoproteins. The three molecular complexes made by the  and  chains of HLA glycoproteins and the Invariant chain transit from the endoplasmic reticulum to the post-Golgi vesicles moving toward the cell membrane. During this journey, these vesicles fuse with phagolysosomes.

Fig. 7.10. WHERE PEPTIDES FROM ENDOCYTED STRUCTURES MET CLASS II HLA GLYCOPROTEINS? During their journey towards cell membranes, post-Golgi vesicles containing immature Class II HLA glycoproteins are guided by the Invariant chain to fuse with an acid phagolysosome containing peptides of the endocyted structures. The acidic pH and the action of peptidases cleave the Invariant chain leaving only the groove sealed by Clip. Subsequently, the association of nascent Class II glycoproteins with DM and DO chaperone proteins allows Clip dislodgment and favors the binding of peptides. DM and DO chaperone proteins are coded by additional Class II genes (See Fig. 6.20). Mature Class II glycoproteins displaying peptides from the endocyted structures are then inserted on the cell membrane. As the half-life of membrane HLA class II glycoproteins is of about 6 hours, peptides from the structures recently endocyted are displayed on the cell surface. The interaction established by the peptide with the floor and walls of the groove of Class II molecules are similar to those illustrated on Fig. 7.6. Here, however, the groove is made by two chains (the ď Ą and ď ˘ chains). It is longer and more flexible

Fig. 7.11. ENDOCYTED PEPTIDES END UP IN THE GROOVE OF CLASS II HLA GLYCOPROTEINS. A phagolysosome (Orange in the Fig.) containing peptides (Red) deriving from endocyted proteins fuses with a post-Golgi vesicle transporting newly synthesized HLA Class II glycoproteins towards the cell membrane. In the post-Golgi vesicles, the Class II HLA glycoproteins interacting with the Invariant chain have the groove sealed by the Clip fragment of the invariant chain (A in the Fig). Phagolysosome acid proteases degrade the Invariant chain (B) and the Class II HLA glycoproteins with the groove still sealed by the Clip fragment (C) binds the DM glycoprotein. DM and DO (not shown) are other HLA Class II glycoproteins not expressed on the cell surface but only in the cytoplasmatic vesicles (See Fig. 5.19). The interaction with DM stabilizes the HLA Class II glycoproteins, permits the release of Clip (D) and the binding of the peptide.

CHAPTER 8. ď Ą-ď ˘ T CELLS

Fig. 8.1. A DIFFERENT STRATEGY: THE ADAPTIVE IMMUNITY

Fig. 8.2. ADAPTIVE IMMUNITY: MAIN FEATURES. T and B cells are the key cells of the adaptive immunity. Roughly, in a person, there are 1012 T and B cells. Each of those virgin T or B cells expresses many copies of a single unique, individual receptor specific for a foreign molecule, which is called antigen. As almost every lymphocyte expresses a single unique receptor different from that of the others, the total repertoire of different antigen receptors of T and B cells of a person exceed 1011. This means that the T and B population of a person is able to recognize more than different 1011 different targets.

Fig. 8.3. ADAPTIVE IMMUNITY. II: CLONAL SELECTION. Adaptive immunity is a defense strategy based on: A. Cell membrane receptors recognizing with high precision (high specificity) the molecular features of their target (here defined as the antigen, red triangle in the Fig.); B. Proliferation (clonal expansion) of the lymphocyte whose receptor better interact with the antigen; C. Competition among lymphocytes expressing receptors able to bind the antigen with different strength (different affinity, see Fig. 16.5).

Fig. 8.4. CELLS OF ADAPTIVE IMMUNITY. T and B cells selectively interact with the specific cognate antigen at high precision (high affinity) and orchestrate the activation of multiple and complex defense reactions.

Fig. 8.5. THE SELECTIVE ACTIVATION OF A T AND B CELL. In the dark of the interior of our body, the life of T and B lymphocytes is modulated by a large series of membrane receptors perceiving environmental signals. Following the capture of its ligand, a receptor transduces the signal to the nucleus. The integration of multiple signals captured by distinct receptors modulates gene activation and cell activities. Unlike cells of innate immunity (See Fig. 5.2), every T and B cell expresses many copies of a unique individual antigen-specific receptor: The T Cell Receptor (TCR) and B Cell Receptor (BCR). The TCR expressed on the membrane of a virgin T cell are different from those of all the other virgin T cells. Similarly, the BCR on the membrane of a virgin B cell is different from those of all the other virgin B cells. The selectivity in the activation of a particular T cell rests on the interaction of its individual TCR with the cognate HLA-p complex (See Figs. 8.10, 8.12). On the other hand, the selective activation of a particular B cell rests on the interaction of its individual BCR with the cognate antigenic molecule (See Figs. 14.10 and 14.11). Signals provided by the other receptors guide the T and B cell maturation, differentiation, and homing and concur to cell activation. Once they are selectively activated, T and B cells give rise to a large clone of effector/memory cells, all expressing the same individual TCR (or BCR) of the cell initially activated (See Fig. 8.3).

Fig. 8.6. THE T CELL. Ultrastructural (A) and histological (B) aspects. A. The image derived from an electron micrograph shows a T cell with a round nucleus surrounded by a thin rim of cytoplasm containing several ribosomes, scattered mitochondria, scarce endoplasmic reticulum, and very few lysosomes. Short microvilli are present at the cell surface. B. At light microscopy, a T cell displays a round nucleus surrounded by an almost imperceptible rim of cytoplasm. T cells are morphologically identical to B cells. The antigen receptor expressed by T cells on the cell membrane is called T cell receptor (TCR). The TCR does not interact with foreign antigens in their natural conformation. The - TCR binds only HLA glycoproteins presenting an antigen peptide in their groove (HLA-p, See Figs. 8.12 and 10.8).

Fig. 8.7. ORIGIN AND MATURATION OF - T CELLS. As all White Blood Cells, T cells originate from bone marrow maturing passing through characteristic developmental stages.

Fig. 8.8. MATURE VIRGIN (OR NAIVE) - T CELLS.

Fig. 8.9. MAIN SUBSETS OF HUMAN − T CELLS. The large - T cell population present in the human body can be subdivided in subsets on the basis of both their surface markers (the so-called CD, See Fig. 22.7) and their main function. T cells expressing the CD4 marker (the CD4+ lymphocytes) interact with Class II HLA glycoproteins and peptides entangled in their groove (HLA-p). The majority of CD4+ T cells display helper activity (See Chapter 12). However, a few CD4+ T cells have instead a cytotoxic activity and kill HLA Class II-positive target cells. On the contrary, T cells expressing the CD8 marker (the CD8+ T cells) interact with Class I HLA-p. The majority of CD8+ T cells display cytotoxic activity (See Chapter 11). However, a few CD8+ T cells may have a T helper activity. Double negative (CD4-, CD8-) invariant NK cells display a TCR interacting with CD1 HLA Class I glycoprotein (See Fig. 6.19) presenting microbial lipids and glycolipids entangled in their groove. The immense diversity of the TCR repertoire and function of − T cell populations ensures that the immune system can respond to almost any peptide in a highly specific manner.

Fig. 8.10. ACTIVITY OF COMMON - T CELLS. T cells mediate their action though direct, antigenspecific contact with the target cells. Every T cell encodes a unique TCR that recognize 8-20 amino acidlong peptides presented on the groove of HLA glycoproteins (HLA-p). The - T cell receptor (TCR) does not interact with foreign antigens but binds only HLA-p. T cells in circulation in the body tissues and in the lymphatic organs continuously interact with body cells. Once an interaction is established, the - TCR expressed on the T cell membrane probes Class I and Class II HLA-p glycoproteins on the surface of the target cells. Class II HLA-p are scanned by the - TCR of CD4+ T cells while Class I HLA-p glycoproteins are scanned by the - TCR on CD8+ T cell membrane. Peptides generated from normal proteins are ignored (See chapter 22) whereas anomalous peptides will trigger T cell response.

Fig. 8.11. THE TCR OF - T CELLS. Both  and  chains are members of the Ig superfamily (See Fig. 14.5). Each chain is constituted by two globular extracellular domains, a transmembrane domain made by hydrophobic amino acids and a short intracytoplasmic tail, unable to transduce the signal to the cell nucleus. The amino acid sequence of the most external domain (the V domain) is typical of each virgin T cell. This individual difference allows the TCR of each virgin T cell to react with a different HLA-peptide complex. By contrast the C domain is identical in all - T cells.

FIG. 8.12. THE - TCR OF CD3+, CD8+ T CELL SCANS THE CLASS I HLA GLYCOPROTEINS AND PEPTIDES IN THEIR GROOVE (HLA-p). A few portions of the variable (V) domains of both  and  chains of the TCR (the complementary determining regions CDR1 and CDR2, see Fig. 8.13) should have a molecular conformation allowing a close interaction with the more external structures of HLA glycoproteins. A distinct portion of the TCR (the hypervariable portion, CDR3) should have a conformation allowing a close interaction with the shape of the peptide entangled in the groove of HLA glycoproteins. When the reciprocal molecular complementarity allows a high affinity interaction of the TCR with the HLA-p complex, activation signals are transduced to the nucleus of the T cell.

Fig. 8.13. WHAT A - T CELL RECOGNIZES. Two portions (CDR1 and CDR2 2) of the TCR mainly interact with the amino acids of the HLA molecule. The third portion, the CDR3, the more variable portion interacts with the peptide entangled in the groove of the HLA molecule. A similar three-component interaction guides the activation of all - T cells. iNK cell, however, display a TCR interacting with CD1 HLA Class I glycoprotein (See Fig 6.19) presenting microbial lipids and glycolipids (and not peptides!) entangled in their groove.

Fig. 8.14. TCR SIGNAL TRANSDUCTION DEPENDS ON SEVERAL MONOMORPHIC CHAINS ( CHAINS and CD3 COMPLEX) ASSOCIATED WITH THE TCR. When the TCR on T cell surface interacts with high affinity with the HLA-p complex, activation signals are transduced to the nucleus of the T cell. However, the TCR does not transduce directly the signal to the nucleus. High affinity interaction of the TCR with the HLA-p complex leads to the activation of four transducer chains, the  (zeta), the  (gamma),  (delta) and  (epsilon) chains that are defined as members of the CD3 complex, a typical marker of T cells. Delta () and  chains associate to form the CD3 heterodimer,  and  chains to form the CD3 heterodimer, while the  chains form the CD3ζζ homodimer.

Fig. 8.15. TRANSDUCTION OF THE TCR SIGNAL. High affinity interaction of TCR with the HLApeptide (HLA-p) complex leads to the activation of CD3ζζ homodimers, CD3 and CD3 heterodimers that are the members of the CD3 complex. The two tyrosines of a peculiar amino acid sequence called ITAM (Immuno-receptor Tyrosinebased Activation Motif) on the  chain and on the three CD3 chains are phosphorylated and form the docking site of the signaling Zap70 ( chain Associated Protein 70) (See Figs. 10.9. and 10.11. For ITAM see also Fig. 20.9). Each CD3 chain expresses one ITAM, whereas the  chains expresses three ITAM.

Fig. 8.16. MEMBRANE MARKERS OF T - CELLS.

Fig. 8.17. SCHEMATIC DRAWING OF HOW CD3+ CD8+ T CELLS SCAN CLASS I HLA GLYCOPROTEINS AND PEPTIDES IN THEIR GROOVE. The CD8 co-receptor interacts with a monomorphic (invariant) sequence 3 domain of the  chain of class I HLA glycoprotein.

Fig. 8.18. SCHEMATIC DRAWING OF HOW CD3+ CD4+ T CELLS SCAN CLASS II HLA GLYCOPROTEINS AND PEPTIDES IN THEIR GROOVE. The CD4 co-receptor interacts with a monomorphic (invariant) sequence of the 2 domain of the  chain of a class II HLA glycoprotein

Fig. 8.19. INVARIANT NK T CELLS (iNK T cells) form a distinct but heterogeneous population of lymphocytes expressing a ď Ą-ď ˘ T Cell Receptor with a limited variability (defined as invariant). iNKT cells predominantly are found at barrier sites (For details see also Fig. 9.16).

CHAPTER 9. THE THYMIC EDUCATION OF T CELLS. Fig. 9.1. THE HUMAN THYMUS. I: ANATOMICAL LOCATION. The thymus is the lymphoid organ where precursor T cells are induced to differentiate into diverse type of mature T cells and are “educated” to recognize foreign molecules and tolerate self-molecules. Children born with a thymic defect may display a major immunodeficiency since normal T cell maturation is impaired (See Chapter 28). Fig. 9.2. T CELL FAMILIES DEVELOPING IN THE THYMUS. T cells precursors mature in the bone marrow. Then, in the absence of Notch fate signals, they exit from the bone marrow and are attracted to the thymus where they differentiate in T cells. The multiple and intimate contacts that each precursor T cell establishes with the various niches of thymic stromal cells shown in the previous figures trigger gene recombination events allowing each precursor T cell to both acquire its individual T Cell Receptor (TCR) and differentiate in one of the five main T cell populations: a) -, CD3+ and CD4+ cells; b) -, CD3+ and CD8+ T cells; c) NK T cells; d) -, CD3+ T cells; d) Mucosal-Associated Invariant T (MAIT) cells.

Fig. 9.3. THE HUMAN THYMUS. I. In the thymus two anatomically and functionally distinct regions can be identified, the outer cortex (up in the Fig) and the inner medulla. Each of these two regions contains different thymic stromal cells (connective cells, epithelial cells, Dendritic Cells, macrophages, cells similar to taste chemosensory tuft cellsâ&#x20AC;ŚSee Fig. 4.5) which provide the microenvironment required for the development and selection of different T cell populations. Fig. 9.4. THE HUMAN THYMUS. II HISTOLOGICAL SECTION OF THE THYMUS STAINED WITH HEMATOXYLIN AND EOSIN. The thymus is surrounded by a thin fibrous capsule. Fibrous trabeculae create numerous thymic lobules. The thymic cortex appears darker because of the much higher number of precursor T cells-thymocytes than of medulla. The blue staining of the nuclei of the crowded thymocytes hides the endodermal-derived epithelial cells, forming a reticular meshwork. These cells contain long processes creating an interconnected network and a framework of supporting cells. In addition, the thymus harbors macrophages, mainly located in the cortex, and Dendritic Cells, mainly located in the medulla. In their maturation, thymocytes move from the subcapsular region into the medulla. With ageing, the thymus is progressively replaced by adipose tissue.

97 Fig. 9.5. THE HUMAN THYMUS. III HISTOLOGICAL SECTION OF THE THYMUS STAINED WITH AN ANTI-CD3 MONOCLONAL ANTIBODY. Staining with monoclonal antibodies (See Chapter 22) binding CD3 (See Fig. 8.14), a cell membrane marker of the most common T cell population shows that the vast majority of the cells in the thymus are CD3 positive (CD3+) lymphocytes. These cells are so numerous that they hide the reticular epithelial meshwork.

Fig. 9.6. THE HUMAN THYMUS. IV: HISTOLOGICAL SECTION OF THE THYMUS STAINED WITH AN ANTI-Ki-67 MONOCLONAL ANTIBODY. Antibodies binding Ki-67, a protein expressed by proliferating cells, disclose an intense proliferative activity in the thymus. About a week after their arrival in the thymus, precursor T cells enter a phase of intense proliferation (positive selection) followed by a complex differentiating process. This process leads to the survival of only a minor population of mature T cells expressing a T Cell Receptor (TCR) able to bind with low-affinity self HLA glycoproteins presenting self-peptides.

98 Fig. 9.7. THE HUMAN THYMUS. VI: IMAGE OBTAINED BY SCANNING ELECTRON MICROSCOPY. I. The extremely numerous developing thymocytes (the spherical cells in Fig) occupy the small niches and interstices formed by the thymic epithelial cells. This and the following picture (See Fig. 9.7) illustrate the close cell-to-cell contacts leading to the interactions of developing thymocytes and thymic cells.

Fig. 9.8. THE HUMAN THYMUS. VII: IMAGE OBTAINED BY SCANNING ELECTRON MICROSCOPY. II. The fragment of the human thymus was washed in a tissue culture medium before the scanning electron microscopy procedure. When the majority of thymocytes are washed out, the extensive network of epithelial cells becomes evident. This and the previous picture show clearly the intimate and continuous interaction taking place between the membranes of developing thymocytes and thymus epithelial and lymphoid cells. These interactions form the basis of negative and positive selections of thymocytes

Fig. 9.9. THYMIC MATURATION OF DISTINCT T CELL POPULATIONS

Fig. 9.10. THYMIC MATURATION OF − CD4+ AND − CD8+ T CELLS. I. − CD4+ and −− CD4+ AND − CD8+ T CELLS. I. − CD4+ and − CD8+ T cells are by large the main T cell populations. Their thymic educational program occurs in both the thymus cortex and medulla. Both compartments are characterized by the presence of specialized thymic stromal cells which provide the signals and checkpoints required for their T cell development and selection.

100 Fig. 9.11. THYMIC MATURATION OF − CD4+ AND − CD8+ T CELLS. II. STEPS OF THE EDUCATIONAL PROGRAM. The education of these T cell populations is based on a complex series of massive cell deaths (negative selections) alternate to intense cell proliferations (positive selections). In the thymus, CD4- CD8-doublenegative precursor T cells which do not yet express the TCR or the CD4 and CD8 coreceptors undergo the rearrangements of – T Cell Receptor genes. Cells that rearrange successfully the TCR gene become CD3+. These cells start to express both the CD4 and the CD8 co-receptors and are called double-positive cells (CD4+, CD8+). Their first selection takes place in the thymus cortex: double positive thymocytes that have generated a - TCR unable to interact with self HLA glycoproteins presenting peptides in their groove (HLA-p) expressed on the surface of cortical epithelial cells die by neglect. By contrast, those that have generated a TCR interacting with self HLA-p are induced to proliferate (positive selection). Moreover, cells with a TCR binding HLA class I glycoproteins retain the expression of the CD8 but no longer express the CD4 and become CD3+, CD8+, CD4-. Cells with a TCR binding HLA class II glycoproteins retain the expression of the CD4 but no longer express the CD8, and become CD3+, CD8+, CD4+. In this way, a double positive CD4+ and CD8+ cell becomes a single positive cell, CD4+ or CD8+. These single positive cells migrate to the thymus medulla. Here, the cells with a TCR binding self HLA-p with high affinity are induced to commit apoptosis. This negative selection is a dramatic event since the majority of single positive thymocytes disappear. The few surviving thymocytes express a TCR binding self HLA-p with low-affinity. These thymocytes are induced to proliferate again and eventually migrate out to the periphery as mature virgin T cells.

Fig. 9.12. THYMIC MATURATION OF − CD4+ AND − CD8+ T CELLS. III. INITIAL STEPS. These early steps of precursor T cell differentiation take place independently from interaction with HLA glycoproteins and the peptides in their groove (HLA-p) expressed on the surface of thymic epithelial-stromal cells.

101 Fig. 9.13. THYMIC MATURATION OF − CD4+ AND − CD8+ T CELLS. IV. DEATH BY NEGLECT AND POSITIVE SELECTION. Until the double positive (CD4+, CD8+) stage T cell differentiation continues to take place independently from the interaction with HLA glycoproteins. By contrast, starting from this differentiation stage, the further differentiation of immature T cells (differentiation of thymocytes) is critically modulated by the interactions of the newly formed T Cell Receptor (TCR) with HLA glycoproteins presenting peptides in their groove (HLA-p). At this differentiation stage, the strength of the signal transmitted by the interactions between the TCR and HLA-p decides whether a developing T cell is selected to die or survive. TCR-HLA-p signaling strength below the minimum (for example, non-functional TCR) causes death by neglect. By contrast, strong signals cause cell survival and cell proliferation. Signal strength is also influenced by the recruitment of T cell co-receptors (CD4 and CD8, See Figs 10.12 and 10.13).

Fig. 9.14. THYMIC MATURATION OF − CD4+ AND − CD8+ T CELLS. V. THE NEGATIVE SELECTION. In the next step of the educational program, T cells are selected for a low T Cell Receptor (TCR) signaling strength: self HLA-p recognition can result in both clonal deletion of the developing T cell and its diversification into a natural Treg cell (for

102 natural Treg cells see Fig. 26.10). Negative selection: Thymocytes that have generated a TCR interacting at high affinity with self HLA glycoproteins presenting self-peptides in their groove (HLA-p) either undergo apoptosis (Central tolerance, See Fig. 26.6 – 26.9) or become a natural T regulatory (Treg) cell (See Fig. 26.10). Thus, apoptotic cell death becomes an important and dramatic occurrence since it involves the majority of thymocytes. The utmost importance of this negative selection of the TCR repertoire rests in the deletion of autoreactive cells with a TCR interacting at high affinity with self HLA-p. Role of AIRE in the negative selection. To provide a broader negative selection of potentially autoreactive T cells, thymic epithelial cells express the Auto Immune Regulator (AIRE) gene. This gene encodes a transcriptional factor enabling epithelial thymic cells to express proteins that normally are expressed only by peculiar peripheral body tissues. Single positive thymocytes. Thymocytes that did not die by neglect and escaped negative selection by self HLA glycoproteins with self-peptides in their groove (HLA-p) are CD4+ CD8+ double positive cells expressing a TCR that interacts with self HLA-p with low-affinity. Depending on the HLA-p recognized by their TCR, double positive thymocytes become single positive: those with a TCR recognizing Class I HLA-p glycoprotein will keep the CD8 co-receptor expression while they repress the expression of the CD4 co-receptor, whereas those with a TCR recognizing Class II HLA-p will keep CD4 co-receptor expression. Exit from the thymus. Following this complex and dramatic selection of the TCR repertoire, T cells emerging from the thymus express a TCR that recognizes self HLA-p at low-affinity. The interaction of their TCR with self HLA-p is strong enough to transduce anti-apoptotic signals from the environmental cells but is not sufficient for T cell activation. These surviving cells enter the blood as mature virgin (naïve) T cells. This cell population expresses a huge repertoire of different TCR since every T cell has generated its own TCR. A TCR binding self HLA-p at low-affinity may interact with high affinity with self HLA glycoproteins expressing a foreign peptide or against foreign HLA glycoproteins. Foreign peptides and foreign HLA glycoproteins display only a few amino acid differences from self HLA-p and these differences may allow the establishment of a high affinity interaction with the TCR expressed by virgin T cells.

Fig. 9.15. THYMIC MATURATION OF − CD4+ AND − CD8+ T CELLS. VI. FEATURES OF MATURE VIRGIN - CD3+ T CELLS EMERGING FROM THE THYMUS.

103

Fig. 9.16. THYMIC MATURATION OF − CD4+ AND − CD8+ T CELLS. VII. CELL DIFFERENTIATION FOLLOWED THROUGH THE EXPRESSION OF CD3, CD4 AND CD8 MEMBRANE PROTEINS.

Fig. 9.17. THYMIC MATURATION OF iNKT CELLS. Invariant NK T (iNKT) cells form a minor population of either CD4+, CD8+ (double positive) or CD4- CD8(double negative) lymphocytes expressing a − T cell receptor (TCR) with very limited repertoire, called invariant TCR. This invariant TCR is positively selected to interact with CD1, a nonpolymorphic family of Class Ib HLA glycoprotein (See Fig. 6.19) involved in the presentation of microbial lipids and glycolipids. The development and polarization of iNK T cells take place in particular thymus niches rich in epithelial cells similar to tuft taste sensory cells releasing IL25. At the barrier sites, iNKT cells recognizing conserved molecules of commensal microbes (Nformyl peptides presented by CD1 glycoproteins) that normally colonize barrier surfaces such as those of the skin and gut do not directly retaliate against the bacterium but instead aids tissue repair. In this way, the immune response elicited control microbial containment rather than driving bacterial elimination which is usually accompanied by inflammation and tissue damage. REFERENCES: JL Linehan et al, Cell 2018,172:784; P. Klenerman and G. Ogg, Nature 2018, 555:594; C.N. Miller et al, Nature2018,559:627.

104

Fig. 9.18. THYMIC MATURATION OF - T CELLS.

Fig. 9.19. THYMIC MATURATION OF GAMMA-DELTA (-) T CELLS. The cells of this family of T lymphocytes express a T Cell Receptor (TCR) resulting from the rearrangement of - genes. The - TCR expressed by the majority of these cells recognizes directly (i.e. without the presentation by HLA molecules) microbial metabolites, molecules rapidly appearing following a microbial infection and other forms of cell stress.

105

Fig. 9.20. HOW - TCR RECOGNIZES PHOSPHO ANTIGENS. Phosporilated metabolites (phosphoantigens) are produced by microbes and overexpressed by cancer cells or rapidly appear following other forms of cell stress. Recent data suggest that phospho antigens trigger the modulation of butyrophilin proteins on the surface of Antigen Presenting Cells (APC). The interaction of - TCR (See Fig. 9.17) with modulated butyrophilins directly triggers the proliferation of - T cells, killer activity and their release of pro-inflammatory cytokines (IL17, IFN…) and chemokines. REFERENCE: M Rigau, Science 2020,367:643.

106

Fig. 9.21. THYMIC MATURATION OF MUCOSAL-ASSOCIATED INVARIANT T CELLS (MAITC).

Fig. 9.22. MUCOSAL-ASSOCIATED MUCOSAL-ASSOCIATED INVARIANT T CELLS (MAIT) CELLS . As shown in Fig. 5.37, MAIT cells are a specialized population of T cells present at mucosal surfaces. All MAIT cells express an identical (invariant) antigen receptor. Once activated by antigen presenting cells (see Chapter 7) these MAITC recognize and respond to microbial metabolites and play an important role in tissue repair, wound healing and anti-microbial defense. REFERENCE: J Oh and D Unutmaz, Science, 2019,366:429.

