Transcriptional repressor Blimp-1 is essential for T cell ... - Nature

© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

ARTICLES

Transcriptional repressor Blimp-1 is essential for T cell homeostasis and self-tolerance Axel Kallies, Edwin D Hawkins, Gabrielle T Belz, Donald Metcalf, Mirja Hommel, Lynn M Corcoran, Philip D Hodgkin & Stephen L Nutt T cell homeostasis is crucial for a functional immune system, as the accumulation of T cells resulting from lack of regulatory T cells or an inability to shut down immune responses can lead to inflammation and autoimmune pathology. Here we show that Blimp-1, a transcriptional repressor that is a ‘master regulator’ of terminal B cell differentiation, was expressed in a subset of antigen-experienced CD4+ and CD8+ T cells. Mice reconstituted with fetal liver stem cells expressing a mutant Blimp-1 lacking the DNA-binding domain developed a lethal multiorgan inflammatory disease caused by an accumulation of effector and memory T cells. These data identify Blimp-1 as an essential regulator of T cell homeostasis and suggest that Blimp-1 regulates both B cell and T cell differentiation.

The size of the peripheral T cell pool, comprising naive, effector and memory T cells, is very stable in adult mice because of a homeostatic balance of cell survival, proliferation and differentiation. An inability to control peripheral T cell numbers through loss of important negative regulatory molecules1,2 or defects in apoptosis3,4 results in inflammation and autoimmune disease. The specificity of peripheral T cells is also tightly controlled, as autoreactive T cells that have escaped thymic deletion are kept in check by mechanisms of peripheral self-tolerance such as deletion and anergy5. Finally, regulatory T cells (Treg cells) provide another essential means of preventing autoimmunity by limiting immune responses to self and foreign antigens6,7. The homeostasis of T cells is known to involve intrinsic factors that guide T cell differentiation and activation as well as extrinsic factors such as cytokines of the common g-chain family. Whereas interleukin 2 (IL-2) controls T cell numbers indirectly by maintaining Treg cells in the periphery8,9, IL-7 and IL-15 are directly involved in the generation and survival of memory T cells10. Transcription factors that intrinsically control peripheral T cell homeostasis include LKLF and Foxo3a, which are required for T cell quiescence11,12. Once CD4+ T cells are activated, other transcription factors, including GATA-3 and T-bet, are important for their differentiation into the T helper (TH) cell lineages13,14. Beyond those relatively proximal responses to T cell stimulation, little is known about the transcriptional regulation of later stages of T cell maturation and the formation of memory. Only a few transcription factors, such as T-bet, eomesodermin and Bcl-6, are known to be required for the generation and maintenance of CD8+ T cell memory15,16. The transcriptional regulation of the differentiation of B cells into antibody-secreting plasma cells, in contrast, is relatively well understood and is known to require the transcriptional repressor B

lymphocyte–induced maturation protein 1 (Blimp-1)17. Blimp-1 is expressed in all plasma cells, whose ontogeny can be defined by quantitative changes in Blimp-1 (ref. 18). Mice with B cell–specific deletion of the gene encoding Blimp-1 (Prdm1) lack mature plasma cells and have considerably reduced serum immunoglobulin titers19. On the molecular level, Blimp-1 is postulated to drive plasma cell differentiation by repressing Pax5 and Bcl-6, factors that control B cell identity and the germinal center reaction, respectively17. Blimp-1 is also thought to be essential for the extinction of Myc expression and exit from the cell cycle, a characteristic of terminal differentiation20. Those data collectively indicate that Blimp-1 is a ‘master regulator’ of plasma cell differentiation. Blimp-1 is also reported to be involved in myeloid cell differentiation21 and is widely expressed during embryogenesis, in which it is required for germ cell formation22–24. Those studies raise the possibility that Blimp-1 is more broadly involved in cellular differentiation. Here we show that Blimp-1 was expressed in a defined subset of effector and memory T cells of both the CD4 and CD8 lineages. Blimp-1 deficiency resulted in dysregulated T cell homeostasis and a lethal inflammatory disease with autoimmune characteristics as well as impaired apoptosis of effector T cells in vitro. These data demonstrate that Blimp-1 is a key regulator of the later stages of T cell differentiation. RESULTS Inflammatory syndrome in Blimp-1-deficient mice A ‘knock-in allele’ consisting of the genes encoding Blimp-1 (Prdm1), an internal ribosomal entry site and green fluorescent protein (GFP) has been shown to serve as a precise reporter of Prdm1 expression in antibody-secreting cells18. That allele expresses GFP and a truncated

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia. Correspondence should be addressed to S.L.N. ([email protected]). Received 17 January; accepted 15 February; published online 26 March 2006; doi:10.1038/ni1321

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CD8+

470

80 Survival (%)

CD4+

gfp/gfp (n = 24) +/+, +/gfp (n = 19)

60

Rag2–/– gfp/gfp (n = 6)

40

198

0

0

20

CD44

0

b

20

40 60 80 100 120 Time after reconstitution (d)

Lung

Liver

140

d Colon +/+

80 IFN-γ (ng/ml)

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60 40 20 0

+/+ gfp/gfp

B220

F4/80 Lung gfp/gfp

Blimp-1 protein that is deficient in DNA binding and nonfunctional. Embryos homozygous for that allele die during late gestation. Reconstitution of adult hematopoiesis with fetal liver stem cells from Prdm1gfp/gfp embryos fully recapitulated the phenotype reported for a B cell–specific Prdm1 mutation19, characterized by a lack of plasma cells and considerably reduced serum immunoglobulin18 (data not shown). After initially normal hematopoiesis, Rag1–/– mice reconstituted with Prdm1gfp/gfp fetal liver cells (called ‘Prdm1gfp/gfp mice’ here) developed substantial weight loss, ruffled coat and diarrhea and were killed between 4 and 18 weeks after reconstitution (Fig. 1a). Histological analysis of these mice showed a complex phenotype with autoimmune and inflammatory disease characteristics, including extensive lymphocyte infiltration, tissue destruction and inflammation of a variety of organs, including lung, liver and gut (Fig. 1b). The mice also had increased splenic cellularity (wild-type, 8.1 107 ± 1.4 107 cells; Prdm1gfp/gfp, 15.6 107 ± 5.5 107 cells; n ¼ 6) and mesenteric lymph node cellularity (wild-type, 2.25 107 ± 0.3 107 cells; Prdm1gfp/gfp, 12.6 107 ± 2.8 107 cells; n ¼ 4 of each genotype). Analysis of lymphoid organs and livers of the reconstituted mice showed substantial population expansion of CD4+ and CD8+ T cells

