Martins et al 2006 transcriptional repressor blimp 1 regulates t cell homeostasis and function by magnusdottir

ARTICLES

Transcriptional repressor Blimp-1 regulates T cell homeostasis and function Gislaˆine A Martins1,7, Luisa Cimmino2,7, Miriam Shapiro-Shelef1,7, Matthias Szabolcs3, Alan Herron3,4, Erna Magnusdottir5 & Kathryn Calame1,6 The B lymphocyte–induced maturation protein 1 (Blimp-1) transcriptional repressor is required for terminal differentiation of B lymphocytes. Here we document a function for Blimp-1 in the T cell lineage. Blimp-1-deficient thymocytes showed decreased survival and Blimp-1-deficient mice had more peripheral effector T cells. Mice lacking Blimp-1 developed severe colitis as early as 6 weeks of age, and Blimp-1-deficient regulatory T cells were defective in blocking the development of colitis. Blimp-1 mRNA expression increased substantially in response to T cell receptor stimulation. Compared with wild-type CD4+ T cells, Blimp-1deficient CD4+ T cells proliferated more and produced excess interleukin 2 and interferon-c but reduced interleukin 10 after T cell receptor stimulation. These results emphasize a crucial function for Blimp-1 in controlling T cell homeostasis and activation.

B lymphocyte induced maturation protein-1 (Blimp-1) is a zinc finger–containing transcriptional repressor1 that is necessary2 and sufficient1,3 for the generation of terminally differentiated antibodyproducing plasma cells4. However, Blimp-1 is also expressed in non–B cell lineages. Blimp-1 is expressed at specific stages and in specific cells in the mouse embryo5 and is required for embryo viability after days 10.5–11 and for the formation of primordial germ cells6. In adult mice, Blimp-1 is expressed in several differentiated cell lineages, including granulocytes, macrophages7, epithelial cells8 and retinal neurons (unpublished data). Because Blimp-1 functions as a ‘master regulator’ of plasma cell differentiation, identification of its gene targets9 has provided important insights into the regulatory networks controlling terminal B cell differentiation and function. Blimp-1 is a key component of a network of plasma cell transcription factors that repress other factors, such as Bcl-6 and Pax5 (ref. 10), which are required for the function of activated B cells. Bcl-6 and Blimp-1 repress each other and establish mutually exclusive B cell and plasma cell gene expression programs, respectively1. Blimp-1 inhibits proliferation and induces immunoglobulin secretion, thereby promoting the generation of a plasma cell phenotype9. Blimp-1 may also regulate developmental checkpoints in other lymphocyte lineages. Here we report that Blimp-1 was expressed in T cells and that mice lacking Blimp-1 specifically in the T cell lineage had more effector CD4+ and CD8+ cells in the periphery and developed severe colitis. Naive Blimp-1-deficient CD4+ cells were hyperproliferative in response to T cell receptor (TCR) stimulation and produced more interferon-g (IFN-g) and less interleukin 10

(IL-10) than did their wild-type counterparts. Bcl-6 repression was impaired in Blimp-1-deficient CD4+ effector T cells. In addition, Blimp-1-deficient CD4+CD8+ double-positive (DP) thymocytes showed survival defects. Thus, in the T cell lineage, Blimp-1 is important for survival in the thymus as well as homeostasis and function in the periphery. RESULTS T cell Blimp-1 represses colitis To determine if Blimp-1 (encoded by Prdm1) has a function in T cells, we crossed mice with loxP-flanked Prdm1 (Prdm1flox/flox mice)2 with mice expressing a Cre transgene under control of the Lck proximal promoter11 and analyzed the resulting ‘conditional knockout’ (CKO) Prdm1flox/floxCre+, heterozygous Prdm1flox/+Cre+ and control (Prdm1+/+Cre+, Prdm1flox/+Cre– or Prdm1flox/floxCre–) littermates. In mice with the Lck-Cre transgene, Cre-dependent deletion (as assessed by green fluorescent protein (GFP) expression from a ‘ROSA26-stopfloxed-GFP’ reporter) is first detected in CD4–CD8– double-negative (DN) CD44–CD25+ (DN3) thymocytes but is not complete until the CD44–CD25– DN4 or DP stage (H. Gu, personal communication). We analyzed thymocyte DNA by Southern blot and found deletion of more than 90% of Prdm1 in the CKO mice (Fig. 1a). There was also almost complete deletion in purified peripheral T cells, by quantitative PCR analysis (Fig. 1b). CKO mice were born at the expected mendelian frequency, but as early as 4 weeks of age they were noticeably smaller than their littermate controls, and they became severely wasted and moribund with age (Fig. 1c). There was colitis, demonstrated by thickened colons containing lymphocytic and

1Department

of Microbiology, 2The Institute of Human Nutrition, 3Department of Pathology, 4Institute of Comparative Medicine, 5Department of Biological Sciences, and of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA. 7These authors contributed equally to this work. Correspondence should be addressed to K.C. (klc1@columbia.edu). 6Department

Received 24 October 2005; accepted 14 February 2006; published online 26 March 2006; doi:10.1038/ni1320

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Deleted

30 Body weight (g)

Nondeleted

Males

Females

b Relative floxed Blimp-1 DNA

re – F/ re + F C F/+ re + C F/F re – F C /F re + C F/ re + + F/ F

1.2 1.0

Irregular crypts

0.8 0.6 0.4 0.2 0.0

25 20 20 15

Crypt abscesses

15 10

3 4 5 6 7 8 910

3 4 5 6 7 8 9 10

Control

CKO

Erosion

Time (weeks)

Figure 1 The generation and phenotype of CKO mice. (a) Southern blot analysis of thymocyte DNA from two sets of Prdm1flox/floxCre– (Cre– F/F), Prdm1flox/+Cre+ (Cre+ F/+) and Prdm1flox/floxCre+ (Cre+ F/F) mice. (b) Quantitative PCR analysis of Prdm1 in CD4+ T cells from Prdm1+/+Cre+ mice (gray bar) and Prdm1flox/floxCre+ mice (open bar), normalized to results obtained for cyclophylin. (c) Total body weights of male and female CKO mice (open boxes) and control mice (filled boxes) of various ages (horizontal axes), presented as mean ± s.e.m. (n ¼ 3–5 mice per age). (d) Half a colon from a 20-week-old CKO mouse and a littermate control mouse. (e–h) Hematoxylin-and-eosin staining of colon sections from 6- to 16-week-old control (e) or CKO (f,g) mice. Original magnification, 20 (e,f), 100 (g) and 200 (h). Data are representative of three mice per group (b) or 18 control and 21 CKO mice (e–h).