107

CHAPTER 10. ACTIVATION OF VIRGIN T CELLS.

Fig. 10.1. THE NATURAL LIFE STORY OF T CELLS. The signals delivered by VISTA receptor upon the interaction with Lselectin commonly expressed on cell membranes act as a brake restraining inappropriate virgin T cell activation against self antigens (See Chapter 26). REFERENCES: C Brown and A Rudensky, Science 2020,367:247; MA EITanbbouly et al, Science 2020, 367:264.

Fig. 10.2. THE CONCURRENCE OF NUMEROUS SIGNALS IS REQUIRED TO ACTIVATE A VIRGIN T CELL. The activation of a virgin T cell is triggered by specialized cells, the antigenpresenting cells (APC). Only these cells provide the multiplicity of signals required for virgin T cell activation. This complexity of triggering signals secure against incorrect activation of T virgin cells. Once activated, a virgin T cell quickly generates a clone of effector/memory T cells that will persist almost indefinitely (See Chapter 24). Therefore, an erroneous T cellâ&#x20AC;&#x2122;s activation against self-antigens can lead to a persisting and devastating autoimmune disease (See Chapter 27).

108 Fig. 10.3. ROLES OF ANTIGENPRESENTING CELLS (APC) IN VIRGIN T CELL ACTIVATION. The conversion of an APC from a cell focused in antigen capture into a cell dedicated to antigen presentation to a T cell is clearly evident with Dendritic Cells (DC). Through the expression of Pattern Recognition Receptors (PPR, see Figs. 4.4) immature DC perceive the presence of foreign bodies. Their enhanced phagocytic activity allows an efficient antigen capture. Then, DC present the peptides derived from the captured foreign structure in the groove of Class II HLA glycoproteins. Antigen phagocytosis along with alarm and inflammatory signals induces the maturation of immature DC into mature DC. DC travel to the lymphatic organs where they home into T cell areas (See Figs. 10.5, 18.4, and 19.3). Fig. 10.4. ANTIGEN CAPTURE AND PEPTIDE PRESENTATION TO T CELLS. Pattern Recognition Receptors (PPR, See Fig. 4.4) allow antigenpresenting cells (APC) to sense the presence of the intruder, to capture it and transform it into peptides to be associated with HLA glycoproteins (HLA-P), the only antigenic signal that T lymphocytes are able to perceive. Furthermore, the type of PPR that interacted with the intruder molecules guide the choice of lymphocytes that APC associates with the presentation of the peptides.

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Fig. 10.5. ANTIGEN CAPTURE AND PRESENTATION TO T CELLS. Then, alarm signals and proinflammatory cytokines induce APC maturation and mobilization. From the tissues where the antigen capture has taken place, mature APC migrate to local lymphatic vessels and then slowly travel towards the draining lymph node (Figs. 18.4 and 19.3) where they present peptides of the captured antigen in the groove of their HLA glycoproteins.

Fig. 10.6. THE JOURNEY OF ANTIGENPRESENTING CELLS (APC) ENDS IN THE T CELL AREAS OF DRAINING LYMPH NODES. Afferent lymphatic vessels collect and channel interstitial fluid and APC from the periphery to the subcapsular sinus of the lymph node (See Fig. 19.2). During their long journey to the lymph node, APC digest the captured antigen and present antigen peptides in the groove of their HLA glycoproteins (HLA-p).

110 Fig. 10.7. T CELLANTIGEN-PRESENTING CELL (APC) INTERACTION. In lymphatic organs, numerous T cells packed in the T cell area scan the HLA glycoproteins and the peptides (HLAp) on the membrane of APC. A T cell first interacts with the APC through non-specific adhesion glycoproteins. Then, the TCR probes the HLA-p glycoproteins expressed on the APC membrane.

Fig. 10.8. FIRST ROLE OF ADHESION MOLECULES. The binding of adhesion molecules expressed by T cells with the corresponding molecules on antigenpresenting cells (APC) allows an initial T cellAPC interaction. Among the adhesion molecules expressed on T cell surface we have: Lymphocyte FunctionAssociated antigens (LFA-1 and LFA2/CD2), integrins connected with the cell cytoskeleton; The Intracellular Adhesion Molecule-3 (ICAM-3), a member of the Ig super-family molecules (See Fig. 14.5). These adhesion molecules interact with other ICAM, LFA molecules, and C-type lectins (DC-SIGN, Dendritic Cell Specific Icam-3 Grabbing Non integrin) expressed on the APC cell membrane. These multiple and non-specific interactions lead towards the formation of a temporary immunological synapse between the APC and the T cell.

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Fig. 10.9. TCR SCAN HLA GLYCOPROTEINS AND PEPTIDES ON ANTIGEN-PRESENTING CELL (APC). Receptors and ligands involved in T cell activation cluster in the area of T and APC membrane contact. Here, the TCR expressed by T cells starts to probe HLA glycoproteins with peptides in their groove (HLA-p) displayed by the APC. In most of the cases, the TCR does not bind the HLA-p with high affinity and T cell and APC separate after a short period. By contrast, if the TCR binds HLA-p with high affinity, the LFA-1 adhesion molecule acquires a new conformation, binds ICAM-1 with higher affinity and the T cell remains in contact with the APC for several hours. During this prolonged cell-to-cell interaction, the APC and the T cell deliver and receive multiple signals leading toward the activation of the T cell. The high affinity binding of TCR with HLA-p is the central event on which rests the specificity of T cell activation. It is due to the spatial complementarity of TCR complementary determining regions (CDR1, 2 and 3, See Figs. 8.12) and HLA-p (See Fig. 8.13). A single amino acid difference may transform an activating, high affinity binding into a non-activating interaction. In the case of high affinity interaction, the TCR repeatedly binds the HLA-p, delivers activation signals and dissociates from the HLA-p. Signals coming from these repeated TCR bindings put the virgin T cell in a state of initial activation. Accessory costimulatory signals are then required to further progress in the T cell activation, clonal expansion, and differentiation. In Fig. the alpha chain of the TCR is in dark blue; the beta chain of TCR in light blue; the alpha chain of Class I HLA in green; the beta-2 microglobulin in brown; the peptide on the HLA groove in red.

112 Fig. 10.10. T CELL RECEPTOR AND HLA GLYCOPROTEINS MOVE TO THE LIPID RAFT. Following the first high affinity interactions, TCR moves to a special microdomain of the membrane (the lipid raft, in orange in the Fig.) rich in saturated lipids and cholesterol. This move is critical for the transduction of TCR signals to the nucleus since the Lck and Fyn Src kinases involved in T cell activation home in this area. During the multiple interactions between TCR and HLA-p, the zeta chain of CD3 (CD3 z) dimerize to initiate a downstream signaling cascade. CD45, a tyrosine phosphatase, is activated. Activated CD45 removes phosphate groups of inactive Lck and Fyn. Once activated, Lck and Fyn phosphorylate the tyrosines of ITAM regions of the  chain and CD3. Then, phosphorylated ITAM act as the docking site for ZAP 70 (the Zeta Chain Associated Protein kinase). Next, ZAP 70 activates LAT and SLP-76 adaptor molecules. In the Fig. Lck and Fyn are in black when inactive and in orange when active; yellow circles enclosing a P: phosphorylate ITAM domains. Fig. 10.11. TCR SIGNALING. The tyrosine kinase ZAP-70 phosphorylates scaffold proteins leading to the activation of phospholipase C-gamma. It moves to the cell membrane and cleaves phosphatidyl inositol biphosphate in inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 causes the release of Ca2+ from endocellular stores and opens Ca channels allowing the intracellular entrance of extracellular Ca2+. DAG diffusing into the cytoplasm activates protein kinase C  and Mitogen Activated Protein (MAP) kinases. MAP kinases are also activated by ZAP-70 kinase. Finally, the transcription factors of the NF-B, NFAT and AP-1 families trigger the activation of both the IL2 gene and the gene of the IL2 receptor  chain (CD25) (See Figs. 2.14 - 2.20 and 10.11).

113 Fig. 10.12. ACTIVATED T CELL. The final step in the activation of a T cell leads to the expression on the cell membrane of the IL2 receptor  chain (CD25), the formation of a trimeric high affinity IL2 receptor and the secretion of IL2. When CD25 is expressed on a T cell membrane, tiny amounts of the IL2 secreted by the T cell are autocrinally caught. Then, phosphorylated  and  chains of IL2 receptor transduce signals through the JAK- STAT pathway and induce T cell clonal proliferation and differentiation in effector/memory T cells (See also Figs. 2.12 - 2.20). Fig. 10.13. T CELL COSTIMULATION BY CD4. For the activation of a virgin T cell, the high affinity interaction between TCR and HLA-p and the signal transduction schematically shown in Fig. 10.9 are not enough. Efficient signal transduction of TCR signals, T cell survival, and differentiation require multiple accessory signals delivered by several costimulatory molecules and receptors. On the cell membrane of CD3+ CD4+ T cells, the costimulatory molecules CD4 should bind a conserved domain of HLA Class II glycoprotein. This binding stabilizes the interaction of TCR with Class II HLA-p. Moreover, CD4 bound to Class II HLA-p activates Src kinases Lck and Fyn (see Fig.9.8) enhancing the phosphorylation of ITAM regions of the CD3 and  chains. The lack of these costimulatory signals induces a state of T cell anergy, the T cell become dysfunctional (See also Fig. 26.13). Fig. 10.14. T CELL COSTIMULATION BY CD8. On the cell membrane of CD3+ CD8+ T cells, the costimulatory molecule CD8 binds a conserved domain of HLA Class I glycoproteins. This binding stabilizes the interaction of TCR with Class I HLA-p. Moreover, CD8 bound to Class I HLA-p activates Src kinases Lck and Fyn enhancing the phosphorylation of ITAM regions of the CD3 and  chains. The lack of costimulatory signals induces a state of anergy, the T cell becomes dysfunctional (See Fig. 26.13).

114 Fig. 10.15. T CELL COSTIMULATION BY B7. B7.1 (CD80) and B7.2 (CD86) molecules are homodimers of the Ig superfamily (See Fig. 13.5) expressed by activated antigenpresenting cell (APC). Their interaction with the costimulatory receptor CD28 expressed on the cell membrane of T cells induces receptor phosphorylation. Phosphorylated CD28 activates phospholipase Cgamma to produce IP3. In addition, signals delivered by TCR and CD28 induce the expression on T cell of the CD40 ligand (CD40L), another costimulatory molecule that binds CD40 receptors expressed by APC. The signals delivered by the activated CD28 are of critical importance for the clonal expansion of virgin T cells and their survival. Also, in this case, the lack of B7-CD28 costimulatory signal induces a state of anergy (See Fig. 26.13).

Fig. 10.16. T CELL COSTIMULATION BY CD40 LIGAND. Signals delivered by TCR, CD4 or CD8 and CD28 induce a pre-activated T cell to express on its cell membrane the CD40 ligand (CD40L) a trimeric membrane cytokine of the Tumor Necrosis Factor (TNF) family. This costimulatory molecule binds CD40, a receptor expressed by antigenpresenting cells. A ligand activated CD40 receptor delivers signals inducing APC to provide T cell costimulatory signals more efficiently and to increase their expression of HLA and B7 molecules and the secretion of cytokines (See Fig. 18.9). 17.9).

Fig. 10.17. T CELL COSTIMULATION BY MEMBRANE TUMOR NECROSIS FACTOR (TNF). The expression of a trimeric form of the cytokine TNF-ď Ą on the cell membrane of activated APC is perceived by the TNF receptor II (TNFR II) expressed on the surface of T cells. On the contrary of what takes place with the costimulation by CD40L expressed by pre-activated T cells, the TNF expressed on APC cell membrane binds the TNF receptor on the T cell surface. Through the TRAF-2 (TNF Receptor Associated Factor) the TNFR II activates the AKT pathway promoting T cell survival, the NF-ď ŤB pathway enhancing T cell mitotic activity and the JNK and AP1 pathways enhancing IL2 secretion by T cells. All these transduction pathways have important roles in helping activation and clonal expansion of a virgin T cell.

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Fig. 10.18. THE COSTIMULATION OF A VIRGIN T CELL. When the TCR of a virgin T cell interacts with high affinity with an HLA-p complex, multiple receptors and ligands costimulate T cell activation. These costimulatory signals have a critical importance in the full activation of a virgin T cell whereas they are disposable in the activation of effector/memory T cells. The interaction of TCR with an HLA-p in the absence of costimulatory signals induces T cell anergy: Anergic or dysfunctional T cells normally circulate in the body but are unable to mount an immune response even when HLA-p recognition is accompanied by the correct costimulatory signals. The absence of a concomitant costimulatory signal may also drive Th cells1 to differentiate into induced Treg cells (iTreg, transdifferentiation). In a few cases, the TCR interacting at high affinity with an HLA-p complex is extracted from the T cell membrane and remain on associated with the HLA-p on the target cell membrane (an event called Trogocytosis).

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Fig. 10.19. THE SUPRA MOLECULAR ACTIVATION CLUSTER (SMAC). When TCR binds HLA glycoproteins presenting peptides (HLA-p) with high affinity, T cell, and antigen-presenting cells (APC) establish a longlasting interaction that can be viewed as a prolonged cell to cell kiss.

The clustering in the area of cell-cell contact of molecules involved in T cell activation gives rise to a

SMAC. SMAC molecules repeatedly interact and deliver signals. Then, are endocytosed and become reexpressed.

As the cell-cell interaction progresses, this continuously renewing molecular cluster acquires a

characteristic spatial organization. On the T cell, TCR, CD3 and ď ş chains and the costimulatory CD4 or CD8 and B7 molecules occupy the central area (c-SMAC). Adhesion and costimulatory molecules (LFA-1, CD2-LFA-3; CD4 or CD8) home around it (p-SMAC). The adhesion molecule CD43, the CD44 marker, and the tyrosine phosphatase CD45 gather in a more external area. On the APC, the corresponding ligands and receptors acquire a corresponding localization.

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Fig. 10.20. STEPS OF VIRGIN T CELL ACTIVATION. Specialized sentinel cells (APC) in the body periphery sense, capture, digest the antigen and present antigen peptides in the groove of HLA glycoproteins. Activated by the antigen, danger signals and cytokines, APC travel to T cell areas of lymphoid organs where their HLA glycoproteins and peptides (HLA-p) are probed by the TCR of numerous T cells (See Figs. 10.4 and 10.5). If the TCR of one of these T cells binds their HLA-p with high affinity, the T cell reaches an initial pre-activation stage. Then, multiple costimulatory signals delivered by APC lead the T cell to express the  chain (CD25) of the IL2 receptor (See Figs. 2.12-2.18) and secrete IL2. The fully activated T cells proliferate giving rise to a clone of effector/memory cells whose differentiation is modulated by the cytokines secreted by the APC. In a week, an activated virgin T cell may generate more than 5 x 104 daughter cells. A few of these activated effector/memory cells continually recirculate throughout the body searching for target cells expressing the same peptide in the groove of the same HLA molecule. They exit from the spleen into blood, and from lymph nodes end Peyer’s patches into the lymph, travel in the circulatory fluid and they re-enter into lymphatics organs. This journey is guided by the chemoattractant sphingosine 1-phosphate (S1P), whose concentration is higher in the body fluids than in lymphatic organs, and by the S1P receptor, that is differently expressed by resting and activated T cells. Other effector/memory cells seed in the skin and mucosae where they form a very sensitive and specific sentinel system. Is the TGF (See Fig. 25.6) secreted by activating Dendritic Cells that induces the expression of the CD 103 integrin and sensitize CD8+ effector /memory T cells for the homing in the skin and mucosae. REFERENCE: V Mani et al, Science 2019,366:202

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Fig. 10.21. SPHINGOSINE 1-PHOSPHATE AND ITS RECEPTORS REGULATE THE TRAFFICKING AND MIGRATION OF IMMUNE CELLS. Reference: A Cartier and T Hla, Science 2019, 366:323.

119

CHAPTER 11. T KILLER CELLS.

Fig. 11.1. THE KILLER T CELL. Once correctly activated, a virgin T cell quickly generates a clone of effector/memory T cells. Commonly, a virgin CD8+ cell generates a clone of effector/memory killer T cells (Tk cells), all with the same TCR as the virgin T cell.

Fig. 11.2. THE KILLER ACTIVITY OF EFFECTOR/MEMORY CYTOTOXIC T CELL. Effector/Memory Tk cells patrol the body in the search of the same HLA-p complex that triggered the activation of the progenitor virgin T cell. Following a first non-specific interaction (1), the high affinity binding of the T Cell Receptor (TCR) with an HLA-p displayed on the membrane of a cell (2) is sufficient to trigger their killer activity (3). During the short Tkâ&#x20AC;&#x201C;target cell interaction (the so-called Kiss of Death) the Tk cell releases lytic molecules that cause the death of the target cells. While the target cell dies, the Tk cell detaches and is ready to engage another target (4)

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Fig. 11.3. THE LYTIC SYNAPSE. Following a first, non-specific interaction due to an adhesion molecule, T Cell Receptors (TCR) of an effector/memory Tk cell repetitively scan the HLA-p expressed on the surface of the target cell. The target cell can be any cell of the body and not necessarily an antigen-presenting cell. If the TCR interacts with the HLA-p with high affinity, the signal transduced by the ď ş chain and the CD3 molecules triggers the killing program of the Tk cell.

121 Fig. 11.4. THE KISS OF DEATH. A temporary synapse takes place when the T Cell Receptor (TCR) of a T killer (Tk) cell interacts with high affinity with a complementary HLAp displayed by a target cell. During this short interaction, lasting about 5 minutes, the repeated TCR signals activate the killer program of the Tk cell. The Tk cell re-organizes microtubules and Golgi apparatus to have a localized and polarized exocytosis of perforins and granzymes in the area of cell to cell contact

Fig. 11.5. THE DYING OF THE TARGET CELL. The polarized secretion of perforin and granzymes and other lytic molecules stored within lytic granules in the cytoplasm of the T killer (Tk) cell leads to the death of the target cell. Membrane pores made by perforin allow granzymes to enter into the target cell and induce nuclear fragmentation and chromatin condensation. Dying apoptotic cells do not release their cellular constituents into the surrounding tissue since their fragments (apoptotic bodies) are always enclosed within a plasma membrane. These bodies are subsequently taken up by macrophages without production of inflammatory substances. The killing of virus-infected cells by Tk cells is the most important mechanism of healing from viral diseases. The importance of this defense mechanism is illustrated by the fact that the evolution has endowed a variety of pathogenic viruses with the mean of blocking it. For example, Herpes virus simplex produces a protein that binds to TAP1 and TAP2 (See Fig. 7.5) and blocks the transport of peptides to the endoplasmic reticulum. Cytomegalovirus encodes molecules which induce the degradation of Class I HLA glycoproteins by the proteasome.

122 .

Fig. 11.6. PERFORIN AND GRANULYSIN. Lytic granules of T cells hold several cytolytic proteins including perforin, granzymes, granulysin, and other lysosomal proteins.

Fig. 11.7. THE GRANZYME FAMILY.

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Fig. 11.8. WHY IT IS THAT PERFORIN, GRANULYSIN AND GRANZYMES DO NOT KILL ALSO THE TK CELL? The membrane damaging proteins released by a Tk cell during the Kiss of Death could also lead toward autocrine damage of the Tk cell membrane. The unidirectional killing that takes place seems to be due to the simultaneous release of cathepsin B, Lamp-1 (CD107a) and other molecules that protect the Tk cell membrane in the area of the cell to cell contact. Cathepsin B is a protease. By adhering to the Tk cell membrane, its enzymatic activity degrades perforin and the other membrane damaging proteins.

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Fig. 11.9 EXHAUSTED OR DYSFUNCTIONAL CD8 T KILLER CELLS. T cell exhaustion is a common feature of many chronic infections and cancers. The transcription factor TOX is a central regulator of the early epigenetic events that drive the fate commitment of T ex cells. REFERENCE: O Khan et al, Nature 2019, 571:211

125

CHAPTER 12. T HELPER CELLS.

Fig. 12.1. T HELPER CELLS. The activation of a virgin T cell depends on antigen-presenting cells (APC) displaying in the groove of HLA glycoproteins peptides of the captured antigen (HLA-p). Once correctly activated, a virgin CD4+ cell generates a clone of helper (Th) cells, all with the same TCR as the virgin cell. However, depending on the cytokines secreted by the APC and signals that are present during its activation, a virgin T cell selects one among the Th differentiation programs and generates a particular clone of effector/memory Th cells. In effect, in our body, there are several Th cell subpopulations that modulate and bias the progression of the immune response by secreting distinct combinations of cytokines. These various Th cell populations are competing among themselves and the initial activation and expansion of a particular Th cell population inhibits the development of the other populations, polarizing the immune response being activated. The importance of the role of effector/memory Th cells in the regulation of immune responses is dramatically shown by patients infected by the Human Immunodeficiency Virus (HIV): the progressive decrease of CD4+ cells caused by the HIV infection results in a severe immunodeficiency incompatible with patient survival (See Fig.28.8).

126 Fig. 12.2. THE ANTIGENPRESENTING CELL (APC) INDUCES AND BIASES Th CELL DIFFERENTIATION. Different antigens (bacteria, parasites, foreign or altered cells…) are recognized and captured by antigen-presenting cells (APC) through distinct cell membrane receptors (Pattern Recognition Receptors, PPR; See Fig. 4.4). These various PPR2transduce signals that induce APC to release distinctive cytokine combinations and express particular co-receptors on their cell membrane. For example, cytokines and co-receptors expressed by and APC following the capture of a bacterium are different from those expressed by an APC that has captured a parasite. The peculiar combination of cytokines and co-receptors expressed by an APC during HLA-p presentation to a virgin CD4+cell guides the polarization of the generated clone of Th cells towards Th1, Th2, Th17, induced Treg… (For iTreg see Fig. 25.5). Fig. 12.3. ANTIGENPRESENTING CELLS (APC) DECIDE THE TRANSDIFFERENTIATION OF EFFECTOR/MEMORY Th CELLS. APC bias the activation of the most appropriate immune reaction against the captured antigen by secreting a distinctive combination of cytokines and expressing particular co-receptors (For induced Treg see Fig. 25.5). In this way they guide the conversion from one Th cell type to another, a passage called transdifferentiation.

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Fig. 12.4. ANTIGEN-PRESENTING CELLS (APC) BIAS Th0 TRANSDIFFERENTIATION. Dendritic Cells and macrophages capture the invading antigen perceived through their Pattern Recognition Receptors (PPR) and digest it. Then, antigen peptides are associated with Class II HLA glycoprotein and expressed on the APC surface. Moreover, the different kind of APC, the distinct PPR involve in antigen capture, the molecular structure of the captured antigen and the concomitant environmental signals concur to induce APC to release a particular combination of cytokines and to express a particular set of costimulatory molecules. In response to this combination of signals, the Th0 cell transdifferentiate towards Th17, Th1 or Th2. Transdifferentiation is a complete and stable change in cell identity. The Th0 cell activates one of its gene expression programs. One program is winning out while alternative programs are extinguished. Triggered transcription factors bind to specific sequence motifs within the promoter, enhancer and silencer gene regions, and recruit also epigenetic regulators to modulate the activation state of the genes, activation state that is then transmitted to subsequent cell generations. REFERENCE: E Laurent and B Gottgens, Nature 2018,553:418.

128

Fig. 12.5. Th17 CELLS. Immediately after the capture of bacteria and fungi the antigen-presenting cells secrete TGBď ˘, IL23, and IL6, a combination of cytokines that bias the transdifferentiation of Th0 cells towards Th17. Also, the epithelial cells lining the intestine and sensing tight attachment of microbes respond by producing proteins that guide the transdifferentiation of Th0 cells into Th17 cells. The high salt diet of the Western world may favor Th17 differentiation and thus contribute to both hypertension and autoimmunity. REFERENCE: N Wilck et al, Nature 2018,551:585. On the other hand, transdifferentiation of Th17 in iTreg cells is an important physiological mechanism leading towards the resolution of the inflammation. Metabolic factors regulate the transdifferentiation of Th17 towards iTreg by controlling the expression of the Foxp3 gene. REFERENCE: T Xu al, Nature 2017,548:228. Fig. 12.6. THE INFLAMMATORY RESPONSE INDIRECTLY ACTIVATED BY Th17 CELLS. Th17 cells play a prominent role tissue inflammation and autoimmunity. IL17 and the various proinflammatory cytokines secreted by Th17 cells stimulate fibroblasts, epithelial cells, endothelial cells, and keratinocytes to release anti-microbial factors (See Fig. 3.4) and several cytokines and chemokines, including IL6, IL8, G-CSF and GM-CSF. These factors attract and activate neutrophils and macrophages on epithelial and mucosal surfaces.

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Fig. 12.7. Th1 CELLS. The polarization of the cell clone deriving from the activated virgin Th cell towards Th1 rests on the combination of cytokines and membrane ligands expressed by the antigen-presenting cells displaying the complementary HLA-p. Following the capture of certain antigens, APC expresses distinct ligands of Notch receptors. Notch are a special family of receptors that specify cell differentiation and cell fate. The expression on the APC of the Delta ligand of Notch receptors favors the differentiation of the clone deriving from a virgin Th0 cell towards Th1 whereas the expression of Jagged, an alternative Notch ligand, favors clonal differentiation towards Th2cell.

