© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
A dominant function for interleukin 27 in generating interleukin 10–producing anti-inflammatory T cells Amit Awasthi1,5, Yijun Carrier2,5, Jean P S Peron3, Estelle Bettelli2, Masahito Kamanaka4, Richard A Flavell4, Vijay K Kuchroo2, Mohamed Oukka1 & Howard L Weiner2 Regulatory T cells (Treg cells) expressing the transcription factor Foxp3 are key in maintaining the balance of immune homeostasis. However, distinct induced T regulatory type 1 (Tr1) cells that lack Foxp3 expression also regulate T cell function, mainly by producing the immunosuppressive cytokine interleukin 10 (IL-10). However, the factors required for the induction of IL-10-producing suppressive T cells are not fully understood. Here we demonstrate that dendritic cells modified by Treg cells induced the generation of IL-10-producing Tr1 cells. The differentiation of naive CD4+ T cells into IL-10-producing cells was mediated by IL-27 produced by the Treg cell–modified dendritic cells, and transforming growth factor-b amplified the generation of induced IL-10+ Tr1 cells by IL-27. Thus, IL-27 and transforming growth factor-b promote the generation of IL-10-producing Tr1 cells.
T cell activation and differentiation are tightly regulated events, as naive T cells can differentiate through the T helper type 1 cell (TH1 cell), TH2 cell or interleukin 17 (IL-17)–producing T helper cell (TH-17 cell) pathway to exert specific effector functions during an immune response1. Exaggeration of these pathways can lead to inflammation and tissue injury. T regulatory cells (Treg cells) positive for expression of the transcription factor Foxp3 have a dominant function in active immune suppression and the maintenance of immune homeostasis2, although other regulatory T cells such as TH3 cell and T regulatory type 1 (Tr1) cells also contribute substantially to active suppression in the periphery3,4. Tr1 cells that produce mainly interleukin 10 (IL-10) are efficient regulators of inflammation and autoimmunity5. IL-10, a potent anti-inflammatory cytokine, ‘antagonizes’ TH1 responses by inhibiting the production of interferon-g (IFN-g)6 and also inhibits antigen presentation7. IL-10 deficiency leads to spontaneous colitis8 due to excessive activation of dendritic cells (DCs) and Toll-like receptor signaling pathways9. Conversely, IL-10-modified DCs maintain an immature phenotype and support IL-10 production by T cells10,11. IL-10-producing Tr1 cells can be generated in vitro by repetitive antigen stimulation in the presence of IL-10 (ref. 12). These cells secrete large amounts of IL-10 and variable amounts of IFN-g, IL-2, IL-5 and transforming growth factor-b (TGF-b) but no IL-4. In addition, Tr1 cells generated in vitro in the presence of the immunosuppressive drugs vitamin D3 and dexamethasone do not express Foxp3 but suppress T cell functions in an IL-10-dependent way13,14. Thus, lack of expression of Foxp3, predominant production of IL-10
and suppression of immune responses are hallmarks of Tr1 cells. In contrast to naturally occurring Foxp3+ Treg cells, whose antigen specificity is often unknown, Tr1 cells by definition are antigen specific and therefore may provide therapeutic advantages over Foxp3+ T cells. In addition, the therapeutic efficacy of the Foxp3+ Treg cells is compromised in the presence of inflammatory cytokines because effector T cells may become refractory to Foxp3+ Treg cell–mediated suppression15,16. Thus, IL-10-producing Tr1 cells may provide an advantage relative to Foxp3+ Treg cells in mediating suppression of ongoing inflammation. Progress in the study of Tr1 cells has been hampered because of lack of knowledge about growth factors capable of promoting the generation of IL-10-producing Tr1 cells. Although IL-10 can induce Tr1 differentiation, the resulting Tr1 cells do not proliferate because of the suppressive nature of IL-10. This observation suggests that factors other than IL-10 might be important for the differentiation and population expansion of IL-10-secreting Tr1 cells. Here we used induced Treg cells (iTreg cells) to demonstrate that DCs modified by TGF-b-induced Treg cells elicited the generation of Tr1-like cells that produced large amounts of IL-10 and IFN-g and suppressed T cell responses in a Foxp3-independent way. The modified DCs were fully mature and had a plasmacytoid-like phenotype (CD11cintCD11bloCD8a–CD45RBhiB220hi). In addition to IL-10, the modified DCs produced IL-27 and TGF-b. IL-27, a member of the IL-12 family, was the dominant factor involved in the induction of IL-10-producing T cells and worked together with TGF-b to further enhance Tr1 differentiation.
1Center
for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA. 2Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. 3Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil CEP 05508-900. 4Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. 5These authors contributed equally to this work. Correspondence should be addressed to M.O. (
[email protected]) and H.L.W. (
[email protected]). Received 22 June; accepted 17 October; published online 11 November 2007; doi:10.1038/ni1541
1380
VOLUME 8
NUMBER 12
DECEMBER 2007
NATURE IMMUNOLOGY
RESULTS DCs modified by iTreg cells induce Tr1 differentiation Foxp3+ Treg cells can be induced from naive T cells by TGF-b. However, such iTreg cells are ‘unstable’, because they lose Foxp3 expression and their suppressive activity in the absence of TGF-b (unpublished observations). To circumvent that problem, we used TGF-b-transgenic (b-Tg) mice17 in which TGF-b expression is controlled by the Il2 promoter. In such T cells, TGF-b produced by activated transgenic T cells induces Foxp3 expression in an autocrine way17. To obtain antigen-specific iTreg cells, we crossed the b-Tg mice with mice expressing a myelin oligodendrocyte glycoprotein (MOG)– specific T cell receptor transgene (2D2)18. Most naive CD4+CD25– CD62L+ T cells (about 70%) in the resultant ‘2D2 b-Tg’ double-transgenic mice expressed Foxp3 within 5–7 d of in vitro
activation in the presence of the cognate antigen MOG(35–55) (amino acids 35–55 of MOG); it has been shown before that such Foxp3+ T cells can suppress antigen-specific T cell responses17. Growing evidence indicates that Treg cells directly interact with DCs, but not effector T cells, to mediate their suppressor function19, which indicates that Treg cells modify DCs to render them tolerogenic. The use of 2D2 b-Tg mice thus provided us with a tool to investigate how Treg cells modify DCs and suppress immune responses. To begin, we primed 2D2 T cells or 2D2 b-Tg T cells with wildtype antigen-presenting cells (APCs) in the presence of MOG(35–55) (producing ‘p.2D2 T cells’ or ‘p.2D2 b-Tg T cells’, respectively), then incubated splenic DCs from naive wild-type C57BL/6 mice in primary cultures with the p.2D2 T cells or p.2D2 b-Tg T cells. After 12 h of coculture, we sorted the splenic DCs (called ‘modified DCs’ here) and then added them to secondary cultures with CD4+CD25– naive T cells
P = 0.015
c
250
103 c.p.m.
100
30 20
1.3
0.69
d
T (recipient) 10,000
103
0.67
26.6
70.1
1 0.021
3.18
0.68
103
10,000
WT,α-CD3 + α-CD28
101
102
1,000
3.03
0.48
48.9
1.14
88.6
7.85
0.97
46.3
8.26
3.44
0.81
44.2
1.21
86.6
9.17
3.61
1.48
82.8
12.1
100
β-Tg,IgG
100
10 0.049
2.65
1
2.89
103
10,000
102
1.52
31.1
9.38
55.3
4.26
1,000 β-Tg,α-CD3 + α-CD28
CD4
IFN-γ
100 100 101 102 103 104 CD4
IL-10
101
100 101 102 103 104
10 10 0 1, 00 10 0 ,0 00
IL-4 1
0 1, 00 10 0 ,0 00
10
IL-10
10
10 0 ,0 00
1,
102
00
1
1
1
IFN-γ
10
0
2.35
10
17.10
0.064
103
100 Foxp3
100
10
101
p.2D2 × β-Tg
7.38
4.33
1.72
10,000
104
42.6
1 0.055
101
p.2D2
3.25
10
103
104
94.6
100
100
n.WT
1.05
0.54
1,000
102
104
0.29
10
100
MOG
1.89
100
WT, IgG
101
104
2.75
1,000
102
–
W
(re T no
p. 0.19
T,
pi ci
× 2 2D
no 104
Ig G T + ,α α- -C C D D 3 28 βTg ,I gG βTg + , α- αC CD D 28 3
g
2
βT
2D p.