neutrophilic infiltration, irregular crypts, crypt abscesses and erosions (Fig. 1d–h), in 20% of CKO mice at 4 weeks of age, 70% of CKO mice at 6–10 weeks of age and 83% of CKO mice at 13–21 weeks of age (Table 1). These mice were of mixed 129 and C57BL/6 genetic backgrounds, which may have contributed to variations in disease progression in individual mice12. There was no colitis in heterozygous or control (Prdm1+/+Cre+, Prdm1flox/+Cre– or Prdm1flox/floxCre–) mice. Additionally, thorough histopathological examination of mice 16–20 weeks of age showed that some CKO mice had nonspecific inflammation in bronchus-associated lymphoid tissue (four of five mice) and liver (two of five mice); however, salivary glands, stomachs, small intestines, pancreata, hearts and kidneys were histologically normal in all mice. Colon pathology in the absence of chronic inflammatory changes in other organs strongly resembles human inflammatory bowel disease.

ducibly increased at least 20-fold (Fig. 2d). Anti-CD3e alone and IL-2 alone but not anti-CD28 alone induced smaller increases in Blimp-1 mRNA expression (data not shown). Blimp-1 induction was gradual and slow, occurring mainly between days 3 and 6 after stimulation. The amount of Blimp-1 mRNA in ‘day-6’ activated CD4+ cells was similar to that in lipopolysaccharide-activated splenic plasma cells (Fig. 2e). Thus, Blimp-1 mRNA is present in thymocytes and in naive peripheral T cells, but Blimp-1 expression is higher in memory, effector and regulatory T cell populations and is induced after activation of naive CD4+ T cells in vitro by TCR and/or IL-2 stimulation.

Blimp-1 expression in the T lineage The CKO mouse pathology prompted thorough analysis of steadystate Blimp-1 mRNA in the T cell lineage. Early studies did not detect Blimp-1 mRNA in pre–T cell or T cell lines3, but more recent microarray studies have reported induction of Blimp-1 mRNA during the DN-to-DP transition13. We analyzed purified ex vivo thymocyte populations by quantitative RT-PCR to measure steady-state Blimp-1 mRNA. We detected relatively little Blimp-1 mRNA in thymocytes. DN thymocytes, CD4+ single-positive thymocytes and peripheral naive CD4+ T cells had similar amounts of Blimp-1 mRNA and expressed threefold more Blimp-1 mRNA than did DP thymocytes (Fig. 2a). CD44hiCD62Lhi memory T cells had more Blimp-1 mRNA than did naive T cells, but CD44hiCD62Llo effector T cells had the most Blimp-1 mRNA (Fig. 2b). CD4+CD25+ T cells also had more Blimp-1 mRNA than did naive CD4+ T cells (Fig. 2c). When we stimulated purified naive CD4+CD44loCD62Lhi T cells in vitro with plate-bound antibody to CD3e (anti-CD3e), anti-CD28 and IL-2, Blimp-1 mRNA was repro-

4 weeks old Cre– (n ¼ 4)

75%

25%

Prdm1flox/+Cre+ Prdm1flox/floxCre+ (n ¼ 5)

NA 40%

NA 20%

6–10 weeks old Cre- (n ¼ 9)

78%

22%

Prdm1flox/+Cre+ (n ¼ 4) Prdm1flox/floxCre+ (n ¼ 10)

50% 10%

50% 20%

0% 70%

60% 100%

40% 0%

0% 0%

458

Table 1 Development of colitis in CKO mice

Normal

Nonspecific inflammation

Inflammatory bowel disease

13 or more weeks old Cre– (n ¼ 5) Prdm1flox/+Cre+ (n ¼ 2) Prdm1flox/floxCre+ (n ¼ 6)

17%

0% (Prdm1flox/floxCre

83% Prdm1flox/+Cre–)

Sections of colon from CKO, heterozygous and control or mice were stained with hematoxylin and eosin and were analyzed for the presence of colitis. Inflammatory bowel disease (colitis) was defined by the presence of crypt abscesses, erosions or crypt irregularities (evidence of chronic disease); nonspecific inflammation was defined as the accumulation of neutrophils or lymphocytes (not in lymphoid tissue) without any signs of chronic crypt damage. NA, not analyzed.

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or y Ef fe ct or

ai ve

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400 300 200 100 0

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c 1,000

Relative Blimp-1 mRNA

CD8

1,000

100

ai ve

DN DP SP4 Naive CD4

100

CD4

100

b 1,000 Relative Blimp-1 mRNA

Thymus

Relative Blimp-1 mRNA

a 1,000

103

102

101

3 4 Time (d)

CD4 CD4 PCs naive day 6

Figure 2 Blimp-1 transcripts in T lineage cells. Blimp-1 mRNA was measured by quantitative RT-PCR and was normalized to 18S RNA. (a) Blimp-1 mRNA in purified thymocyte subsets and peripheral CD4+CD44loCD62Lhi naive T cells. SP4, single-positive CD4. Representative of four experiments. (b) Blimp-1 mRNA in purified naive, memory and effector CD4+ (left) and CD8+ (right) peripheral T cell subsets (n ¼ 3). (c) Blimp-1 mRNA in purified naive CD4+ T cells and Treg cells (n ¼ 3). (d) Blimp-1 mRNA in purified naive CD4+CD44loCD62Lhi T cells stimulated for various times (horizontal axis) in vitro with plate-bound anti-CD3e, anti-CD28 and IL-2 (n ¼ 5). (e) Blimp-1 mRNA in naive and stimulated CD4+ T cells and in splenic B cells treated with lipopolysaccharide to cause plasma cell formation PCs. Error bars indicate 1 s.d.