130

Fig. 12.8. THE STRAIGHTFORWARD Th1 RESPONSE. The release of TNF, IFN- and IL2 by Th1 cells triggers a complex and powerful immune response. These cytokines help B cell activation and drive activated B cells to produce opsonizing and Complement-fixing antibodies endowed with a powerful antibacterial activity (See Figs. 19.11, 19.12). Macrophages activated by pro-inflammatory Th1 cytokines are defined M1 macrophages (See Fig. 3.18): their metabolism is dramatically enhanced. Macrophage phagocytic activity, their ability to digest ingested microbes, their secretion of antimicrobial substances such as nitric oxide (NO2), superoxide (O2-) and other Reactive Oxygen Radicals (ROS) are markedly increased.

Fig. 12.9. Th1 CELLS HELP THE EFFICIENT INDUCTION OF Tk CELL ACTIVITY. A Th1 cell activated by an antigen-presenting cell displaying the complementary Class II HLA-p and costimulatory molecules (B7, CD40…) secretes IL2 and IFN in its microenvironment. These cytokines are instrumental in promoting the efficient activation followed by enhanced survival of neighboring CD3+ C8+Tk cells which recognize the complementary Class I HLA-p.

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Fig. 12.10. Th1 CELL: FEED-BACK ACTIVATION CIRCUITS. The Th1 biased immune response is markedly enhanced by the pro-inflammatory cytokines released by the cells recruited in the reaction. M1 macrophages, NK cells and Tk cells activated by IFNď §, TNF and IL2 released by Th1 cells not only acquire a powerful and differentiate killer activity but also start secreting a large array of pro-inflammatory cytokines that further enhance the reaction triggered by Th1 cells.

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Fig. 12.11. Th2 CELL. Both the factors released by barrier epithelial cells (See Figs. 1.4 and 1.5) and the features of captured antigens (worms, parasitesâ&#x20AC;Ś) induce antigen-presenting cells (APC) to mount a Th2 cell-mediated immune response. APC secrete IL4, IL10 and express the Jagged ligand of Notch receptors. Notch are a special family of receptors that specify cell differentiation and cell fate. The expression of the Jagged ligand favors the transdifferentiation of the clone deriving from a virgin Th0 towards a peculiar Th2 cell. In addition, the secretion of IL10 by APC inhibits the clone polarization towards Th1.

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Fig. 12.12. THE Th2 RESPONSE: A REFINED ANTI-PARASITE REACTION. The polarization of the cell clone deriving from activated virgin Th cell towards Th2 rests on the combination of cytokines (IL4â&#x20AC;Ś) and ligands expressed by the antigen-presenting cells (APC) and cytokines released by Innate Lymphoid Cells (ILC). Following the interception with certain intruders, APC express the Jagged ligand of Notch receptors that favors the transdifferentiation of the clone deriving from a virgin Th0 cell towards Th2. IL4 and IL13 released by activated Th2 cells help B cell activation and drive activated B cells to produce IgG1 and IgE antibodies. Moreover, IL4 and IL13 secreted by activated Th2 cells induce peculiar macrophage activation (alternatively activated macrophages or M2 macrophages). M2 macrophages (See Fig. 5.21) are different from M1 macrophages as they mostly release arginase which induces tissue remodeling and contractility of intestinal smooth muscles. IL13 intensifies the renewal of epithelial cells. The combination of IL4 and IL13 increases the production of mucus and induces the hyperplasia of mucus-secreting goblet cells. The elevated mucus production and smooth muscle hypercontractility triggered by IL4 and IL13 may result in worm expulsion.

134

Fig. 12.13. THE Th2 REACTION TO PARASITIC WORM INFECTION. Parasitic worms, also known as helminths, are a major global health and social burden since nearly one-third of the human population is infected with these parasites, primarily in tropical regions. Therefore, parasitic worms are among the most prevalent infectious agents in the world, and they are responsible for many debilitating diseases and syndromes. However, regions where helminth parasites are endemic record much lower prevalence of allergies and autoimmune diseases, suggesting that the immune reaction against parasites bias the Th0 cell response towards a Th2 prevalence and protects against some allergic syndromes. Sentinel phase: Following a worm infection sentinel epithelial cells of mucosal barriers (tuft cells, See Fig. 3.8) secrete IL25 that activates Innate Lymphoid Cells (ILC) (See Fig. 4.11-4.12). Worms are also perceived by antigen-presenting Cells (tissue macrophages and Dendritic Cells). Cytokines released by Innate Lymphoid Cells and antigen-presenting cells and the peculiar differentiation stage acquired by tissue macrophage (M2 macrophage differentiation, See Fig. 5.20) and Dendritic Cells concur to induce the transdifferentiation of Th0 lymphocytes in Th2 cells. Effector phase: Activated Th2 cells orchestrate a complex and multicellular immune response to parasitic worms by releasing IL4 and IL13 cytokines that further induce M2 macrophage differentiation. a. Hostile micro-environment. Activated M2 macrophages secrete arginase, an enzyme of urea cycle that promote fibrosis and tissue repair. In addition, M2 macrophages produce extracellular matrix components that along with arginase causes tissue remodeling. Moreover, M2 macrophages further bias the transdifferentiation of activated Th0 cells in Th2 cell. The combination of IL4, IL9 and

135 IL13 cytokines released by Th2 and innate lymphoid cells induce the hyperplasia of mucus-secreting goblet cells, enhances mucus production and changes its composition. In addition, IL4, IL9 and IL13 cytokine combination triggers smooth muscle hypercontractility. This hostile micro-environment may result in the expulsion of worms. b. Direct killing. Tissue basophils and mast cells activated by Th2 cytokines enhance local blood supply and the extravasation of eosinophils. IL5 induces eosinophil chemotaxis and activation (See Fig. 5.15). Eosinophils directly attack worms by releasing cytotoxic molecules and Major Basic Protein. In addition, Th2 cells switch the production of IgE antibodies. IgE bound on the cell membrane of basophils and mast cells may induce their degranulation (See Fig. 17.26, 20.10). Moreover, IgE bound on the surface of parasitic worms may impair worm activities and guide the killer activity of eosinophils (Antibody Dependent Cellular Cytotoxicity, ADCC) (See Fig. 5.32).

Fig. 12.14. THE FOLLICULAR T HELPER (TFh) CELL. There are two broad divisions of effector CD4+T cells: non-TFh effector cells (Th17, Th1, and Th2 cells) and T follicular helper (TFh) cells. TFh cells are programmed to interact with B cells within lymphoid tissues to support the production of high affinity, class-switched antibodies. TFh cells home in the follicles of the lymphoid organs (See Figs.18.4-18-10). TFh transdifferentiation is guided by the combination of the secretion of IL6 by the activating antigenpresenting cell (APC) expressing high amounts of ICOS ligands and by a strong T Cell Receptor signaling (REFERENCE: D DiToro et al, Science,2018,361).

ICOS ligands are members of the family of B7 costimulatory molecules expressed by APC, while ICOS (Inducible CO-Stimulator) is a receptor of the TNF receptor family expressed by T cells. IL6 and ICOS ligands induce Th cells to express the chemokine receptors that guide their homing at the interface of B cell follicle and T cell zone. For the role of TFh in B cell activation see Chapter 18.

136

CHAPTER 13. B CELLS.

Fig. 13.1. B CELL ORIGIN AND ACQUISITION OF THE ANTIGEN RECEPTOR, THE SO-CALLED B CELL RECEPTOR (BCR). For bone marrow differentiation of B cells see Figs. 3.4 and 3.5).

Fig. 13.2. ANTIGEN INDEPENDENT BONE MARROW PRODUCTION OF B CELLS. The first phase of B cell maturation is called Antigen Independent, since is taking place independently from any antigen stimulation.

137

Fig. 13.3. BONE MARROW MATURATION OF B CELLS. This cartoon shows a red bone marrow extravascular space between the sinusoids in the hemopoietic medullary cavity of a bone (See Fig. 5.4). Hemopoietic stem cells (HSC) are located close to both osteoblasts (In orange in the Fig.) and endothelial cells of sinusoids (In light purple). During differentiation, HSC move towards a particular kind of stromal cell (reticular stromal cells, in green). This contact induces the maturation to pro-B cell stage. Pro-B cells adjoining other stromal cells secreting interleukin 7 (IL7) mature in pre-B cells. Subsequently, pre-B cells leave stromal cells and express the cell membrane IgM B Cell Receptors (BCR): B cells at this stage of differentiation are called immature B cells. Then, these cells may express also an IgD BCR, exit the bone marrow, enter the blood and become peripheral virgin mature B cells.

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Fig. 13.4. STROMAL CELLS GUIDE B CELL MATURATION. in some ways, this figure is a schematic representation of the events illustrated in Fig. 13.3. Cytokines and membrane molecules expressed by bone marrow stromal cells guide the progressive passage of Hematopoietic Stem Cells (HSC) into pro-B, pre-B and immature B cells. Then, immature B cells leave the bone marrow and home in various areas of lymphoid organs (transitional virgin B cell). During this maturation period and independently from any antigen stimulation, every maturing B cell acquires a single unique individual BCR made by monomeric IgM. Every B cell expresses numerous copies (about 104) of BCR, all with the same binding site. As every maturing B cell generate a distinct binding site, different from that of the BCR of other maturing B cells, an extremely large repertoire of distinct binding sites is generated. The generation of this extremely large repertoire of distinct binding sites is made possible by a unique cut and paste of the genes encoding the binding site of the BCR. This cut and paste takes place in every B cell maturing in the bone marrow (See Chapter 15). This gene recombination is a difficult task, and very often B cells are unable to perform it successfully. In other cases, B cells generate a binding site that binds self-antigens expressed on the surface of bone marrow stromal cells. In this latter case, the B cell will try to change the binding site (receptor editing). If it is not possible, the B cells with a binding site that binds self-antigens will become anergic (unable to mount an immune response) or will undergo apoptosis (See Chapter 26).

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Fig. 13.5. GENERATION OF DISTINCT B CELL POPULATIONS. Signals delivered by bone marrow stromal cells on immature B cells and signals received in peripheral lymphoid organs by transitional virgin B cells (the first B cells which migrate from bone marrow to peripheral blood) influence transitional virgin B cell differentiation in B1 and B2 B cells. Signals received by Notch receptors on B B2 cell membrane induce their differentiation in Marginal Zone B (MZ B) cells. Notch are a special family of receptors that specify cell differentiation and cell fate (See Fig.12.7). Here the presence or the absence of Notch signalling plays a key role in the maturation of hematopoietic stem cells. In the absence of Notch signals, B2 cells differentiate into Follicular (Fo) B2 B cells. Then, the survival and homing of Fo B2 B cells in lymphoid organs is driven by the expression of the receptor for the B cell activating factor (BAFF), a cytokine of the TNF family produced in the lymphoid follicles.

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Fig. 13.6. B1 B CELLS. These cells form a peculiar population of B cells predominantly found in peritoneal and pleural cavities. B1 B cells express a B Cell Receptor (BCR) with low levels of somatic mutations and junctional diversity Once activated, B1 cells differentiate in plasma cells producing low-affinity and poorly specific (cross-reactive) IgM. These antibodies are called natural antibodies since they may be produced without a specific antigenic stimulation. Natural IgM provide a â&#x20AC;&#x153;first line of defenseâ&#x20AC;? against common bacterial pathogens. Moreover, natural IgM reacting against a variety of antigens and autoantigens contribute to the clearance of apoptotic cells and oxidized lipids. However, natural antibodies reacting with self-antigens can be implicated in several autoimmune pathologies. Stress of intestinal epithelial cells triggers the thymus-independent activation of peritoneal B1 B cells that differentiated in IgA secreting plasma cells homing in the sub-mucosal space. These polyreactive IgA secreted at mucosal surfaces regulate microbial commensalism and are protective against enteric inflammation. CD5+ B1 cells are also the normal counterpart of CD5+B chronic lymphocytic leukemia.

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Fig. 13.7. ANTIGENS RECOGNIZED BY B1 B CELLS. The BCR of B1 B cells interacts with low affinity with many different antigens, such as self-antigens and common bacterial polysaccharides. B1 B cells produce the majority of natural immunoglobulin M (IgM) and A (IgA). These natural antibodies are largely encoded by germline immunoglobulin genes (not re-arranged genes, See Chapter 14). Because of their ability to bind different target antigens (poly-reactivity) and their ability to recognize large repetitive structures, B1 B cells provide a first line of defense against pathogens such as encapsulated polysaccharide-expressing bacteria in the gut mucosa and respiratory tract.

Fig. 13.8. AUTONOMOUS ACTIVATION OF B1 B CELLS.

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Fig. 13.9. THE ORIGIN OF MARGINAL ZONE (MZ) B CELLS

Fig. 13.10 MARKERS AND FUNCTIONS OF MARGINAL ZONE B CELLS.

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Fig. 13.11. ANTIGENS RECOGNIZED BY MARGINAL ZONE B CELLS.

Fig. 13.12. LOW-AFFINITY IgM PRODUCED BY MARGINAL ZONE B CELLS TRIGGER A HIGH AFFINITY ANTIBODY RESPONSE.

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Fig.13.13. FOLLICULAR B2 B CELLS.

Fig.13.14. Th CELLS ARE REQUIRED FOR THE ACTIVATION OF FOLLICULAR B2 B CELLS.

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CHAPTER 14. B CELL RECEPTOR AND ANTIBODIES. Fig. 14.1.THE B CELL RECEPTOR (BCR) AND IMMUNOGLOBULIN (Ig). The binding site is the domain of BCR (and of Ig) interacting with the antigen. All BCR expressed by a B cell on the cell membrane and all the Ig that this cell will produce once activated and differentiated in plasma cells share the same binding site.

Fig. 14.2.BCR and IMMUNOGLOBULINS: BASIC STRUCTURE. The Fc fragment of BCR is longer than that of the Ig. It contains an extra transmembrane domain made by hydrophobic amino acids and a short intra-cytoplasmic tail (See Fig. 14.7).

Fig. 14.3.BCR AND IMMUNOGLOBULINS. MOLECULAR FEATURES. I. The binding site is a critical region of the BCR, and Ig made by the NH2 terminus of both H and L chains. The extremely variable sequence of the amino acids of this region creates billions of different binding sites. This large repertoire of binding sites allows a few of them to establish a very precise (specific) interaction with the antigen. The repertoire of BCR is very large in the B cell population, however it should be noted that every B cell expresses BCR and produces Ig with a binding site of a unique specificity, the one randomly acquired among the billions of distinct binding sites expressed by the B cell populations (See Chapter 15).

146 Fig. 14.4. BCR AND IMMUNOGLOBULINS. MOLECULAR FEATURES. II. BCR and Ig are made by four chains, two identical H chains and two identical L chains. These chains are linked by inter-chain disulfide bridges (S-S). The L chain is made by two domains, the variable light domain (VL) and the constant light (CL) domain. The H chain is made by a variable domain (VH) and by three or four constant heavy domains (CH). The VL and VH make the binding site. The binding site of BCR and the Ig should interact with the natural conformation of unpredictable antigens. Therefore, size and flexibility of the BCR are features of critical importance. By contrast, these features have a limited importance with the T Cell Receptor (TCR) that constantly interacts only with HLA-p that displays little conformational variability. Fig. 14.5. BCR AND IMMUNOGLOBULINS: THE Ig SUPERFAMILY. Several immune molecules are made by repeats of the Ig basic molecular structure of 110 amino acids and an S-S bridge. All the molecules made by replicas and slight modifications of this basic molecular structure are considered members of the Ig superfamily. Numerous molecules of the Ig superfamily are expressed on the cell membrane by the cells of the immune system

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Fig. 14.6. B CELL RECEPTOR (BCR) ON THE SURFACE OF A FoB2 B CELL. On the cell membrane, virgin B1 and MZ B cells express a BCR made only by monomeric IgM. By contrast, mature virgin FoB2 B cells have a BCR made by monomeric IgD and IgM. Note that IgM has an extra heavy chain domain and thus is longer than IgD (See Figs.14.8 and 17.18).

Fig. 14.7. BCR AND SOLUBLE MONOMERIC IMMUNOGLOBULINS.

148 Fig. 14.8. THE ASSEMBLY OF A B CELL RECEPTOR (BCR). On a resting B cell, BCR are mobile monomers floating on the cell membrane. The BCR of virgin FoB2 B cells is composed by both monomeric IgM and IgD and the dimeric Ig alpha (Igď Ą) and Ig beta (Igď ˘) transducer molecules. By contrast, in the BCR of B1 and MZ B cells the BCR is made by IgM only, associated with the transducer Ig alpha and Ig beta. The specificity of the BCR signal rests on the ability of the binding site of IgD and IgM to interact with a specific antigen (Orange arrow in the Fig.). Then, the Ig alpha and Ig beta uniformly transduce the signal to the cell nucleus.

Fig. 14.9. THE BCR: SIGNAL TRANSDUCTION. When the binding site of a BCR encounters the specific antigen (in brown in the Fig.), BCR aggregate in micro-clusters that migrate to a lipid raft, a special microdomain of the membrane (Polar head groups are in orangeyellow) rich in saturated lipids and cholesterol. This move is critical for the transduction of BCR signals to the nucleus since the Lck, Fyn, and Blk Src kinases involved in B cell activation home in this area. CD45, a tyrosine phosphatase is the first to be activated. Activated CD45 removes phosphate groups of inactive Lck, Blk, and Fyn and thus activates them. Once activated, Lck and Fyn phosphorylate the tyrosines of ITAM regions of the Ig alpha and Ig beta. In this drawing the cell membrane is depicted as two layers of lipids, their orange and light green polar head groups separate the yellow-orange hydrophobic tails from the aqueous cytosolic and extracellular environments.

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Fig. 14.10. BCR SIGNALING. On the lipid raft, the ITAM of the intracellular portion Ig alpha and Ig beta phosphorylated by Lck, Fyn, and Blk kinases act as a docking site for SYK. The SYK tyrosine kinase then phosphorylates scaffold proteins leading to the activation of phospholipase C-gamma. It moves to the cell membrane and cleaves phosphatidyl-inositol bi-phosphate in inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 causes the release of Ca2+ from endocellular stores and opens Ca2+ channels allowing the intracellular entrance of extracellular Ca2+. DAG diffusing into the cytoplasm activates protein kinase C and Mitogen Activated Protein (MAP) kinases. These molecular events are sufficient to lead to the full activation of B1 and MZ B cells. By contrast, in FoB2 B cells the triggering of the transcription factors of the NF-kB, NFAT and AP-1 families activates the gene of IL2 alpha (ď Ą) chain receptors (CD25) (See Figs. 2.14 - 2.20 and 10.11) and the receptors of other cytokines, making the FoB2 B cells ready to receive the helper signals delivered by TFh cells.

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Fig. 14.11. B CELL COSTIMULATION BY COMPLEMENT. When C3b fragments of Complement cascade adhere on the antigen surface (See Chapter 21), B cell activation is greatly facilitated (costimulated). The C3b Complement fragments adhering on the antigen surface bind the Complement Receptor 2 (CR2, CD21) (See Fig. 21.8). CR2 in cooperation with CD19 and CD81 membrane proteins transduce signals that enhance the transduction of BCR activating signals. Many microbes directly express molecules that activate the Complement cascade through the Lectinic (See Fig. 21.3) and Alternative pathway (See Fig. 21.4). These activation pathways are important mechanisms of natural immunity, quickly activated following a microbial invasion, which costimulate a specific B cell activation. Thus Complement, a key mechanism of natural immunity, plays an important role in the stimulation of antibody response.

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CHAPTER 15. GENERATION OF B and T CELL RECEPTOR REPERTOIRE. Fig. 15.1. THE GENERATION OF THE REPERTOIRE OF BCR (and TCR) BINDING SITES. Each B cell generates a BCR with an individual binding site and expresses on the cell membrane around 104 BCR molecules all with this identical binding site. The acquisition of the individual BCR (and TCR, for T cells) comes from the recombination of three gene segments (V, D, and J) that fuse to form a new unique coding gene. The mechanism of this gene rearrangement leading to the generation of the individual binding site is similar for the BCR and the T Cell Receptor (TCR) and it will be illustrated in detail for the BCR only. In both cases, however, less than two hundred genes recombine and code billions of BCR (and TCR) each with a different binding site. The rearrangement of BCR genes is taking place during B cell maturation in the bone marrow and is driven by signals delivered by stromal bone marrow cells, while the rearrangement of TCR genes is taking place during T cell maturation in the thymus and is driven by stromal and epithelial cells of the thymus (See Chapter 9). In order to understand the mechanisms resulting in the immense repertoire of different binding sites (roughly 1011) expressed by virgin B (and T) cells of our body we have first to realize that the binding site of the BCR is made by the variable domain of both the H (VH) and L (VL) chains. Rearranged H and L chains freely associated with them. Both the H and the L chain are coded by multiple genes: The constant region of the H chain (CH) is coded by a single gene, whereas the various domains of the VH region are coded by three genes, the V, the D, and the J gene. Similarly, the constant region of the L chain (CL) is coded by a single gene, whereas the various domains of the VL region are coded by two genes, the V and J gene. The immense repertoire of BCR binding sites is the result of the combination among the various V, D and J genes coding the V region (Combinatorial diversity). Moreover, the junction of selected V with the selected D and J genes is an error-prone process allowing the formation of new DNA sequences (Junctional diversity). In Fig. the antigen is shown as a green arrow while the area of the binding site in orange.

152 Fig. 15.2. INVENTORY OF THE GENES CODING FOR THE BCR. The various domains of the VH regions are coded by three genes, the V, D, and J gene. In our genome, we have around 65 genes that may code the V domain, 27 that may code the D domain and 6 genes that may code the J domain. The domains of the VL region are coded by the V and J genes. In our genome we have 70 genes that may code the V domain and 9 genes coding the J domain. Each B cell maturing in the bone marrow randomly selects one of the various V, D and J genes to shape the VH and VL region forming the binding site. The CH and the CL regions are coded by a single gene. However, the CH gene can be coded by one of five genes (, , , ,  genes); the CL gene can be one of the two types, the  and  genes.

Fig. 15.3. WHERE ARE GENES CODING FOR THE BCR LOCATED? Genes coding for the H chain are on the human chromosome 14 (CR14), those coding for the L chain of  type on chromosome 2 (CR2) and those coding for the L chain of  type on chromosome 22 (CR22). Long introns are intermingled among these gene loci.

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Fig. 15.4. RANDOM GENE COMBINATIONS RESULT IN A LARGE REPERTOIRE OF DIFFERENT BINDING SITES. A computation of the possible combinations among the various V, D, and J genes shows that there are 320 possible VL regions and 10.530 VH regions. Supposing that any VH can be combined with any VL there are millions of different binding sites. During the antigen-independent maturation driven by the molecules expressed and cytokines secreted by bone marrow stromal cells, every maturing B cell selects and combines these genes to make its own individual binding site. In Fig. the antigen is shown as a green arrow while the area of binding site in orange.

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Fig. 15.5. THE REARRANGEMENT OF GENES CODING FOR THE H CHAIN. Signals delivered by stromal bone marrow cells induce early pro-B cells to open the chromatin, to modify the histones and to express special enzymes and molecules (See Fig. 13.4) involved in the cutting and pasting (recombination) of the DNA, a process which takes place only during B cell maturation. In a random way, each pro-B cell selects one of the 27 D genes and attaches it to one of the 5 J genes. To do so, the double-strand DNA must be cut, the two fragments should be pasted whereas the long DNA fragment between the two cuts is eliminated as an episome. Then, the pasted DJ genes should be attached to one of the 65 V genes. As before, the doublestrand DNA is cut, the two fragments are pasted whereas the long DNA fragment between the two cuts is eliminated as an episome. Therefore, the rearranged DNA of the B cell is shorter than the germline DNA, since it will miss long regions eliminated as episomes. Following a differential splicing, the mRNA codes an H chain with its own particular variable domain. C genes (Cď , Cď ¤, in pink) are two of the various genes coding the constant region of the H chain. In Fig., L: The leader sequence.

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Fig. 15.6. ASSESSMENT OF THE H CHAIN. The H chain coded by the V, D and J genes randomly rearranged should be tested before a pro-B cell proceeds to rearrange the genes of the L chain. In effect, in many cases, the protein coded by the genetic sequence deriving from these gene rearrangements may not be functional. To test if the H chain is functioning, the new H chain is associated with an invariant surrogate L chain made by two segments: VpreB and Lambda5. The H chain (in green in the Fig.) associated with VpreB and Lambda5 (in red) is exposed on the cell membrane of pro-B cells. Here it spontaneously dimerizes with another H chain. The two H chains associated with VpreB and Lambda5 join another similar two H chains molecule and interact with ligands expressed by bone marrow stromal cells. If everything is correct, the signals stemming from the interaction of the H chain associated with VpreB and Lambda5with stromal bone marrow cells are transduced to the nucleus by the Brutonâ&#x20AC;&#x2122;s tyrosine kinase (BTK). This signal blocks any further rearrangement of the genes coding for the H chain. On the contrary, if the H chain is unable to interact with stromal bone marrow cells, the residual genes on the same chromosome 14 are rearranged again in the attempt to code a new function H chain. If also this further attempt fails, the cell rearranges the genes of the other allelic chromosome 14. If again the H genes are not correctly rearranged, the cell undergoes apoptosis. On the contrary, the pro-B cell finally able to express a correctly signaling H chain proliferates giving rise to a clone of pre-B cells (See Fig. 13.4) all expressing the same H chain. In this way, the H genes of only one of the two allelic chromosomes 14 are arranged to code the H chain (allelic exclusion). Then, each of these pre-B cells will start to rearrange independently the genes of the L chain and thus will generate a BCR with an individual binding site. A genetic defect of BTK causes the X-linked agammaglobulinemia, an immunodeficiency due to the inability of pro-B cells to mature in pre-B cells. This block in B cell maturation impedes the antibody production (See Fig. 28.3).

