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. Author manuscript; available in PMC: 2009 Jul 13.
Published in final edited form as: Immunity. 2008 May 29;28(6):870–880. doi: 10.1016/j.immuni.2008.03.018

Two functional subsets of Foxp3+ regulatory T cells in human thymus and periphery

Tomoki Ito 1,1,#, Shino Hanabuchi 1,#, Yi-Hong Wang 1, Woong Ryeon Park 1, Kazuhiko Arima 1, Laura Bover 1, F Xiao-Feng Qin 1, Michel Gilliet 1, Yong-Jun Liu 1,*
PMCID: PMC2709453  NIHMSID: NIHMS55824  PMID: 18513999

Summary

Previous studies suggest that thymus produces a homogenous population of natural regulatory T cells (TR) that express a transcriptional factor Foxp3 and control autoimmunity through a cell contact-dependent mechanism. We found two subsets of Foxp3+ natural TR defined by ICOS-expression in the human thymus and periphery. While the ICOS+Foxp3+ TR use IL-10 to suppress dendritic cell function and TGF-β to suppress T cell function, the ICOS−Foxp3+ TR use TGF-β only. The survival and proliferation of the two subsets of TR are differentially regulated by signaling through ICOS or CD28 respectively. We suggest that the selection of natural TR in thymus is coupled with TR differentiation into two subsets imprinted with different cytokine expression potentials and use both cell-contact-dependent and independent mechanisms for immunosuppression in periphery.

Introduction

CD4+CD25+, naturally occurring regulatory T cells (TR) constitute 5–10% of peripheral CD4+ T cells, which play an essential role in the active suppression of autoimmunity in both humans and rodents. TR appear to differentiate as a unique T-cell lineage from the developing T cells in the thymus at either the CD4+CD8+ double-positive (DP) thymocyte or CD4+CD8− single-positive (SP) thymocyte stage. It has become increasingly clear that the intrathymic development of TR depends on signaling through T-cell receptor (TCR) with medium to high affinity for self-antigens, interleukin 2 (IL-2) and signaling through the co-stimulatory receptor CD28. Foxp3, a member of the forkhead transcriptional factor family, has been demonstrated to be the master regulator of TR development in the thymus, as well as TR suppressive function (Fontenot et al., 2003; Hori et al., 2003; Khattri et al., 2003). However, the molecular mechanism by which the Foxp3+ TR-mediate immunosuppression has remained elusive. Although in vivo experiments suggest that IL-10 and TGF-β may be involved in the TR-mediated immunosuppresion (Asseman et al., 1999; Belkaid et al., 2002; Fahlen et al., 2005; Green et al., 2003; Nakamura et al., 2001), conflicting in vitro data presented as to whether IL-10 is involved in the TR-mediated immunosuppresion (Jonuleit et al., 2001; Piccirillo et al., 2002; Taams et al., 2001; Thornton and Shevach, 1998). Currently, three major types of CD4+ TR have been proposed, the CD4+Foxp3− IL-10-producing TR or Tr1 cells that are generated during immune responses in the periphery (Vieira et al., 2004); The TGF-β-expressing TH3 cells originally identified in mice after oral tolerance induction to myelin basic protein (MBP) (Chen et al., 1994; Weiner, 2001); and natural occurring Foxp3+ TR generated in the thymus (Annunziato et al., 2002; Stephens et al., 2001). In this study, we report the identification of two subsets of natural occurring Foxp3+ TR generated in thymus according to their differential expression of a costimulatory receptor ICOS. While the ICOS+Foxp3+ TR use IL-10 to suppress dendritic cell (DC) function and TGF-β to suppress T cell function, the ICOS−Foxp3+ TR use TGF-β mainly. The survival and proliferation of the two subsets of TR are regulated by signaling through ICOS or CD28, respectively. Our study suggest that the molecules used by the ICOS+Foxp3+ and ICOS−Foxp3+ natural occurring TR to mediate immunosuppression mirrors that used by the peripheral Tr1 cells and TH3 cells, respectively. The selection of the Foxp3+ TR in thymus is likely coupled with their differentiation into the ICOS+Foxp3+ imprinted with the high IL-10-producing capacity and the ICOS−Foxp3+ imprinted with the high TGF-β-expresion capacity.