Blimp-1 function in thymocytes We compared thymocyte development in CKO mice and Prdm1+/+Cre+ control mice. Young CKO mice (3.5–4.5 weeks of age) had higher frequencies of DN thymocytes and gd TCR+ thymocytes, but DN thymocytes expressed normal quantities of CD25 and CD44 (Fig. 3a). Total thymocyte numbers in CKO mice were reduced to about 35% that of the littermate controls (Fig. 3b, left), and this reduction was more apparent in older mice. Total numbers of gd TCR+ and DN thymocytes were normal (Fig. 3b,c), suggesting that the reduced thymocyte numbers were the result of smaller DP (36% of control) and single-positive (50% of control) populations (Fig. 3b). To further investigate that thymic defect, we analyzed the expression of developmental stage–specific surface markers. At each developmental stage, CKO thymocytes expressed normal quantities of CD5, CD69, TCRb and CD24 (Fig. 3c and data not shown). That indicated normal maturation of each thymocyte subset. To assess thymocyte proliferation, we injected mice with 5-bromodeoxyuridine (BrdU) and measured BrdU incorporation in each thymocyte subset. Control and CKO thymocyte subsets incorporated similar amounts of BrdU (Fig. 3d). However, when we assessed cell death by staining with annexin V, we noted twofold more apoptotic DP thymocytes in CKO mice than in littermate control mice when we analyzed these ex vivo and after in vitro stimulation with anti-CD3e and anti-CD28 (Fig. 3e). These data suggest that Blimp-1 is necessary for normal thymocyte survival.

Blimp-1 in colitis Because abnormal CD4+ T cell regulation and/or function can result in colitis12,14, we analyzed the CKO regulatory T cell (Treg cell) compartment. Control and CKO mice had equivalent numbers of CD8–CD4+CD25+ Treg cells in the thymus (Fig. 5a). The frequency and total number of CD4+CD25+CD62Lhi Treg cells was similar in young control and CKO mice (Fig. 5b), but Treg cell numbers increased as colitis progressed in older mice (data not shown). Purified control and CKO Treg cells suppressed the proliferation of control and CKO naive CD4+ cells in vitro after stimulation with soluble antiCD3e and antigen-presenting cells (APCs; Fig. 5c). Purified wild-type Treg cells protect wild-type mice from acute colitis induced by oral administration of dextran sodium sulfate (DSS)15. Wild-type but not CKO Treg cells blocked colitis in DSStreated mice (Fig. 6a). Thus, although CKO Treg cells seemed normal in vitro, they were defective in vivo in this model of colitis. We also tested the ‘colitogenic’ activity of naive CD4+ T cells in a colitis model involving adoptive transfer into lymphopenic hosts16. Both control and CKO naive CD4+ T cells caused colitis in recombination-activating gene 1–deficient recipient mice. However, CKO T cells induced more weight loss, produced higher pathology scores and led to the killing of two of six recipient mice before the end of the experiment (Fig. 6b). We conclude that CKO naive CD4+ T cells are hyper-responsive and abnormally ‘colitogenic’ and that CKO Treg cells are defective in protecting against colitis in vivo. Both defects probably contribute to the spontaneous development of colitis in CKO mice.

Effector T cells in CKO mice We compared frequencies and total numbers of peripheral T cell subsets in young (4–5 weeks of age) CKO and Prdm1+/+Cre+ control mice. Total numbers of splenic CD4+ and CD8+ T cells were similar in control and CKO mice, and CKO mice had a modest increase (of about 20%) in lymph node CD4+ cells. Lymph node CD8+ T cell numbers were similar in wild-type and CKO mice (data not shown). However, in the CD4+ and CD8+ splenic and lymph node compartments, the frequency of naive CD44loCD62Lhi T cells was decreased (Fig. 4a,b), whereas the frequency and total number of effector CD44hiCD62Llo T cells was increased (Fig. 4a,c). Wild-type and CKO mice had similar numbers of memory CD44hiCD62Lhi T cells (Fig. 4). Thus, CKO peripheral T cells have an abnormally activated phenotype.

Hyper-responsive Blimp-1-deficient T cells The hyperactivated state (Fig. 4) and increased ‘colitogenic’ properties (Fig. 6) of CKO CD4+ T cells prompted further analysis. CKO and control (Prdm1+/+Cre+) CD4+ T cells were similarly susceptible to activation-induced cell death (Fig. 7a,b). We also investigated the ability of CKO and control naive CD4+ T cells to proliferate in response to activation signals. We stained purified naive CD4+CD44loCD62Lhi T cells with a division-sensitive dye and activated the cells using various conditions (Fig. 7c–e). Naive CKO CD4+ T cells were hyperproliferative compared with control T cells when plated at very low density (Fig. 7c). CKO T cells were more proliferative than control T cells in response to stimulation with antiCD3e (Fig. 7d), but stimulation at higher cell density decreased the differences between control and CKO cells. Stimulation with a

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CD4

104 103

103

102

101

100 5.3 ± 0.6 100 101

2.4 ± 0.3 102

CKO 104

85.5 ± 1.2

7.0 ± 0.7

103

9.0 ± 0.8

9.3 ± 0.9 100 104 100 101

78.5 ± 0.2

Total cells (× 106)

3.1 ± 0.3 102

103

104

CD8

104

γδ TCR

103

104 0.6 ± 0.2

1.0 ± 0.2

103

102

101

100

101

102

103

104

100

1.0

0.5

0.0 Total

160

γδ

100

101

102

103

3 2

102

101

102

103

104

100

101

102

103

104

10 100 101 102 103 104

100 101 102 103 104

SP4 0

100 101 102 103 104

0 100 101 102 103 104 350 280 210 140 70 0 70 60 50 40 30 20 10 0

15 10 5 0 DN

100 101 102

100 101 102 103 104

TCRβ

Ex vivo

100 101 102 103 104

200 150

9.0 ± 1.4

100 0 100 101 102 103 104 250

240

19 ± 3.3

180

27 ± 2.8 M1

24 h

0 100 101 102 103 104

103 104

300

120

2.5 ± 0.3

15 10

120

100 101 102 103 104

5.4 ± 0.2

180

100 80 60 40 20

250

240

500 400 300 200 100 0 100 101 102 103 104

100 101 102 103 104

300

100 101 102 103 104

SP8

CKO

SP4

Control 90 1.3 ± 0.3

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SP8

CD5

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BrdU incorporation (%)