156 Fig. 15.7. REARRANGEMENT OF THE GENES CODING FOR THE L CHAIN. Once the very difficult task of rearranging the genes of the H chain is achieved, the pre-B cell starts to rearrange the genes of the L chain. This is a less errorprone task since there is only one recombination event (V-J). Moreover, a pre-B cell can rearrange the V and J genes not only on the two alleles of chromosome 2 (ď Ť L chain) but also on the two alleles of chromosome 22 (ď Ź L chain). Here too, the first correct rearrangement of L genes blocks any further rearrangement of the L chain genes. In this way, only a chromosome displays a correct L gene rearrangement (allelic exclusion). The newly coded L chain substitutes VpreB e Lambda5 and the cell expresses a definitive BCR. At this maturation step VpreB and Lambda 5 genes are inhibited. The immature B cell expressing the definitive BCR down-regulates the expression of adhesion molecules, exits the bone marrow and homes into peripheral lymphoid organs.

Fig. 15.8. REARRANGEMENT OF GENES CODING THE V DOMAINS OF H AND L CHAIN. The signals delivered by bone marrow stromal cells guide the nonhomologous recombination among the V, D and J genes coding the V domains of both the H (VDJ) and L (VJ) chains. These unique gene recombination events involve only genes that are flanked by particular invariant sequences (the Recombination Signal Sequences, RSS).

157 Fig. 15.9. THE RECOMBINATION SIGNAL SEQUENCES (RSS). RSS permitting gene recombination flank V genes at 3’, D genes at 5’ and 3’, and J genes at the 5’. The 12 base pair spacer allows the DNA helix to make one turn while the 23 base pair spacer allows it to make two turns. A gene flanked by a 12 base pair spacer can be joined only with a gene flanked by a 23 base pair spacer (12/23 rule). This 12/23 rule provides the specific orientation of gene recombination

Fig. 15.10. THE ALIGNMENT OF RECOMBINATION SIGNAL SEQUENCES (RSS). VDJ gene recombination starts with two RSS alignment. This alignment is first made possible by the extrusion of a long DNA loop through a ring formed by cohesin, a ring-shaped DNA-entrapping adenosine triphosphatase (ATPase) protein complex that regulate chromosome functions (REFERENCE: Zang Y et al, Nature 2019,573:600).

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Fig. 15.11. THE CUTTING OF DOUBLE STRANDED DNA. During the dynamic process of alignment driven by cohesin, RAG1 and RAG2 (Recombination Activating Gene coded proteins) are recruited at the alignment site. These enzymes cleave double stranded DNA exactly at the end of the Eptamer. They are expressed only during the pro-B cell maturation in the bone marrow.

D, E. RAG1 and RAG2 proteins along with the HMG1 protein (not shown) interact with RSS and pair a gene flanked by a 12 base pair spacer (In blue in the Fig.) with a gene flanked by a 23 base pair spacer (in green). Following this 12/23 rule, RAG1, RAG2 and HMG1 proteins pair the two Eptamers (7-7, in yellow) and the two Nonamers (9-9, in orange). Then, RAG1 and RAG2 cut the double-stranded DNA at the end of the Eptamer sequence (zig zag in purple.

F. Magnification of the cutting site of the double-stranded DNA. The bases of D gene are in black; those of the Eptamer in red.

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G H

FIG. 15.12. THE REMOVAL OF THE EPISOME AND THE JOINING OF DNA BREAKS. I. G. The double stranded DNA is cut by RAG1 and RAG2 enzymes. The long episome comes off and goes away. H. 1. The double stranded DNA is cut by RAG1 and RAG2 enzymes at the end of Eptamer sequence (In the Fig. zigzag in purple). The long cut off portion of DNA is removed as an episome (in yellow). 2. The –OH groups of DNA bases at the end of the fragments form a hairpin loop (in blue) leaving a blunt double-strand DNA break. 3. Several proteins (Ku factors, protein kinases DNA dependent, Artemis endonuclease…) bind the blunted DNA breaks. Then, randomly Artemis nuclease (in pale blue) cuts one of the DNA filaments. 4. Following the Artemis cut, DNA bases that were located in one filament give rise to a new sequence (sequence P, Palindromic, in green) on the other DNA filament

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Fig. 15.12. THE JOINING OF DNA BREAKS. II. The joining of DNA breaks generates new coding sequences. 5. Following the random Artemis cut (Pale blue arrows in the Fig. 15.11), DNA bases located on one filament move to the other filament giving rise to new P coding sequences (in green). Moreover, the enzyme Terminal Deoxynucleotidyl Transferase (TDT) randomly adds new nucleotides in a template free way at the 3â&#x20AC;&#x2122; terminus of single strand ends. This TDT activity gives rise to the N â&#x20AC;&#x201C;newregions (in red). 6. Finally, the DNA coding ends are ligated by the DNA ligase IV and XRCC4 protein while DNA polymerase and DNA repair enzymes remove and add nucleotides (in blue). The P and N regions and the activity of DNA repair enzymes create an enormous junctional diversity making about 1016 (!) new coding DNA sequences.

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Fig. 15.13. THE BINDING SITE. The binding site encompasses the area of BCR that interacts with the antigen (Green arrow in the Fig.). It consists in framework and hypervariable sequences of amino acids at the NH2 terminus of both the H and L chain. The framework sequences are not too different among various B cells whereas hypervariable sequences are markedly different. These hypervariable sequences are those that directly bind the regions of the antigen establishing multiple and different bonds. These regions are defined as Complementary Determining Regions (CDR) (See Figs. 8.12, 8.13, 10.8 and 16.1).

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CHAPTER 16. BINDING SITE-ANTIGEN INTERACTION.

Fig. 16.1. FEATURES OF THE BINDING SITE.

The binding site is made by the amino acids of the NH2 terminus of both the H and L chains. The

external loops (In light brown and pink in the Fig.) are called Complementary Determining Regions (CDR) since they interact with complementary conformations of the antigen. Somewhat similar CDR are evident on the TCR (See Figs. 7.6, 8.11, 10.8, 16.4).

The variability of the amino acid sequences of the binding site is very high. In a person, BCR and

antibodies express over 1011 different binding site sequences. However, the variability of the amino acid sequences at the binding site is not constant: On both the H and L chains, hypervariable regions (in brown) where the variability of amino acid sequences is very high are alternated by conserved framework regions (in pink).

163 Fig. 16.2. WHAT IS IT THAT THE BINDING SITES BINDS TO? In most cases, antigens are large molecules, large molecular aggregates, microbes or even foreign cells. In these cases, the binding site of a BCR (and of an Ig) binds only a small portion of the antigen, the epitope. A large antigen expresses numerous identical and different epitopes. In most cases, epitopes recognized by the binding site are made by an amino acid sequence. However, binding sites may interact with peculiar sugar and lipidic sequences.

Fig. 16.3. LINEAR AND CONFORMATIONAL EPITOPES. The epitope interacting with the binding site of a BCR and an antibody can be formed by a sequence of amino acids (linear epitope, upper panel) or by discontinuous amino acid sequences making a peculiar tridimensional structure (conformational epitopes, lower panel). In the Fig. in grey, a portion of the antigen; In light blue the amino acids sequences making linear and the conformational epitopes. These are the amino acid sequences interacting with the Complementary Determining Regions (CDR) of the binding site (See Figs. 7.6, 8.11, 10.8, 16.1, 16.4).

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Fig. 16.4. ANTIGEN-ANTIBODY INTERACTION. When a BCR (or an antibody) interacts with an antigen, the amino acids of the Complementary Determining Regions (CDR) of the binding site establish multiple non-covalent bonds with the epitope. The strength and the persistence of the interaction (Affinity) depend on the spatial (conformational) complementarity between the amino acid sequences of the CDR and the epitope. When there is a poor complementarity, the affinity is poor (low affinity). A good complementarity secures that the strength of the interaction is high, and the interaction is more persistent (high affinity). However, it should be noted that the interaction between the binding site and the epitope is reversible: its Association/Dissociation Constant (Affinity) rests on the strength and multiplicity of the non-covalent bonds occurring between the amino acids of the CDR and those of the epitope. In the Fig. in grey, the antigen; In blue the antibody. The complementary determining regions of the L and H chain are shown in light pink and yellow. The amino acid sequences of the epitope are in light blue.

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Fig. 16.5. AFFINITY. The strength by which a single binding site of a BCR or an antibody interacts with an antigen expressing a single epitope (monovalent antigen) is evaluated by equilibrium dialysis. This test measures the amount of a monovalent antigen (in moles) required to bind a percentage (33-50%) of the binding sites of an antibody. The higher the Ig affinity for that special antigen, the lower the amount of antigen required to bind the determined percentage of the binding sites will be. To perform this equilibrium dialysis test, a small dialysis chamber full of a liquid medium with two compartments separated by a dialysis membrane is employed.

A. A small monovalent antigen (red dots) is added to the medium of one compartment of the dialysis chamber.

The small monovalent antigen goes through the dialysis membrane and reaches the same

concentration in the two compartments of the dialysis chamber.

C. Antibodies (pale blue Y) are added to one compartment. These remain confined in the compartment since they are too large to cross the dialysis membrane.

The antigen bound by the antibodies is removed from the equilibrium, while the residual free

antigen again reaches the same concentration in the two compartments. The difference in antigen concentration in the two compartments of the dialysis chamber is due to the amount of antigen bound by the binding sites that is removed from the equilibrium. The higher the antibody affinity for that special antigen the lower the amount of antigen required to bind the selected percentage of the antibody binding sites will be.

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Fig. 16.6. IN WHICH WAY DO THE AMINO ACIDS OF THE BINDING SITE INTERACT WITH THE EPITOPE? The chemical bonds resulting in the binding site-epitope interaction are weak and non-covalent. These bonds require a strict structure matching. The presence of this kind of bond allows associations and dissociations between the binding site and epitope.

Fig. 16.7. STRENGTH OF THE INTERACTIONS. The strength of the multiple and diverse bonds between the amino acids of the binding site and the epitope is influenced on log scale by the distance between interacting structures (spatial proximity). Therefore, space proximity between the binding site and the epitope is of extreme importance for the force and persistence of the interaction.

Fig. 16.8. AFFINITY VS. AVIDITY. The strength by which a binding site interacts with a monovalent antigen is called affinity. However, a BCR (and antibodies) expresses two or more identical binding sites, each one of which has the same affinity for a given epitope. When a BCR (and an antibody) interacts with multiple identical epitopes on a multivalent antigen the binding strength is higher. The stronger bindingâ&#x20AC;&#x2122;s strength resulting from multiple binding sitesâ&#x20AC;&#x201C;epitope interactions is called Avidity. In the Fig. in grey, a multivalent antigen; In blue the antibody. The complementary determining regions of the L and H chains are shown in light pink and yellow. The amino acid sequences of the epitope are in light blue.

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CHAPTER 17. THE ANTIBODIES. Fig. 17.1. MEMBRANE BCR VERSUS SOLUBLE MONOMERIC IMMUNOGLOBULINS (Ig). As already shown in Fig. 14.7, when a B cell is activated, it changes its genetic program and instead of producing BCR to be inserted on the cell membrane it produces and secretes a modified version of the BCR: The Antibody or Immunoglobulin (Ig) lacks the transmembrane domain and the intracytoplasmatic tail. While structurally different, the BCR and the Ig produced by the same B cell share the same binding site. An activated B cell producing, and secreting Ig acquires a distinct morphology and is defined as a plasma cell (See Fig. 18.18).

Fig. 17.2. Ig AS GAMMA GLOBULINS. The Ig secreted by activated B cells are so numerous that they form a distinct group of serum proteins, the gamma (ď §) globulins. Following an active immunization their concentration in serum increases significantly (red dotted line in the Fig.). This electrophoretic profile is obtained when electrical current is applied to serum proteins at pH 8.6 in a support medium. All the major serum proteins migrate towards the anode traveling in accord to the size and electrical charge and five major protein bands become evident. The diffuse band formed by gamma-globulins shows that they are not exactly identical. What are the differences among them?

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Fig. 17.3. MOLECULAR DIFFERENCES AMONG Ig. The diffuse electrophoretic band formed by Ig is due to their molecular differences. A distinct biological role corresponds to each of these structural differences.

Fig. 17.4. THE MAIN FEATURES OF IgG.

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Fig. 17.5. SCHEMATIC DRAWING OF THE MOLECULAR STRUCTURE OF THE FOUR IgG SUB-CLASSES. The difference in the location and the number of S-S cysteine bridges connecting the IgG chains markedly affect the flexibility of the molecule and its ability to bind two epitopes variously located in the space.

Fig. 17.6. DIRECT AND Fc MEDIATED ACTIVITY OF ANTIBODIES. Direct Ig activities: Numerous activities of antibodies are directly due to the Ig ability to bind the target antigen (See also Figs. 19.1-19.6) Indirect Ig activities: Several other important biological functions of Ig are mediated by the peculiar structure of the Fc fragment (See Figs 19.1, 19.7-1912). The Fc of some Ig classes (IgM and IgG) activates the Complement cascade through the Classical pathway (See Fig. 19.11, 19.12) and interacts with specific receptors expressed on the cell membrane of various immune cells. The peculiar structure of the Fc: a) Separates Ig in classes and subclasses; b) Influences the half-life of the Ig; c) Allows Ig to form dimers or pentamers; d) Lets Ig diffuse into intravascular sites and cross the placenta and the gut mucosa; e) Allows the activation of the Complement cascade; f) Permits it to opsonize the target; g) Guides Antibody-Dependent Cellular Cytotoxicity (ADCC) (See Figs. 5.32; 20.8); h) Guides cell activities; i) Decides cell survival or cell apoptosis.

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Fig. 17.7. Fc RECEPTORS. Receptors for the Fc fragment (FcR) of the various Ig classes form a large family of molecules expressed on the surface of immune and epithelial cells. FcR are made ether by a single chain able to bind the Fc of the Ig and transduce the signal to the cell nucleus or by multiple chains. In most of the cases, the receptor chain binding the Fc belongs to the super-family of Ig (See Fig. 14.5). Each FcR binds the Fc of a certain class (isotype) of Ig. A few FcR bind Ig at high affinity (10-10 M) and are able to immobilize a single Ig (upper panel) whereas other FcR bind the Fc at low affinity (10-6 M). In the latter case, several FcR are required to immobilize large Ig â&#x20AC;&#x201C; antigen aggregates (immunocomplexes, lower panel). The FcR transduce activating, inhibiting, survival or dead signals into the cell.

171 Fig. 17.8. THE BIOLOGICAL ROLE OF THE Fc GAMMA FRAGMENT. Several other important biological functions of IgG are mediated by the Fc fragment (See Fig. 14.2). The Fc interacts with specific receptors expressed on the cell membrane of various kinds of cells. A few of these receptors are of high affinity and strongly bind a single IgG. Numerous other receptors are of low-affinity and cannot immobilize a single IgG. However, a multiple of these weakly bonds can immobilize large immunocomplexes. Following the interaction with an IgG, FcR transduce the activating signal and trigger several cell effector functions. Fig. 17.9. THE TRANSPORT OF IgG ACROSS THE PLACENTA. The interaction of Fc with the FcRn receptor expressed on the placenta is instrumental for the passage of maternal IgG to the fetus. Mother IgG have an important protective role during the first three months of life. However, these antibodies may also cause major diseases: The passage of mother autoantibodies against thyroid can cause thyroiditis in the fetus. Moreover, a Rh negative (Rh-) mother can transmit IgG reacting against the Rh blood group to the fetus and cause hemolytic disease (erythroblastosis fetalis) in a Rh positive (Rh+) fetus.

Fig. 17.10. INHIBITION OF ANTIBODY PRODUCTION.

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Fig. 17.11. IgA: MOLECULAR FORMS. IgA oligomerize to form dimers, though they may form trimers, tetramers and pentamers.

Fig. 17.12. IgA: SUB-CLASSES OF IgA.

Fig. 17.13. Fc ALPHA: BIOLOGICAL ROLE.

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Fig. 17.14. DIMERIC AND SECRETORY IgA. The joining chain (J chain), MW 15kD links two up to five IgA monomers. At epithelial surfaces, secretory IgA are an important contribution to gut barrier function (See Fig. 19.10).

Fig. 17.15. SECRETORY IgA: Secretory IgA play a critical role in the intersection between host immunity and microbiota, the entirety of microbes colonizing mucosal surfaces. Polymeric IgA secreted by mucosal plasmacells cross the epithelium (transocytose) and reach the luminal space releases secretory IgA. A proteolytic cleavage of the pIg receptor releases secretory IgA (sIgA).

Fig. 17.16. SECRETORY IgA. The secretory component of sIgA protects the IgA from being degraded by proteolytic enzymes present on mucosal surfaces. Thus, sIgA survive in the harsh gastrointestinal tract environment and provide protection against microbes present in body luminal spaces and secretions. sIgA coat and agglutinate microbes and antigens to prevent their direct interaction with mucosal epithelial cells.

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Fig. 17.17. IgA DEFICIENCY.

Fig. 17.18. IgM: MOLECULAR STRUCTURE. IgM is the first kind of immunoglobulin probuced after B cell activation. The J chain polymerize IgM monomers in pentamers.

Fig. 17.19. PENTAMERIC IgM: BIOLOGICAL FEATURES. I. The polymeric Ig receptor (See Figs. 17.14 and 17.15) transports pentameric IgM to mucosal luminal surfaces.

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Fig. 17.20. PENTAMERIC IgM: BIOLOGICAL FEATURES. II.

Fig. 17.21. IgM: NATURAL ANTIBODIES.

Fig. 17.22. IgM: ANTIBODIES TO A, B, 0 GROUPS. These IgM are induced by a natural reaction against sugars of the cell wall of bacteria normally present in the gut and bronchi. The antibody response against carbohydrate antigens is of IgM class only since the majority of Th cells does not recognize carbohydrates and do not deliver isotype switching signals (See Fig. 18.13). Moreover, these IgM are mostly produced by B1 B cells (See Fig. 13.6).

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Fig. 17.23. IgE: MOLECULAR STRUCTURE.

Fig. 17.24. THE TETRAMERIC FcR EPSILON.

Fig. 17.25. FcR EPSILON: BIOLOGICAL ACTIVITIES.

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Fig. 17.26. THE TETRAMERIC FcR EPSILON. The interaction of IgE with the tetrameric Fcď Ľ receptor extends the lifespan of basophils and mast cells (See Fig. Fig. 5.7) and makes these cells reactive against the antigen recognized by the IgE. The reaction elicited protects against parasites, nematodes especially. However, when the antigen recognized by an IgE is an innocuous antigen the reaction activated by the degranulation of basophils and mast cells is defined as allergic reaction. Allergic reactions can be localized or systemic. Localized reactions (immediate hypersensitivity) may cause itching of the eyes or involve the skin, the gastrointestinal and respiratory tract with clinical symptoms of varying severity. Systemic degranulation of basophils and mast cells causes a potentially life-threatening reaction marked by swallowing and breathing difficulties, abdominal pain, vomiting, diarrhea, hives, angioedema and dramatic decrease of the blood pressure.

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Fig. 17.27. THE TRIMERIC Fc RECEPTOR EPSILON.

Fig. 17.28. THE LOW-AFFINITY LECTINIC Fc RECEPTOR EPSILON (CD23).

Fig. 17.29. THE MOLECULAR STRUCTURE OF IgD.

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CHAPTER 18. ACTIVATION OF B CELLS.

Fig. 18.1. B1 B CELL ANTIBODY RESPONSE. The distinct antibodies produced by the three major B cell populations (B1, MZB, FoB2 B cells, See Fig. 13.5) sequentially counteract the invasion of microbes and antigens. In most instances, natural antibodies produced by B1 B cells (See Fig. 13.6 and Fig. 17.21) are already present before microbe or antigen invasion (Natural Antibodies). These natural antibodies are also present in persons who apparently had not been previously exposed to the corresponding antigen. In other cases, their production is boosted by the intruders. Natural antibodies are of IgM class. By interacting at low-affinity with many distinct epitopes expressed by common bacterial polysaccharides (See Fig. 13.7) they provide a first line of defense against systemic blood-borne intruders. Some natural antibodies react at low affinity with self-antigens. The role of these self-reactive natural antibodies is not yet defined. Perhaps they may have a homeostatic role and housekeeping functions, such as recognition and removal of senescent and altered cells. The IgM against A, B, 0 blood groups are an example of these natural IgM produced by B1 B cells (See Fig. 17.22)

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Fig. 18.2. MARGINAL ZONE (MZ) B CELL ANTIBODY RESPONSE. When the invasion by microbes or antigens is not cleared by natural antibodies and cells of innate immunity, persisting intruders are drained to adjacent lymph nodes (See Fig. 10.5 and 19.2) which drain the fluid from the infected tissue. Here, the first reactive response is made by Marginal Zone B (MZB) cells homing in the marginal zone of the spleen and lymph nodes (See Fig. 13.9). BCR on the cell membrane of MZB cells recognize glycolipid and other microbial antigens bound to the C3b component of Complement cascade (See Fig. 21.2) and develop an early (< 3 days) IgM antibody production (See Fig. 13.10). These IgM bind their target epitopes at low-affinity and form a second line of defense. Moreover, the immunocomplexes formed by these early IgM linked to the invading antigen are captured by Follicular Dendritic Cells (FDC) (See Figs. 18.10 and 18.11) homing in the center of the follicle. The antigen trapped as immunocomplex by FDC plays a critical role in the induction of the high affinity antibody response made by B2 B cells.

181 Fig. 18.3. A SIMPLIFIED OUTLINE OF THE STEPS OF FoB2 B CELL ACTIVATION. The third line of defense against persisting intruders rests on the production of high affinity antibodies of various classes, the kind of reactive response made by activated FoB2 B cells. However, the activation of a FoB2 B cell is a complex process that requires the collaboration of Follicular Th cells and Antigen-Presenting Cells (APC). These complex requirements are probably due to the important and persisting consequence of FoB2 B cell activation: a) The induction of a powerful, high affinity and long-lasting antibody response; b) The induction of an immune memory that may last several decades. These two outcomes may be catastrophic in the case of the induction of an erroneous immune response. The steps of FoB2 B cell activation summarized here are presented in detail in the following Figs. 1. The first step of FoB2 B cell activation rests on the arrival of the specific antigen in the spleen and lymph node. A soluble antigen with its natural conformation is drained to the B cell areas of lymph nodes and spleen (See Fig. 18.4). Here, antigen epitopes may be recognized by BCR on a FoB2 B cell membrane and prime the FoB2 B cell. 2. In the periphery, the antigen is also captured by APC (See Fig. 18.4). During their journey to the T cell areas of lymphoid organs, APC mature, digest the antigen and display antigen peptides associated with Class II HLA glycoproteins (HLA-p, See Chapter 6). Once in the T cell area, APC activate a Th cell with a TCR interacting at high affinity with HLA-p displayed on the APC membrane. An activated Th0 cell gives rise to an expanded clone of differentiated effector Th cells (See Chapter 12). 3. Following a first non-specific encounter, effector Th cells establish a long interaction with the antigen primed FoB2 B cell. 4. A FoB2 B cell first primed by the antigen and then activated by effector Th cells proliferates and gives rise to a spherical area rich in blast cells (a Germinal Center), the site for antibody diversification and affinity maturation. 5. Then, a Dark Zone made by proliferating B cell blasts (Centroblasts) and a Light Zone made by not proliferating B cell blasts (Centrocytes) becomes evident in the Germinal Center. 6. During successive migrations of Centroblasts to the Light Zone and Centrocytes to the Dark Zone, blast cells display hyper-mutation of the genes coding the binding site of their BCR, change the class of the antibody they produce (Isotype switch) and become plasma cells. 7. Blast cells that have generated a hyper-mutated BCR interacting at poor affinity with the antigen displayed by Follicular Dendritic Cells die whereas those with a hyper-mutated BCR interacting with the antigen at high affinity survive and give rise to plasma cells and long-living Memory B cells. A subsequent invasion by the same antigen triggers memory B cells to produce a quicker and more intense production of antibodies interacting with the antigen at high affinity.

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Fig. 18.4. ANTIGEN-PRIMED FoB2 B CELLS. Going into more detail through the phases of FoB2 B cell activation, when an invasion endures, the antigen in its native structural conformation is drained to the lymphoid organ where it may be recognized by a FoB2 B cell expressing on the cell membrane a BCR able to interact at high affinity with antigen epitopes. The same intruder antigen is captured by Dendritic Cells, macrophages and other antigenpresenting cells (APC, in green in the Fig.) that shuttle it to the T cell areas of lymphoid organs. During their journey to lymphoid organs, APC mature, digest the antigen and associate a few antigen-derived peptides to the groove of Class II MHC glycoproteins. Once in T cell areas, APC present antigen peptides to Th cells (See Fig. 12.4).

183 Fig. 18.5. ANTIGEN PRIMING OF FoB2 B AND T CELLS. In lymphoid organs there are distinct areas where T cells (In the Fig. T cell area is in pale blue) and FoB2 B cells home (In the Fig. B cell area is in pale pink, see also Figs. 19.5 and 19.6). In B cell areas, FoB2 B cells form spherical aggregates (Primary follicles over a net of peculiar cells, the Follicular Dendritic Cells (See Figs 19.7 and 19.8). If an antigen interacts at high affinity with the BCR of a FoB2 B cell, the cell internalizes the BCR, processes the antigen and displays antigen peptides on the groove of MHC Class II glycoproteins (See Chapter 7). At the same time, arrive in T cell areas antigen-presenting cells (APC, in green) that have captured the same antigen in the periphery, Here, APC presenting antigen peptides on the groove of HLA Class II glycoproteins activates TFh cells expressing the cognate TCR. The TFh cell activated by APC proliferates and gives rise to an expanded clone of effector TFh cells (intense blue).