0.6

with an activated or memory phenotype (Fig. 1c and data not shown). Phorbol 120.2 myristate 13 acetate and ionomycin stimula0 tion of CD4+ T cells isolated from those mice +/+ gfp/gfp resulted in considerable interferon-g (IFN-g) 3 production and low IL-4 and IL-10 secretion, which is indicative of a TH1 inflammatory 2 response (Fig. 1d). In contrast, Rag1–/– mice 1 reconstituted with recombination-activating gene 2–deficient (Rag2–/–) Prdm1gfp/gfp fetal 0 +/+ gfp/gfp liver cells remained healthy for the time during which they were monitored (over 12 months), indicating the dependence of the phenotype on T cells and/or B cells (Fig. 1a). 0.4

Blimp-1 in activated and memory T cells Analysis of Prdm1+/gfp mice showed GFP expression in a subset of peripheral CD4+ and CD8+ T cells, whereas thymocytes lacked R1

a

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12.6

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72.6

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9.30

11.5

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77.7

CD25

+/gfp 0.03

0.02

75.9

gfp/gfp 0.64

1.64

17.6

8.04

49.8

CD62L

Figure 2 Blimp-1 is expressed in and controls the number of effector and memory CD4+ T cells. (a) Surface expression of activation markers (along margins) on splenic CD4+ cells gated for GFP. CD4–GFPhi cells (left) are antibody-secreting cells with a mean fluorescence index six times higher than that of CD4+GFP+ cells. Similar results were obtained in three independent experiments. (b) Expression of GFP in and CD62L on splenic CD4+ cells of age-matched mice (genotypes, above dot plots). Results are representative of at least ten independent experiments. Numbers in quadrants indicate percentage of cells in each.

68.5

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Figure 1 Blimp-1-deficient mice develop a lethal lymphocyte hyperproliferative syndrome. (a,b) Survival curves (a; genotypes and number of mice in key) and histological examination (b) of Rag1–/– mice reconstituted with fetal liver cells. Histology staining (b): top and middle rows, hematoxylin and eosin; bottom row, hematoxylin and various antibodies (above images). Original magnification, 100 (lung and liver, top two rows), 50 (colon) and 100 (lung, bottom row). Data in b are representative of four wild-type and twelve Prdm1gfp/gfp mice. (c) Surface phenotype of splenic T cells (solid lines, Prdm1gfp/gfp; dashed lines, wild-type). Data are representative of six experiments. (d) Cytokine production by splenic CD4+ T cells stimulated ex vivo with phorbol 12-myristate 13 acetate and ionomycin. Similar data were obtained in two experiments. gfp/gfp, Prdm1gfp/gfp; +/gfp, Prdm1+/gfp; +/+, Prdm1+/+.

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139

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176

CD62L

d 265

CD122

2444

0

1

Gated CD8+ +/+

+/gfp

gfp/gfp

CD95

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Figure 3 Blimp-1 is expressed in and controls the number of effector and memory CD8+ T cells. (a) Surface phenotype of gated splenic CD8+ T cells (genotypes, above dot plots). Data are representative of six experiments. (b) GFP expression in splenic CD8+ memory T cells (CD44+CD122+) from Prdm1+/gfp mice (solid line) and wild-type mice (dashed line). Similar results were obtained in two independent experiments. (c) Expression of surface markers (above histograms) on Prdm1gfp/gfp (solid line) and wild-type (dashed line) CD8+ T cells. Results are representative of two to ten independent experiments. (d) GFP expression in antigen-specific effector CD8+ T cells. Mice were infected with HSV and were analyzed after 7 d for CD8+ T cells specific for the dominant gB(498–505) epitope with a gB-specific tetramer. Results were similar for four mice of each genotype. (e) Top, cytotoxic function of cultured gB-specific CD8+ T cells against EL4 targets pulsed with the gB peptide. Results are representative of two independent experiments. Bottom, tetramer-binding CD8+ cells, counted for each individual mouse at 7 or 55 d after infection. *, P ¼ 0.0146.

GFP (Figs. 2 and 3 and data not shown). GFP+CD4+ T cells were CD44hi and mainly CD62Llo, a cell surface phenotype indicative of effector and memory CD4+ T cells (Fig. 2). Consistent with Prdm1 expression in effector and memory T cells, GFP+CD4+ T cells were heterogeneous for CD25 and had high expression of other activation markers such as CD122 and GITR (Supplementary Fig. 1 online). In healthy heterozygous mice, the splenic GFP+CD4+ population increased with age from less than 3% in 6-week-old mice to up to

17.6

Ly5.2

46.9

18.8

45.3

24.0

11.9

c

**

7 6

130

5

120

4 3 2 1 0

7

+/+, +/gfp (n = 8) gfp/gfp (n = 8)

7 Cells/spleen (×106)

11.8

CD44

23.7

Gated CD8+

10% in mice older than 6 months (Fig. 2b and data not shown). GFP expression in the CD8+ T cell lineage was similarly restricted to CD44hiCD122hi effector and memory cells (Fig. 3a,b). Prdm1gfp/gfp mice had a considerable expansion of the GFP+ T cell population and a substantial increase in the proportion and total number of effector and memory T cells of both the CD4+ and CD8+ T cell lineages, with most peripheral T cells displaying a CD44hiCD62Llo phenotype (Supplementary Fig. 1). As in the heterozygous mice, GFP+