102

100 100

104

100 101 102 103 104

HSA

Counts

CD44

DP 103

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Counts

1.5

103

SP8

150

CD25

104

2.0

120

CD3

200

0 100 101 102 103 104

Annexin V

Figure 3 Thymocyte development in CKO mice. (a) Flow cytometry of Thy-1.2+-gated thymocytes from 4-week-old control mice (Prdm1+/+ Cre+; left) and CKO mice (right). Numbers in quadrants and above boxed areas indicate the frequency of subpopulations (mean ± s.e.m.; n ¼ 8). (b) Numbers of thymocyte subsets (horizontal axes) in 4-week-old control mice (gray bars) and CKO mice (open bars), presented as mean ± s.e.m. (n ¼ 8). SP4, single-positive CD4; SP8, single-positive CD8. (c) Flow cytometry of differentiation markers in thymocyte subsets (left margin) of control mice (black lines) and CKO mice (gray lines). Representative of four experiments. (d) In vivo BrdU incorporation in thymocyte subsets (horizontal axis) of 4-week-old mice control mice (gray bars) and CKO mice (open bars), presented as mean ± s.e.m. (n ¼ 5 mice per group). (e) Annexin V staining of thymocytes from control mice (left) and CKO mice (right) stimulated with anti-CD3 and anti-CD28 (times, right margin). Numbers above bracketed lines indicate percent annexin V–positive cells (mean ± s.e.m.; n ¼ 4 mice per group).

combination of anti-CD3e, anti-CD28 and IL-2 induced equal proliferation of CKO and control T cells (Fig. 7e). Measurements of [3H]thymidine incorporation also demonstrated hyperproliferation of CKO T cells (data not shown). We measured CKO CD4+ T cell IL-2 production by intracellular cytokine staining. Fivefold more CKO CD4+ T cells than control CD4+ T cells produced IL-2 after ex vivo treatment with phorbol 12myristate 13-acetate (PMA) and ionomycin (Fig. 7f,g). After 3 d of in vitro stimulation, 2.5-fold more CKO CD4+ cells produced IL-2

460

(Fig. 7f,g). Thus, when CKO naive CD4+ T cells are stimulated via the TCR, more cells produce IL-2 and proliferate. Blimp-1 in cytokine production Next we analyzed the production of other cytokines by CKO T cells. After stimulation with PMA and ionomycin, more CKO effector CD4+ T cells produced IFN-g and fewer CKO CD4+ T cells produced IL-4 or IL-10 (Fig. 8a,b). After 6 d of stimulation in neutral conditions, 2.5fold more CKO naive CD4+ T cells than control naive CD4+ T cells

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51 ± 4

CD4

103

102

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101 27 ± 6 100

CD62L

104

13 ± 2

103

100

CD8+

CKO

104

101

102

36 ± 1

103

104

100

103

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101

101 15 ± 5 100

101

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13 ± 2

47 ± 8 100

104

40 ± 5

103

100

33 ± 7

Frequency (CKO/Ctrl)

101

102

26 ± 4

103

104

40 ± 6

30 ± 8 100

101

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103

Spleen

CD4

CD8

0 N

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CD4

CD8

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CD4

CD8

0 N

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CD4

CD8

104

CD44

0 N

Figure 4 Hyperactivation of CKO peripheral T cells. (a) Flow cytometry of control (left) and CKO (right) splenic CD4+ and CD8+ T cells. Numbers in quadrants indicate frequencies of naive (CD44loCD62Lhi), effector (CD44hi, CD62Llo) and memory (CD44hiCD62Lhi) T cell subsets (mean ± s.e.m.; n ¼ 4). (b) CKO/control ratio (CKO/Ctrl) of the frequency (b) and number (c) of naive (N), effector (E) and memory (M) CD4+ and CD8+ T cells in the spleens and lymph nodes of 4- to 5-week-old mice, presented as mean ± s.e.m. (n ¼ 4). Horizontal lines indicate no difference between CKO and control.

102

CD4+

CD4

Bcl-6 expression in CKO T cells To begin defining target genes regulated by Blimp-1 in CD4+ T cells, we quantified Bcl-6 and c-Myc transcripts, whose expression is repressed by Blimp-1 in B cells. CKO CD4+

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CKO 104 103 102

3.9 ± 0.8

101

100

100 100 101 102 103 104

100 101 102 103 104

0 Ctrl

103

104 7.6 ± 0.4

103

102

101

7.2 ± 0.9

100 100 100 101 102 103 104 100 101 102 103 104

Total Treg cell number (×106)

104

CD62L

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CD25

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Inhibition (%)

Figure 5 Treg cells in CKO mice. (a,b) Flow cytometry of Treg cells. Numbers in dot plots (left) indicate frequency; total numbers are in graphs (right). Data represent mean ± s.e.m. for three 4- to 6-week-old mice per group. (c) In vitro suppressor activity of control and CKO Treg cells. Left, representative dot plot of sorted CD4+CD25+ T cells indicates 97% purity. Right, effector T cells were stimulated with T cell– depleted, mitomycin C–treated syngeneic splenic cell samples (APCs) and soluble anti-CD3e (1 mg/ml) in the presence of increasing numbers of Treg cells (wedges). Effector T cell proliferation was assessed by [3H]thymidine incorporation during the last 8 h of a 72-hour culture; the percentage of suppression in the presence of Treg cells was calculated as 100 [1 – (count with Treg cells / counts without Treg cells)]. Data points represent means of triplicate wells and errors bars represent s.d.

effector T cells had twice the abundance of Bcl6 mRNA transcripts as did control effector cells, but there was no difference in Bcl-6 transcripts in CD4+ memory T cells comparing control and CKO mice (Fig. 8f). We detected no reproducible differences in c-Myc mRNA expression in cells from control and CKO mice (data not shown). The gene encoding IL-5 is a direct target of Bcl-6. Consistent

Total Treg cell number (×106)

produced IFN-g and fewer CKO CD4+ T cells than control naive CD4+ T cells produced IL-10 (Fig. 8c,d). Further analysis of acutely stimulated effectors showed that whereas both CD25– and CD25+ CD4+ T cell subsets demonstrated reduced IL-10 production, the defect was greater in the CD25+ subset, which included Treg cells and CD25+ effector T cells (Fig. 8e). That was consistent with inability of CKO Treg cells to block colitis in vivo (Fig. 6a). These results suggest that Blimp-1 Ctrl is important for regulating cytokine produca 104 tion and that its absence results in a skewing 103 toward stimulatory T helper type 1 responses.