Fig. 18.6. FULL ACTIVATION OF ANTIGEN-PRIMED FoB2 B CELLS. The antigen primed FoB2 B cell starts to overexpress the chemokine receptor CCR7 and migrates towards the boundary with the T cell area attracted by chemokine ligands for CCR7 which are secreted by stromal and Dendritic Cells in the T cell area. This positioning of antigenprimed FoB2 B cells at the interface with T cell area favors the scanning of HLA-p displayed on their cell membrane by the numerous effector TFh cells previously expanded upon the recognition of the same antigen presented by APC.

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Fig. 18.7. THE FoB2 B CELL AS A PROFESSIONAL ANTIGEN-PRESENTING CELL (APC). When the BCR of a FoB2 B cell interacts with the antigen, the B cell internalizes the BCR, processes the antigen as a professional APC, and presents antigen peptides in the groove of Class II HLA glycoproteins (HLA-p) (Figs. 7.8-7.10). Moreover, antigen-primed FoB2 B cells start to express special chemokine receptors and these cells are attracted at the interface of T-B cell areas. The positioning of ANTIGEN-PRIMED FoB2 B cells at the follicle-T zone interface favors their interaction with TFh cells. Initially, adhesion molecules establish multiple and not specific interactions between the leading towards the formation of a temporary immunological synapse between antigen primed FoB2 B and TFh cells. Once the two cells are in close contact, TCR on the membrane of TFh cell repeatedly scan the HLA-p displayed on the membrane of the FoB2 B cell. If TCR bind HLA-p with high affinity, the two cells remain in contact for several hours. During this prolonged interaction B cell acts as a professional APC: B cell not only displays on the groove of HLA Class II glycoproteins the peptides of the antigen initially bound by the BCR but also provides the required costimulatory molecules and receptors. While B cells are very effective APC, they are also peculiar APC since they present only peptides of the antigen that has been specifically captured by their BCR. In Fig. the antigen is in red, the BCR in orange and purple.

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Fig. 18.8. THE TRIGGERING OF A FoB2 B CELL PROLIFERATION. Once a T and a B cell have established a first not specific interaction through adhesion molecules (in gray in the Fig.), TCR scan HLA class II glycoproteins and peptides (HLA-p) displayed on the cell membrane of the B cell. If TCR interact at high affinity with the HLA-p, several accessory molecules deliver a complex series of costimulatory signals to both the T and B cells. These accessory signals are the same as those delivered by Th cells and professional antigen-presenting cells (See Chapter 10). The final outcome of this prolonged Th and FoB2 B cell interaction is both the expression of a trimeric high affinity IL2 receptor (See Fig. 10.11 and 2.11-2.18) on the FoB2 B cell membrane and the secretion of IL2 by the Th cell.

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Fig. 18.9. FoB2 B CELL-TFh CELL INTERACTION. ICAM, LFA, and CD2 adhesion molecules (in gray in the Fig.) establish a first non-specific interaction between a FoB2 B cell and an effector Th cell resident in the follicle (TFh cell). Then the TCR on TFH cell membrane starts to scan HLA class II glycoproteins and peptides (HLA-p) on the cell membrane of the FoB2 B cell. If a high-affinity interaction between TCR and HLA-p is taking place: a) These interacting molecules move to a lipid raft (See Figs. 10.9 and 10.10). b) CD3 and Zeta (ď ş) chain molecules (See Fig. 8.15) transduce first activating signals to the TFh cell; c) TCR signals increase the expression of adhesion molecules making the FoB2 Bâ&#x20AC;&#x201C;TFh cell interaction tight and persistent. d) TFh cell secretes dopamine molecules that interact with the DRD1 dopamine receptor on the FoB2 B cell membrane. e) The signal transduced by the DRD1 dopamine receptor induces B cell to express ICOS (Inducible T cell costimulatory ligand). f) The interaction of ICOS with the newly expressed ICOS ligand enhances the expression of CD40 ligand (in red) REFERENCE: I Papa et al, Nature 2017,547:318; g) The CD40 ligand binds the CD40 co-receptor on the surface of the FoB2 B cell (See Fig. 10.15). This CD40-CD40L interaction is of critical importance for triggering FoB2 B cell clonal expansion, gene hypermutation, and immunoglobulin class switching. CD40-CD40L interaction also induces (blue arrow) the overexpression of molecules of the B7 costimulatory family (in green).

187 h) The interaction of B7 molecules with the members of CD28 receptor family provides additional important costimulatory signals (See Fig. 10.14). i) The final outcome of this long TFh and FoB2 B cell interaction is both the expression of cytokine receptors on the FoB2 B cell membrane and the secretion of different combinations of cytokines by TFh cells (See Chapter 12). The IL2 released by TFh cells and captured by the high affinity IL2 receptor triggers B cell proliferation and induces its clonal expansion and the formation of a Germinal Center.

Fig. 18.10. THE GERMINAL CENTER: A TRANSIENT ANATOMICAL SITE TO REFINE AND AUGMENT THE ANTIBODY RESPONSE. Following a long-lasting interaction with TFh cells, antigen primed FoB2 B cells are induced to proliferate by the IL2 secreted by TFh cells and captured by their high affinity IL2 receptor (See Figs. 2.11-2.16). Actively proliferating FoB2 B cells give rise to a sphere-shaped aggregate of large blast cells (Germinal Center). Follicles in which a Germinal Center is evident are known as Secondary Follicles. The presence of Secondary Follicles shows that an immune response is going on. Despite being made up of motile B cells, Germinal Centers are tightly confined over a network of Follicular Dendritic Cells (FDC) (See Figs. 19.7, 19.8). B cell blasts express a receptor (P2RY8) that mediate the inhibition of migration. The activity of the ligand of this inhibitory receptor is controlled by FDC. The expression of this receptor is downregulated in memory B cells and plasma cells that exit from Germinal Centers. REFERENCE: E Lu at al, Nature 2019,567:244. FDC display antibody-antigen aggregates (immunocomplexes) on long elongations of their cell membrane. These immunocomplexes are made mostly by IgM, produced by plasma cells (PC) deriving from MZ B cells during their earlier response to the same antigen (See Fig. 13.9-13.12). In Fig. antigenpresenting cells are in green, B cells in pale pink, T cells in pale blue and the antigen is shown as solid red circles

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Fig. 18.11. HOW TO INCREASE ANTIBODY SPECIFICITY? As the immune response continues, an affinitybased B cell competition takes place to generate plasma cells producing antibodies reacting at high affinity with the antigen. in the germinal centers distinct Dark and a Light zone become evident. The Dark Zone is made by packed blast cells in active proliferation (Centroblasts). Centroblasts undergo hyper-mutations of their BCR. Regularly, Centroblasts migrate from the Dark Zone to the Light Zone and become Centrocytes. Centrocytes are non-proliferating blast cells or blast cells proliferating to a low degree that express high levels of the hyper-mutated BCR. Centrocytes are apoptosis-prone cells which compete to get anti-apoptotic signals by the antigen displayed by Follicular Dendritic Cells. Only those Centrocytes that have acquired a hyper-mutated BCR interacting at high affinity with the antigen of the immunocomplexes on the surface of FDC win the competition and survive. Surviving Centrocytes re-enter in the Dark Zone and again become highly proliferating Centroblasts. Reiterated passages of blast cells from Light to Dark zone (Centroblasts-CentrocytesCentroblastsâ&#x20AC;Ś) are instrumental for the hypermutations of the binding site of the BCR and the selection of cells that have acquired a hypermutated BCR interacting with the highest affinity with the antigen. Every time, a few blast cells do not re-enter the Dark Zone but differentiate into plasma Cells (PC) secreting high amounts of antibodies.

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Fig. 18.12. KEY EVENTS TAKING PLACE DURING BLAST CELL MIGRATIONS FROM DARK TO LIGHT ZONE.

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Fig. 18.13. SWITCHING THE PRODUCTION OF IgM AND IgD TO IgG, IgA or IgE. Virgin FoB2 B cells produce IgM and IgD to be inserted into their cell membrane as BCR. Under the influence of cytokine combinations secreted by TFh cells, Centroblasts change the class of Ig they produce (Ig class switching) through a process of irreversible DNA recombination. Upstream from genes coding the constant part of the H chain (, , , ) there is the gene promoter and e long, repetitive and conserved nucleotide stretches, called Switch regions (Shown as an orange star in the Fig.). The alignment of two Switch regions is made possible by the extrusion of a DNA loop through a ring formed by the cohesin protein complex (REFERENCE: Zang Y et al, Nature 2019,575:385).

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Fig. 18.14. ACTIVATION INDUCED DEAMINASE (AID) RECRUITMENT AND ACTIVATION. When the two Switch regions are dynamically paired through the extrusion of a DNA loop across the cohesin protein ring (See Fig 15.10), double strand DNA is open to allow the access of the DNA-mutating enzyme called AID. AID deaminates cytidines in the Switch regions (See Fig. 18.15). General DNA repair pathways trigger the removal of the lesion made by AID to double-stranded breaks: Through the combined action of other repair enzymes, the double strand DNA is cut at the two Switch regions. In this way, the DNA intervening between the Switch regions is excised. The free DNA ends of the Switch regions are rejoined through a non-homologous recombination mechanism and the DNA ring goes away as an episome. The deletion of the intervening DNA allows the selected constant region to localize adjacent to the recombined VDJ. (REFERENCE: Zang Y et al, Nature 2019,575:385) Fig. 18.15. HYPERMUTATION OF THE GENES CODING THE BINDING SITE. This is another process of utmost importance which takes place in Centroblasts. Through the action of Activation Induced Deaminase (AID), the same enzyme responsible for the initiation of Ig Class Switching, cytosines present in the rearranged genes segments coding for the Binding Site (VDJ/VJ), are deaminated to uracil. The anomalous presence of uracil in the DNA molecule triggers a series of repair mechanisms to respond to that damaged DNA sequence. These numerous mutations (hypermutations) can either increase or decrease the affinity of the binding site for the antigen.

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Fig. 18.16. PROGRESSIVE INCREASE IN AFFINITY OF THE BINDING SITE (AFFINITY MATURATION). As the immune response continues, the affinity of Ig for the antigen increases. This increase in affinity rests on the combined action of hyper-mutations in the gene segments coding for the antibody binding site and a subsequent selection of Centrocytes expressing the hyper-mutated BCR. Centroblasts with a hyper-mutated BCR move from the Dark Zone to the Light Zone and become Centrocytes. Centrocytes express the hyper-mutated BCR at high density (Pink cells in the Fig.) and compete for the antigen (red circle) displayed as immunocomplexes by Follicular Dendritic Cells (FDC). Only Centrocytes that have acquired a BCR able to interact at higher affinity with the antigen win the competition, bind the antigen and receive anti-apoptotic signals by FDC. These Centrocytes either differentiate into plasma cells secreting the antibody or return to the Dark Zone and again, as Centroblasts, proliferate and hyper-mutate their BCR. After each round of hyper-mutations and competitive selection, the affinity and specificity of the BCR of surviving blasts continually increases (Affinity maturation). By contrast, those Centrocytes that have acquired a BCR interacting at poor affinity with the antigen are induced to die in the absence of surviving signals. The extremely numerous dead cells evident in Germinal Centers are rapidly eliminated by macrophages that acquire a characteristic histological aspect (Tingible body macrophages).

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Fig. 18.17. STEPS OF FoB2 B CELL ACTIVATION. 1. The antigen in its natural conformation (In red in the Fig.) interacts with the BCR of a FoB2 B cell (red circle). 2. An antigen-presenting cell (APC) displaying antigen peptides on Class II HLA glycoprotein is recognized by a TFh cell, which then generates a clone of activated effector TFh cells (dark blue). 3. The antigen-primed B cell expresses antigen peptides in the groove of Class II HLA glycoprotein (HLAp) and migrates to the boundaries of B (pink) and T (pale blue) cell areas. Here, HLA-p displayed by the FoB2 B cells may interact with the TCR of antigen-activated effector TFh cells. 4. As a result of this FoB2 B and TFh cell interaction, the FoB2 B cell starts to proliferate and gives rise to a spheroidal area full of blast cells, the Germinal Center. 5. Progressively, both a Dark and Light Zone become evident in the Germinal Center. In the Dark Zone, blast cells (called Centroblasts) actively proliferate while BCR genes undergo hypermutations. The Centroblasts move to the Light Zone and differentiate in Centrocytes. Centrocytes compete for the antigen displayed as immunocomplexes by Follicular Dendritic Cells. Those Centrocytes expressing a BCR able to interact at high affinity with the antigen move to the Dark Zone and proliferate again. By contrast, those which have acquired a hypermutated BCR interacting poorly with the antigen undergo apoptotic death. 6. Reiterating passages of blast cells from the Light to the Dark Zone result in a progressive increase of the affinity of antibodies produced by Centrocytes. 7. At each stage of this reiterating Centrocyte - Centroblast recycling, a few blast cells differentiate into both plasma cells (PC) producing antibodies of various classes and into Memory Cells. The kind of antibody class produced by a plasma cell is then regulated by the combination of cytokines secreted by TFh cells.

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Fig. 18.18. OUTCOMES OF FoB2 B CELL ACTIVATION. FoB2 B cells primed by the antigen and activated through a selective interaction with TFh cells start to proliferate as Centroblasts. Centroblasts hypermutate the binding site of their BCR, differentiate in Centrocytes and express numerous copies of the new BCR. Only those with a BCR able to bind the original antigen with higher affinity receive antiapoptotic signals, survive, hyper-mutate the binding site of their BCR again and proliferate. Rounds of Centroblasts-Centrocytes passages continually increase the affinity of the BCR. Progressively, Centrocytes expressing a BCR binding the antigen with high affinity differentiate into plasma cells and memory cells. At each round, the Centrocytes with a hyper-mutated BCR binding the antigen at poor affinity, die by apoptosis. Fig. 18.19. MAIN FEATURES OF PLASMA CELLS. Several plasma cells migrate from the germinal center to the bone marrow where they survive for different period of time. Others migrate to the medullary cords of lymph nodes or into splenic red pulp. Plasma cells display absent or poor production of BCR but a massive production of secreted Ig. BCR H chains have a transmembrane domain of about 25 amino acids and a short cytoplasmatic tail (See Fig. 13.7). The passage from transmembrane BCR to secreted Ig depends on alternative RNA splicing at two different adenylation sites.

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Fig. 18.20. MAIN FEATURES OF MEMORY B CELLS. Following a subsequent arrival of the antigen, longlived memory B cells quickly differentiate in plasma cells producing large amounts of antibodies interacting with the antigen with high affinity and specificity. Memory B cells differentiate from a Centrocyte with a hyper-mutated BCR of a switched immunoglobulin class and inherit the genetic changes occurred in the Germinal Centers. Their activation (secondary immune response) rests on the cooperation with memory TFh cells and triggers the production of antibodies of the switched isotype reacting at high affinity with the antigen. The rapid and specific response of reactivated memory B cells effectively protects the body against subsequent infections by the same pathogen. These may be so mild as to be unnoticed. The protection afforded by vaccines rests on the induction and maintenance of memory B cells. The secondary antibody response made by the reactivation of memory B cells markedly differs from a primary response. For the memory of adaptive immunity cells see Chapter 23.

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Fig. 18.21. KINETICS OF PRIMARY AND SECONDARY ANTIBODY RESPONSE. The antibody production is a protective mechanism which acts far away from the production site. During the primary response, an initial protective response is based on the secretion of relatively poor affinity IgM. Slowly, a more efficacious response takes place. The switch of antibody classes leads to a more appropriate response at peculiar anatomical sites. Moreover, because of the hypermutations of the genes coding for the binding site, antibodies become highly specific and able to interact at high affinity with the antigen. A. Kinetics of the antibody class switch during a primary immune response. B. Comparison of the phases of a primary and a secondary antibody production. 1. Lag phase. In the primary response, antibodies become detectable between 3-4 days to 2 weeks after antigen arrival, depending on the kind of antigen. The Lag phase is shorter or virtually absent in the secondary response. 2. Log phase. The titer of antibodies increases exponentially doubling every 6 hours. This exponential increase lasts several days in the primary response. A shorter and more intense log phase is evident in the secondary response. 3. Steady stage. The number of antibodies produced and catabolized is almost identical. In a secondary response, this phase is reached quickly and lasts longer. 4. Decrease phase. It lasts much longer in the secondary response.

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CHAPTER 19. SECONDARY LYMPHOID ORGANS. Fig. 19.1. SECONDARY LYMPHOID ORGANS. Usually, microbes and antigens enter the body through the skin, gastrointestinal and respiratory tracts. Secondary lymphoid organs act as a series of immunological filters monitoring the content of body fluids. The incoming antigen diffuses in the secondary lymphoid organs where its epitopes may directly interact with the B Cell Receptor (BCR) on the membrane of B cells or be captured by local antigenpresenting cells (APC) localized in T cell areas and around B cell follicles. Moreover, APC that have captured the antigen in the periphery at the site of entry travel through lymphatic vessels to the T cell areas of draining lymph nodes (Fig. 10.4). Here, many T cells rapidly fan out to scan APC for HLA-p. When the T cell Receptors (TCR) of a T cell interact at high affinity with the antigen, the T cell rapidly proliferate. Thus, following an antigen challenge dividing B and T cells markedly increase lymphoid organ cellularity leading to their enlargement. Tissue fluids are filtered by numerous small lymphoid aggregates and by larger lymphoid structures (Tonsils, Peyer's patches and Appendix) forming the Mucosa Associated Lymphoid Tissue (MALT). The blood is filtered by the spleen, the body's largest lymphatic organ. The spleen is also important for the elimination of aged blood cells. The interstitial fluid from peripheral body tissues is filtered by the lymph nodes. The various secondary lymphoid organs are anatomically different. However, they share a common basic microscopic architecture: A spherical B cell aggregate (B follicle) surrounded by a T cell area where numerous APC (macrophages, Dendritic Cellsâ&#x20AC;Ś) are present. The selective homing of APC, T and B cells in distinct areas of the lymphoid organs is guided by constitutive chemokines (See Fig. 2.26) released by stromal, Dendritic Cells and Follicular Dendritic Cells (FDC). Activation of an immune response may take place at the site of microbe or antigen invasion and in any district of the body. However, the specialized anatomical features of secondary lymphoid organs, the local interplay between chemokine, cytokine gradients and cell adhesion molecules markedly favor both cell to cell contacts and the interaction with the antigen. These features turn secondary lymphoid organs into a specialized site for the efficient activation of T and B cells.

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Fig. 19.2. THE LYMPH NODE: SCHEMATIC DIAGRAM. Lymph nodes are the meeting places for B cell and antigen, T cells and antigen-presenting cells (APC) and activated T and B cells. In our body, there are thousands of lymph nodes strategically localized where antigen invasions are more common. In many cases, they are aggregated in clusters. A lymph node is a complex, encapsulated structure present in mammals only. A lymph node weighing about 1 g contains around 2 x 107 lymphoid cells. Afferent lymphatic vessels collect and channel interstitial fluid into the subcapsular sinus (in pink). From here, the lymph is drained towards medullary sinuses. The lymph node is constituted of an outermost cortex where FoB2 B cells are organized into lymphoid follicles and inner paracortical areas made up mainly of T cells and APC as Dendritic Cells and macrophages. When an immune response involving B cells is underway, some of the primary follicles enlarge and display central areas of intense B cell proliferation called Germinal Centers and the follicles are known as secondary lymphoid follicles. About 2 x 107 lymphoid cell/hour leave the lymph node through the efferent lymphatic vessels: About 75% of these are T cells, 25% B cells, and only 1% macrophages and Dendritic Cells. 90% of the lymphoid cells leaving the lymph node have arrived through the blood and about 6% are locally generated cells.

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Fig. 19.3. THE LYMPH NODE: CELL ARRIVAL.

ANTIGEN-PRESENTING Cells (APC): Free antigen (In red in the Fig.) and APC (Dendritic Cells,

macrophagesâ&#x20AC;Ś, in green in the Fig.) arrive from peripheral body districts to the draining local lymph node through afferent lymphatics. Every hour, about one million cells arrive. Of these, 75% are T cells, 6% B cells, and 15% are APC. In the presence of inflammatory cytokines, APC that have captured an antigen in the periphery decrease their adhesiveness to the tissues, migrate and localize in T cell areas. Immature Dendritic Cells (DC) in the body peripheral sites expresses surface receptors allowing the capture of microbes which are taken up by micropinocytosis and receptor-mediated phagocytosis (See Fig. 5.22). Then, DC leave the epithelium and migrate via the blind-ending afferent lymphatics to regional lymph nodes, where they arrive as fully matured DC expressing high levels of Class II HLA glycoprotein and efficiently present antigen peptides to TFh cells. In the T cell rich paracortical area of the lymph node, mature DC select virgin specific TFh cells from those arrived from blood vessels (See below). DC stimulation of antigen-specific virgin TFh lymphocytes leads to their clonal expansion.

B. Lymphocytes: Once in the lymph node parenchyma, the afferent artery divides into smaller arterioles that become High Endothelium Venules (HEV) within the T cell areas. HEV consist of cuboidal endothelial cells with numerous lymphocytes within the walls. Blood lymphocytes, attracted by a combination of chemokine gradients produced by HEV and lymph node stromal cells increase their expression of L-Selectin, a homing receptor expressed on the cell membrane of virgin T and B cells, and cross the walls of HEV. In this way about 25% of virgin T and B cells exit (extravasate) in lymph node parenchyma. Only virgin lymphocytes extravasate since activated lymphoid cells do not express L-selectin anymore. Once in the lymph node parenchyma, other chemokine combinations segregate lymphocytes into the T and B cell area following a network of fibroblastic reticular cells: B cells move at the speed of 6 microns/minute while T cells at the speed of 12 microns/minute.

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Fig. 19.4. THE LYMPH NODE: HISTOLOGICAL APPEARANCE OF SECONDARY FOLLICLES.

Fig. 19.5. THE LYMPH NODE: B CELL AREAS. Exploiting a monoclonal antibody (mAb, See Chapter 22) specific for a surface marker of B cells (the anti-CD20 mAb), B cells are selectively stained in brown. This technique (immune-histo-chemistry) shows the particular homing of B cells in lymph nodes where they give rise to spheroidal aggregates (follicles).

Fig. 19.6. THE LYMPH NODE: T CELL AREA. Exploiting a different monoclonal antibody (mAb, See Chapter 22) specific for a surface marker of T cells (the anti-CD3 mAb, See Fig. 8.15); T cells are selectively stained in brown. Immunohistochemistry shows that the majority of T cells localize around the B cell follicles. However, a significant population of T cells is also dispersed inside B cell follicles (TFh).

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Fig. 19.7. THE LYMPH NODE: FOLLICULAR DENDRITIC CELLS (FDC).

Exploiting a monoclonal antibody (mAb, See Chapter 22) specific to a surface marker highly

expressed on the cell membrane of FDC (the anti-BLC mAb) a network of FDC becomes evident at the center of B cell follicles.

B. FDC display long cytoplasmatic elongations (dendrites). Several surface markers of both lymphoid and myeloid cells are expressed on FDC cell membrane. Moreover, FDC display numerous receptors for the Fc domain of antibodies of different classes (FcR, in blue in the Fig.) and for Complement components (CD21, CD35, See Fig. 21.8). FDC also express numerous adhesion molecules (LFA-1 and other integrins, in gray) that favor their interaction with ICAM-1 and other adhesion molecules on the surface of B cells and Centrocytes.

C. Filiform dendrites of certain FDC develop multiple beads coated by immunocomplexes (iccosomes). D. Iccosomes are spherical bodies coated by immune complexes formed by antigen-antibodies trapped in follicular Dendritic Cells. Iccosomes can be shed from FDC and be captured by FoB2 B cells and Centrocytes. FDC do not derive from bone marrow, and TNF and Lymphotoxin play a crucial role in their development. Chemokines released by FDC guide the homing of T and B cells and are crucial for the normal architecture of secondary lymphoid organs.

202 Fig. 19.8. THE LYMPH NODE: ANTIGENPRESENTATION BY FOLLICULAR DENDRITIC CELLS (FDC). FDC capture the antigenantibody complexes (immunocomplexes) through their receptors for the Fc domain of antibodies (FcR). Antigens and immunocomplexes bound by Complement components (opsonized antigens; See Fig. 21.6) are captured by Complement receptors (CR) (See Fig. 21.6). A captured antigen is not internalized but remains intact on the cell membrane. In this way, FDC act as longterm repositories of antigens to be presented to FoB2 B cells and Centrocytes and to maintain B cell memory. Centrocytes with a hyper-mutated BCR compete for the antigens displayed by FDC and the antiapoptotic signals they deliver (See Fig. 18.11).

Fig. 19.9. THE SPLEEN. Despite its complex structure, the basic microanatomy of the white pulp of the spleen is similar to that of the other secondary lymphoid organs: T cells are clustered around arterioles (PALS); FoB2 B cells give rise to spherical follicles, some of which may display Germinal Centers; B cell follicles are surrounded by peri-follicular areas rich in antigen-presenting cells (macrophages and Dendritic Cellsâ&#x20AC;Ś), non-circulating B cells and T cells. Follicular Dendritic Cells are at the center of B follicles while High Endothelial Venules are in the peri-follicular areas. As in lymph nodes and other secondary lymphoid organs, the homing of various cell populations is guided by chemokines.