Body weight (%)

b Gated CD4+

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a

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55

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6

5

10

15

20

25

30

+/+ gfp/gfp

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+/+ +/gfp gfp/gfp Figure 4 Blimp-1 intrinsically regulates the homeostatic population 69 61 40 expansion of T cells. (a) Flow cytometry of splenic T cells 19 weeks CD4+ after reconstitution of Rag1–/– (Ly5.1+) mice with a 1:1 mixture of 100 Prdm1gfp/gfp (Ly5.2+) and C57BL/6 (Ly5.1+) fetal liver cells. Numbers 80 in quadrants indicate the percentage of cells in each. Results are 0 0 0 representative of ten mice. (b) Percentage of tetramer-specific cells in 60 75 56 75 the Ly5.1+ and Ly5.2+ CD8+ T cell populations of chimeric mice CD8+ 40 generated as described above and infected with HSV, assessed 7 and 55 d after infection. The corresponding values from individual recipient 20 mice (wild-type (Ly5.1+), open circles; Prdm1gfp/gfp (Ly5.2+), filled circles) 0 0 0 0 are connected by a line. **, P ¼ 0.0052. (c) Percent change in body GFP +/+ gfp/gfp weight (left) and splenic T cell numbers (right) after adoptive transfer of 6 + 3 10 CD4 cells (genotypes, key (left) or horizontal axis (right)) into Rag2–/– recipients. Data represent two experiments (left) and three experiments (right; each dot represents an individual mouse). (d) GFP expression in various T cell populations (above histograms) 3 weeks after transfer into Rag2–/– recipients. (e) Splenic T cell numbers after adoptive transfer of a mixture of 1.5 106 Prdm1gfp/gfp (Ly5.2+) and 1.5 106 Prdm1+/+ (Ly5.1+) CD4+ cells into Rag2–/– recipients. Splenic T cells of each genotype were assessed after 4 weeks and data are presented as average ± s.d. for nine mice. Total events


GFP

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CD44

CD44

R1

75

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180

160 120

103 c.p.m.

100

80

60

40

0.20

Myc

80

Foxp3

Foxp3

+/+ CD25+

p gf p/

Il10 Ctla4

0 1

2 4 8 16 32 64 128 CD25–/CD25+

gfp/gfp CD25+

1

4 Colitis score

3 2 1 0

CD25

gfp/gfp –

+

CD25–

CD25

4 8 16 32 64 128 – + CD25 /CD25

Fasl

e

+/+ CD25–

2

CD4+ T cells (105)

–

d

4 8 16 32 64 128 Dilution factor

gf

Ifng 40

– +/+ /gfp – +/+ /gfp p p gf gf

+/+

gfp/gfp

140

Body weight (% max)

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+

Irf4

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120

20 CD4


7.6

Hprt1 Prdm1

140 29.4

+/

gfp/gfp CD25–, gfp/gfp CD25+

CD25+

gf

gfp/gfp CD25–, +/+ CD25+

+/+ CD25–, +/+ CD25+ +/+ CD25–, gfp/gfp CD25+

+

+/+ CD25– gfp/gfp CD25– +/+ CD25+ gfp/gfp CD25+

0.24

gfp/gfp

CD25–

c

p/

b

+/

2.6

14.1

gf

+/+

a

80 60 40 20 0

CD25+ CD25–

– +/+ /gfp – +/+ /gfp p p gf gf

+/+

gfp/gfp

100 95 90 85 80 75 0

CD25+ CD25–

– +/+ /gfp – +/+ /gfp p p gf gf

+/+

gfp/gfp

Figure 5 Blimp-1 is dispensable for Treg cell function. (a) Flow cytometry of Foxp3 expression in splenic CD4+ T cells from wild-type or Prdm1gfp/gfp mice. Numbers in quadrants indicate percent CD4+Foxp3– (top left) or CD4+Foxp3+ (top right). Results are representative of two independent experiments. (b) Analysis of the suppression of in vitro proliferation by Treg populations. CD25+ and CD25– CD4+ T cells from Prdm1gfp/gfp or wild-type mice were sorted and were cultured alone or together (keys). Triplicate wells were pulsed with [3H]thymidine at 72 h and incorporation was measured 8 h later. Data represent the average ± s.d. of triplicate wells and are representative of three experiments. (c) Semiquantitative RT-PCR of cDNA from CD4+ T cells from Prdm1gfp/gfp or wild-type mice, sorted according to CD25 expression. Hprt1 encodes hypoxanthine guanine phosphoribosyl transferase 1; Irf4 encodes interferonregulatory factor 4. Data are representative of two independent experiments. (d,e) Histology of colon (d), colitis score (e, left), number of CD4+ T cells in the mesenteric lymph nodes (e, middle) and body weight (e, right) of Rag1–/– mice injected with various CD4+ T cell populations (left margin and above images) (d) or below horizontal axis (e). Transferred CD25– cells were CD45RBhi, and CD25+ cells were CD45RBlo. Original magnification (d), 50; data are representative of at least five mice for each treatment. Body weight (e) is presented as percent of maximum; data are from two independent experiments, with each dot representing an individual mouse.

Prdm1gfp/gfp cells had high expression of CD122 and GITR (Supplementary Fig. 1 and Fig. 3c), whereas expression of the early activation marker CD69 was increased only slightly (Supplementary Fig. 1). Thus, Prdm1 was expressed in a subset of CD4+ and CD8+ T cells with the characteristics of effector and memory cells, and loss of Blimp-1 function led to expansion of that population. The GFP expression profile suggested that Blimp-1 was induced in antigen-experienced T cells. To directly test that hypothesis, we infected wild-type, Prdm1+/gfp and Prdm1gfp/gfp mice with herpes simplex virus (HSV) and quantified virus-specific CD8+ T cells using a major histocompatibility class I tetramer comprising an immunodominant peptide derived from amino acids 498–505 of HSV glycoprotein B (gB(498–505)) in complex with H-2Kb (ref. 25). This demonstrated similar population expansion of antigen-specific effector CD8+ T cells that expressed Prdm1 (Fig. 3d). Furthermore, these experiments showed that Prdm1gfp/gfp mice mounted an appropriate immune response and indicated that antigen-specific Prdm1gfp/gfp CD8+ cells were able to lyse target cells (Fig. 3e). Kinetic analysis demonstrated a small but significant increase in number of CD8+ tetramer-binding memory cells in Prdm1gfp/gfp mice at day 55 after infection with HSV (P ¼ 0.0146; Fig. 3e), indicating that the increased numbers of effector and memory cells seen in polyclonal T cell populations were also found in antigen-specific Blimp-1-mutant CD8+ T cell populations.