W n.
M
O
T
G
–
) C (D
T
0 T (recipient):
t)
10
0 T (DC):
W
103 c.p.m.
150
50
T (DC)
P = 0.004
40
200
b
P = 0.0005
50
P = 0.001
en
a
Foxp3
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
IL-10
100 101 102 103 104
Figure 1 DCs modified by b-Tg T cells induce the generation of IL-10-producing suppressive T cells. (a) Proliferation of naive responder CD4+CD25–CD62L+ T cells obtained from 2D2 mice and cultured with T(DC) cells previously cultured with unmodified DCs (–), DCs pulsed with MOG(35–55) alone (MOG) or DCs pulsed with MOG(35–55) and incubated with naive wild-type T cells (n.WT), p.2D2 T cells (p.2D2) or p.2D2 b-Tg T cells (p.2D2 b-Tg), assessed by [3H]thymidine incorporation. (b) Flow cytometry of the T(DC) cells in a after restimulation with PMA plus ionomycin. (c) Proliferation of naive responder cells: splenic DCs from wild-type (WT) or b-Tg mice that had previously been injected with isotype control (IgG) or anti-CD3 and anti-CD28 (a-CD3 + a-CD28) were pulsed with MOG(35–55) and injected intravenously into naive 2D2 recipient mice; 3 d later, CD4+ T cells from recipient mice were isolated and restimulated for 5 d with MOG(35–55) in the presence of irradiated wild-type spleen cells (T(recipient)) and then added to cultures of naive 2D2 responder T cells, which were analyzed by [3H]thymidine incorporation. (d) Flow cytometry of the T(recipient) cells in c after restimulation with PMA plus ionomycin. Numbers adjacent to boxed areas or in quadrants (b,d) indicate percent positive cells in each. Data represent one of two (a,c) experiments (mean ± s.d. of triplicate wells) or one of four (b) or two (d) experiments.
NATURE IMMUNOLOGY
VOLUME 8
NUMBER 12
DECEMBER 2007
1381
ARTICLES by p.2D2 b-Tg T cells acquired suppressive properties; for example, such T(DC) cells inhibited the proliferation of naive responder 2D2 CD4+ cells exposed to MOG(35–55) plus irradiated wild-type splenocytes as APCs (Fig. 1a). These suppressive T(DC) cells induced by p.2D2 b-Tg T cell–modified DCs did not express Foxp3, ruling out the possibility of involvement of Foxp3+ Treg cells in the inhibition of responder T cell proliferation (Fig. 1b). In addition, a much higher percentage of T(DC) cells induced by p.2D2 b-Tg T cell–modified
0
40
60 40
60 40
20
20
20
0
0
0
0
0
40
60 40
60 40
5
10
1×
60 40 20 0
100 90.2% 708 80
100 56.6% 312 80
% of Max
60
% of Max
40
% of Max
% of Max
60
100 85.2% 30 80
00
40
20
100 88.6% 404 80
100 97.3% 80 180
60
20
100 84.2% 180 80
0
100 96.9% 3,302 80
100 95.2% 80 1,635
% of Max
60
% of Max
40
% of Max
% of Max
60
20
100 96.6% 104 80
60 40
60 40
500
0
0.0
1,500
400
500
–
n.WT p.2D2 p.2D2 × β-Tg 600
500 None α-CD40
400
LPS
200
None α-CD40 LPS
IL-10
300
1,
0
0
400
1
200 100
100 0
0
300 1,000
TGF-β
200
10 0 ,0 00
0.5
0
300
10
1.0
500 400
00
1.5 1,000
IL-27p28
IL-6 (pg/ml)
200
1,
2.0
400 300
10
10 0 ,0 00
10
00 1,
1,
10
00 ,0
0
10
00
10
d
1,500
1
0
0
0 10
0 1
0 00 10 0 ,0 00
0
0
20
0
10
20
1
20
0
20 10
20 1
20
00
0
,0
0
10
0
0
0
40
0
20
10
20
60
00
20
100 79.0% 28 80
00
40
20
100 76.4% 80 273
,0
60
10
40
20
100 93.9% 80 235
10
100 0
5
1×
10
0
00
00
,0 10
100 0
60
% of Max
40
CD40 100 89.9% 85 80
% of Max
60
CD86 100 75.8% 80 1,028
% of Max
40
% of Max
60
CD80 100 76.4% 937 80
% of Max
CCR7 100 76.5% 25 80
1,
00
10
,0
00
0
0
1×
1,
10
10
1
5
10
00
00
,0
CD83 100 80.7% 523 80
% of Max
40
10
10 0 0
% of Max
60
1,
10
1×
00
00
,0 10
1,
100 73.6% 80 164
0
0
5
0
0
20
0
0
40
0
TNF (pg/ml)
IL-12 (pg/ml)
60
1,
40
100
IL-10 (pg/ml)
% of Max
60
0
500 IFN-γ (pg/ml)
40
67.7% 80 2,2184.0
20
10
c
60
B220 100
39.1% 969.0
80
20
1
p.2D2 × β-Tg
40
CD11b 100
88.9% 4.4
80
20
10
% of Max
p.2D2
60
100
20
10
% of Max % of Max
n.WT
89.0% 5,838.0
80
% of Max
% of Max
40
0 0
% of Max
60
100
I-ab
b
CD8α
CD45RB 78.5% 445.0
80
% of Max
CD11c 100
1,
a
0 1, 00 10 0 ,0 00
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
(called ‘responder T cells’ here) from 2D2 mice (Supplementary Fig. 1a online). DCs modified by p.2D2 b-Tg T cells did not support the proliferation of naive responder T cells, whereas DCs modified by wild-type or p.2D2 T cells did (Supplementary Fig. 2a online). The lower T cell proliferation induced by DCs modified by p.2D2 b-Tg T cells was not due to induction of apoptosis of the responder T cells (data not shown). In contrast, T cells isolated from DC secondary cultures (called ‘T(DC) cells’ here) that were modified
200
100 0 –
n.WT
0
p.2D2 p.2D2 × β-Tg
–
n.WT
p.2D2 p.2D2 × β-Tg
Figure 2 Characterization of iTreg cell–modified CDs. (a) Surface expression of CD11c, CD45RB, CD8a, CD11b and B220 on splenic DCs cultured for 12 h together with p.2D2 b-Tg T cells (open histograms). (b) Surface expression of I-Ab, CD83, CCR7 CD80, CD86 and CD40 on splenic DCs cultured for 12 h together with naive wild-type, p.2D2 or p.2D2 b-Tg T cells (open histograms); histograms are gated on CD11c+ cells. Numbers in plots (a,b) indicate frequency of positively stained cells in gate (top) and mean fluorescent intensity (bottom); shaded histograms are freshly isolated splenic DCs. (c) Cytokines produced by DCs modified by various T cell subsets (horizontal axis) after overnight treatment with anti-CD40 (a-CD40) or lipopolysaccharide (LPS). (d) Quantitative RT-PCR of the expression of IL-27p28, TGF-b and IL-10 mRNA, relative to the expression of mRNA encoding glyceraldehyde phosphate dehydrogenase. Data represent one of three (a), six (b) or two (c,d) experiments (mean ± s.d. of triplicate wells, c,d).