203

Fig. 19.10. PEYER’S PATCHES. The Mucosa-Associated Lymphoid Tissue (MALT) is formed by numerous small lymphoid aggregates and by larger encapsulated lymphoid structures (Tonsils) all sharing the basic micro-anatomy of the other secondary lymphoid organs. Peyer’s patches are 100-200 small lymph node-like structures lying beneath the gut mucosa. They consist of a central dome made by B cell follicles and Germinal Centres. TFh cells and antigenpresenting cells (APC), Macrophages, Dendritic Cells… occupy the area around the follicles. Peyer’s patches collect antigens and fluids under the epithelial gut surface. Moreover, specialized epithelial cells (Microfold cells or M cells) channel by transcytosis antigens and microbes from the gut lumen. Lymphoid cells enter the Peyer’s patches from High Endothelial Venules (HEV) and exit through efferent lymphatics to travel to mesenteric lymph nodes and, then, to the blood. Local cytokines (TGF-, TNF and Lymphotoxin, (LT) and Dendritic Cells induce the differentiation of CD4+T cell into TFh cells. CD40 ligand and IL-21 from TFh cells induce the expression of Activation Induced Deaminase (AID) in B cells and promote IgA class-switch recombination (See Fig. 18.13). Dimeric IgA secreted by plasma cells (PC) generated in the Peyer’s Patches are trapped by the poly IgA receptor1 (pIgR1) and transported by transcytosis to the gut luminal surface. Following a proteolytic cleavage, dimeric IgA associated with the pIgR1 fragment (secretory IgA) are released into luminal spaces (See Figs. 17.14-17.16).

204

CHAPTER 20. DIRECT AND INDIRECT ANTIBODY ACTIVITIES.

Fig. 20.1. DIRECT AND INDIRECT ACTIVITIES OF ANTIBODIES.

Fig. 20.2. TOXIN NEUTRALIZATION. Every year snakebites kill about 80,000-140,000 persons, mostly agricultural workers in poor rural areas of the world. The preparation of stockpile of antibodies able to neutralize snake venoms is one of WHOâ&#x20AC;&#x2122;s priorities.

205 Fig. 20.3. VIRUS NEUTRALIZATION. Following a viral infection, antibodies are produced against many epitopes of viral proteins. A few of these antibodies may neutralize the virus while other antibodies may be ineffective. Frequently antibodies neutralize viral infectivity by blocking virus absorption. Other antibodies may prevent the uncoating of the virus genomes in endosomes or aggregate (agglutinate) virus particles. In addition, enveloped viruses may be lysed (destroyed) by Complement activated by anti-viral antibodies (the indirect activity of antibodies).

Fig. 20.4. IMMUNOCOMPLEXES. An immunocomplex activates the Complement cascade. The adhering Complement fragments C4b and C3b bind to the CR1 Complement receptor (See Fig. 21.8) on the surface of red blood cells. In this way, red blood cells capture the immunocomplexes and transport them to the liver. Liver macrophages capture and destroy the immunocomplex without damaging the red blood cell.

206

Fig. 20.5. AGGLUTINATION.

Fig. 20.6. AGGREGATION AND INTERNALIZATION OF MEMBRANE MOLECULES AND RECEPTORS. Antibodies against cell membrane molecules and receptors cause their aggregation and internalization. Once they have interacted with the antibody, membrane molecules form several small aggregates (patches) that aggregate together (cap). The capped molecules are then internalized and temporarily the membrane becomes free from these molecules (stripped). Autoantibodies directed against membrane receptors can cause major diseases. For example, myasthenia gravis is caused by autoantibodies stripping acetylcholine receptors. Another example is provided by monoclonal antibodies (See Chapter 22) against the membrane product of the Her-2 (neu) oncoantigen (Herceptin). Herceptin mediated aggregation and internalization of Her-2 receptors inhibit the proliferative activity of Her-2 positive neoplastic cells. Other antibodies do not aggregate membrane receptors but inhibit their function by blocking their binding site.

207 Fig. 20.7. IMMUNE PHAGOCYTOSIS: TARGET OPSONIZATION. A phagocytic cell can recognize a foreign body through a series of Pattern Recognition Receptors (See Fig. 4.4) which trigger the emission of pseudopods that engulf the particle in a phagosome (Fig. 5. 21) (Left side in the Fig.). However, the same target antigen is recognized and engulfed much better when it is coated by antibodies (opsonized antigen) (Right side, in blue). The Fc domain of the antigen-bound antibodies allows the cross-linking of Fc receptors (FcR, in orange, mainly Fc gamma RI, CD64 and Fc alpha RI, CD89) on the phagocytic cell membrane. The signal delivered by cross-linked FcR activates macrophages and other phagocytic cells and increases the phagocytosis and the destruction of engulfed particles. The same antibodies not bound to their antigen (free antibodies) link the FcR with low-affinity and cannot activate the phagocytic cell. A similar increase of phagocytosis (opsonization) is observed when the target antigen is coated by components of the Complement cascade. In this case, Complement receptors are involved in the opsonization (See Fig. 21.8).

Fig. 20.8. ANTIBODYDEPENDENT CELLULAR CYTOTOXICITY (ADCC). The killer activity of an NK cell (See Fig.5.32 and 20.9), a macrophage, a granulocyte or a T cell can be activated and guided by receptors for the Fc domain of antibodies (FcR, in red in the Fig.). The cross-linking of various FcR on a killer cell membrane by the antibodies bound to antigens (triangles) on the surface of a target cell

208 triggers the cytolytic activity of the cell. This killer activity is dependent on antibodies (Antibody-Dependent) but is fully mediated by the killer cell (Cellular Cytotoxicity). ADCC mediated by NK and T cells is an important defense mechanism against viral infections and tumor cells. ADCC mediated by eosinophils and neutrophils controls infection from parasites that are too large to be endocyted.

Fig. 20.9. THE Fc GAMMA RECEPTOR III (FcRIII, CD16). In numerous cases, the ADCC (See Fig. 5.32 and 20.8) is mediated by the FcRIII. This important receptor binds the Fc domain of IgG1 and IgG3 antibodies. It is expressed on the cell membrane of NK cells, neutrophils, eosinophils and macrophages. Like the majority of receptors for the Fc, the FcRIII is a molecule of the Ig-superfamily (In dark red in the Fig.) (See Fig. 14.5). The cross-linking of multiple FcgRIII induces the phosphorylation of the ITAM sequences (See Fig. 8.15) present on the transducer  and  chains. Phosphorylated ITAM sequences (P) provide docking sites for the activation of Zap 70 and Syk that begin the signal transduction.

209

Fig. 20.10. DEGRANULATION OF MAST CELLS AND GRANULOCYTES. Granulocytes, basophils, mast cells and Dendritic Cells express a set of receptors for the Fc domain of the Ig (FcR) which bind IgE at high affinity (FcRIII, in purple in the Fig.). In a few individuals, FcR of basophil granulocytes and mast cells are bound to numerous IgE (in blue) specific for a special antigen (allergen). IgE remain bound for a very long time to cells that receive anti-apoptotic signals by the bound IgE. When the allergen binds IgE on FcR, the granulocyte basophil and the mast cell immediately degranulate, i.e. release the content of cytoplasmatic granules. Numerous mediators are suddenly released: vasoactive amines (histamine) and arachidonic acid metabolites (prostaglandins), cytokines (TNF, IL-4…) and platelet-activating factor (PAF). A massive degranulation of mast cells induces the often-life-threatening phenomenon of systemic anaphylaxis or the anaphylactic shock.

210

Fig. 20.11. THE ROLE OF DENDRITIC CELLS (DC) IN THE ANAPHYLACTIC SHOCK. The anaphylactic reaction (from Greek, lack of protection) is caused by the rapid exposure to several environmental antigens (allergens) derived from certain foods, drugs, insect venoms, latex, and immune sera injections. In people who have IgE specific for that allergen bound to Fcď ĽR on the membrane of mast cells and basophils, the presence of these allergens in the blood (Yellow oval in the Fig) causes a massive, immunoglobulin E (IgE) antibodyâ&#x20AC;&#x201C;mediated release of immune mediators (Black blots) from tissue-resident mast cells (MC) and basophils. To interact with IgE bound to tissue MC, allergens present in the blood should cross endothelial barriers. In this shuttling, DC appear to play a central role. Perivascular DC (In green) project cytoplasmatic extensions (dendrites) which penetrate the endothelial wall and protrude into the vascular lumen of blood vessels. These cytoplasmic extensions capture allergens present in the blood by means of a series of receptors of different kind. The captured allergen is not internalized but is instead passed on to mast cells via microvescicles budding from the DC cell membrane. The massive release of these microvescicles causes the simultaneous degranulation of mast cells in different body locations and leads to the anaphylactic shock (See Fig. 20.10). REFERENCES: HW Choi et al, Science 2018,362:657; F. Levy-Shaffer and J. Scheffel, Science 2018,362:640. In the fig: DC, Dendrits

211

Fig. 20.12. CLASSICAL PATHWAY OF COMPLEMENT ACTIVATION. The Complement is a large family of proteins endowed with powerful and distinct biological activities (See Chapter 21). A few Complement proteins finalize (i.e. complement) the activity of IgM and IgG antibodies. Once bound to an antigen, the Fc domain of IgM and IgG interacts with C1q, a component of this classical Complement activation pathway. When the globular heads of C1q sense conformational changes of the Fc domain (In purple in the Fig.) of a single IgM or several IgG (at least two), C1q becomes activated and recruits C1r and C1s components of the Complement family. The complex C1q, r, s acquires a proteolytic activity and acts on the two subsequent components of the Complement cascade, the C4 and C2. The complex C1q, r, s first cuts the inactive C4 into two active fragments, C4a and C4b. Then it cuts the inactive C2 into two active fragments, C2a and C2b. The C4b fragment exposes a reactive group that binds the antigen surface covalently. Subsequently, the fragment C2a binds C4b. The combined activity of C4b and C2a (C4b2a) acquires an important enzymatic activity called C3 convertase. C3 convertase cuts the inactive C3 into two active fragments (See Chapter 21).

212

Fig. 20.13. COMPLEMENT ACTIVATION: FINAL STAGES. The active group of C3b binds covalently to the surface of most antigens. Acting on C5, C3b generates C5a and C5b fragments. The final stages of Complement activation imply both lethal damage of the surface of the intruder cell (See Fig. 21.5) and the release of soluble Complement fragments (anaphylatoxins) that recruit a local inflammatory reaction (See Fig. 21.7). C5b initiates the assembly of other components of the Complement cascade and forms the Membrane Attack Complex (MAC), inserting it into the membrane of the target cell (See Fig. 21.5).

213

CHAPTER 21. THE COMPLEMENT SYSTEM. Fig. 21.1. THE COMPLEMENT SYSTEM: A CASCADE OF PROTEOLYTIC ACTIVITIES. The Complement system is a crucial component of the immune response to infection and tissue damage. Once activated Complement proteases cleave each other through precise cascade mechanisms. The Complement can be activated by IgM and IgG antibodies bound to an antigen. Alternatively, Complement can be directly activated by foreign sugars expressed on the surface of microbes. Thus, Complement is both a â&#x20AC;&#x153;complementâ&#x20AC;? of the specific activity of antibodies and a key defense mechanism of natural immunity. The final outcomes of Complement activation are: 1) The killing of the foreign intruder; 2) A facilitated phagocytosis (opsonization) of the intruder; 3) The recruitment of a local inflammatory reaction. Fig. 21.2. PATHWAYS OF COMPLEMENT ACTIVATION. The Complement cascade is activated by distinct sensor proteins. Through the classical activation pathway, Complement reactions are activated by IgM and IgG bound to an antigen. In this case, Complement reactions complement (i.e. complete, finalize) the specific reactivity of antibodies (See Figs. 20.13 and 20.14). Alternatively, specialized sensor Complement proteins directly perceive the presence of foreign molecules, mainly microbial sugars. All the three major pathways of Complement activation (the Classical pathway, the Lectinic pathway and the Alternative pathway) converge in the generation of a C3 convertase activity. Once a C3 convertase is generated, a common pathway (In brown in the Fig.) activates Complement effector functions.

214 Fig. 21.3. THE LECTINIC PATHWAY. The Complement components Mannose Binding Proteins (MBP) and Ficolins are typical Pattern Recognition Receptors (See Fig. 2.4). Following the recognition of foreign sugars, these receptors trigger a rapid activation of the destructive and proinflammatory activities of Complement. As a result of this activation pathway, Complement plays a central role in natural immunity. For the Classical pathway of Complement activation see Figs. 20.13 and 20.14. Fig. 21.4. THE ALTERNATIVE PATHWAY. In our body fluids, C3 is spontaneously cleaved into the two fragments C3a and C3b. Normally the C3b fragment has a very short life. However, if microbes are present, C3b may bind the microbe. Once stabilized on the microbial surface, C3b binds Factor B, Factor D and Properdin and generates the Bb-C3 (C3bBb) convertase. Since the C3b fragment is generated continuously, the activation of Complement via the Alternative pathway takes place almost immediately following microbial invasion. In addition, when C3b is generated through the Classical or Lectinic pathway, its binding to Factor B, Factor D, and Properdin on microbial surfaces triggers the Alternative pathway of Complement activation providing an important amplification of Complement activity. Properdin is another soluble Pattern Recognition Receptor (See Figs. 4.4) able to bind both microbes and C3b and, thus, to further amplify Complement activation. Properdin is stored in secondary granules of basophils, mast cells and in granules of neutrophils.

215 Fig. 21.5. THE MEMBRANE ATTACK COMPLEX (MAC). Complement activation through Classical, Lectinic and Alternative pathways triggers a common final cascade reaction that may result in the insertion of the MAC into the cell membrane and the subsequent death of the cell or microbe. C3 is the most abundant Complement component (1.2 mg/ml in the human serum). A single C3 convertase (C4b2a or C3bBb) generates more than 1000 C3a and C3b fragments. Fig. 21.6. FACILITATION OF PHAGOCYTOSIS (OPSONIZATION). The Complement system does not always succeed in inserting Membrane Attack Complexes and making death holes in microbial surfaces. In numerous cases, the microbial surface remains intact even if covered by fragments of Complement components. However, Complement fragments adhering on cell membrane make the microbe (or a particulate antigen) much more susceptible to phagocytosis. In effect, as shown in Fig. 20.7, a phagocytic cell can recognize a target antigen through a series of Pattern Recognition Receptors (See Fig. 4.4) which trigger the emission of pseudopods to engulf the particle in a phagosome (Fig. 5.18) (left side). However, the same target antigen is recognized and engulfed 10100 times better when it is coated by antibodies or by a few Complement fragments (opsonized) (right side of the Fig.). The Complement fragments adhering on the microbeâ&#x20AC;&#x2122;s surface are recognized by many Complement receptors commonly expressed by phagocytic cells (See Fig. 21.8). The signals delivered by these receptors efficiently activate phagocytosis and the subsequent destruction of engulfed particles.

216 Fig. 21.7. INDUCTION OF A LOCAL INFLAMMATORY REACTION. These cleavage fragments of Complement components (anaphylatoxins) bind receptors expressed by granulocytes and macrophages and trigger a local inflammatory response. Acting on mast cells and, directly, on endothelial cells these Complement fragments increase vessel permeability, favor leukocyte extravasation and enhance the local accumulation (edema) of serum fluids rich in molecules of natural immunity, Complement, and antibodies. These fluids are then drained to local lymph nodes where they may trigger a specific immune response.

Fig. 21.8. COMPLEMENT RECEPTORS. The numerous Complement receptors (CR) mediate the multiple activities of Complement. The soluble cleavage fragments C2b, C3a, C4b and C5a bind receptors expressed by granulocytes, macrophages, mast cells, and endothelial cells and trigger a local inflammatory response. By contrast, receptors to fragments which adhere on the cell surfaces (C3b, C4b) trigger an efficient phagocytosis (opsonization), Complement Dependent Cellular Cytotoxicity (CDCC) and the capture and transport of immunocomplexes that have activated the Complement cascade. CR1 expressed on a Follicular Dendritic Cell surface plays a key role in capturing immunocomplexes and in the antigen stimulation of Centrocytes (See Figs. 18.15 and 18.16). The CR2 receptor, by contrast, is an important costimulator of B cell response (See Fig. 14.11). Thus, the activation of Complement through the Lectinic and Alternative pathways provides an important costimulation of the antibody response.

217

CHAPTER 22. MONOCLONAL ANTIBODY (mAb).

Fig. 22.1. DYNAMICS OF A POLY-CLONAL IMMUNE RESPONSE. In general, an antigen displays several different epitopes (See Fig. 16.2). Each epitope is recognized by several B cells (a, b, c, d) expressing distinct BCR interacting with the same epitope with different affinity. Once activated, each of these B cells generates a clone of daughter cells all expressing the identical BCR During the evolution of an immune response, a Darwinian competition among these clones takes place. Clones with a BCR that interacts better (with higher affinity) with the epitope win the competition and overcome poor affinity clones (See Fig. 18.16). Moreover, as time passes, B cells undergo BCR hypermutation (dark blue, orange clones) and may change the class of antibody they produced (isotype switch, See Fig. 18.13). Therefore, an immune response is a dynamic process, based on clonal expansion, clonal competition, and clonal contraction. This means that the immune response mounted by an individual against an antigen, the titer of antibodies produced, their specificity for distinct epitopes, their affinity for the same epitope, and the class of antibodies produced changes continuously. At the end of the immune response, only a few high affinity memory cells will survive (See Fig. 18.16).

218 Fig. 22.2. WHY ARE MONOCLONAL ANTIBODIES PRODUCED? An immune serum is a serum collected from an individual (a person, a mouse, a sheepâ&#x20AC;Ś) after immunization against an antigen. An immune serum contains a mixture of antibodies against the antigen used for the immunization. The titer of antibodies in an immune serum, the percentage of antibodies of different classes and their affinity change continuously and can be markedly different in immune sera collected from various individuals immunized against the same antigen. By contrast a monoclonal antibody constantly displays the same specificity, affinity and isotype. Thus, it provides a specific and invariant tool.

Fig. 22.3. HOW TO PRODUCE A MONOCLONAL ANTIBODY. The technology invented by George Kohler and CĂŠsar Milstein in 1975 (both were awarded the Nobel prize in 1984) is based on the fusion of a shortliving B cell obtained from a mouse immunized against an antigen

219 with a neoplastic plasma cell, potentially immortal but lacking an enzyme necessary to grow in a special culture medium. Thus, both the B cells and these plasmacytoma cells cannot survive in the special culture medium. However, if a short-lived B cell fuses with a potentially immortal plasma cell, the resulting hybrid cells may acquire from the B cells the genes coding the specific BCR and the genes allowing the survival in the special culture medium. At the same time, the hybrid cell may also acquire from the plasma cell both the immortality and the machinery to produce and secrete high amounts of antibodies. Making millions of random cell-to-cell fusions it is possible to generate a significant number of these immortal hybrid cells (hybridoma cells) able to proliferate in the special culture medium and produce antibodies against the antigen used for the immunization.

Fig. 22.4. CELL-TO-CELL FUSION. There are several technologies to favor the fusion between a B cell and a plasma cell cultured together in a special selective medium. In all cases, the fusion is a random event which produces all possible kinds of hybridomas. The majority of cells will not survive the fusion shock. Moreover, independently from a successful integration of cell genomes, hybrids between B and B cells will undergo apoptosis. Also, hybrids between plasma cells and plasma cells will die since they lack the gene necessary to survive in the special culture medium. Only hybrids between a B cell and the defective plasma cell have a chance to survive. Surviving B-plasma cell hybrids will then be placed in micro-culture wells in order to favor their clonal expansion.

220

Fig. 22.5. SELECTION OF HYBRIDOMAS PRODUCING THE DESIRED MONOCLONAL ANTIBODY (mAb). While previous maneuvers are mostly driven by casual events, the selection of the clones to be expanded is based on the intelligence of the researcher. To make this selection, a single hybrid cell will be placed in each micro-culture well. In a few of these wells, the single hybrid cell survives and generates a clone of identical daughter cells producing the identical antibody. Accurate evaluation of the titer, the affinity and the class of the antibody produced in the various wells permits the selection of the best hybridoma producing the desired antibodies. Selected hybridomas are then expanded in larger cultures. Then, one can produce liters and liters of culture medium containing high amounts of the antibody produced by a large clone descending from a single hybrid cell (mAb). A few cells of the selected hybridoma can be stored in liquid nitrogen for an almost indefinite period of time in order to re-start the clonal expansion and the production of the identical antibody on demand. mAb are now essential reagents in any cell biology lab, hospital diagnostic services and therapy.

221 Fig. 22.6. FEATURES OF MONOCLONAL ANTIBODIES (mAb). Commonly, mAb are produced with mouse cells. However, there are mAb variously engineered to have part of the binding site or the constant domains of human origin. Humanization of murine antibodies is important when mAb are used in therapy since repeated administrations of mouse antibodies to a human patient may elicit the patient’s immune reaction. The patient antibodies against mouse immunoglobulins may neutralize the mAb activity. As mAb are specific and invariant tools that can be variously exploited in diagnosis and therapy, several new techniques (phage display, genetic engineering…) have been set up for their production. mAb conjugated with a fluorescent dye or an enzyme are currently widely used in immunohistochemistry (See Figs. 9.4, 9.5, 19.5-18.7). mAb conjugated with gold particles are exploited in immune-electron-microscopy. mAb conjugated with toxins, radioisotopes, and drugs are used in therapy. Fig. 22.7. CLUSTERS OF DIFFERENTIATION (CD). Monoclonal antibodies (mAb) reacting against antigens expressed on the cell membrane of human leukocytes have been produced (and are still currently produced) in various parts of the world. So numerous mAb recognizing the same or different epitopes of the same membrane molecule with various affinities have been made available. An international standardization has allowed and is still allowing the molecule recognized by these groups (cluster) of mAb on the immune cell surface to be defined as Differentiation Antigen recognized by a cluster of mAb. Thus, molecules on the surface of immune cells are now denoted by the acronym CD (a Differentiation molecule recognized by a Cluster of mAb) followed by a number (CD1, CD2, CD3, CD4…CD125…).

222

Fig. 22.8. TECHNOLOGICAL MANIPULATIONS OF MONOCLONAL ANTIBODIES (mAb). mAb can be variously engineered to better exploit their ability to bind a target antigen. The (Fab)â&#x20AC;&#x2122;2 fragment maintains the ability to bind two identical epitopes and to form antigen aggregates (immunocomplexes). When it is engineered to recognize two different epitopes [(Fab)â&#x20AC;&#x2122;2 bispecific] it can be exploited to put in contact two different cells, e.g. a killer cell and a target cell. A bi-specific diabody is a smaller version able to better diffuse in the body tissues. Diabodies, Triabodies, and Tetrabodies are small interconnected antibody fragments able to bind two, three and four epitopes. Despite the removal of the constant domains, these small molecules may maintain the specificity of the original mAb. Alternatively, they can be engineered to express different binding sites. Monovalent Fab is another small fragment of the antibody molecule that may freely diffuse in the body tissues. The scFv is an even smaller engineered molecule (Arrows indicate the binding sites; in purple peptides connecting antibody fragments.

223

CHAPTER 23. THE MEMORY RESPONSE. Fig. 23.1. THE IMMUNE MEMORY. The cells of the immune system learn to respond differently when they meet the same invader or the same antigen for a second time. The particular training consisting of the first response teaches immune cells to respond either with greater efficiency (positive memory) or with a more attenuated response (negative memory). A more effective response to a subsequent invasion of the same microbe makes it improbable to get sick again until the positive immune memory lasts. There are, however, also numerous situations where it is important to learn how to decrease the intensity of the reaction, reducing the risk of altering body tissues and going towards autoimmune diseases.

Fig. 23.2. IMMUNE CELLS REMEMBER. The capacity to learn how to respond with an increased or decreased efficacy to a second encounter with the same invader, the so-called immune memory, is a crucial and diffuse feature of the immune system.

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Fig. 23.3. INFLAMMATORY MEMORY OF EPITHELIAL STEM CELLS. An inflammatory memory of the event in the form of changes of the chromatin (epigenentic changes) facilitates the accessibility of selected groups of genes to the transcription machinery. This enables epithelial cells to respond more rapidly and strongly to a subsequent inflammatory challenge. REFERENCES: S Naik et al., Nature 2017,550:475; X Dai, Nature 2017,550:460.

Fig. 23.4. EPIGENETIC IMMUNE MEMORY OF MACROPHAGES. For decades it was believed that innate immune cells do not acquire a memory against a specific danger. However, recent studies discovered that beyond the initial innate response, macrophages retain a positive or a negative memory of previous stimuli, a concept known as innate immune memory. In a memory response, macrophages respond differently upon a second encounter with the same danger or intruder. Several stimuli train blood monocytes and peripheral macrophages to either become more prone to react to a second stimulus (positive memory), improving tissue surveillance, or less reactive to stimulation (negative memory) and consequently avoid extensive tissue damage. Prolonged exposure to microbial products such as lipopolysaccharide can induce a form of negative memory that blunts subsequent macrophage responses. Various microRNA that affect gene promoters have been identified as responsible for the functional reprogramming of macrophages during negative memory. REFERENCE: JJ Seeley et al., Nature 2018,559:114. Moreover, temporary changes in the chromatin due to the new expression of histones and DNA methylation results in epigenetically mediated increased or decreased accessibility to a few regions of DNA and gene enhancers boosting or inhibiting the expression of selected gene clusters. Because of these different mechanisms, primed macrophages may respond in a different way to a subsequent inflammatory challenge. REFERENCES: R Medzihitov, Nature 2017,550:460; AC Wendeln, Nature 2018,556:33.

225 Fig. 23.5. EPIGENETICS. Epigenetic changes are due to molecular mechanisms that regulate gene expression without changing DNA sequences. Gene expression can be modulated by regulating the access of transcription factors and enhancers to DNA. The addition of CH3 (methyl) groups to Cytosine, the density and positioning of histones (the proteins around which DNA coils to form chromatin strands), Polycomb- end Tritorax-group proteins, and noncoding RNA may influence the ability of cellular machinery to access the DNA.