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Intrinsic activation of Blimp-1-mutant T cells To assess whether the population expansion of effector and memory T cells in Rag1–/– mice reconstituted with Blimp-1-mutant stem cells was T cell intrinsic, we used mixed fetal liver reconstitution (1:1 ratio, C57BL/6 to Prdm1gfp/gfp). Although the presence of wild-type hematopoietic cells resulted in reduced lethality (2 of 14 chimeric mice versus 24 of 24 Prdm1gfp/gfp mice died within 19 weeks), a higher percentage of Prdm1gfp/gfp T cells than wild-type T cells had cellintrinsic maintenance of the effector and memory phenotype (Fig. 4a). The proportion of Prdm1gfp/gfp (Ly5.2+) CD4+ T cells with a CD62Llo effector and memory phenotype was on average 3.8-fold higher than the proportion of C57BL/6 (Ly5.2–) CD4+ T cells with such a phenotype, and the proportion of activated Prdm1gfp/gfp CD44+CD8+ T cells was 2.9-fold higher than the proportion of activated wild-type cells (n ¼ 10; Fig. 4a). That finding was supported by the consistently higher percentage of mutant tetramer-positive CD8+ T cells present in HSV-infected chimeric mice, suggesting a cell-intrinsic advantage of Blimp-1-mutant memory cells (Fig. 4b). Mixed reconstitution into Rag1–/recipients using a 1:1 ratio of Rag2–/– to Prdm1gfp/gfp fetal liver cells demonstrated pathology indistinguishable from that of Prdm1gfp/gfp mice, indicating that the alleviation of the disease in C57BL/6–Prdm1gfp/gfp chimeras was mediated by lymphocytes (data not shown).

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a

CD4 +/+

+/gfp

Total events Total events

gfp/gfp 217

344

344

TH1

0

0

0

304

321

317

TH2

0

0

0

GFP

c 160

Blimp-1 t-Blimp-1

80

Zap70

40

IgM

0

PU.1

1 2 3 4 5 6 7 8 9 10 11 12

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50 40 30 20 10 0

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40 t-Blimp-1

20 0

+ gf /+ p/ gf p

IL-4 (ng/ml)

300

TH2 IL-10 (ng/ml)

TH1

Actin

+ gf /+ p/ gf p

MFI

120

d

+/+ TH1

C D 62 + D ay 1 D ay D 3 ay 5 D ay 7 D ay D 9 + ay /g fp 9 AS C M

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Figure 6 Blimp-1 expression in vitro. (a) GFP expression in CD62L+CD4+ cells grown for 14 d in TH1- or TH2-polarizing conditions. (b) Time-course analysis of GFP induction in differentiating Prdm1+/gfp cells in TH1promoting conditions. (c) Immunoblot for Blimp-1 protein expression in wild-type CD4+CD62+ cells ex vivo (CD62+) or cultured in TH1-polarizing conditions (time, above lanes). Blots with anti-Zap70 (T cells) and anti-actin serve as loading controls; blots with anti-IgM and anti-PU.1 demonstrate the absence of contaminating cells. ASC, antibody-secreting cells; M, macrophages. (d) Cytokine production of cultured TH1 and TH2 cells, measured by ELISA. (e) Immunoblot of full-length and truncated Blimp-1 protein expression in TH1 cells collected after 10 d. t-Blimp-1, truncated Blimp-1 lacking exons 7 and 8. Results are representative of two to three independent experiments.

+

Zap70

The expanded pool of activated T cells in the Prdm1gfp/gfp mice suggested aberrant responsiveness to antigen stimulation or dysregulated homeostasis. To assess the homeostatic regulation of Blimp-1deficient T cells, we adoptively transferred purified CD4+ or CD8+ T cells from C57BL/6 or Prdm1gfp/gfp mice into Rag1–/– recipients and assessed splenic T cell numbers after 3–4 weeks. Whereas wild-type T cell populations responded to the lymphopenic environment by undergoing limited homeostatic expansion, Prdm1gfp/gfp CD4+ and CD8+ T cells showed substantially enhanced population expansion and induced weight loss similar to that seen in irradiated mice reconstituted with Prdm1gfp/gfp fetal liver stem cells (Fig. 4c). Homeostatically expanding Prdm1+/gfp and Prdm1gfp/gfp T cell populations expressed GFP and, like their wild-type counterparts, had an activated phenotype (Fig.4d and data not shown). Enhanced expansion also occurred in the presence of C57BL/6 CD4+ cells, indicating that wild-type T cells were not sufficient to control the proliferation of the Blimp-1-mutant CD4+ cells (Fig. 4e). Thus, Blimp-1-mutant T cells underwent dysregulated population expansion that resulted in multiorgan infiltration and lethality, linking Blimp-1 to the control of T cell homeostasis. Limited involvement of Blimp-1 in Treg cell function Similar inflammatory syndromes to that described here have been associated with the loss of Treg cells expressing the transcription factor Foxp3 (refs. 26,27). As wild-type T cells were able to reduce the severity of the wasting disease, we speculated that Blimp-1 was involved in Treg cell function. Moreover, we noted GFP expression in a fraction of CD4+CD25+ cells known to contain Treg cells28 (Fig. 2b). Intracellular staining for Foxp3, however, indicated that Foxp3+ Treg cells were formed in the absence of Blimp-1 (Fig. 5a). To test whether Blimp-1-mutant Treg cells were functional, we sorted Prdm1gfp/gfp and wild-type CD4+CD25+ T cells and analyzed them by