1382
VOLUME 8
NUMBER 12
DECEMBER 2007
NATURE IMMUNOLOGY
ARTICLES Table 1 CD11cintB220+PDCA-1+ cells in vivo
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
Mice (treatment)
Frequency among total lymphocytes (%)
Frequency among CD11c+ cells (%)
WT (naive) WT (IgG)
0.24 ± 0.01 0.29 ± 0.05
2.66 ± 0.32 3.09 ± 0.83
WT (a-CD3 + a-CD28) b-Tg (IgG)
0.25 ± 0.02* 0.33 ± 0.01
4.04 ± 0.24** 3.17 ± 0.11
b-Tg (a-CD3 + a-CD28)
0.40 ± 0.01*
4.35 ± 0.22**
‘Seven-color’ flow cytometry of CD3–CD19–NK1.1–CD11bneg–loCD11cintB220+PDCA-1+ cells (n ¼ 3 mice per group) in the spleens of naive wild-type, wild-type or b-Tg mice injected with isotype control antibodies (IgG) or anti-CD3 and anti-CD28 a-CD3 + a-CD28). *, P ¼ 0.027; **, P ¼ 0.030 (Student’s t-test). Data represent one of two independent experiments (mean ± s.d.).
DCs produced more IL-10 than did T(DC) cells induced by naive wild-type T cells (n.WT) or p.2D2 T cell–modified DCs. These results indicated that DCs modified by p.2D2 b-Tg T cells could induce the generation of Foxp3– IL-10-producing Tr1-like T cells (T(DC) cells) in vitro. To confirm those in vitro observations, we attempted to generate ‘modified DCs’ in vivo by injecting antibody to CD3 (anti-CD3) and anti-CD28 into b-Tg or wild-type mice20 (Supplementary Fig. 1b). This treatment doubled the amount of Foxp3+ CD4+ T cells in b-Tg mice (about 9.58%) compared to mice that received isotype control antibodies (about 4.97%; data not shown) and allowed us to study modified DC function in vivo. To begin, we isolated in vivo–modified splenic DCs, pulsed them with MOG(35–55) and then injected the modified, pulsed DCs into naive 2D2 recipient mice. We then isolated CD4+ T cells from the recipient 2D2 mice and restimulated the cells with MOG(35–55) and syngenic APCs (producing ‘T(recipient) cells’), then added the T(recipient) cells to cultures of naive 2D2 responder T cells. Only 2D2 T(recipient) cells from mice injected with DCs modified by activated b-Tg T cells inhibited the proliferation of naive responder T cells (Fig. 1c). Similar to the T(DC) cells generated in vitro by induction with p.2D2 b-Tg T cell–modified DCs, a higher percentage of 2D2 T(recipient) cells from mice injected with DCs modified by b-Tg T cells produced IL-10 but did not express Foxp3 (Fig. 1d). Likewise, 2D2 T(recipient) cells from mice injected with DCs modified by b-Tg T cells inhibited the proliferation of naive responder T cells in an IL-10-dependent way (Supplementary Fig. 2b online). Of note, these IL-10-producing 2D2 T(recipient) cells also expressed IFN-g but not IL-4 (Fig. 1d). These data confirm the ability of DCs modified by b-Tg T cells to support the differentiation of Tr1-like cells in vivo. Characterization of iTreg cell–modified DCs Next we characterized the phenotype of the DCs that mediated the induction of Foxp3– T cells that produced IL-10 and IFN-g. After 12 h of incubation with p.2D2 b-Tg T cells, 80% of DCs had a plasmacytoid-like CD11cintCD11bloCD8a– CD45RBhiB220hi surface phenotype (Fig. 2a). Furthermore, compared with wild-type mice, b-Tg mice had a higher proportion of plasmacytoid-like DCs (CD11cintB220+CD11bneg–loCD8a–BST2+) among total lymphocytes after in vivo treatment with anti-CD3 plus anti-CD28, which allowed modification of DCs by iTreg cells (Table 1). However, these DCs were fully mature, as their expression of major histocompatibility class II molecules, CD83, CD40 and CCR7 was equivalent to that of DCs modified by either naive wild-type or p.2D2 T cells (Fig. 2b). However, DCs modified by p.2D2 b-Tg T cells expressed less CD80 and CD86. To compare these DCs with previously described tolerogenic
NATURE IMMUNOLOGY
VOLUME 8
NUMBER 12
DECEMBER 2007
DCs11, we examined their cytokine profile. DCs modified by p.2D2 b-Tg T cells produced much more IL-10 than did DCs modified by naive wild-type or p.2D2 T cells; this was further enhanced by stimulation with anti-CD40 or lipopolysaccharide (Fig. 2c). Because the DCs modified by p.2D2 b-Tg T cells had a phenotype and cytokine profile similar to that of the tolerogenic CD11cloCD45RBhi DCs described before10,11 that induce Tr1 differentiation by an IL-10-dependent mechanism, we sought to determine whether IL-10 was required for the Tr1 differentiation induced by p.2D2 b-Tg T cell–modified DCs. We generated Il10+/+ and Il10–/– DCs modified by p.2D2 b-Tg T cells in vitro as described above. Unexpectedly, these DCs generated similar percentages of IL-10producing T cells (Supplementary Fig. 3 online). Thus, modified DC–derived IL-10 was dispensable for Tr1 differentiation. In fact, T cell proliferation and production of IL-4 and IFN-g increased in the absence of DC-derived IL-10 (Supplementary Fig. 3). To find factors other than IL-10 from iTreg cell–modified DCs for Tr1-like differentiation, we measured expression of mRNA encoding cytokines in DCs. We noted much more expression of transcripts encoding IL-27p28 and TGF-b1 in DCs modified by p.2D2 b-Tg T cells than in DCs modified by naive wild-type or p.2D2 T cells (Fig. 2d). Expression of IL-27p28 mRNA was further upregulated by lipopolysaccharide but not by anti-CD40. IL-27 and TGF-b induce IL-10 production in vitro Because the IL-10 produced by DCs modified by p.2D2 b-Tg T cells was not involved in Tr1 differentiation, we investigated the possibility that IL-27 produced by the modified DCs was involved in the generation of IL-10-producing Tr1-like cells. Although IL-27 was initially shown to be important for the induction of T-bet+IFN-g+ TH1 cells21, subsequent studies have indicated that IL-27 exerts inhibitory effects. Like IL-10, IL-27 is now considered an important anti-inflammatory cytokine, as it negatively regulates the generation of TH-17 cells22,23. IL-27 is a member of the IL-12 cytokine family and signals through a receptor containing the common IL-6 receptor chain gp130 and the unique IL-27 receptor chain WSX-1 (ref. 24). Notably, like IL-10-deficient mice, WSX-1-deficient mice develop severe immunopathology after infection with Toxoplasma gondii25,26. The similarity in immune responses in these two models suggested a possible link between IL-27 and IL-10. Thus, we investigated the function of IL-27 alone or in combination with TGF-b in the generation of IL-10-producing T cells. Naive T cells activated in the presence of IL-27 produced IL-10 and small amounts of IFN-g, IL-5 and IL-13 without upregulating Foxp3 (Fig. 3a). The addition of TGF-b together with IL-27 further increased IL-10 production but decreased the production of IL-4, IL-5 and IL-13 by the responding T cells (Fig. 3a). Results obtained with T cells from mice with sequence encoding the reporter green fluorescent protein (GFP) ‘knocked in’ to the Il10 gene showed that IL-27 induced a transient burst of IL-10 production and that the addition of TGF-b was essential for sustained IL-10 production (Fig. 3b). In the presence of TGF-b plus IL-27, a substantial proportion of wild-type but not WSX-1-deficient T cells produced IFN-g and IL-10 together (Fig. 3a,c), supporting the conclusion that interactions of IL-27 with its receptor (IL-27R) were essential for the generation of Tr1 cells. Cytokine production, as measured by enzyme-linked immunosorbent assay and intracellular cytokine staining, correlated with cytokine mRNA expression, as measured by quantitative RT-PCR, in these cells (Supplementary Fig. 4 online). As Foxp3+ T cells produce IL-10 (ref. 15) and can be induced by TGF-b, we measured Foxp3 expression in T cells obtained from mice
1383
ARTICLES
0.11
0.18
0.84
100
TG
0
0
0
75.1
8.52
0
24.9
0
78.5
21.5
1.73
98.1
0.93
82.6
8.94
93.8
3.47
0.36
0.27
0.098
0.27
0.083
0.15
0.31
72 h
0
0
104 92.4
101
0 91.5
7.62
0
0
0
84.8
8.51
0
0
92
15.2
8.02
103 96 h
CD4
102
103 102
IL-10
0 91.5
101 100
WSX-1-KO
101 96.6
0 6.06
102
WT
104 0.14
0
103
2.33
102
98
23.9
48 h
104 93.9
103
100
IL-27 76.1
23.1
101
n F- TG es β F + -β IL -2 IL 7 -2 7
IFN-γ (pg/ml) 0.36
TGF-β + IL-27 76.9
0.45
102
IL-27
5.08
99.5
103
to cy o N
TG
3.39
TGF-β
No cytokines 104 99.7 0.26
ki
ki cy o N
TG
TGF-β + IL-27
b
700 600 500 400 300 200 100 0
to
to cy o N
ne F- TG s β F+ β IL -2 IL 7 -2 7
IL-13 (pg/ml)
400 350 300 250 200 150 100 50 0
ki
ne F- TG s β F + -β IL -2 IL 7 -2 7
IL-4 (ng/ml)
IL-5 (pg/ml) F- TG s β F + -β IL -2 IL 7 -2 7
TGF-β
No cytokines
104 0.21
400 350 300 250 200 150 100 50 0
ne ki to cy o
N
c
2.92
2.15
97.5
100 100 101 102 103 104
100 101 102 103 104
97.2
2.4
96.5
100 101 102 103 104
101 0
0
0
0
0
0
0
0
100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 IL-10 (GFP)
3.05
100 101 102 103 104
IFN-γ
e
Foxp3 (GFP)+ (%)
120
No cytokines 0.35
TGF-β
TGF-β + IL-27
IL-27
22.9
0.26
68
f
120 100
103 c.p.m.