Fig. 23.6. IMMUNE MEMORY OF NK CELLS. The mechanisms of NK immune memory are not only based on possible epigenetic changes but also on a local numerical expansion of the various NK cells expressing activating receptors that interact with anomalous molecules expressed by target cells (poly-clonal expansion). REFERENCE: H Peng and Z Thian, Frontiers in Immunol, 2017,8:1143.

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FIG. 23.7. THE SPECIFIC MEMORY OF T AND B CELLS. When a microbe (or an antigen) enters our body for the first time, it involves T and B cells in a primary immune response. The T and B cells that are activated and expand are those that recognize the antigen with high affinity. When the microbe (or the antigen) is eliminated, this primary cell expansion slowly vanishes. However, immune responses involving the training and expansion of T and B cells leave an important reactive state, called Immune memory, due to the persistence of an imprinted lymphocyte population, the memory T and B cells. Thanks to their prolonged survival, a re-infection by the same microbe (or a second arrival of the same antigen) elicits a secondary immune response due to the rapid and effective re-activation and clonal expansion of memory T and B cells The duration and fading of memory are discussed in Figs. 23.12 and 23.14. In these figures there is no time scale because the duration of the immune memory varies from antigen to antigen. It is greatly influenced by how the antigen entered the immune system, by the cytokines associated with the antigen entrance and from the genetic characteristics of the individual. Anyhow, usually T and B cell memory lasts for many months, years and in some cases for the whole life.

227 Fig. 23.8. B CELL IMMUNE MEMORY IS LONG-LIVED. Following a primary immune response, antigenactivated B cells undergo asymmetrical division, wherein daughter cells mature into plasma cells (antibody-secreting cells) and long-lived memory B cells. Differentiation of activated B cells into plasma cells and memory B cells mostly rests on the expression of the protein Blimp-1. The expression of Blimp-1 controls the activation of many genes important for plasma cell differentiation, including genes inducing the formation of the secretory apparatus necessary for the production of large amounts of antibodies. In addition, Blimp-1 inhibits cell proliferation and maintains the terminally differentiated plasma cells in a post-mitotic state. When Blimp-1 expression is inhibited, activated B cells differentiate into memory B cells. The rapid kinetics of B cell activation and the intensity of the antibody production in memory response (secondary response) are shown on Fig. 18.20.

Fig. 23.9. T CELL MEMORY IS LONGLIVED. Upon activation by an antigen-presenting cell (APC), a T cell generates a clone of Effector/Memory cells. This effector cell population is responsible of the reaction against the antigen. When the antigen is eliminated, progressively this population declines since these T cells, no more stimulated by the antigen, are facing apoptotic death (death by neglect).However, during later

228 phases of the response, these effector/memory T cells undergo asymmetrical divisions, wherein the daughter cell proximal to APC differentiates again in effector/ memory cells while the distal cell differentiate in long-lasting memory T cells. These long-lasting memory T cells express different homing receptors responsible for their selective homing: Central memory T cells are attracted in lymphoid organs while Effector memory T cells are present both in the circulation and in peripheral tissues. In the absence of booster restimulations, after a long maintenance period, also the memory cells slowly decrease as shown in Fig. 23.12 and 23.14.F

Fig. 23.10. WHAT CAN BE SAID ABOUT MEMORY T AND B CELLS? When virgin T and B cells differentiate into effector cells, their DNA methylation profile changes. Methyl groups are added to many genes associated with the virgin state whereas a loss of DNA methylation is evident at genes that encode key components of the effector response. These epigenetic changes enable memory cells to rapidly become a proliferating effector cell upon a re-encounter with the same intruder. For the features of memory B cells see Fig. 18.19. REFERENCE: RS Akondy et al. Nature 2017,552:362; B Youngblood et al, Nature 2017,552:404; KQ Omilusik and AW Goldrath, Nature 2017 552:337.

229 Fig. 23.11. WHY IS THE MEMORY RESPONSE SO AMAZING? The combination of a marked numerical cell expansion with the persistence of epigenetic changes makes the perfect conditions for an extremely effective long-lasting anamnestic immune response.

Fig. 23.12. HOW MANY MEMORY CELLS ARE REQUIRED FOR A SECONDARY IMMUNE RESPONSE? During T and B cell maturation, we generate a large repertoire of TCR and BCR binding sites. This means that there is approximately a T (or B) cell with a specific binding in one every 1011 cell, an extremely low frequency. However, the frequency of lymphocytes reacting to an antigen is much higher since most of the antigens have many epitopes and each epitope is recognized with a different affinity by several virgin cells. Periodically memory cells display expansions (cell proliferation) and contractions (cell apoptosis). The rapid re-activation of these memory cells provides an enhanced response against the reentry of the same antigen. As time elapses, the frequency of memory cells slowly declines. When the frequency of memory cells specific for a given antigen is less than 1 every 700 000 cells, an antigen reentry does not more elicit a significant protective memory reaction.

230

Fig. 23.13. AND WHEN THE MEMORY FADES AWAY?

Fig. 23.14. HOW TO RETAIN A GREAT IMMUNE MEMORY? Often, unnoticed antigen re-stimulations allow keeping an effective memory for a long time. In the absence of these natural boosters, progressively the population of memory cells decreases, and the memory reaction doesn't take place anymore. However, once a T and B memory was triggered, a timely booster induces a particularly effective memory that lasts much longer.

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CHAPTER 24. VACCINES.

Fig. 24.1. THE OUTSTANDING POTENTIAL OF IMMUNE MEMORY. The persistence of both the expanded population of the effector lymphocytes and a high antibody titer that follows the recovery from an infectious disease accounts for the elimination of a subsequent invasion by the same intruder microbe with such an efficacy and rapidity that the re-infection generally goes unnoticed. A suggestive account of the potential of the immune memory was provided by Thucydides, an ancient Greek historian when he describes the plague of Athens during the Peloponnesian war.

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24.2. ATTEMPTS TO INDUCE AN IMMUNE MEMORY. Since when it became clear that the recovery from an infectious disease may confer a persistent protection against the risk of contracting again the same disease, human intelligence has been trying to induce a protective immune memory. An ancient method used in China for the prevention of smallpox, a disease that caused devastating epidemics, was to let children inhale a dust obtained from the smallpox scabs. Variolation (also known as Inoculation) was another preventive method, practiced in the East, especially in the Ottoman Empire but also known in Europe consisting in the introduction into superficial scratches made in the skin of powdered smallpox scabs from pustules of people affected by light cases of smallpox. After living for a while in Turkey, Lady Mary Wortley Montagu, an English aristocrat (1689-1762), sent letters to influential European personalities to promote this primitive way to induce a protective immune memory. However, a rational vaccination against smallpox was first set up in England by Edward Jenner in 1796. Jenner, a country doctor, showed that the inoculation of purulent material obtained from bovine smallpox pustules protected humans from the infection by the human smallpox virus. Since the virus used by Jenner for his successful vaccination was obtained from a cow (Vacca in Latin), this new biotechnology was called vaccination. Thanks to Jenner, empirical practices, often mixed with magic, become a rational procedure, even if the scientific basis of vaccination were ignored. About a century later, Louis Pasteur in France succeeded in making another important scientific quantum jump showing that vaccines based on living microbes that have been weakened so they can not cause disease, induce a significant and long-lasting protection. Beginning from the studies of Pasteur, the vaccines have progressively become a sophisticated and effective biotechnology.

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Fig. 24.3. THE LESSON OF VACCINES. Against some microbes, an effective protection depends only on the vaccineâ&#x20AC;&#x2122;s ability to elicit a T cell mediated immunity. Instead, against other microbes the vaccine should be able to elicit antibodies of certain classes (those able to activate the Complement cascade, those able to reach mucosal surfacesâ&#x20AC;Ś). Current vaccines are based on sophisticated technologies. In several cases, the vaccine contains only the microbial epitopes that are critically important for the survival and proliferation of the microbe. Various attempts to elicit protective immunity against purified microbial epitopes showed that the simple injection of these foreign molecules is often not sufficient to elicit protective immunity. In many cases, the target antigen should be administered in combination with particular substances (the adjuvant, See Fig. 24.5) that elicit the danger signals (See Figs. 4.3-4.4) required to trigger a concomitant reactivity of natural immunity and induce a local inflammatory response. The peculiarities of the invader, the characteristics of the danger signals and of kind of the initial inflammatory reaction decide the characteristics of memory cells, i.e. the choice between different genetic programs of lymphocyte differentiation.Another lesson taught by the study of the vaccine is that to elicit and maintain a protective immunity the vaccine should be re-administered several times at determined intervals. These repeated vaccine administrations boost the immunity and allow a progressive selection and expansion of the T and B cell clones reacting against the microbe at higher affinity.

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Fig. 24.4. KINDS OF VACCINES. A vaccine may be based on a living microbe whose ability to induce a disease has been disabled by culturing it under particular conditions or through genetic engineering (attenuated microbe). Other vaccines are made by microbes that have been killed in various ways but that maintain their immunogenicity. Several effective vaccines are based on toxoids, microbial toxins that have been modified in such a way as to have lost their ability to cause a disease while maintaining their immunogenicity. More sophisticated vaccines are based on the selection of fragments of a toxin or molecules on the surface of a microbe. In a few cases, these vaccines are based on a few small microbial epitopes (See Fig. 16.2). These fragments can be obtained directly from microbes or produced by genetic engineering. Other vaccines exploit the special ability of Dendritic Cells to present peptides to T cells. Since the antigens are taken up by Dendritic Cells and presented as peptide fragments associated with HLA glycoprotein, these vaccines activate the response of T cells only. As the presentation of endocyted antigens takes place mostly on Class II HLA glycoprotein, they elicit a particularly effective Th cell activation. DNA and RNA vaccines are molecularly deďŹ ned reagents that are easy to construct and modify. They consist of plasmids coding for the target protein. A few of these small plasmids injected in the muscle enter the cells and the coded antigen is expressed on the cell membrane. Damaged muscle cells and their fragments are drained to the local lymph node. There they directly prime cognate B cells or are taken up by macrophages and Dendritic Cells and presented to T cells. The consequent B-T cell interaction will lead to the activation of antibody response by B cells.

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Fig. 24.5. ADJUVANTS. Adjuvants are a critical component of vaccines that markedly increase the vaccine capacity to elicit an immune response (antigen immunogenicity). Initially, vaccines made use of live attenuated or killed and inactivated microbes that naturally express on their surface adjuvant molecules recognized by Pattern Recognition Receptors (PPR) expressed on the cell membrane of the cells of innate immunity. By contrast, most vaccines developed in recent years are based on molecules or molecular aggregates rather than the whole microbe. These molecules have to be associated with adjuvants in order to trigger the activation of the cells of innate immunity. An adjuvant may act in many different ways. It may induce a slow release of the antigen, and thus it enhances the persistence of the antigen over a long period of time. It may also aggregate soluble antigens and thus favor their uptake by antigen-presenting cells (APC). An adjuvant may trigger the release of alarm signals (See Fig. 4.3) or may activate innate immunity cells and trigger the differentiation of antigen-presenting cells (APC) and Dendritic Cells (DC). A risk in the use of adjuvants lies in their ability to induce the immune recognition of a selfantigen and trigger an immune reaction against tolerated self-molecules causing autoimmunity and a devastating inflammation.

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Fig. 24.6. VACCINE: KINDS, ADVANTAGES, AND LIMITS. The various kinds of vaccines have distinct advantages and limitations. The selection of the best vaccine is based on a balance between its immunogenicity, cost, storage problems and kind of immunity elicited. World countries and the World Health Organization (WHO) have prepared detailed schedules indicating dose, times, boosters for the administration of compulsory and recommended vaccines for human and veterinary use.

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Fig. 24.7. TRIUMPHS AND DEFEATS OF VACCINES. Vaccination is an exceptionally effective biotechnology of preventive medicine: Probably vaccination is the most successful biotechnology.

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Fig. 24.8. OPINION POLLS AGAINST VACCINES: WHY? Efforts to make vaccines more effective and universally available clash with the passionate anti-vaccination reactions that slither in the population of affluent countries. Until the last century, these movements were minorities and vaccination coverage tended to grow. At present, vaccine-opposing groups found the internet an effective vehicle to spread their positions and thereby we are witnessing a fall in vaccine coverage. The mass media emphasis on hypothetical side effects of vaccines triggers waves of collective fear that mainly concern the false accusation of causing autism, the adjuvant and preservative toxicity, and the weakening of the immune system caused by too many vaccines. While anti-vaccine movements spread their objections with militant enthusiasm, health authorities /often appear unable to convincingly explain the fundamental importance of vaccines. No matter how authoritative the official documents are, it appears extremely difficult to wipe out the suspicion that these documents are the result of a concerned manipulation and global conspiracy.

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CHAPTER 25- NEGATIVE CONTROLS OF THE IMMUNE RESPONSE Fig. 25.1. Positive and negative controls of the immune responses. The immune response is not exhausted by inertia. On the contrary the shutdown of the immune response is an active function, carefully regulated

Fig. 25.2. The dynamic yin-yang control of the immune response. Activation versus suppression are just the two extremes of a continuum of response patterns characterized by specific transcription factors, cytokines and cellular circuits. REFERENCE: AM MUJAL & F Krummel, Science 2019,364:28.

240 Fig. 25.3. THE ABSOLUTE IMPORTANCE OF INHIBITORY RECEPTORS. The regulation and termination of the immune response often rest on signals captured by inhibitory receptors. Errors in the regulation of innate and adaptive immune responses may result in autoimmune disorders. Dysregulated expression of inhibitory receptors promoted autoimmunity, whereas their sustained expression impairs the ability of the immune system to clear infections and control cancer growth. When ITIM-possessing inhibitory receptors interact with their ligand, their ITIM motif becomes phosphorylated and forms the docking site for phosphatase that dephosphorylate molecules involved in cell activation.

Fig. 25.4. A PROVISIONAL LIST OF INHIBITORY RECEPTORS. The control of immune response made by several different inhibitory receptors acting on the cells of innate and adaptive immunity keeps a tight rein on the onset of autoimmune diseases. Dominant inhibitory receptors control the activity of NK cells (See Fig. 5.29). DNGR, Dendritic Cell NK Lectinic Group Receptor; BTLA, B and T Lymphocyte Attenuator; HVEN, Herpes virus entry mediator; REFERENCE: C del Fresno et al. Science 2018,362:35. The Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) is an inhibitory receptor expressed on myeloid and Dendritic cells. Leukemic cells expressing the LILRB4 dampen the anti-leukemia T cell reaction by creating an immunosuppressive microenvironment. APOE: Apolipoprotein E. REFERENCE: M Deng et al, Nature 2018,562:605

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Fig. 25.5 INDUCED Treg (iTreg) CELLS. An antigen-presenting cell (APC) displaying antigen peptides along with the secretion of a high amount of Transforming Growth Factor- (TGF-), IL10 and other cytokines in the absence of IL-6 triggers the activation of the program leading virgin Th0 CD4+ T cell to differentiate towards iTreg cell. On the other hand, the repertoire of cytokines released by APC is dictated by the kind of antigen they have taken up. In many cases, the phagocytosis of microbes triggers a high production of TGF- and IL6 and switches the differentiation of virgin CD4+ Th0 cells toward Th17 cells (See Figs. 12.5) and not toward CD4+ Treg cell. Once activated, iTreg cells plays a pivotal role in the maintenance of immunological self-tolerance. They suppress the activity of other T cells within close proximity by releasing TGF- and IL10. iTreg cells may trigger the production of indoleamine dioxygenase (IDO) by APC. IDO breaks down tryptophan, and the lack of tryptophan is an effective way to block the activation of the lymphocytes present in the microenvironment. The transdifferentiation of Th17 in iTreg cells is an important physiological mechanism for the resolution of the inflammation. Metabolic factors that regulate the transdifferentiation of Th17 towards iTreg regulate the expression of the Foxp3 gene. REFERENCE: T Xu al, Nature 2017,548:228.

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Fig. 25.6. TRANSFORMING GROWTH FACTOR-BETA (TGF-): A MULTIFUNCTIONAL REGULATORY CYTOKINE.

Fig. 25.7. IL10: A SUPPRESSOR CYTOKINE. Alone or in combination with TGF-, IL10 plays a central role in the negative regulation of immune responses. IL10 is directly involved in the induction of unresponsiveness to antigens introduced through oral and respiratory routes.

243 Fig. 25.8. THE RESTRICTED VIRGIN T CELL COSTIMULATION. The specificity of virgin T cell activation rests on the high affinity interaction between the TCR with HLA-p (See Fig. 10.8-10.10). However, to proceed in the activation, a virgin T cell requires the concurrent series of costimulatory signals (B7, CD40â&#x20AC;Ś See Fig. 10.1410.17), the so-called activating checkpoints. The poised T cell waits for checkpoint signals before continuing in the activation process.

Fig. 25.9. VIRGIN T CELLS: ACTIVATION VS. INHIBITION. The absence of the signals delivered by these molecules arrests cell activation and induces either a state of T cell dysfunction (anergy) or triggers T cell apoptosis. Therefore, only a specialized population of cells, the so-called antigen-presenting cells (APC, see Chapter 10) expressing the HLA-p and costimulatory checkpoint molecules are able to trigger the activation of a virgin T cell. By contrast, HLA-p expressed on the surface of the other body cells unable to provide concurrent costimulatory signals blocks T cell activation. Mature autoreactive T cells that may have slipped Central Tolerance do not attack normal body molecules because these cells do not express costimulatory checkpoint molecules.

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Fig. 25.10. CONTROLLING SIGNIFICANCE OF NEGATIVE REGULATORY CHECKPOINTS. CTLA-4 and PD-1 are members of the CD28 family of cell membrane receptors (See Fig. 10.14). Despite being highly homologous to CD28, their expression on the surface of CD4+ and CD8+ mature T cells differs markedly from that of CD28. CTLA-4 and PD-1 are receptors not expressed on the cell membrane of resting T cells. Instead, their expression is up regulated during a later stage of T cell activation. This late expression fits in well with the negative regulatory role that they play in T cell activation. In effect, CTLA-4 and PD-1 do not block the induction of the immune response of virgin mature T cells. On the contrary, these receptors play a critical role in regulating and dampening an ongoing immune response. Both receptors have an important role in inhibiting autoimmune activation of T cells.

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Fig. 25.11. CTLA-4 AND PD-1 INHIBITORY CO-RECEPTORS. In the later stage of activation, T cells express the CTLA-4 and the PD-1 co-receptors that bind B7 and PD1L ligands with higher affinity as compared to CD28 co-receptor and deliver signals that dampen T cell activation. T cells transducing the signals delivered by CTLA-4 and PD-1 release inhibitory cytokines that block the activation of surrounding cells. The late expression of CTLA-4 and PD1 co-receptors allows the physiological regulation of the intensity and persistence of T cell immune response. The interaction of CTLA-4 and PD-1 co-receptors with their ligands serves as a molecular brake, preventing hyperactivity of the T cells of the immune system and, in some cases, preventing autoimmunity (Regulatory or Inhibitory Checkpoints). Activation versus tolerance of T cells is thus the result of a combination of positive costimulatory and inhibitory checkpoint signals. Dominant inhibitory signals induce T cell tolerance or T cell exhaustion. T cell exhaustion is often found in patients with chronic viral infections or cancer. Several human tumors are able to dampen anti-tumor T cell reaction through the expression on their cell membrane the ligands of CTLA-4 and PD-1 receptors (See Fig. 25.12). In these tolerant and exhausted dysfunctional T cells, the suppressive NR4A1 transcription factor is preferentially recruited to the binging sites of AP-1 transcription factor (See Fig. 10.10) and activates tolerance-related genes. REFERENCE: X Liu et al, Nature 2019, 567:525.

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Fig. 25.12. THE BLOCKADE OF REGULATORY CHECKPOINTS UNVEILS THE EFFECTIVENESS OF NATURAL IMMUNE RESPONSE TO CANCER CELLS. Tumors evade immune control by creating a suppressive microenvironment that perturb T cell metabolism and effector functions. Several tumor hijack normal inhibitory checkpoints molecules for protection from immune attack. For example (Fig.) tumor cell may start to express the programmed death-ligand 1 (PD-L1, in black) which interacts with programmed death-1 (PD-1) receptor commonly expressed by activated T cells. In addition, human melanomas may release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. The transduction of the signals delivered by PD-L1 - PD-1 interaction inhibits the activities of T cells reacting against tumor antigens. Activated T cells are also inhibited by tumor infiltrating inflammatory myeloid cells expressing both the PD-1 ligand and the B7 costimulatory molecules. These ligands interact respectively with the

247 PD-1 and CTLA-4 co-receptors, both expressed by activated T cells. These receptors transduce inhibitory signals and thus dampen T cell anti-tumor reaction. Commonly, tumor cells escape T cell attack in this way. A large series of monoclonal antibodies (See Chapter 21) have been now produced to interfere with these immune regulatory checkpoints. Cancer patients receive the administration of monoclonal antibodies against CTLA-4 (In red in the Fig.) or PD-1 receptors (In blue) expressed by activated T cells or monoclonal antibodies against the PD-1 ligand (In black) expressed by both tumor cells and tumor infiltrating myeloid cells. In several cases, treatments of this kind are enough to rescue T cell anti-tumor reactivity, induce tumor shrinkage, and significantly improve patient survival. In these cases, tumor shrinkage depends on immune surveillance by tumor-infiltrating T cells that are no more inhibited by negative signals delivered by PD-1 and CTLA-4 co-receptors. The Nobel Prize in Physiology or Medicine 2018 was awarded jointly to James P. Allison, USA and Tasuku Honjo, Japan for their discovery of cancer therapy based on the inhibition of these negative

Fig. 25.13. THE EXHAUSTION OF T CELLS. The NR4A1 transcription factor plays a key role in repressing effector genes and in activating exhaustion-related genes. REFERENCES: O Khan et al, Nature 2019, 571:211; X Liu et al, Nature 2019, XXX.

248

CHAPTER 26- IMMUNE TOLERANCE

Fig. 26.1. IMMUNE TOLERANCE. The combination of several control mechanisms explains why the immune system reacts to a limitless variety of antigens without inappropriately attacking the bodyâ&#x20AC;&#x2122;s own molecules. Self-tolerance is a first, crucial training process that enables the immune system to discriminate between self and not-self-molecules. REFERENCE: AC Wendeln at al, Nature 556:332,2018.

Fig. 26.2. SELF vs. NOTSELF. Self-molecules are invisible to the cells of innate immunity since these cells do not express receptors interacting with self-molecules. By contrast, the concurrence of distinct central and peripheral training and educational mechanisms contributes to the acquisition and maintenance of T and B cell tolerance towards selfmolecules.

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Fig. 26.3. TO LEARN TO RECOGNIZE SELFMOLECULES.

Fig. 26.4. THE RESULT OF THE IMMUNE TRAINING DEPENDS ON THE MATURATION STAGE OF T AND B CELLS. In addition, the strength of the antigen-receptor interaction and the presence or not of accessory signals dramatically affect the result of the experience. REFERENCE: AC Wendeln et al, Nature 2018,556:332.

250 Fig. 25.5. FOUR MAJOR MECHANISMS SHAPING THE IMMUNOLOGICAL SELF. Since central tolerance (1) is learned during the development of T and B cells, any self or foreign antigen present in the body during the perinatal period cells may induce tolerance. Other central (Natural Treg cells, 2) and peripheral tolerance mechanisms (Anergy, expansion of Induced Treg cells, expression of Inhibitory checkpoints, 3) may impede the elicitation of an immune reaction to selfmolecules. Lastly, the expression of inhibitory checkpoints molecules (4) blocks the functions of activated T and B cells and induces a kind of tolerance. In effect, many aspects of peripheral tolerance and of the role of inhibitory checkpoint molecules are similar or overlap with negative controls dealt with in Chapter 25.

Fig. 26.6. 1. CENTRAL TOLERANCE: THE DELETION OF SELFREACTIVE T CELLS. The generation of the large repertoire of T Cell Receptors (TCR) is based on a random and error-prone gene recombination (See Chapter 15). Because of the casual nature of this process, numerous T cells are autoreactive, i.e. express a TCR interacting at high affinity with self HLAp. These potentially dangerous T lymphocytes have a few differentiation options in order to not cause a harmful selfaggression. Their common outcome is the disappearance or inactivation of lymphocytes expressing a receptor reacting at high affinity with self HLA-p. The receptor re-editing or secondary TCR gene rearrangement (d) is an alternative attempt to build a new TCR taking advantage of VDJ and VJ genes remaining after the primary TCR recombination. Because the key role of Th cell in the immune response and in the activation of Fo B2 B cells, there is a dedicated organ (the thymus) for the purging of autoreactive T cells (See Chapter 9). Because of our individual antigenic peculiarities, Central Tolerance to self-antigens shapes the repertoire of T B cells differently in each person. For natural Treg cells see Figs. 26.10; for anergy see Fig. 26.13.

251

Fig. 26.7. 1. CENTRAL TOLERANCE: THE DELETION OF SELF-REACTIVE B CELLS. The V(D)J genes encoding the BCR randomly recombine in developing B lymphocytes. In about 50% of the cases, the BCR resulting from this gene recombination reacts at high affinity with self-antigens. Among the three major mechanisms purging self-reactive receptors from the B cell repertoire, BCR re-editing is the most frequent. This secondary V(D)J recombination leads to alteration of the BCR specificity. For natural Treg cells see Fig. 26.10; for Anergy see Fig. 26.13.