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an in vitro suppression assay26. Prdm1gfp/gfp and wild-type CD4+CD25+ cells were equally able to supress the proliferation of Blimp-1-mutant and wild-type CD25– responder cells and, as has been reported before for Treg cells, did not respond to stimulation with antibody to CD3 (anti-CD3; ref. 26; Fig. 5b). RT-PCR analysis provided independent confirmation of Prdm1 transcription in a fraction of CD4+CD25+ T cells. The expression of other genes linked to Treg cell function was unaltered in Prdm1gfp/gfp cells (Fig. 5c). An exception was Il10, which was undetectable in Blimp-1-mutant CD4+CD25+ T cells, despite having normal expression in Blimp-1mutant TH2 cells generated in vitro (Figs. 5c and 6 discussed below). IL-10 is an immunosuppressive cytokine essential to the prevention of enterocolitis and contributes to the function of Treg cells in vivo29,30. To test Treg cell function in an in vivo model, we injected CD4+CD25– T cells from Prdm1gfp/gfp and wild-type mice into Rag1–/– recipients to induce colitis31. Wild-type CD4+CD25– T cells caused severe colitis within 6–8 weeks of transfer, and Prdm1gfp/gfp and wild-type CD25+ cells were equally efficient in preventing the disease (Fig. 5d,e). Similarly, Blimp-1-mutant CD4+CD25– T cells induced colitis, but many more T cells accumulated in the mesenteric lymph nodes of these recipients than in the mesenteric lymph nodes of recipients of wild-type CD4+CD25– T cells (Fig. 5e, middle). Although Prdm1gfp/gfp and wild-type CD25+ cells were capable of reducing the severity of the colitis and the number of cells accumulating in the mesenteric lymph nodes, they were unable to fully prevent the disease induced by Blimp1-mutant T cells, as shown by the increased T cell numbers and higher colitis scores of recipients of Blimp-1-mutant CD4+CD25– T cells (Fig. 5e). These data collectively indicate that Blimp-1-deficient Treg cells were present and functional but neither they nor wild-type Treg cells could efficiently control the in vivo population expansion of Blimp-1-mutant effector T cells. Blimp-1 in TH cell differentiation To assess Prdm1 expression during the differentiation of CD4+ T cells into effector cells, we grew CD62L+CD4+GFP– T cells in TH1- or TH2polarizing conditions. GFP analysis indicated that Blimp-1 was induced in effector cells of both TH lineages (Fig. 6a) and showed a concordance between the induction of GFP and Blimp-1 protein in Prdm1+/gfp and wild-type TH1 cultures (Fig. 6b,c). Prdm1gfp/gfp CD4+ T cells were capable of differentiating into TH1 and TH2 effector cells and had secretion of IFN-g, IL-4 and IL-10 similar to that of wild-type effector cells (Fig. 6d), indicating that the lack of IL-4-producing CD4+ T cells in Prdm1gfp/gfp mice ex vivo was not due to an intrinsic inability to generate TH2 cells. These data show that Blimp-1 was induced during the normal differentiation program of T cells into TH1 and TH2 effector cells but was not required for cytokine secretion. Analysis of the GFP fluorescence in TH1 and TH2 effector cells showed that its intensity was notably more than expected in

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+/+

+/gfp

74

0

0

0

54

54

40

+

CD8 primary (total events)

4

Cells (10 )

230

+

CD8 blast

+/+ IL-2 gfp/gfp IL-2

14 12

gfp/gfp

90

10 15 10 8 5 6 0 4 2 0 0

20

30

CD8 blast (total events)

40

15

20

10

0

3.0

+/+ TH2 gfp/gfp TH2

25

d

2 0

1.0

Total cells (%)

Cells (104)

1.5

15 10 5

0.5 0 0

20 40 60 80 100 120 140 Time (h)

0

40 60 80 100 120 140 160 Time (h) +/+ TH1 gfp/gfp TH1

e

20

40

60 80 100 120 140 Time (h)

+/+ TH1 gfp/gfp TH1 +/+ TH2 gfp/gfp TH2

50 40

60 40 20

30 20 10

0 0

20 40 60 80 100 120 140 160 180 Time (h)

0

80

2.5 2.0

20

100

20

8 7.5 5.0 6 2.5 0 4

Cells (104)

+/+ TH1 gfp/gfp TH1

3.5

+/+ IL-15 + IL-21 gfp/gfp IL-15 + IL-21

10

0 0

Cells (104)

12

5

GFP

c

100

4

20

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Figure 7 Blimp-1 regulates T cell numbers in vitro. (a) GFP expression in CD44loCD8+ cells cultured with anti-CD3, anti-CD28 and IL-2 for 7 d (primary) or allowed to ‘rest’ for 3 d and restimulated for 5 d (blast). (b) Total live cell numbers after culture of CD44loCD8+ T cells with anti-CD3, anti-CD28 and various cytokines (keys; left) and restimulation of CD8+ T cell blasts with anti-CD3, anti-CD28 and various cytokines (keys; right). Insets, average cell numbers (± s.d.) of three experiments at 96 h (left) and 140 h (right). (c) Cell numbers, assessed after culture of TH1 or TH2 cells (genotypes, keys) with anti-CD3, anti-CD28 and IL-2 (c). Cell numbers ± s.d. were determined in triplicate, and data are representative of three experiments. (d) Percentages of Ly5.1+ (wild-type) and Ly5.2+ (Prdm1gfp/gfp) TH1 cells, determined at various times (horizontal axis) after Prdm1gfp/gfp and wild-type TH1 cells were mixed at a ratio of 1:1 and cultured in the presence of anti-CD3, anti-CD28 and IL-2. Data are representative of three experiments. (e) Cell numbers, assessed after culture of TH1 cells (genotypes, key) with IL-2. Cell numbers ± s.d. were determined in triplicate, and data are representative of three experiments.