Cells
80
40
80 60 40 20 7
-2
-2 IL
Fβ
+
IL
s ne
Fβ
ki
TG
to cy o
TG
N
s F- TG β F+ β IL -2 7 IL -2 7
ne ki to cy o N
Foxp3 (GFP)
7
0
0
TG
d
Figure 3 TGFb and IL-27 induce Tr1-like cells in vitro. Analysis of the cytokine expression (a,d,e) and proliferation (f) of Foxp3– CD4+CD62L+ cells from mice with sequence encoding GFP ‘knocked in’ to Foxp3 (a,d–f), CD4+CD25–CD44– cells from mice with sequence encoding GFP ‘knocked in’ to Il10 (b) or CD4+CD25– T cells from wild-type or WSX-1-deficient (WSX-1-KO) mice (c,f), activated without cytokines or with IL-27 alone, TGF-b alone, or IL-27 plus TGF-b, in the presence of irradiated syngenic APCs plus anti-CD3 (1 mg/ml). (a) Bead array of cytokines in culture supernatants at 48 h. (b) Flow cytometry of IL-10 expression in CD4+ T cells at 48, 72 or 96 h (assessed as expression of GFP). (c) Intracellular expression of IL-10 and IFN-g in CD4+ T cells at day 4. Numbers in quadrants (b,c) indicate percent positive cells in each. (d,e) Flow cytometry of Foxp3 expression in CD4+ T cells at 72 h (assessed as expression of GFP). Numbers below bracketed lines indicate percent Foxp3+ cells. (f) Proliferation of cytokine-treated wild-type T cells, assessed as [3H]thymidine incorporation (mean ± s.d. of triplicate wells). Data represent one of three experiments.
The transcription factors T-bet, GATA-3, RORgt and Foxp3 are required for the generation of TH1, TH2, TH-17 and Treg cells, respectively. We found that TGF-b induced expression of mRNA encoding Foxp3, whereas IL-27 promoted the expression of T-bet transcripts (Fig. 4a,b). TGF-b in combination with IL-27 further enhanced the expression of T-bet but inhibited Foxp3 expression. Neither TGF-b or IL-27 alone nor a combination of these enhanced the expression of GATA-3 or RORgt (Fig. 4c,d). Notably, IL-27 plus TGF-b failed to trigger upregulation of T-bet in WSX-1-deficient T cells; in contrast, expression of GATA-3 and RORgt was slightly
400
RORγ t
500
2,000 1,500 1,000
WT WSX-1-KO
600
2,500
300 200
500
100
0
0 -2 IL + Fβ TG
Fβ
+
+
IL
IL
-2
7
7
-2 7
T-bet
g
3,000
TG
o
Fβ
to ki ne T s F- GF β + β IL -2 7 IL -2 7 T H1 7
cy
TG
TG
ki to cy N o
TG
cy o N
o
0 Fβ IL -2 7 IL -2 7 T H2
0 +
0
400 200
ne s
0
ki ne T s F- GF β + -β IL -2 7 IL -2 7
2,000
to ki ne TG T s F- GF β + β IL -2 7 IL -2 7 T H1
2,000
600
f
800 700 600 500 400 300 200 100 0
TG
4,000
100
800
β
4,000
e
1,000 RORγ t
6,000
N
6,000
d 1,200
F-
Foxp3
200
c 8,000
cy
T-bet
300
8,000
TG
b
GATA-3
400
to
a
GATA-3
with sequence encoding the reporter GFP ‘knocked in’ to the Foxp3 gene and cultured with TGF-b and IL-27. As expected, TGF-b stimulation increased Foxp3 expression (assessed as expression of GFP; Fig. 3d,e). However, the addition of IL-27 substantially inhibited Foxp3 expression and concomitantly increased IL-10 production from Foxp3– T cells. Moreover, unlike IL-10, TGF-b in combination with IL-27 did not inhibit T cell proliferation and instead expanded IL-10producing T cell populations (Fig. 3f). These data collectively indicate potential involvement of TGF-b and IL-27 as factors promoting Tr1-like differentiation.
N
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
TG
o
cy to ki n F- TG es β F + -β IL -2 IL 7 -2 7
3 2 1 0
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
TG
IL-10 (ng/ml)
7 6 5 4
N
a
Figure 4 Transcription factors induced by TGF-b and IL-27. Quantitative PCR of the expression of mRNA encoding T-bet, Foxp3, GATA-3 and RORgt in naive T cells sorted from wild-type mice (a–d) or wild-type and WSX-1-deficient mice (e–g) and cultured for 48 h with no cytokines, with IL-27 alone, with TGF-b alone, with IL-27 plus TGF-b, under TH1, TH2 and TH-17 culture conditions in the presence of APCs, or with plate-bound anti-CD3 and anti-CD28, presented relative to the expression of mRNA encoding glyceraldehyde phosphate dehydrogenase. Data represent one of two experiments (mean + s.d.).