Fig. 26.8. 1. CENTRAL TOLERANCE OF IMMATURE B CELLS. B cells that have finally acquired their definitive individual BCR during their bone marrow differentiation (Immature B cells, see Figs.13.4 and 13.5) are cells prone to apoptosis. At this differentiation stage (Orange arrow in the Fig.) their survival depends on the strength of downstream signals triggered by BCR interacting with membrane antigens expressed by surrounding cells. Both the attenuation of the signalling strength below minimum signal threshold (for example, in the case of a non-functional BCR) or hyperactivation of the signalling strength above maximum threshold (for example, in the case of BCR reacting at high affinity with the antigen) triggers the BCR re-editing, the acquisition of an anergic state or B cells apoptosis. The common outcome of these three processes is the disappearance or inactivation of B cells reacting at high affinity with self-antigens.

252 Fig. 26.9 1. DISAPPEARANCE OF SELFREACTIVE IMMATURE B CELLS. When the BCR of a B cell interacts at high affinity with an environmental antigen, downstream signals are initiated by the phosphorylation of Immunoreceptor Tyrosine-based Activation Motifs (ITAM) sequences in the cytoplasmatic tail of Ig alpha and Ig beta signaling chains (See Fig. 13.8). When these events take place in immature B cells, hyperactive downstream signaling initiated by a BCR interacting with high affinity with self-antigens induces one of the three programs leading to the disappearance of autoreactive immature B cells. It should be noted that in mature B cells the same signal transduction signaling pathway induces the expression of the  chain of the IL2 receptor (CD25), expression that is instrumental for B cell activation and clonal expansion (See Fig. 18.9). Fig. 26.10. 2. NATURAL Treg CELLS expressing constitutively the alpha () chain of IL2 receptor (CD25) and the transcription factor Foxp3 (CD4+ Foxp3+ Treg cells) are 10-15 % of body CD4 cells. As Treg cells interact at high affinity with self HLA-p and express the high affinity IL2 receptor, they are very sensitive to IL-2 and can be promptly activated to inhibit the immune response against self-antigens. Therefore, natural Treg cells have a pivotal role in the establishment and maintenance of immunological selftolerance, inhibiting autoimmunity and preventing immune responses from becoming uncontrolled. Several potentially lethal autoimmune diseases, allergies, and food intolerance develop when Treg cells fail to limit the effector activity of autoreactive T cells that have escaped thymic negative selection or peripheral inactivation. In addition to maintaining immune tolerance, Treg cells control tissue homeostasis and remodeling. REFERENCE: M Panduro et al, Annu Rev Immuno 2016, 34:609. For their thymic maturation see Chapter 9; for the  chain of the IL2 receptor see Figs.2.13-2.20. Foxp3 is a Forkhead-family transcription factor expressed by Treg cells.

253 Fig. 26.11. 3.VIRGIN T CELL UNRESPONSIVENESS AGAINST SELF-ANTIGENS. During thymic education (See Chapter 9), lymphocytes that generate a T Cell Receptor (TCR) interacting at high affinity with self HLA glycoproteins (HLA-p), may rearrange the TCR or undergo apoptotic cell death (Negative selection, see Fig. 9.13). However, a few mature virgin T cells expressing a TCR reacting at high affinity with

self-molecules escape this negative thymic selection. While these T cells are present in our body, normally self HLAp are not targeted by their attack since, virgin T cells express the VISTA receptor that interacting with L-selectin on the target cell prevent the conversion of virgin T cells to effector T cells. However, the expression of VISTA receptor is down modulated by pro-inflammatory cytokines and costimulatory signals delivered by antigen presenting cells (APC) (REFERENCES: C Brown and A Rudensky, Science 2020,367:247; MA EITanbouly et al, Science 2020, 367:264). In addition, in most of the cases, self-peptides presented by HLA glycoproteins are just ignored. This immune ignorance is due to the fact that most self-peptides are too scarcely expressed on the cell membrane of APC due to the enormous competition of the numerous selfpeptides for the groove of HLA glycoprotein (See Fig. 7.5). Thus, in most of the cases, there are too few HLA glycoproteins expressing the target peptide to reach the activation threshold of a virgin T cell. Consequently, T cells ignore them. Immune ignorance is an important and common way to avoid autoimmune aggressions. However, viral infections, release of interferons and other pro-inflammatory cytokines may induce an over-expression of the HLA glycoprotein or self-peptides and thus may overcome their Immunological Ignorance. This is one of the reasons why infectious diseases may result in autoimmune disorders. Mature virgin T cells may also not respond to an antigen and develop a state of anergy when the peptide is presented by cells unable to provide the costimulatory signals essential for T cell activation (See Figs. 26.13). Moreover, natural Treg cells recognizing the self HLA glycoproteins with self-peptide (HLA-p) at high affinity can block the T cell activation (See Fig. 26.10).

254 Fig. 26.12. 3. B CELL UNRESPONSIVENESS AGAINST SELFANTIGENS. The purging of immature selfreactive B cells during their bone marrow differentiation is a crucial mechanism of tolerance, defined Central Tolerance (Figs. 26.7-26.9). However, this elimination of B cells with a BCR reacting at high affinity with selfmolecules (Central Tolerance) is never complete. Mature autoreactive B cells that have slipped Central Tolerance do not produce dangerous high affinity autoantibodies because are kept under control by several other immune mechanisms in the body periphery. Fig. 26.13. 3. THE ANERGIC STATE OF B AND T CELLS. The lack of reaction of mature B and T cells against a highly expressed target antigens recognized at high affinity by their membrane receptors is defined anergy. This dysfunctional status takes place when the target antigen is first recognized without appropriate costimulatory signals. Following this first peculiar antigen encounter, the lymphocyte becomes dysfunctional (anergic), while it remains alive for an extended period of time.

255 In T cell, the anergic/dysfunctional state appears to be due to an insufficient JAK-STAT signalling pathway (See Fig. 10.12) due to the overexpression of the NR4A1 transcription factor. In addition, NR4A1 represses effector gene expression mediated by the AP-1 transcription factor (See Fig. 10.10). REFERENCE: X Liu, Nature 2019, 567:525.

Specialized antigen-presenting cells are required to trigger T cell activation. Since the majority of body cells display target HLA-p without costimulatory signals (See Chapter 10) the T cell activation is blocked, and the cell may become anergic. FoB2 B cell activation requires the Th cell cooperation. If the antigen priming of a B cell is not followed by a B-Th cell interaction, the B cell activation is blocked, and the B cell may become anergic. However, anergy is a temporary condition. A continuous binding of the target antigen in the absence of costimulatory signals is essential for the maintenance of anergy. Otherwise, anergy can be lost.

Fig. 26.14. THE PARTICULAR TOLERANCE OF THE MOTHER TOWARDS THE FETUS. For the nine months of the, the motherâ&#x20AC;&#x2122;s immune system does not react against fatherâ&#x20AC;&#x2122;s HLA glycoproteins expressed by the placenta of fetal origin. The placental villi invade a portion of maternal uterine mucosa (the decidua) to acquire nutrients and oxygen from maternal blood. Numerous mother immune cells are present in the decidua: Decidual natural killer (dNK) cells, macrophages, dendritic cells (DC), Innate Immunity cells (ILC), T cells (T), regulatory T (TReg) cells... Each of these cell populations plays a distinct role in maintaining the immune tolerance towards the fetal antigens: 1. HLA glycoproteins. Highly polymorphic HLA-A and HLA-B glycoproteins are not expressed by the outer layer of the placenta (syncytiotrophoblast), thereby reducing maternal T cell reaction

256 against paternal HLA glycoproteins. Only the polymorphic HLA-C and invariant HLA-E and HLA-G (See Fig. 6.19) glycoproteins are expressed on the cell membrane of these cells. 2. T cells. Cytokines present in the semen induce maternal T cells to differentiate towards Treg cells (See Fig. 5.29). Then, the release of transforming growth factor (TGF-) by fetal placenta cells promotes their expansion. Both TGF- (See Fig 25.6) and Treg cells (See Fig. 25.5) concur in dampening T cells reactivity against father HLA glycoproteins. 3. dNK cells. The release of IL15 by decidual stromal cells induces the expansion of dNK cells (See Fig. 5.26). dNK cells express various receptors that interact with HLA-C, HLA-E and HLA-G glycoproteins (See Fig. 5.29). The interaction with HLA-E inhibits dNK cells killer activity while other interactions activate dNK cells to release cytokines that induce the differentiation of uterine blood vessels into large spiral arteries. Since villi of the outer layer of the placenta absorb nutrients and exchanging gases within spiral arteries, dNK play a crucial role in promoting placental invasion and fetal growth. 4. ILC. ILC2 are present in the decidua from mid-pregnancy to term, ILC1 increase throughout gestation, and ILC3s remain constant (See Fig. 4.12). Collectively, decidual ILC have a role in tissue remodeling and in dampening immune reactivity. REFERENCE: F Colucci,, Science 2019,365:862.

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CHAPTER 27. AUTOIMMUNITY. Fig. 27.1. AUTOIMMUNITY. I. Autoimmunity is the immune aggression against oneâ&#x20AC;&#x2122;s normal own body molecules, cells, and tissues. Genetic factors, peculiarities of the individual immune system and microbial infections concur in the pathogenesis of autoimmunity. A critical role is also played by the efficacy by which induced Treg cells (See Fig. 25.5) restrain the activation of pro-inflammatory Th17 cells (See Figs. 12.5, 12.6). Chronic inflammatory reactions to microbes driven by Th17 escaping Treg cell control may result in autoimmunity. Fig. 27.2. AUTOIMMUNITY. II. The immune system is a collection of complex and sophisticated mechanisms. Therefore, is not surprising that a few of these may fail to be appropriately controlled. These control mistakes often lead towards an incorrect discrimination between self and not-self and allow the onset of self-aggressive immune reactions. The progressive extension of human life is accompanied by an increased incidence of control mistakes. In wealthy countries, there is a large population of patients suffering from diseases due to aggression against self-antigens. In effect, autoimmune diseases such as diabetes, multiple sclerosis, rheumatoid arthritisâ&#x20AC;Ś result from chronic autoimmune aggressions involving T and B cells reacting against multiple self-epitopes. A central role is played by self-reactive Th cells since they orchestrate the reaction of several other cells of the immune system including B cells, T killer cells, and macrophages.

258 Fig. 27.3. FAILURE OF SELF TOLERANCE CAUSES AUTOIMMUNITY. During their maturation, T and B cells randomly generate their individual antigen receptor (TCR and BCR). The majority of these receptors react with self-molecules (See Chapter 11). Therefore, various control mechanisms of self-tolerance (See Chapter 26) remove or make unreactive (anergic) the B and T cells expressing an antigen receptor reacting at high affinity with self-molecules (autoreactive lymphocytes). In other cases, autoreactive lymphocytes fail to be activated because they encounter insufficient antigen stimulation (Immune ignorance, See Fig.26.11) or fail to receive concurrent costimulatory signals. In several chronic inflammatory diseases, a dysregulated expression of IL-17 (See 12.5 and 12.6) by Treg cells may results in the loss of their physiological suppressive function (See Fig. 27.3.). REFERENCE: KA Remedios et al, Science Immunol 2018,3, 30. Autoimmune reactions are due to a failure of a few of these control mechanisms caused by genetic and environmental factors.

Fig. 27.4. THE FAILURE OF REGULATORY MECHANISMS CAUSES AUTOIMMUNITY. Besides the failure of the various mechanisms of self-tolerance, autoimmunity may arise from a failure of inhibitory checkpoints on which the regulation of immune responses depends (See Fig. 25.10). Major functional defects of the immune system (Immunodeficiencies) are often associated with autoimmune reactions.

259

Fig. 27.5. GENETIC FACTORS PREDISPOSING TOWARD AUTOIMMUNITY. Often autoimmune diseases, such as systemic lupus erythematosus, are characterized by the presence of large numbers of self-reactive antibodies that induce deposition of immune complexes, leading to inflammation and tissue damage. Single-nucleotide polymorphisms (SNP) as well as other genetic mutations may alter checkpoint controls and facilitate antibody over production. REFERENCE X Chen et al, Science 2018: 362,700.

Fig. 27.6. ENVIRONMENTAL FACTORS PREDISPOSING TO AUTOIMMUNITY. Acute rheumatic fever is caused by an autoimmune response to throat infection with Streptococcus pyogenes. Cardiac involvement during acute rheumatic fever can result in rheumatic heart disease, which can cause heart failure and premature mortality.

260

Fig. 27.7. B AND B CELL AUTOIMMUNE DAMAGE. Often reactive T cells invade normal or damaged target tissues and induce a complex selfdestructive inflammatory process.

Fig. 27.8. ANTIBODY AND COMPLEMENT AUTOIMMUNE DAMAGE. For details see: a- Fig. 21.5; b- Figs. 20.9; c- Fig. 20.7; dFig. 20.6; e- The interaction of antibodies with the binding site of a membrane receptor can stimulate the receptor mimicking the ligand; f- Fig. 20.6; g- Fig. 20.4; h- Fig. 21.8; i- Fig. 21.7; l- Fig. 21.8.

261 Fig. 27.9. INFLAMMATORY BOWEL DISEASES: AN INFLAMMATION INDUCED AUTOIMMUNITY. These diseases are due to chronic innate immunity reactions (chronic inflammation) that damage the integrity of the mucosal barriers, increase the susceptibility to microbial infections, and trigger the induction of autoimmune reactions.

Fig. 27.10. THE ONSET OF INFLAMMATORY BOWEL DISEASES MAY DEPEND ON THE DYSREGULATION OF MULTIPLE IMMUNE RELATED GENES. Various HLA alleles are associated with a higher risk of inflammatory bowel diseases. A hyper-activation of Pattern Recognition Receptor (PRR) genes and of genes coding for proinflammatory cytokines may trigger a too intense inflammatory reaction and stress or damage the cells of mucosal barrier. The same damage may be due to a defective release of regulatory/suppressor cytokines. An appropriate production of mucus by goblet cells is critical for the maintenance of mucosal barrier efficiency. Lastly, an increased homing of reactive leukocytes at mucosal surfaces may efficiently protect against microbial incursion but, at the same time, it could cause damage to the cells of the mucosal membranes.

262

Fig. 27.11. PATTERN RECOGNITION RECEPTORS (PRR) ON CELL MEMBRANE OF GUT MUCOSA CELLS TRANSDUCE EITHER ACCOMMODATING OR PROINFLAMMATORY SIGNALS. Due to the enormous quantity of microbes normally present in the gut, microbial incursions crossing intestinal mucosa are a common event. It is therefore essential to implement an appropriate containment, considering that excessive, repeated and chronic inflammatory reactions pave the way to Inflammatory Bowel Diseases. The interaction of microbes with Pattern Recognition Receptors (PRR) on the outer part of epithelial cells of the gut mucosa triggers signals that block inflammation while stimulate an accommodating Type 2 containment. This kind of reaction dampens the “Type 1” innate immunity reaction (See Figs. 2.22 and 27.12) and avoids the inflammation-associated mucosal damage. By contrast, a robust Type I inflammatory reaction is triggered when microbes overcoming the mucosal surface interact with PPR expressed on basal portion of the epithelial cells.

263 Fig. 27.12. DISORDERS OF THE INTESTINAL BARRIER OPEN THE WAY TO INFLAMMATORY BOWEL DISEASES. The two layers of mucus normally impede the contact of microbes with the cells of the intestinal mucosa (upper panel, See also Fig. 3.9). Genetic or acquired defects in the normal production of mucus by goblet cells (second panel) may open the way to microbial incursions. Besides the different signals transduced by Pattern Recognition Receptors expressed on the apical and basal portion of gut epithelial cells (See Fig. 27.11) there are additional mechanisms that concur in the appropriate modulation of the immune reaction against the invaders. Intra epithelial lymphocytes (mostly ILC3) that are present in the Gut Associated Lymphoid Tissue (GALT, See Fig. 3.8) recognizing particular metabolic conditions (anaerobiosis), recurrent metabolites and structures of the microbes making repeated incursions, may acquire an immune memory of a tolerogenic type (See Fig. 23.4). The accommodating signals delivered by PRR along with this peculiar tolerogenic immune memory trigger: a) M2 macrophage differentiation and induces a tolerogenic macrophage memory (See Fig. 5.20); Macrophages sensing of the microbiota produce IL-1Î˛ that activates ILC3 to produce IL-2 (See Fig. 4.12); Subsequently, ILC3derived IL2 supports intestinal Treg cells, immunological homeostasis and oral tolerance. REFERENCE: L Zhou et al, Nature, 2019,568:405.

b) Tolerogenic differentiation of Dendritic Cells; c) Treg cell activation and expansion; d) Mucosal repair mechanisms; e) Mucus hyper production (IL22)

264 f) Production of secretory IgA. This “Type 2” containment of habitual microbes might collectively be called accommodation. REFERENCE: AM Mujal & F Krummel, Science 2019,364:28. By contrast, a massive microbial invasion by certain kind of microbes may trigger a very efficient “Type 1” innate immunity reaction (lower panel). In this case, invading microbes are efficiently destroyed. However, a vicious circle may take place between repeated functional damage of the mucosal barriers, microbial incursions and reactive inflammatory reactions causing additional mucosal damage. The gut-draining lymph aggregates and lymph nodes are key sites for orchestrating these inflammatory, accommodating and tolerogenic reactions. Those more proximal to mucosal epithelia barrier preferentially give rise to tolerogenic responses while those more distal to pro-inflammatory responses. REFERENCE: D Esterhazy et al, Nature 2019,569:126. The importance of the delicate regulation of the containment of gut microbes is empathized by recent data in mice showing that the production of IgA supported by local T helper cells may promote the expansion of some microbe (Clostridia) that protect against obesity. Even a complex metabolic disease as obesity may rest on a defective immune control of the gut microbes (REFERENCE: C Petersen et al, Science 365. XXX, 2019).

Fig. 27.13. MULTIPLE SCLEROSIS. Another paradigmatic example of the unfortunate association of inflammation, HLA alleles and T cell attack. REFERENCE: D Lodygin et al, NATURE, 2019,566:503

265

Fig. 27.14. PARKINSON’S DISEASE. Data in mice suggest a link between bacteria, inflammation and neurodegenerative diseases. An uncontrolled inflammatory reaction may contribute to the onset of Parkinson’ disease. REFERENCE: D Matheud et 2019,571:565

al,

NATURE,

266

CHAPTER 28. IMMUNODEFICIENCIES. Fig. 28.1. IMMUNODEFICIENCIES. The failure of some immune mechanisms often opens the door to infections due to a particular class of bacteria virus or fungi. These infections become recurrent. The kind of microbe responsible for these recurrent infections highlights the peculiar defense role normally played by the immune mechanism that does not work.

Fig. 28.2. INHERITED IMMUNODEFICIENCIES. I.

267

Fig. 28.3. INHERITED IMMUNODEFICIENCIES. II. A FEW DEFECTIVE MECHANISMS. Inherited Immunodeficiencies provide a dramatic illustration of the function of the various components of the immune system. Severe Immunodeficiencies due to the absence of granulocytes are incompatible with life. Due to their central role in the immune system, Immunodeficiencies due to a defective T cell or T and B cell maturation cause Severe Combined Immune Deficiencies (SCID). The common  chain is a transducer chain exploited by numerous distinct cytokine receptors (See Fig. 2.12). Therefore, the lack of a functioning common  chain causes a SCID due to the simultaneous inactivation of multiple immune mechanisms. As the common  chain gene is located on the X chromosome, the immunodeficiency caused by  chain gene mutations is known as an X linked SCID (X-SCID). Genetic defects affecting selectively B cell maturation result in the absence of antibodies, a condition called a-gammaglobulinemia. The first immunodeficiency identified was an X-linked agammaglobulinemia caused by a defective Bruton tyrosine kinase (BTK) which blocks the maturation of B cells following H chain gene rearrangement (See Fig. 15.6). The recurrent infections by staphylococci and streptococci (microbes producing pus, known as pyogenic microbes) displayed by these patients show the importance of antibody-mediated microbe opsonization. Staphylococci and streptococci have a polysaccharide capsule that inhibits phagocytosis. However, when their surface antigens are bound by antibodies, they are picked up efficiently through the binding of the Ig Fc domain to Fc receptors (See Fig. 19.7). A similar clinical situation is caused by a variety of defects in Complement components impairing Complement-mediated opsonization of microbes (See Fig. 21.6).

268

Fig. 28.4. ACQUIRED IMMUNODEFICIENCIES. Acquired Immunodeficiencies may be due to factors of a diverse kind. A decrease in body weight to less than 70% of recommended weight results in a severe immunodeficiency. In 2010 there were 925 million undernourished people. In 2013 undernutrition resulted in 469,000 deaths. Immunodeficiency disorders may result from aging, almost any prolonged serious disease, and advanced cancer. Immunodeficiency may also result from several kinds of medical treatment (chemotherapy, radiation, immunosuppressive drugs administered after organ transplants, glucocorticoids). Immunodeficiencies may be the result of radioactive and nuclear accidents and several kinds of occupational exposure to immunosuppressive agents. Several viral infections result in systemic and local Immunodeficiencies.

269 Fig. 28.5. THE INFECTION BY THE HUMAN IMMUNODEFICIENCY VIRUS (HIV). Currently, more than 35 million people are infected worldwide, with millions new diagnosis each year and 1.6 million deaths. The most prevalent route of HIV infection is across mucosal tissues. Because of the fragility of the barrier offered by the glans penis, vagina (See Fig. 3.10) and rectal mucosa (See Fig. 3.11), mechanical stress, cuts, and microabrasions may open the door to HIV infection.

Fig. 28.6. HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION OF CD4+ T CELLS. Anatomical localization and membrane receptor characteristics make tissue antigen-presenting cells (APC: Macrophages and Dendritic Cells) expressing both CD4 co-receptors and other co-receptors (DC-Signâ&#x20AC;Ś) the primary targets of HIV infection. Then, infected APC present HIV peptides on the groove of HLA glycoproteins. CD4+ cells with a TCR which recognizes viral peptides establish a close interaction with HIV infected APC. The HIV exploits this close APC-T cell interaction to bind the CD4 glycoprotein on T cell membrane.

Fig. 28.7. CD4 IS THE PRIMARY HUMAN IMMUNODEFICIENCY VIRUS (HIV) RECEPTOR. The first step of the infection of host cell by HIV-1 virus is the binding of the trimeric glycoprotein (gp) 120 to CD4 on the surface of CD4+ macrophages, dendritic cells and CD4+ T cells. Before the binding, gp120 is in the native conformation. Following the binding to CD4 gp120 acquires an open conformation and can interact with CCR5 and CXCR4 chemokine receptors.

270 Fig. 28.8. THE OPEN CONFORMATIONAL ACQUIRED BY gp120 ALLOWS ITS INTERACTION WITH CHEMOKINE RECEPTORS. The open conformation acquired by gp120 of the HIV-1 following its interaction with CD4 receptor allows its interaction with the CXCR4 and CCR5 chemokine receptors. Then, the fusogenic portion of gp41 allows the fusion of the membranes and subsequent entry of the viral capsid. A few persons express the 32 variant of the gene of the CCR5 receptor that does not bind the HIV gp120. The gp120 inability to bind the -32 variant hampers or impedes the HIV infection (See Fig. 2.29 and 28.9).

Fig. 28.9. HIV-1 REMISSIONS FOLLOWING CCR532 HEMATOPOIETIC STEM CELL TRANSPLANT. The non-functioning CCR532 receptor does not bind the Human Immunodeficiency Virus-1 (HIV-1) gp120 glycoprotein (See Fig. 2.29). Two patients with leukemia or Hodgkin lymphoma and infected with HIV-1, who have received allogeneic hematopoietic stem-cell transplant from donors with the homozygous -32 gene mutations are experiencing a persistent remission of the HIV-1 infection. REFERENCE: RK Gupta et al, Nature 2019,569:244.

271

Fig. 28.10. FROM HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION TO ACQUIRED IMMUNODEFICIENCY (AIDS).

1. Once the HIV has entered the body, tissue antigen-presenting cells (Macrophages and Dendritic Cells) are these first cells to be infected (See Fig. 28.6). The HIV escape the RIG-like receptor recognition (See Fig 4.7) and the RIG-like receptor activated innate reaction by hijacking cellular proteins that methylate the HIV virus RNA and hide it from cellular host detection. REFERENCE: Ringeard M et al, Nature 2019,565:500.

2. Infected APC present HIV peptides on the groove HLA glycoproteins. 3. CD4+ Th cells with a TCR which recognizes viral peptides establish a close interaction with HIV infected APC. HIV exploits this interaction to bind CD4 and CXCR4 and CCR5 chemokine receptors on the membrane of CD4+ cells and infect them (See Fig. 2.27). Thanks to the activation of CD4 T cells and B cell, antibodies are produced against the HIV (Serum conversion). This production of antiviral antibodies

4. HIV infected CD4+ Th cells release virus particles that are now so numerous as to infect other CD4+ cells directly.

5. HIV infected CD4+ Th cells release HIV virions and die. Moreover, the fuosogenic domain of HIV stemming from the membrane of infected cells favors the formation of large syncytia of CD4+ Th cells. Lastly, the body mounts an immune reaction against the HIV and HIV infected cells. Tk cells recognizing viral peptides expressed by infected CD4+ Th cells kill them. Steadily a slow decrease in the number of CD4+ Th cells takes place for a period ranging from two to twelve years (or more). Then the equilibrium between destroyed CD4+ Th cells and those newly produced is lost and the number of CD4 + Th cells decreases progressively. Only when the number of CD4+Th cells is below 200 per square microliter, a severe combined immunodeficiency is acquired (AIDS).

272

Fig. 28.11. THE NATURAL DECLINING OF IMMUNE FUNCTIONS: THE FRAILTY OF OLDER ADULTS.