Prdm1gfp/gfp versus Prdm1+/gfp cells (Figs. 2b, 3a and 6a). Immunoblot analysis, however, did not demonstrate substantial overexpression of the truncated Blimp-1 protein compared with that of full-length Blimp-1 protein in differentiated Prdm1+/gfp or Prdm1gfp/gfp TH1 cells (Fig. 6e). This indicated that the large amounts of GFP found in effector and memory T cells in Prdm1gfp/gfp mice reflected an accumulation of the GFP protein rather than artificially high expression from the mutated Prdm1 allele. Blimp-1 controls T cell proliferation and apoptosis To determine if the dysregulated homeostasis of Blimp-1-mutant T cells in vivo was manifested as enhanced proliferation in vitro, we cultured CD44loCD8+ T cells in the presence of anti-CD3, anti-CD28 and cytokines known to regulate T cell proliferation and homeostasis (IL-2, IL-15 and IL-21)32. CD8+ T cells grown in those conditions had little GFP expression and cumulative cell numbers were similar in wild-type and Prdm1gfp/gfp cultures (Fig. 7a,b, left). In contrast, subsequent stimulation of long-term-cultured CD8+ T cell blasts in the presence of IL-2 or IL-15 and IL-21 resulted in GFP expression and a substantially higher number of Prdm1gfp/gfp cells compared with wild-type control cells (Fig. 7a,b, right). The increase was most prominent in the presence of IL-15 and IL-21, cytokines known to support the population expansion of CD8+ memory T cells32. As with the CD8+ T cell lineage, there were no differences in proliferation or cell numbers when naive CD44loCD62L+CD4+ T cells were cultured in the presence of anti-CD3, anti-CD28 and

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IL-2 (data not shown). However, Prdm1gfp/gfp TH1 and TH2 cells generated more cells than did wild-type TH1 and TH2 cells after restimulation (Fig. 7c), indicating a conserved function for Blimp-1 in controlling effector cell numbers in the CD8+ and CD4+ T cell lineages. The increase in cell number was not due to differential production of cytokines, as Prdm1gfp/gfp TH1 cells maintained their advantage over wild-type cells even in mixed cultures (Fig. 7e). Cumulative cell numbers were similar when effector CD4+ or CD8+ T cell populations were expanded in IL-2 only (Fig. 7d and data not shown), as were proliferation rates, as measured by 5-bromodeoxyuridine (BrdU) incorporation during restimulation experiments (Fig. 8a). We therefore speculated that the increased numbers of Prdm1gfp/gfp cells in the restimulation experiments were due to diminished activation-induced cell death. Staining with annexin V showed a much higher proportion of wild-type than Prdm1gfp/gfp T cells undergoing apoptosis in cultures restimulated with anti-CD3 and anti-CD28 (Fig. 8b), indicating that a lower death rate of Blimp-1-mutant cells was the cause of their increased numbers. Prdm1gfp/gfp cells also had delayed cell death in a standard cytokine withdrawal assay (Fig. 8c). These data demonstrate that impaired cell death resulted in the increased numbers of Blimp-1-mutant T cells noted in vitro and suggest a similar mechanism might contribute to the expanded effector T cell compartment found in Prdm1gfp/gfp mice in vivo. To determine if Blimp-1 directly regulates genes known to be involved in effector T cell survival, we used RT-PCR analysis of

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Figure 8 Blimp-1-mutant T cells are less susceptible than wild-type cells to apoptosis. (a) Percentage of TH1 and CD8+ blasts incorporating BrdU after 3 d of restimulation (genotypes, below graph). Data are the average ± s.d. of three independent experiments. (b) Annexin V staining of propidium iodide–negative cells in TH1 and TH2 or CD8+ blast T cell cultures 3 d after restimulation (Prdm1gfp/gfp, solid lines; wild-type, dashed lines). (c) Time course of TH1 viability after cytokine withdrawal. (d) RT-PCR of various transcripts in naive CD4+ T cells (CD62L+) or in vitro–differentiated TH1 and TH2 cells. Bcl2l1 encodes Bcl-xL. Results are representative of two to three independent experiments.

ex vivo or cultured CD4+ T cells. These studies did not demonstrate substantial differences in expression of the gene encoding Myc, a Blimp-1 target in B cells, or in the expression of genes encoding other key regulatory molecules such as Bcl-2, Bcl-xL and CTLA-4, suggesting that Blimp-1 does not regulate those pathways of T cell survival (Figs. 5c and 8d). Similarly, the accumulation of activated T cells could be attributed to a failure of the Fas–FasL death receptor pathway, but RT-PCR and flow cytometry detected expression of both Fas (CD95) and FasL on Blimp-1-deficient T cells (Figs. 2c, 3c and 5c). Future gene expression screens will be needed to identify the target genes of Blimp-1 in T cells. DISCUSSION Blimp-1 is required and sufficient for the terminal differentiation of activated B cells into antibody-secreting plasma cells. The data presented here have demonstrated a previously unappreciated function for Blimp-1 in controlling the later stages of T cell maturation and homeostasis. Loss of Blimp-1 function led to an accumulation of T cells with an effector and memory phenotype and to a severe inflammatory wasting disease that resembled systemic autoimmunity. In healthy mice, Blimp-1 is restricted to a small subset of antigen-experienced CD4+ and CD8+ T cells. As that is the precise population that is expanded as a consequence of Prdm1 mutation, our data suggest that control of these effector and memory T cells represents the essential function for Blimp-1 in T cell homoeostasis. Similar defects in T cell homeostasis have been reported for mice deficient in a variety of regulatory molecules, including members of the NFAT family of transcription factors33, CTLA-4 (ref. 1), transforming growth factor-b2, IL-10 (ref. 29) and IL-2, as well as components of the IL-2 receptor complex34. Although all of those molecules are widely expressed in the T cell compartment, the pathology associated with their deficiency has mainly been attributed to a lack of Treg cell function6,7,35–37. Treg cells are distinct CD4+ T cells that require the transcription factor Foxp3 for their development and function. Notably, mice and humans lacking Foxp3 fail to produce Treg cells and develop a systemic autoimmune pathology6,7. Blimp-1, in contrast, is expressed only in some Treg cells and seems to be dispensable for the development and function of this lineage, as Blimp-1-deficient Treg cells functioned adequately in vitro and in an in vivo model of induced enterocolitis. Further support for the Blimp-1 independence of Treg cells function came from the analysis of chimeric mice, which showed that the activated phenotype of