1384
VOLUME 8
NUMBER 12
DECEMBER 2007
NATURE IMMUNOLOGY
ARTICLES
10,000
3.41
T cells
0.12
2.86
b
0.26
DC
10,000
modification
1,000
– 1
–
93.7
2.75
96.7
0.19
1.45
0.044
1.03
0.042
10,000
100
–
95.8
0.03
92.4
2.48
94.9
1.22
19.8
6.49e-3
16.6
3.57
9.12
0.087
0.089
98.8
1
80.2
0.019
73.5
6.4
90.3
0.47
16.9
0.043
12.1
14.8
9.33
0
5.62
3.55
2.8
1.14
1 90.7
0
80.3
10.5
89.1
6.96
1,000 p2D2 × β-Tg
100
100
+
Foxp3
100
0.23
100 0
1×105
0
1,000
99.4
0.094
96.7
0.56
10 0 ,0 00
0
00
10
1,
1
10
0.016
10,000 WSX-1-KO
1,000 100 0
98.8
0.63
Foxp3
IFN-γ
IL-4
IL-17
1,
T + g, α- αC CD D 28 3
gG ,I Tg
0.39
0 10 0 1, 00 10 0 ,0 10 00 0, 00 0
0 10 0 00 10 0 ,0 10 00 0, 00 0
100 0
0 10 0 1, 00 10 0 ,0 10 00 0, 00 0
99.5
0.5
10,000
WT
1,000
0 10 0 00 10 0 ,0 10 00 0, 00 0
100 0
0.18
0.051
0 10 0 1, 00 10 0 ,0 10 00 0, 00 0
1
1×105
0
1,000
97.3
β-
W 100 0
0 10 0 1, 00 10 0 , 10 000 0, 00 0
0.32
10,000
APC-A: IL-10
1,000
2.9
10,000
0 10 0 1, 00 10 0 , 10 000 0, 00 0
98.6
0.49
1×105
0.31
β-
G Ig T, W 1,000 100 0
1×105
0.056 APC-A: IL-10
1×105 0.31
96.6
2.18
10,000
APC-A: IL-10
100 0
1×105
0.37
1,000
0.17
10,000
2.58
10,000
0 10 0 1, 00 10 0 ,0 10 00 0, 00 0
96.9
10 0 ,0 00
4
1,
1,000 100 0
1×105
0.049
APC-A: IL-10
2.9
APC-A: IL-10
1×105 10,000
0
8
0
IFN-γ
APC-A: IL-10
d
WT WSX-1-KO
3
10 c.p.m.
0
00 10 0 ,0 00
10
P = 0.0002
+ T, α α- -C C D D 3 28
Foxp3
1,
1
0 00 10 0 ,0 00
10
2.32
1,
1
10
95.7 10
3.28
93.3
1
IL-4
IFN-γ
12
c
IL-10
10
00
0.65
10
1.3
1,000
1,
0.38
1
3.07
10,000
10
11.1
10 0 ,0 00
62
00
0.39
1
1
82.7
0
10
10
IL-10
1.92
1,000
WSX-1-KO
0.26
100
10,000
10,000
p2D2 × β-Tg
3.67
10 96.6
1
IL-10
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
p2D2 × β-Tg
10
WT
0.67
1,000
1,000
p2D2 × β-Tg
4.43
10 1
10,000
WSX-1-KO
0
100
100 WT 10
–
4.19
1,000
10
–
β-IH
1,
DC modification
10
a
Figure 5 DCs modified by iTreg cells induce Tr1-like cells by producing IL-27 and TGF-b. (a,b) Flow cytometry of wild-type and WSX-1-deficient CD4+ T cells (a) and 2D2 CD4+ T cells (b) cultured for 3 d with unmodified DCs (–) or DCs modified by 2D2 b-Tg T cells without (a) or with (b) the Smad inhibitor b-IH. (c,d) Proliferation, as assessed by [3H]thymidine incorporation (c), and flow cytometry of intracellular expression of Foxp3 and cytokines (d), of cells from wild-type and WSX-1-KO mice that received DCs on days –10 and –3 from wild-type or b-Tg mice injected previously with isotype control antibody (c) or anti-CD3 and anti-CD28 (c,d); CD4+ T cells obtained from recipient mice on day 0 were restimulated for 48 h with MOG(35–55) in the presence of naive syngenic APCs. Numbers in quadrants (a,b,d) indicate percent positive cells in each. Data represent one of three (a,b) or two (c,d) experiments (mean ± s.d. of triplicate wells, c).
higher in WSX-1-deficient than in wild-type T cells (Fig. 4e–g). These observations suggest that IL-10 production induced by TGF-b plus IL-27 did not depend on GATA-3, a transcription factor required for the generation of TH2 cells. As exogenous IL-27 and TGF-b induced the differentiation of IL-10-producing T cells, we investigated the function IL-27 and TGF-b released from modified DCs in the induction of Tr1-like cells. Wild-type DCs modified by p.2D2 b-Tg T cells triggered IL-10 production in a substantial proportion of naive wild-type T cells but in very few naive WSX-1-deficient T cells (Fig. 5a). IFN-g production was also reduced from 25.9% in wild-type to 2.97% in WSX-1-deficient T cells. Moreover, blocking TGF-b signal transduction in T cells with an inhibitor of phosphorylation of the signal
NATURE IMMUNOLOGY
VOLUME 8
NUMBER 12
DECEMBER 2007
transducer Smad also substantially reduced the differentiation of IL-10-producing T cells induced by iTreg cell–modified DCs (Fig. 5b). As expected, the numbers of T cells producing IFN-g and/or IL-4 also slightly increased when TGF-b was inhibited in the system. These results show that IL-27 produced by DCs is critical in inducing IL-10producing T cells and that TGF-b acts in synergy with IL-27 to enhance the generation of these Tr1-like cells. We further demonstrated the importance of DC-derived IL-27 by comparing the differentiation of IL-10-producing T cells in WSX-1deficient or wild-type mice that had received modified DCs isolated from either wild-type or b-Tg mice, with or without anti-CD3 and anti-CD28 treatment. We restimulated CD4+ T cells isolated from all recipient mice in vitro with and without MOG(35–55) and APCs.
1385
ARTICLES Table 2 In vivo induction of IL-10-producing T cells in wild-type and WSX-1-deficient mice Donor (treatment)
Recipient
Foxp+
IL-10+
IFN-g+
IL-4+
IL-17+
IL-2+
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
MOG(35–55) WT (IgG) WT (a-CD3 + a-CD28)
WT WT
0.70 ± 0.73 0.88 ± 0.96
0.89 ± 0.17* 1.12 ± 0.13*
1.06 ± 0.39** 1.00 ± 0.34**
0.23 ± 0.16 0.15 ± 0.11
3.13 ± 1.05 2.65 ± 0.49
4.81 ± 0.64 3.75 ± 1.19
b-Tg (IgG) b-Tg (a-CD3 + a-CD28)
WT WT
1.63 ± 0.16 0.98 ± 0.78
1.64 ± 0.23* 2.35 ± 0.33*
0.74 ± 0.48** 0.57 ± 0.13**
0.20 ± 0.11 0.22 ± 0.11
1.92 ± 0.38 1.61 ± 0.50
4.24 ± 0.33 3.76 ± 0.33
WT (IgG) WT (a-CD3 + a-CD28)
WSX-1-KO WSX-1-KO
0.61 ± 0.33 0.86 ± 0.67
0.48 ± 0.18* 0.72 ± 0.31*
0.71 ± 0.17** 0.75 ± 0.31**
0.15 ± 0.10 0.17 ± 0.09
2.05 ± 0.48 1.96 ± 0.91
4.92 ± 1.65 3.41 ± 0.22
b-Tg (IgG) b-Tg (a-CD3 + a-CD28)
WSX-1-KO WSX-1-KO
1.16 ± 0.46 0.89 ± 0.43
0.70 ± 0.27* 0.39 ± 0.09*
0.71 ± 0.22** 0.61 ± 0.32**
0.17 ± 0.12 0.08 ± 0.07
1.61 ± 0.23 2.22 ± 0.13
3.13 ± 0.43 3.72 ± 0.33
OVA(323–339) WT (IgG)
WT
0.29 ± 0.21
0.78 ± 0.18
0.96 ± 0.37
0.35 ± 0.21
2.72 ± 0.25
4.55 ± 0.01
WT (IgG)
WSX-1-KO
0.45 ± 0.45
0.92 ± 0.25
0.79 ± 0.34
0.16 ± 0.11
2.95 ± 0.39
4.48 ± 0.35
CD4+
Flow cytometry of T cells from wild-type or WSX-1-deficient recipients (n ¼ 4 mice per group) of DCs modified by treatment with IgG or with anti-CD3 plus anti-CD28 (in parentheses at left) and restimulated in vitro with MOG(35–55) or OVA(323–339) (ovalbumin peptide of amino acids 323–339) and irradiated wild-type splenocytes, then stimulated with PMA and ionomycin 5 d later. Values indicate percent postitive cells among CD4+ cells (mean ± s.d.). *, P o 0.05; **, P 4 0.05 (Student’s t-test). Data represent one of two independent experiments.