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Our analyses have demonstrated that Blimp-1 mRNA is present in varying amounts in thymocytes and peripheral T cells. Why has Blimp-1 mRNA not been noted before in T cell lines or primary T cells3? Why have analyses of mice expressing a GFP reporter gene in the Prdm1 locus failed to demonstrate Blimp-1 expression in splenic T cells17? Perhaps the Blimp-1 transcripts in thymocytes and naive T cells were below the level of detection of the RNA blotting used in previous studies. Subsequent microarray studies have detected Blimp1 mRNA in thymocytes13. In addition, Blimp-1 mRNA is greatest in effector CD4+ and CD8+ T cells, which are often present at sites of inflammation and therefore may have been overlooked during previous studies. Detection of Blimp-1 protein by immunohistochemistry in extrafollicular CD3e+ T cells in normal human tonsils and in T cells that infiltrate B cell lymphomas18 is consistent with the large quantities of mRNA we found here in effector T cells. Increased apoptosis of CKO thymocytes showed that even though there was relatively little Blimp-1 mRNA in thymocytes, that amount was functionally important for normal survival. Thymocytes lacking Blimp-1 proliferated normally, but Blimp-1deficient DP thymocytes were more susceptible to apoptosis. It seems likely that positive and/or negative selection is defective in Blimp-1deficient thymocytes, but more work is needed to understand precisely if and how Blimp-1 functions during selection. A function for Blimp-1 in developing T cells was unexpected, because the only known function for Blimp-1 in B cells is in terminal differentiation1. This previously unknown aspect of Blimp-1 activity suggests that

CKO naive

80 60 40

n=6 Average CS = 2.4

0 1 2 3 4 5 6 7 8 9

Time after injection (weeks)

Figure 6 Blimp-1 in colitis. (a) Weight loss of mice injected with control (Prdm1+/+Cre+) or CKO CD4+CD25+ (CD62Lhi and CD62Llo, 1:1 ratio) Treg cells or PBS that received drinking water containing 3% DSS at 8–12 h after cell transfer. Data are representative of five experiments (PBS, n ¼ 18; control Treg cells, n ¼ 6; CKO Treg cells, n ¼ 8). (b) Weight loss in C57BL/6 recombination-activating gene 1–deficient mice injected with control (left; n ¼ 5) or CKO (right; n ¼ 6) naive CD4+CD62LhiCD44loCD45RBhi T cells. Body weight was measured weekly and mice were monitored for clinical symptoms. Mice with severe disease (none of five receiving control cells; two of six receiving CKO cells) were killed before the end of the experiment. CS, colitis score, determined using histological sections of colons removed from mice on the day they were killed or at the end of the experiment. Error bars represent s.e.m.

with their high expression of Bcl-6 mRNA, CKO effector CD4+ T cells had less IL-5 transcripts than did control effector CD4+ T cells (data not shown). Thus, we conclude that Blimp-1 directly or indirectly inhibits Bcl-6 expression in CD4+ effector T cells.

462

0 100 101 102 103 104

0 100

50 40

104

160

0 100 101 102 103 104

120 80 40 0 100

100

Ctrl

120

101

102

103

104

Anti-CD3 + anti-CD28 + IL-2

0 Ctrl

CKO

Ctrl

CKO

Ctrl

CKO

100

CKO

80 50

60 40 Counts

20 0 100

101

102

103

104

CFSE

103

200

Annexin V

102

72%

101

Anti-CD3

Counts

CKO

100

67%

Control 40

Anti-CD3 + anti-CD28 + IL-2

Annexin V–positive 7-AAD– cells (%)

Figure 7 Hyper-responsive CKO CD4+ T cells. (a,b) Representative flow cytometry (a) and frequency (b) of annexin V–positive 7-aminoactinomycin D–negative (7AAD–) naive CD4+CD44loCD62Lhi T cells at a 24-hour time point (a) or various times after stimulation (horizontal axis, b). Numbers above bracketed lines (a) indicate frequency of annexin V–positive cells in the 7-amino-actinomycin-D–negative cell gate. The annexin V gate was based on the fluorescence of unstained cells (dotted lines). (c–e) Proliferation of CFSE-labeled control and CKO naive CD4+ T cells in response to stimulation (above histograms); cells were plated at low (c) and high (d,e) density. Graphed data represent the frequency of cells in the live gate that divided more than two times. (f) Frequency of control (gray bars) and CKO (open bars) CD4+ T cells producing IL-2 after treatment with PMA and ionomycin (PMA + iono; left) or plate-bound anti-CD3e, anti-CD28 and IL-2 (CD3 + CD28; right), determined after five and three experiments, respectively. (g) Representative flow cytometry of IL-2 intracellular cytokine staining from the experiment in f. Numbers in boxed areas indicate percent IL-2+ cells.

6 IL-2-producing cells (relative to control)

DISCUSSION Mice lacking Blimp-1 in the T cell lineage show excessive DP thymocyte apoptosis and have fewer DP and single-positive thymocytes. Blimp-1-deficient mice have defects in T cell homeostasis and function that are manifested by decreased frequencies of naive and increased frequencies of effector T cell subsets, defective Treg cell function, altered cytokine production and development of spontaneous colitis.