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Prdm1gfp/gfp T cells persisted in the presence of wild-type T cells. That is in contrast to the hyperactivation and autoimmune phenotypes of CTLA-4-deficient or IL-2-deficient mice, which can be ameliorated by the presence of wild-type T cells34,37. Those data suggest that Blimp-1 does not regulate the homeostasis of T cells via Foxp3-expressing Treg cells but has a function intrinsic to effector and memory T cells. However, as the wasting disease seen in Prdm1gfp/gfp mice was substantially alleviated in the presence of wild-type lymphocytes, we cannot exclude the possibility that Blimp-1 is required for other, lesswell-defined regulatory populations such as type 1 regulatory T cells38. Those cells have substantial secretion of IL-10 and are thought to arise during persistent antigen stimulation such as in the gut or during unresolved infections39. The reduced amounts of Il10 mRNA found in Blimp-1-deficient T cells may indicate a defect in the generation of type 1 regulatory T cells. During an immune response, a range of different intrinsic and extrinsic signals dictates the differentiation program of T cells. Common g-chain cytokines are known to be involved in controlling the ‘burst size’ of antigen-specific T cells as well as the generation and maintenance of T cell memory10. IL-15 is crucial for the generation and homeostasis of memory CD8+ T cells, whereas IL-21 increases effector T cell turnover, resulting in lymphopeniainduced homeostatic proliferation and diabetes in nonobese diabetic mice40. Notably, IL-15 and IL-21 have the dual properties of being essential for T cell homeostasis and being potent inducers of Prdm1 transcription in vitro, thus providing a direct link between the extrinsic and intrinsic regulation of T cell homeostasis. IL-21 also stimulates Prdm1 expression during B cell differentiation41, suggesting a common function for this cytokine in B cell and T cell differentiation. Blimp-1 was initially described as a virally induced repressor of IFN-b in fibroblasts42. Although the relevance of that finding to the adaptive immune system is unknown, type I interferons are also known to be involved in immune homeostasis and autoimmunity43. Many transcription factors, most notably T-bet and GATA-3, are essential for the differentiation and polarization of TH cells13,14, whereas others, including Foxo3a and LKLF11,12, are responsible for aspects of T cell quiescence. Notably, all of those factors are expressed either in naive T cells or shortly after activation, whereas Blimp-1 is induced at a later point in T cell maturation. That expression profile demonstrates that Blimp-1 regulates a distinct phase of T cell differentiation well after the initial activation and polarization events.

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ARTICLES Effector T cells are short-lived and either die or convert into memory cells after antigen exposure. Those events seem to be ‘genetically programmed’, as they do not depend on the dose or persistence of antigen44. The T-box transcription factors T-bet and eomesodermin as well as Bcl-6 are required for memory formation and maintenance in the CD8 lineage15,16. RT-PCR analysis, however, indicated that the quantities of Tbx21 (encoding T-bet) and Bcl6 were unchanged in the absence of Blimp-1, suggesting that those factors are either ‘parallel to’ or ‘upstream of’ Blimp-1. In B cell differentiation, Bcl-6 and Blimp-1 mutually antagonize each other’s expression17. It is will be useful to test whether they also have this relationship in the T cell lineage. We propose that the late expression of Blimp-1 delineates a distinct developmental stage of T cell maturation in which Blimp-1 itself regulates the transcriptional program that controls the number of effector and memory T cells, allowing only a few cells to progress to the memory T cell compartment. Notably, Blimp-1-mutant T cells had a salient defect in activation-induced cell death in vitro, thus providing a possible explanation for increased effector T cell pool and the disturbed T cell homeostasis in vivo in the absence of Blimp-1. B lymphocytes and T lymphocytes have many features in common during their development, including a shared progenitor, antigenreceptor gene recombination and developmental checkpoints. Whereas the terminal differentiation of B cells to either short-lived plasmablasts or long-lived plasma cells are distinct functional end points that are Blimp-1 dependent17, the late stages of T cell ontogeny (after the acquisition of effector function) are less well defined. The evidence presented here that Blimp-1 is essential for the regulation of the homeostasis of the main T cell lineages raises the possibility that Blimp-1 is a conserved regulator of the terminal differentiation program in all lymphocytes. METHODS Mice. Prdm1+/gfp mice were produced and maintained on a C57BL/6 background and fetal liver chimeras were generated as described18. Rag1–/– and Rag2–/– mice were backcrossed for more than nine generations onto the C57BL/6 genetic background. Animal experiments were done according to Melbourne Health Animal Ethics Committee guidelines. Flow cytometry and enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies to CD4 (GK1.5), CD8 (53.6.7), CD69 (H1.2F3), TCRb (H57597) and Ly5.2 (ALI-4A2) were purified from hybridoma supernatants and were conjugated to biotin (Pierce), allophycocyanin or phycoerythrin (ProZyme). Biotinlyated or phycoerythrin-conjugated anti-CD25 (7D4) and anti-CD122 (Tm-b1) and phycoerythrin-conjugated anti-CD44 (IM7), antiCD45RB (16A), anti-CD62L (MEL-14) and anti-CD95 (Jo2) were obtained from BD Biosciences. Anti-Foxp3–allophycocyanin (FJK-16s) was from eBiosciences. Biotinylated annexin V and anti-GITR were provided by D. Huang and Y. Zhan (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia). Biotinylated monoclonal antibodies were visualized with streptavidin-phycoerythrin or indodicarbocyanine (Southern Biotech). Cells were analyzed on a LSR flow cytometer (BD Biosciences) and cells were sorted with high-speed flow cytometers (Moflo cytomation and BD Biosciences). ELISA for IFN-g and IL-10 was done as described45. The IL-4 ELISA used monoclonal antibody BVD4-1D11 as a capture reagent and biotinylated BVD6-24G2.3 for detection. ELISAs were done in triplicate and were quantified with recombinant protein. CD62L+CD44loCD4+