In the absence of MOG(35–55) restimulation, CD4+ T cells from WSX-1-deficient recipient mice underwent threefold more proliferation than did CD4+ T cells from wild-type recipient mice (data not shown), and this trend of increased proliferation continued in the presence of MOG(35–55) (Fig. 5c). In contrast, CD4+ T cells from wild-type recipients of DCs modified by activated b-Tg T cells proliferated less after exposure to MOG(35–55) than did CD4+ T cells from other recipients, a diminished proliferation that was associated with more production of IL-10 (Fig. 5d and Table 2). Compared with T cells restimulated with a nonspecific ovalbumin(232–339) peptide, IL-10-producing T cells were induced by restimulation with MOG(35–55) in wild-type but not WSX-1deficient recipients of DCs modified by b-Tg T cells. In fact, there were fewer IL-10-producing T cells in the WSX-1-deficient recipients after restimulation with MOG(35–55) than after restimulation with the nonspecific ovalbumin(323–339) peptide. Wild-type IL-10-producing T cells did not express Foxp3 or secrete IL-4 or IL-17, although about 10% of these cells expressed IFN-g together with IL-10 (Fig. 5d and Table 2). These observations suggest that iTreg cell–modified DCs promote in vivo generation of Tr1-like cells by producing IL-27. DISCUSSION IL-10 produced by cells of the innate and adaptive immune systems is an important factor in the downmodulation of TH1 immune responses. Macrophages and DCs produce IL-10 when triggered through the Toll-like receptor pathway27, and IL-10 production by T cells is important in downmodulating inflammation and T cell responses during antigen-specific immune responses. IL-10 was originally described as a cytokine produced by TH2 cells, but it soon became evident that this cytokine is also produced by TH1 cells28 as well as by T cell clones specific to an altered ligand peptide of proteolipid protein. These IFN-g+ and IL-10+ T cell clones confer protection from EAE29. However, the source of IL-10 from T cells remained elusive until the description of IL-10-producing Tr1 cells; in those studies, human or mouse CD4+ T cells were shown to differentiate into IL-10 producers with chronic stimulation in the presence of IL-10. Tr1 cells produce large amounts of IL-10 and variable amounts of IFN-g but no IL-4 and are able to suppress in vitro T cell responses. In vivo administration of Tr1 cells has been shown to be protective in a colitis model12. Nonetheless, the generation of Tr1 cells remains difficult because in vitro
1386
culture with IL-10 inhibits the population expansion of these cells. Immunosuppressive drugs have also been used to generate Tr1 cells. Of note, Tr1 cells can suppress T cell proliferation in a Foxp3independent way13,14. Here we found that iTreg cell–modified DCs induced the generation of suppressive T cells that secreted large amounts of IL-10 and suppressed T cell proliferation both in vitro and in vivo. Immature or tolerogenic DCs support the generation Tr1 cells both in vitro and in vivo10,11. We found that iTreg cell–modified DCs had a plasmacytoidlike phenotype (CD11cintCD8a–CD45RBhiB220hi), similar to the tolerogenic DCs described before, and stimulated the production of IL-10-producing Tr1-like cells by an IL-27-dependent mechanism. Plasmacytoid DCs support both TH1 and TH2 differentiation. Notably, cells infected by viruses produce large quantities of type 1 interferon that, among other things, promotes the generation of plasmacytoid DCs. Plasmacytoid DCs induced after viral infection can stimulate naive T cells to produce IL-10 and IFN-g30. The induction of plasmacytoid DCs may represent one of the mechanisms used by viruses to promote tolerance and to limit the development of an effective antiviral immune response. In agreement with that hypothesis, CD8+ T cells primed with plasmacytoid DCs produce substantial IL-10 but little IFN-g and inhibit the proliferation of naive CD8 T cells by an IL-10-dependent mechanism31. Here we found that iTreg cell–modified DCs induced IL-10producing T cells that suppressed naive T cell function by an IL-10dependent mechanism. In addition to generating IL-10-producing T cells, plasmacytoid DCs can also induce T cell tolerance by promoting the generation and population expansion of Foxp3+ Treg cells32. Studies have also shown that DCs from the lamina propria of the small intestine can induce the ‘conversion’ of naive T cells into Foxp3+ Treg cells by a mechanism involving TGF-b and the vitamin A metabolite retinoic acid33. This subset of lamina propria DCs expresses the surface markers CD11b, CD11c and CD103. Because the iTreg cell–modified DCs we reported here expressed cell surface markers different from those expressed by the lamina propria DCs, we conclude that the two cell types are different. However, it is likely that the lamina propria DCs are continuously exposed to iTreg cells in the gut and therefore may share some characteristics with iTreg cell– modified DCs. It remains to be determined whether the lamina propria DCs produce IL-27 and whether they can promote the generation of Tr1 cells. These observations suggest that plasmacytoid
VOLUME 8
NUMBER 12
DECEMBER 2007
NATURE IMMUNOLOGY
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES DCs or tolerogenic DCs can induce T cell tolerance by inducing Foxp3+ T cells. These Foxp3+ Treg cells may then further modify DCs to produce IL-27, which then promote the generation of IL-10producing T cells. The generation of Tr1 cells by tolerogenic DCs is reported to be mediated through IL-10 produced by the tolerogenic DCs11. Our data indicate instead that the IL-10 production by iTreg cell–modified DCs is not sufficient to induce IL-10-producing T cells. Our results suggest the possibility that other factors derived from DCs are needed to support the generation of IL-10-producing T cells. IL-27, a cytokine of the IL-12 family, was first described as a factor that supports T cell proliferation and TH1 differentiation via the STAT1 and T-bet pathways21. Subsequently, the idea of a regulatory function for IL-27 was supported by several studies, as it can inhibit the development of TH1, TH2 and TH-17 cells34. The idea of an antiinflammatory function for IL-27 has been supported by many findings. WSX-1 (IL-27R)–deficient mice have greater T cell and natural killer cell responses. IL-27R-deficient mice have normal production of CD4+ and CD8+ cells and IFN-g in many infectious models25. Furthermore, in response to T. gondii infection, IL-27R-deficient clear mice develop a normal TH1 response and infection but subsequently die because of an exuberant TH1 response that cannot be controlled. Many studies have shown that WSX-1-deficient mice resemble IL-10-deficient mice in that they have dysregulated TH1 responses, which mediate immune pathology during T. gondii infection26. IL-10 has been described as an anti-inflammatory cytokine and a potent inhibitor of TH1 immune responses; in contrast, IL-27 supports T cell proliferation and TH1 development. Despite their contrasting functions, there seems to be a link between these two regulatory cytokines. Like other IL-12 family cytokines, IL-27 is produced by DCs35. Similarly, our data presented here have shown that iTreg cell–modified DCs had higher IL-27p28 expression, which was further upregulated by lipopolysaccharide stimulation. In addition, these DCs also produced TGF-b. Thus, our data suggest a model in which IL-27 and TGF-b produced by iTreg cell–modified DCs induce IL-10-producing T cells, which limits T cell–mediated pathology in infection and autoimmune disease. T cells express lineage-specific transcription factors, with TH1, TH2 and TH-17 cells expressing T-bet, GATA-3 and RORgt, respectively36–38. However IL-10-producing Tr1 cells have not been shown to express a lineage-specific transcription factor. Similarly, we were unable to detect the expression of a lineage-specific transcription factor, except for the expression of T-bet induced by TGF-b and IL-27. It might be that T-bet supports IFN-g induction in IL-10+ T cells, as T-bet deficient mice have increased IL-10 production during mycobacterium infection39, thus ruling out possible involvement of T-bet in IL-10 induction. The production of IL-10 in macrophages has been shown to be regulated by mitogen-activated protein kinase40, whereas Jun kinase has been linked to IL-10 production by TH2 cells. A study has shown a requirement for GATA-3 in stabilizing and remodeling the Il10 locus41. Our data have shown that neither IL-27 nor IL-27 in combination with TGF-b induced GATA-3 expression. As both TGF-b and IL-27 ‘antagonize’ GATA-3 expression42,43, our finding supports the idea that a transcription factor other than GATA-3 is required for the generation of Tr1 cells. STAT proteins are important factors, as STAT4 and STAT6 were originally described as being required for the TH1 and TH2 differentiation, respectively44. IL-27 activates STAT1, STAT3, STAT4 and STAT5 (ref. 43). IL-27 needs STAT1 for TH1 induction but inhibits TH-17 differentiation45. Notably, the Il10 promoter has a STAT1binding site, suggesting possible involvement of STAT proteins in the generation of IL-10 via IL-27 and TGF-b46.