Cells with more than two divisions (%)

Control naive

24 Time (h)

PMA + iono

104 103

4 CD3 + CD28

5 3

IL-2

100

b Weight (% of initial)

Weight (% of initial)

a 110

4 2

1 1

104

103

CKO 22% Total

102

101

100 100 101 102 103 104

104

103

Control

13%

103

27%

102

101

100 100 101 102 103 104

Naive

0 Total

Naive

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IFN-γ

104

21%

CKO 36%

103

102

101

4% 100 100 101 102 103 104

IL-4 104

1.0

104

16%

103

0.5

IFN-γ

IL-4

IL-10

Cytokine-producing cells (relative to control)

1.5

0.0

IL-10

103

102

101

100 100 101 102 103 104

CD4

104

Control

104

103

102

IFN-γ

101

CKO 17%

101

100 100 101 102 103 104

IL-4 104

104

25%

103

IL-10

Cytokine-producing cells (relative to control)

IFN-γ

IL-4

IL-10

103

102

101

17%

100 100 100 101 102 103 104 100 101 102 103 104

CD4

104

Control 5%

11%

104

103

102

101

CKO 3%

100 100 100 101 102 103 104 100 101 102 103 104

400 Relative Bcl-6 mRNA

IL-10

2.0

Control

104

300

200

100

CD25 0 Memory Effector

possible involvement of Blimp-1 in early B cell development19 should be reinvestigated. Attenuation of TCR signaling is important in both resting and activated T cells20. TCR signaling is modulated by the intensity of the signal, the amount of costimulation, the quantities of intracellular signaling mediators and the activation of transcription factors21–24. Transcription factors such as Foxo3a25 and Foxj1 (ref. 26) inhibit activation in resting T cells, and their absence results in T cell hyperproliferation and multiorgan inflammation. Blimp-1 also modulates TCR activation but, in contrast to Foxo3a and Foxj1, which have high expression in naive cells and are downregulated after TCR activation, Blimp-1 is present in low concentrations in naive cells and is induced after TCR activation. Thus, Blimp-1 is likely to function at time points later than Foxo3a and Foxj1. In particular, Blimp-1 may attenuate TCR responses of activated T cells. Colitis in Blimp-1 CKO mice is consistent with a failure to regulate activated T cells. We do not yet know how Blimp-1 mRNA is induced in response to TCR signals or why the process is slow. In B cells, transcription factors AP-1 (ref. 27), NF-kB (unpublished data) and STAT3 (ref. 28) activate Prdm1 transcription, whereas Bcl-6 represses Prdm1 transcription1,27,29. Those regulators may also operate in T cells. AP-1, NFAT and NF-kB are activated after TCR stimulation, and many cytokines that activate STAT3 are important in T cell effector function30. It seems likely AP-1, NF-kB and STAT3 may induce Prdm1 transcription in T cells, whereas the involvement of transcription factors NFAT

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Figure 8 IFN-g, IL-4 and IL-10-production in CD4+ CKO T cells. (a,b) Frequency (a) and representative flow cytometry (b) of cells producing IFN-g, IL-4 and IL-10 in sorted effector CD44hiCD62LloCD4+ T cell populations after stimulation for 4 h ex vivo with PMA and ionomycin. (c,d) Frequency (c) and representative flow cytometry (d) of cells producing IFN-g, IL-4 and IL-10 from purified naive CD44loCD62LhiCD4+ T cell populations stimulated for 6 d in vitro with anti-CD3, anti-CD28 and IL2. Data (a,c) are control (gray bars) and CKO (open bars), presented as the mean and s.d. from five (a) or seven (c) experiments. Numbers in top left and bottom right quadrants (b,d) indicate the frequency of IFN-g+ and IL-4+ cells, respectively; boxed numbers indicate the frequency of IL-10+ cells. (e) Representative intracellular cytokine staining for IL-10, showing CD25 expression. Data are representative of three experiments. (f) Steady-state expression of Bcl-6 mRNA (normalized to 18S RNA) in control (gray bars) and CKO (open bars) purified memory (CD44hiCD62Lhi) and effector (CD44hiCD62Llo) CD4+ T cells. Data are representative of two experiments.

and T-bet or other transcription factors induced by TCR stimulation remains to be investigated. NFAT is an intriguing candidate regulator of Prdm1 transcription, as deletion of different NFAT family members results in thymic defects, T cell hyperproliferation or T cell hyperactivation31–34. The slow kinetics of Blimp-1 mRNA induction, however, are not consistent with a simple model in which Blimp-1 induction depends solely on transcription factors directly activated by TCR signaling. Although products of genes induced by those transcription factors might be involved in Prdm1 expression, we suspect that Prdm1 transcription is partially repressed in naive and recently activated T cells and that this repression must be relieved before transcription can be fully induced after TCR stimulation. Bcl-6 is a likely candidate for the repression of Prdm1 transcription in T cells, as it directly represses Prdm1 transcription in B cells27,29,35. We found a reciprocal relationship between Blimp-1 and Bcl-6 mRNA in DP thymocytes, Treg cells, CD4+ and CD8+ naive and CD8+ effector T cells (L.C. and K.C., unpublished data), which is consistent with Bcl-6-mediated repression of Prdm1 transcription. However, analysis of Bcl-6-deficient mice will be needed to prove if and when Bcl-6 represses Prdm1 transcription in T cells. Our observation that Blimp-1 repressed Bcl6 expression in T cells and B cells is notable, and dysregulated Bcl6 expression may contribute to the phenotype of CKO mice. Although no involvement of Bcl-6 in T cell proliferation has been reported, Bcl-6 is expressed in activated T cells36, competes with STAT6 to repress cytokine gene transcription37,38 and is required for the generation and maintenance of CD8+ memory T cells39. It will be useful to explore more thoroughly the function of Bcl-6 in T cells and to study the settings in which Blimp-1 represses Bcl6 expression. However, complete understanding of the molecular mechanism of Blimp-1 action in T cells awaits microarray analyses to identify target genes in thymocytes and peripheral T cell subsets. TCR responses vary with developmental stage and strength of signal40,41. TCR signals control positive and negative selection of thymocytes40,42. The thymic defects in CKO mice suggest that Blimp-1 may modulate the threshold of TCR responsiveness during selection. TCR and costimulatory signals regulate the activation and delicate homeostasis of naive, effector and memory T cell subsets by balancing self-renewal, differentiation and death40,43. In addition, TCR signals induced in the periphery by complexes of self peptide and major histocompatibility complex are critical for the survival and homeostatic proliferation of T cells43,44. Impaired survival of CKO DP thymocytes and decreased numbers of CKO single-positive thymocytes might result in decreased thymic output to the periphery