Cell culture. T cells were sorted by flow cytometry to more than 99.5% purity and were cultured in the presence of 100 U/ml of recombinant IL-2 (R&D Systems) and 2 mg/ml of anti-CD28 (37.51) on plates coated with 10 mg/ml of anti-CD3 (145-2C11). Cultures included IL-12 (5 ng/ ml; R&D Systems) and anti-IL-4 (10 mg/ml) for TH1 development or IL-4 (20 ng/ml; R&D systems) and anti-IFN-g (10 mg/ml) for TH2 development. For restimulation experiments with differentiated TH1 or TH2 cells, cultures were

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split on day 7 and were collected on day 14. Cells were then washed and restimulated. CD44loCD8+ T cells were purified by flow cytometry and were cultured with IL-2, soluble anti-CD28 and plate-bound anti-CD3. To obtain CD8+ T cell blasts, CD44loCD8+ T cell populations were expanded for 10 d as described above, were allowed to ‘rest’ for 3 d in IL-2 and were reseeded in soluble anti-CD28 and plate-bound anti-CD3 with either IL-2 or IL-15 and IL-21 (50 ng/ml; R&D Systems). In restimulation experiments, dead cells were removed by Histopaque separation (Sigma). For cell proliferation assays, cultures were pulsed for 3–4 h with BrdU (Sigma) and uptake was measured with monoclonal anti-BrdU (Molecular Probes). For cytokine withdrawal assays, cell populations were expanded for 10 d as described above, were allowed to ‘rest’ for 3 d in 100 U/ml of IL-2, were washed three times and were cultured in media alone. Viable cell numbers were determined with propidium iodide exclusion and CaliBRITE beads (BD Biosciences). Immunoblot and RT-PCR. Total protein extracts were produced from equivalent numbers of cells, followed by immunoblot with monoclonal anti-Blimp-1 (ref. 18). Protein loading and cell purity were confirmed with anti-Zap70 (99F2; Cell Signaling), anti-IgM (Southern Biotechnololgy), anti-actin and anti-PU.1 (T21; Santa Cruz). RT-PCR was done as described46. Primer sequences are in the Supplementary Methods online. HSV infection and tetramer staining. Mice were infected with 4 105 plaqueforming units of HSV-1 (KOS strain) by injection into the hindleg hock25. Spleen and popliteal lymph nodes of infected mice were collected for analysis at day 7 or day 55 after infection. Virus-specific CD8+ T cells were identified by flow cytometry with tetramers of H-2Kb and gB(498–505) (SSIEFARL)25. For gB-specific cytotoxic T lymphocyte assays, splenocytes were cultured for 5 d with 1 108 irradiated (1,000 Gy), gB(498–505)-coated C57BL/6 spleen cells. Cytotoxicity was assessed by conventional 51Cr-release assay with EL4 (H-2b) target cells pulsed with 1 mM gB(498–505)47. Histology and immunohistochemistry. Organs were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned and were stained with hematoxylin and eosin. Immunohistochemistry was done as described48. Homeostatic proliferation. CD4+ T cells were isolated with CD4-specific beads, and CD8+ T cells were isolated with a biotinylated anti-CD8 and antibiotin beads (Miltenyi). Purity, as determined by flow cytometry, was more than 95%. In some experiments, cells were sorted to 99.8% purity by flow cytometry. Rag1–/– recipients were injected intravenously with 3 106 cells and were monitored for weight loss. Mice were analyzed 3 weeks after injection or after weight loss of more than 10%. In vitro suppression assays. These assays were done as described26. For proliferation assays, CD4+ T cells (2 104 per well) were stimulated for 72 h with ‘titrated’ amounts of anti-CD3 in the presence of 8 104 T cell– depleted irradiated splenocyte samples as antigen-presenting cells, and were pulsed with 1 mCi per well of [3H]thymidine for the final 8 h of culture. Suppression assays were done in triplicate with 2 104 flow cytometry–sorted CD25–CD4+ T cells as responders, 8 104 irradiated splenocytes and a 1:2 ‘titration’ of various CD25+CD4+ T cells as suppressors at a starting concentration of 2 104 cells/well in the presence of 5 mg/ml of anti-CD3. Colitis model. Colitis was induced in Rag1–/– mice by injection of 5 105 flow cytometry–sorted CD45RBhiCD25–CD4+ T cells with or without 1 105 CD45RBloCD25+CD4+ T cells31. Mice were monitored for weight loss and were killed after 6–8 weeks or a loss of body weight of more than 10%. The severity of disease was assigned a score between 1 and 5 as described49: 0–1, no colitis; 2, mild colitis; 3–5, severe colitis. Statistics. The two-tailed paired or unpaired Student’s t-test was used for statistical analysis. ACKNOWLEDGMENTS We thank J. Carneli, K. D’Costa, L. Di Rago and J. Brady for assistance; D. Huang, G. Davey, Y. Zhan, J. Dromey, W. Heath, D. Tarlinton and A. Strasser for reagents, advice and critical reading of the manuscript; and S. Read (Bio21 Molecular Science and Biotechnology Institute, Melbourne, Victoria, Australia) for help in setting up and analyzing the colitis model. Supported by The

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ARTICLES Walter and Eliza Hall Institute (Metcalf Fellowship to S.L.N.), the Deutsche Forschungsgemeinschaft (A.K. and M.H.), the Leukaemia Foundation of Australia (A.K.), the National Institutes of Health (CA22556 to D.M.), the Wellcome Trust (G.T.B.), Howard Hughes International Fellowship (G.T.B.) and the National Health and Medical Research Council of Australia.


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