NATURE IMMUNOLOGY
VOLUME 8
NUMBER 12
DECEMBER 2007
TGF-b is not only essential for maintaining the naturally occurring Treg cell population but also generates iTreg cells47,48. We suggest a possible positive feedback loop exists in which TGF-b-induced Foxp3+ Treg cells ‘instruct’ DCs to produce IL-27 and TGF-b, which supports the generation of IL-10-producing Tr1 cells. It is possible, however, that IL-10 might further enhance its own induction through STAT3 activation49. During a natural autoimmune reaction, at the time of recovery, CD4+Foxp3– T cells are also reported to produce IL-10 concomitant with an increase in the frequency of Foxp3+ Treg cells. We postulate that the production of IL-10 by Foxp3– T cells during recovery from autoimmune disease might be due to modification of DCs by natural Foxp3+ Treg cells15. The antiinflammatory properties of IL-10 make it an important regulator during various parasitic infections and also in autoimmune disease. In conclusion, our data identify a previously unknown pathway by which IL-27, which ‘preferentially’ acts on T cells, induces anti-inflammatory pathways by eliciting IL-10-producing Tr1 cells. Thus, our results provide a new tool not only for the generation of Tr1 cells in large quantities but also for the study of the basic biology of these important regulatory T cells. METHODS Mice. C57BL/6 wild-type and Il10–/– mice were from Jackson Laboratories. b-Tg mice17, 2D2 mice18, mice with sequence encoding the GFP ‘knocked in’ to Foxp3 (Foxp3-GFP.KI mice)48 and mice with sequence encoding the GFP ‘knocked in’ to Il10 (IL-10-GFP tiger mice)50 have been described. Mice were housed in conventional, pathogen-free facilities at the Center for Neurologic Diseases (65 Landsdowne Street, Cambridge, Massachusetts) and at the Harvard Institute of Medicine. All experiments were in accordance with guidelines from the Committee on Animals at Harvard Medical School. Generation of iTreg cell–modified DCs in vitro. Cells were cultured in DMEM with 10% (vol/vol) FCS supplemented with 1 nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 0.05 mM b-mercaptoethanol, 100 U/ml of penicillin and 100 mg/ml of streptomycin. The p.2D2 and p.2D2 b-Tg T cells were generated in vitro as described17. DCs were obtained from spleens digested for 30–45 min at 37 1C in 7% CO2 with collagenase D (1 mg/ml; Roche) and were isolated with CD11c microbeads (Miltenyi Biotec) and further sorting by flow cytometry using antibodies for CD11c (HL3), CD3 (1452C11), CD19 (1D3) from BD BioSciences and CD49b (DX5) from eBioscience. The resulting CD11c+CD3– CD19–CD49b– DCs were then cultured for 12 h with MOG(35–55) (25 mg/ml) alone or with naive wild-type CD4+ T cells or p.2D2 or p.2D2 b-Tg T cells. These modified DCs were used for the induction of Tr1-like cells or were further stimulated with anti-CD40 (10 mg/ml; clone 3/23, BD Biosciences) or lipopolysaccharide (500 ng/ml) for cytokine measurement. Generation of iTreg cell–modified DCs in vivo. Wild-type or b-Tg mice received 2 mg anti-CD3 (145-2C11) and 5 mg anti-CD28 (PV-1) or 2 mg hamster immunoglobulin G1 (IgG1) k-chain (A19-3) and 5 mg hamster IgG2 l-chain (Ha4/8) as isotype controls (BD BioSciences) in 200 ml PBS intravenously 48 h before DC isolation20. DCs from individual donors sorted by flow cytometry were pulsed for 3 h with MOG(35–55) (50 mg/ml) before being injected intravenously into naive recipient mice (approximately 2 105 to 4 105 DCs per mouse). Generation of IL-10-producing T cells. For the generation of IL-10-producing T cells in vitro, CD4+CD25–CD62L+ cells purified from naive 2D2 mice were cultured together with modified DCs in the presence of MOG(35–55) (25 mg/ml) for 3 d before flow cytometry and for 5 d before suppression assays. In some experiments, the Smad3-phosphorylation inhibitor ALK5 SB-431542 (Sigma Aldrich) was added at a concentration of 5 mM. For the generation of IL-10-producing cells in vivo, modified DCs were injected once into naive 2D2 mice (on day –3) and twice into wild-type or WSX-1-deficient mice (on day –10 and day –3). Then, 3 d after last injection (day 0), CD4+ T cells from
1387
ARTICLES
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
recipient mice were isolated with CD4 microbeads (Miltenyi Biotec) and were restimulated with MOG(35–55) at a concentration of 25 mg/ml for 2D2 cells or 100 mg/ml for wild-type or WSX-1-deficient cells, in the presence of irradiated naive wild-type spleen cells. In vitro T cell proliferation and suppression assays. For analysis of T cell proliferation, 2.5 105 sorted CD4+CD62L+Foxp3– cells (with Foxp3 assessed as GFP) were cultured with irradiated syngenic APCs in the presence of TGF-b (3 ng/ml), IL-27 (100 ng/ml) or both. For suppression assays, 2.0 104 Tr1 cells generated in vivo or in vitro were cultured in triplicate wells together with naive CD4+ 2D2 responder cells at a ratio of 1:2 in the presence of MOG(35–55) (25 mg/ml) and syngenic APCs. In some experiments, neutralizing anti–mouse IL-10 (JES 052A5; R&D Systems) was added at a concentration of 10 mg/ml. Cells were pulsed with 1 mCi [3H]thymidine per well for the final 16 h of incubation and [3H]thymidine incorporation was measured with a MicroBeta liquid scintillation counter (PerkinElmer). Intracellular cytokine staining. Cells were restimulated for 4 h at 37 1C in 10% CO2 with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma), ionomycin (1 mg/ml;Sigma) and GolgiStop (1 ml/ml; BD Biosciences), followed by surface and intracellular staining according to the manufacturer’s instructions (BD Biosciences). Cells were analyzed on a FACSAria (BD Biosciences). Measurement of cytokines by enzyme-linked immunosorbent assay and quantitative RT-PCR. Cytokines in culture supernatants were measured by either enzyme-linked immunosorbent assay or cytometric bead array (BD Biosciences). RNA was extracted with RNAeasy columns (Qiagen) after 48 h of in vitro stimulation and was analyzed by quantitative RT-PCR according to the manufacturer’s instructions (Applied Biosystems). Primer-probe mixtures for mouse IL-4, IL-10, IL-21, IL-27 p28, IFN-g, TGF-b and Foxp3 were from Applied Biosystems; catalog numbers and primer and probe sequences for T-bet, GATA-3 and RORgt are in Supplementary Table 1 online. Statistical analysis. Student’s t-test (two-tailed distribution, paired samples) was used for determining the significance of all experiments. P values of less than 0.05 were considered statistically significant. Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS We thank C. Saris (Amgen) for WSX-1-deficient mice; D. Kozoriz, R. Chandwaskar and D. Lee for cell sorting and technical assistance; and W. Gao and P. Putheti for technical support for quantitative PCR analysis. This work was supported by the National Institutes of Health (NS038037 and AI043458 to H.L.W.; R01AI073542-01 to M.O.; and 1R01NS045937-01, 2R01NS35685-06, 2R37NS30843-11, 1R01A144880-03, 2P01A139671-07, 1P01NS38037-04, 1R01NS046414 and a Javits Neuroscience Investigator Award to V.K.K.), the National Multiple Sclerosis Society (RG-2571-D-9 to V.K.K.; RG-3882-A-1 to M.O.; and a postdoctoral fellowship to A.A.), the Juvenile Diabetes Research Foundation Center for Immunological Tolerance at Harvard Medical School, and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Ensino Superior (CAPES; fellowship to J.P.S.P.). AUTHOR CONTRIBUTIONS A.A. and Y.C. designed experiments, did experiments, collected data and contributed to the writing of the manuscript; J.P.S.P. provided help in performing in vivo experiments; E.B. provided advice and helped edit the manuscript; V.K.K., M.O. and H.L.W. supervised the project and edited the manuscript; M.K. and R.A.F. provided IL-10 GFP reporter mice. Published online at http://www.nature.com/natureimmunology Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Reiner, S.L. Development in motion: helper T cells at work. Cell 129, 33–36 (2007). 2. Kim, J.M., Rasmussen, J.P. & Rudensky, A.Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 8, 191–197 (2007). 3. Weiner, H.L. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18, 335–343 (1997).