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ARTICLES of CKO mice, which could trigger homeostatic proliferation and differentiation of naive CKO T cells43,44. Thus, loss of Blimp-1mediated TCR signal attenuation might affect various stages of T cell development. Although CKO mice had normal numbers of Treg cells that acted like control Treg cells in vitro, CKO Treg cells failed to block colitis in vivo. Although we injected equal ratios of CD62Lhi and CD62Llo Treg cells in those experiments, the possibility of different frequencies of newly activated CD4+ T cells in the control and CKO CD62Llo subsets cannot be excluded. However, such differences would be minor and would probably not interfere with Treg cell function. Thus, we conclude CKO Treg cells were defective in the setting we tested. Defective IL-10 production might contribute to impaired CKO Treg cell function, as IL-10 is important for Treg cell function in vivo but not in vitro45. Transforming growth factor-b responses might also be impaired in CKO mice. Mice expressing a truncated transforming growth factor-b receptor in the T cell lineage have Treg cells that are functional in vitro but fail to block DSS-induced colitis15 or naive T cell transfer–induced colitis in vivo46. Colitis can result from excessive effector T cell function, impaired Treg cell function47 or defective thymocyte development48 or selection49. Lymphopenia can also produce a predisposition to colitis50. CKO mice had hyper-responsive naive CD4+ cells, increased T helper type 1 cytokine concentrations and defective Treg cells. In addition, defects in thymocyte selection and early lymphopenia resulting from decreased thymic output may occur. A combination of these abnormalities is likely to contribute to the development of colitis in CKO mice. Our work here has identified the Blimp-1 transcriptional repressor as a notable and previously unknown regulator in T cells. We have also demonstrated that Blimp-1 regulates thymocyte survival and T cell homeostasis and function. Our data provide a basis for further work to elucidate signals that induce Blimp-1 transcription and to identify Blimp-1-induced T lineage gene expression programs. METHODS Mice. Prdm1flox/flox mice2 were crossed with Lck-Cre mice11 to generate Prdm1flox/floxCre+, Prdm1flox/+Cre+, Prdm1+/+Cre+, Prdm1flox/floxCre– and Prdm1flox/+Cre– mice. Mice were housed in the barrier facility of Columbia University (New York, New York). All experiments were approved by the institutional animal care and use committee of Columbia University. Southern blot. Genomic DNA was isolated from the thymus and digested with SacI, and Southern blot analyses were done according to published methods2. Flow cytometry. Lymph node, spleen and thymus cell suspensions were collected and erythrocytes were lysed. Cells were counted and were stained with fluorochrome-conjugated antibodies specific for CD3e, CD4, CD5, CD8, CD25, CD44, CD62L, gd TCR, HSA, TCRb, Thy-1.2 (all from eBioscience), BrdU, IFN-g, IL-2, IL-4 or IL-10 (BD Pharmingen). Histology. Mice were killed at various ages, colons were flushed and tissues were fixed with 10% formalin for sectioning, followed by hematoxylinand-eosin staining. Slides were analyzed by a pathologist ‘blinded’ to sample identity. Cell purification and CFSE (carboxyfluorescein diacetate succinimidyl diester) labeling. Naive CD4+CD62LhiCD44lo T cells were purified from spleen and pooled lymph node suspensions with a CD4– isolation kit (Dynal Biotech) according to the manufacturer’s instructions, with the addition of anti-CD44, to allow depletion of effector and memory CD4+ T cells. The isolated fraction was more than 90% CD4+CD62LhiCD44lo. Labeling with CFSE or CFDASE (carboxyfluorescein diacetate succinimidyl ester; Molecular Probes) was done essentially as described51.