1388
4. Lohr, J., Knoechel, B., Nagabhushanam, V. & Abbas, A.K. T-cell tolerance and autoimmunity to systemic and tissue-restricted self-antigens. Immunol. Rev. 204, 116–127 (2005). 5. Battaglia, M., Gregori, S., Bacchetta, R. & Roncarolo, M.G. Tr1 cells: from discovery to their clinical application. Semin. Immunol. 18, 120–127 (2006). 6. de Waal Malefyt, R. et al. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J. Exp. Med. 174, 915–924 (1991). 7. Beissert, S., Hosoi, J., Grabbe, S., Asahina, A. & Granstein, R.D. IL-10 inhibits tumor antigen presentation by epidermal antigen-presenting cells. J. Immunol. 154, 1280–1286 (1995). 8. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K. & Muller, W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75, 263–274 (1993). 9. Rakoff-Nahoum, S., Hao, L. & Medzhitov, R. Role of toll-like receptors in spontaneous commensal-dependent colitis. Immunity 25, 319–329 (2006). 10. Steinbrink, K., Wolfl, M., Jonuleit, H., Knop, J. & Enk, A.H. Induction of tolerance by IL-10-treated dendritic cells. J. Immunol. 159, 4772–4780 (1997). 11. Wakkach, A. et al. Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 18, 605–617 (2003). 12. Groux, H. et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389, 737–742 (1997). 13. Barrat, F.J. et al. In vitro generation of interleukin 10-producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J. Exp. Med. 195, 603–616 (2002). 14. Vieira, P.L. et al. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J. Immunol. 172, 5986–5993 (2004). 15. Korn, T. et al. Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat. Med. 13, 423–431 (2007). 16. Pasare, C. & Medzhitov, R. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299, 1033–1036 (2003). 17. Carrier, Y., Yuan, J., Kuchroo, V.K. & Weiner, H.L. Th3 cells in peripheral tolerance. I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-b T celltransgenic mice. J. Immunol. 178, 179–185 (2007). 18. Bettelli, E. et al. Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop spontaneous autoimmune optic neuritis. J. Exp. Med. 197, 1073–1081 (2003). 19. Tang, Q. et al. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat. Immunol. 7, 83–92 (2006). 20. Greve, B. et al. The diabetes susceptibility locus Idd5.1 on mouse chromosome 1 regulates ICOS expression and modulates murine experimental autoimmune encephalomyelitis. J. Immunol. 173, 157–163 (2004). 21. Takeda, A. et al. Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment. J. Immunol. 170, 4886–4890 (2003). 22. Stumhofer, J. et al. Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat. Immunol. 7, 937–945 (2006). 23. Batten, M. et al. Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nat. Immunol. 7, 929–936 (2006). 24. Pflanz, S. et al. WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27. J. Immunol. 172, 2225–2231 (2004). 25. Villarino, A. et al. The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity 19, 645–655 (2003). 26. Gazzinelli, R.T. et al. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-g and TNF-a. J. Immunol. 157, 798–805 (1996). 27. Re, F. & Strominger, J.L. IL-10 released by concomitant TLR2 stimulation blocks the induction of a subset of Th1 cytokines that are specifically induced by TLR4 or TLR3 in human dendritic cells. J. Immunol. 173, 7548–7555 (2004). 28. O’Garra, A. & Vieira, P. TH1 cells control themselves by producing interleukin-10. Nat. Rev. Immunol. 7, 425–428 (2007). 29. Nicholson, L.B., Greer, J.M., Sobel, R.A., Lees, M.B. & Kuchroo, V.K. An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis. Immunity 3, 397–405 (1995). 30. Kadowaki, N., Antonenko, S., Lau, J.Y. & Liu, Y.J. Natural interferon a/b-producing cells link innate and adaptive immunity. J. Exp. Med. 192, 219–226 (2000). 31. Gilliet, M. & Liu, Y.J. Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells. J. Exp. Med. 195, 695–704 (2002). 32. Ochando, J.C. et al. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat. Immunol. 7, 652–662 (2006). 33. Sun, C.M. et al. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 Treg cells via retinoic acid. J. Exp. Med. 204, 1775–1785 (2007). 34. Kastelein, R.A., Hunter, C.A. & Cua, D.J. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu. Rev. Immunol. 25, 221–242 (2007).
VOLUME 8
NUMBER 12
DECEMBER 2007
NATURE IMMUNOLOGY
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES 35. Molle, C. et al. IL-27 synthesis Induced by TLR ligation critically depends on IFN regulatory factor 3. J. Immunol. 178, 7607–7615 (2007). 36. Szabo, S.J. et al. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100, 655–669 (2000). 37. Zheng, W. & Flavell, R.A. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587–596 (1997). 38. Ivanov, I.I. et al. The orphan nuclear receptor RORgt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133 (2006). 39. Sullivan, B.M. et al. Increased susceptibility of mice lacking T-bet to infection with Mycobacterium tuberculosis correlates with increased IL-10 and decreased IFN-g production. J. Immunol. 175, 4593–4602 (2005). 40. Mathur, R.K., Awasthi, A., Wadhone, P., Ramanamurthy, B. & Saha, B. Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses. Nat. Med. 10, 540–544 (2004). 41. Shoemaker, J., Saraiva, M. & O’Garra, A. GATA-3 directly remodels the IL-10 locus independently of IL-4 in CD4+ T cells. J. Immunol. 176, 3470–3479 (2006). 42. Gorelik, L., Fields, P.E. & Flavell, R.A. Cutting edge: TGF-b inhibits Th type 2 development through inhibition of GATA-3 expression. J. Immunol. 165, 4773–4777 (2000).
NATURE IMMUNOLOGY
VOLUME 8
NUMBER 12
DECEMBER 2007
43. Lucas, S., Ghilardi, N., Li, J. & de Sauvage, F.J. IL-27 regulates IL-12 responsiveness of naive CD4+ T cells through Stat1-dependent and -independent mechanisms. Proc. Natl. Acad. Sci. USA 100, 15047–15052 (2003). 44. Kaplan, M.H. & Grusby, M.J. Regulation of T helper cell differentiation by STAT molecules. J. Leukoc. Biol. 64, 2–5 (1998). 45. Neufert, C. et al. IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1. Eur. J. Immunol. 37, 1809–1816 (2007). 46. Ziegler-Heitbrock, L. et al. IFN-a induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3. J. Immunol. 171, 285–290 (2003). 47. Marie, J.C., Liggitt, D. & Rudensky, A.Y. Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factorbeta receptor. Immunity 25, 441–454 (2006). 48. Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006). 49. Staples, K.J. et al. IL-10 induces IL-10 in primary human monocyte-derived macrophages via the transcription factor Stat3. J. Immunol. 178, 4779–4785 (2007). 50. Kamanaka, M. et al. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse. Immunity 25, 941–952 (2006).
1389