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Lymphocyte proliferation assays. CFSE-labeled purified CD4+CD44loCD62Lhi cells were cultured on plates coated with 5 mg/ml of anti-CD3 and 2.5 mg/ml of anti-CD28 (3 105 cells/well in 500 ml per well in 48-well plates or in 200 ml per well in 96-well round-bottomed plates). Where indicated, recombinant human IL-2 (25 IU/ml) was added. Cells were maintained for 3 d at 37 1C in a humidified atmosphere of 5% CO2. In vitro suppression assay. Sorted CD4+CD44loCD25– effector T cells were cultured for 72 h at 37 1C in 5% CO2 in 96-well plates with an equal number of APCs, 1 mg/ml of anti-CD3e and various numbers of sorted CD4+CD25+ Treg cells. Cells were pulsed with methyl-[3H]thymidine (0.5 mCi per well) for the last 8 h of culture. The percentage of suppression was calculated 100 [1 – (count with Treg cells / counts without Treg cells)]. Assessment of activation-induced cell death. Naive CD4+ T cells (2 106 cells/well in a volume of 1.5 ml in 24-well plates) were cultured with APCs plus 2 mg/ml of soluble anti-CD3e. After 48 h, T cells were separated from APCs and debris with Histopaque-1083 gradient (Sigma), were washed three times and then were cultured for another 24 h in the presence of 50 U/ml of IL-2. Cells were collected, were washed again and then were plated (2 105 cells/well in a final volume of 200 ml) in flat-bottomed microwell tissue culture plates coated with anti-CD3e. Apoptosis was assessed 8 and 24 h later by staining with 7-amino-actinomycin D and annexin V (Pharmingen). The frequency of annexin V–positive cells was determined in the 7-amino-actinomycin D–negative subset. Total RNA isolation, quantitative PCR and RT-PCR analysis. Total RNA was isolated from flow cytometry–purified thymocyte, CD4+ and CD8+ T cell subsets and in vitro–activated naive CD4 T cells with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA (1 mg) was reverse-transcribed with Superscript III (Invitrogen) and cDNA was diluted fivefold for amplification. Genomic deletion of loxP-flanked Prdm1 in peripheral T cell subsets and RT-PCR analyses were done with the ABI7700 Sequence Detection System (Applied Biosystems; primer sequences, Supplementary Table 1 online). In vivo BrdU labeling. Mice 4 weeks of age were killed 1 h after intraperitoneal injection with 100 ml of a 10-mg/ml solution of BrdU. BrdU incorporation was analyzed with the BD Pharmingen BrdU Flow Kit according to the manufacturer’s instructions. Intracellular cytokine staining. Total and effector CD4+ T cells were stimulated for 2 h at 37 1C in 5% CO2 with 20 ng/ml of PMA and 1 mg/ml of ionomycin (Sigma) and for an additional 4 h with monensin (GolgiStop; BD Pharmingen) for blockade of cytokine secretion. Naive CD4+ T cells were stimulated for 3 d in the presence of 5 mg/ml of anti-CD3e, 2.5 mg/ml of antiCD28 and 25 units/ml of recombinant human IL-2 and were allowed to ‘rest’ for 3 d before being restimulated for 6 h with 2.5 mg/ml of ant-CD3e and 1.25 mg/ml of anti-CD28, with monensin added for the final 4 h. Cells were fixed with 4% paraformaldehyde and were made permeable with 0.5% saponin for intracellular staining. Colitis models. The induction of colitis by transfer of naive CD4+ T cells was done as described52 with modifications. C57BL/6 recombination-activating gene 1–deficient mice were injected intraperitoneally with 4 105 purified naive CD4+CD62LhiCD45RBhiCD44loCD25– cells in 200 ml of PBS. DSS (MP Biochemicals) with a molecular weight of 36–50 kDa was dissolved in distilled autoclaved water to provide a working solution of 3% (weight/volume). For colitis induction, mice were given free access to this solution for the duration of the experiment. Mice were weighed weekly and were inspected for clinical signs of disease. Mice with clinically severe disease were killed according to the guidelines of the Columbia University animal care and use committee. Adoptive transfer of Treg cells. Treg cells were sorted from CD8-depleted spleen and lymph node cell suspensions from control or CKO mice. Some CKO mice showed altered distribution of the CD62Lhi and CD62Llo subsets. To avoid transfer of different ratios of the CD62Lhi and CD62Llo Treg cell subpopulations from control and CKO mice, we sorted these two populations separately and transferred equal numbers of each population, totaling 7.5 105 Treg

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Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank H. Gu and Y.-R. Zou for advice and for critical reading of the manuscript; the Calame laboratory for advice; J. Liao for assistance with the mice; and D. Savitsky for help with the DSS colitis experiments. Supported by the National Institutes of Health (RO1 AI50659 and RO1 AI43576 to K.C.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/natureimmunology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Shapiro-Shelef, M. & Calame, K. Regulation of plasma cell development. Nat. Rev. Immunol. 5, 230–242 (2005). 2. Shapiro-Shelef, M. et al. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory cells. Immunity 19, 607–620 (2003). 3. Turner, C.A., Jr., Mack, D.H. & Davis, M.M. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 77, 297–306 (1994). 4. Shaffer, A.L. et al. XBP1 acts downstream of Blimp-1 to expand the secretory apparatus, promote organelle biogenesis, and increase protein synthesis during plasma cell differentiation. Immunity 21, 81–93 (2004). 5. Chang, D.H. & Calame, K.L. The dynamic expression pattern of B lymphocyte induced maturation protein-1 (Blimp-1) during mouse embryonic development. Mech. Dev. 117, 305–309 (2002). 6. Vincent, S.D. et al. The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Development 132, 1315–1325 (2005). 7. Chang, D., Angelin-Duclos, C. & Calame, K. BLIMP-1: trigger for differentiation of myeloid lineage. Nat. Immunol. 1, 169–176 (2000). 8. Angelin-Duclos, C., Cattoretti, G., Lin, K.I. & Calame, K. Commitment of B lymphocytes to a plasma cell fate is associated with blimp-1 expression in vivo. J. Immunol. 165, 5462–5471 (2000). 9. Shaffer, A.L. et al. Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program. Immunity 17, 51–62 (2002). 10. Lin, K.I., Angelin-Duclos, C., Kuo, T.C. & Calame, K. Blimp-1-dependent repression of Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma cells. Mol. Cell. Biol. 22, 4771–4780 (2002). 11. Takahama, Y. et al. Functional competence of T cells in the absence of glycosylphosphatidylinositol-anchored proteins caused by T cell-specific disruption of the Pig-a gene. Eur. J. Immunol. 28, 2159–2166 (1998). 12. Bouma, G. & Strober, W. The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 3, 521–533 (2003). 13. Tabrizifard, S. et al. Analysis of transcription factor expression during discrete stages of postnatal thymocyte differentiation. J. Immunol. 173, 1094–1102 (2004). 14. Powrie, F., Correa-Oliveira, R., Mauze, S. & Coffman, R.L. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179, 589–600 (1994). 15. Huber, S. et al. Cutting edge: TGF-b signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells. J. Immunol. 173, 6526–6531 (2004). 16. Powrie, F. Immune regulation in the intestine: a balancing act between effector and regulatory T cell responses. Ann. NY Acad. Sci. 1029, 132–141 (2004). 17. Kallies, A. et al. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J. Exp. Med. 200, 967–977 (2004). 18. Cattoretti, G. et al. PRDM1/Blimp-1 is expressed in human B-lymphocytes committed to the plasma cell lineage. J. Pathol. 206, 76–86 (2005). 19. Angelin-Duclos, C., Johnson, K., Liao, J., Lin, K.I. & Calame, K. An interfering form of Blimp-1 increases IgM secreting plasma cells and blocks maturation of peripheral B cells. Eur. J. Immunol. 32, 3765–3775 (2002). 20. Liu, J.O. The yins of T cell activation. Sci. STKE 2005, re1 (2005). 21. Dykstra, M., Cherukuri, A., Sohn, H.W., Tzeng, S.J. & Pierce, S.K. Location is everything: lipid rafts and immune cell signaling. Annu. Rev. Immunol. 21, 457–481 (2003).

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