RESULTS

Identification of ICOS+ and ICOS− two subsets of Foxp3+ TR in human thymus

During a study on the expression of costimulatory molecules in the human thymus, we found that Foxp3+ TR within the thymic medulla (Fig 1a) were grouped into two subsets, ICOS+ and ICOS− (Fig 1b). Because signaling ICOS primes CD4+ T cells to produce IL-10 (Akbari et al., 2002; Hutloff et al., 1999; Lohning et al., 2003, Ito et al., 2007; Janke et al., 2006), we questioned whether the ICOS+ and ICOS− TR were functionally different. The CD25+ICOS+ and CD25+ICOS− subsets were isolated from the CD4+CD8− thymocytes and both were found to express Foxp3 (Fig 1c). The ICOS+Foxp3+ TR acquired the ability to produce more IL-10 (9–20% by intracellular IL-10 staining) than the ICOS−Foxp3 TR did (3–6%) after priming with anti-CD3 or anti-CD3 plus ICOS-ligand (ICOSL) (Fig 1d). By contrast, the ICOS−Foxp3+ TR expressed higher levels of membrane-bound TGF-β1 (mTGF-β) than the ICOS−Foxp3+ TR (Fig 1e). Both subsets were anergic and have the ability to suppress CD4+CD25− T-cell proliferation in responses to allogeneic stimulation (Fig 1f). These data suggest that the human thymus may generate two functionally distinct Foxp3+ TR subsets.

Figure 1.

Figure 1

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Two subsets of Foxp3+ TR defined by ICOS expression in thymus. a, Double staining of Foxp3 (red) and ICOS (blue) in the human thymus showing the Foxp3+ICOS+ and Foxp3+ICOS− two TR subsets located in the thymic medulla at a low magnification (100×). The scale bar, 50 μm. b, Double immunofluorescence staining of Foxp3 (red) and ICOS (green) performed on the same section of thymus further confirmed the two Foxp3+ T-cell populations: nuclear Foxp3+ cytoplasmic ICOS+ T cells and nuclear Foxp3+ cytoplasmic ICOS− T cells. c, Flow cytometric analysis of ICOS and CD25 expression on CD4+CD8− thymocytes and Foxp3 expression in sorted two TR subsets. d, Flow cytometric analysis of intracellular cytokines in each thymic TR subset after two rounds of culturing with anti-CD3 antibody in the presence of IL-2 and IL-7 on parental L cells or ICOSL-L cells for 5 days. e, Flow cytometric analysis of membrane TGF-β on each thymic TR subset after culturing with anti-CD3 antibody in the presence of IL-2 and IL-7 on parental L cells or ICOSL-L cells for 5 days. The numbers in the histograms indicate the mean fluorescence intensity, which is calculated by subtraction of mean fluorescence intensity with the isotype control from that with the anti-TGF-β antibody. f. Proliferative response of CD4+ CD25− T cells and thymic CD25high ICOS+ CD4+ TR or CD25high ICOS− CD4+ TR primed with anti-CD3 antibody in the presence of IL-2 and IL-7 on ICOSL-L cells for 5 days and their mixtures in response to T cell-depleted peripheral blood mononuclear cells was assessed by [3H]thymidine incorporation. Ratio of TR to CD4+ CD25− T cells is 1 to 2. Error bars represent the s.e.m. of triplicate wells. Similar results were observed in four (a, b) and three (c–f) experiments, and the results of a representative experiment are shown.

ICOS+Foxp3+ and ICOS−Foxp3+ two subsets of TR present in peripheral lymphoid tissues and blood

The question was whether the ICOS+Foxp3+ TR and ICOS−Foxp3+ TR generated in the thymus exist in the periphery. We found that human tonsils and lymph nodes indeed contained ICOS+Foxp3+ and ICOS−Foxp3+ two subsets of TR in the T cell rich and sub-epithelial cell areas (Fig 2a, b, c, d). The germinal center (GC) contained large numbers of ICOS+ follicular T helper cells, few of them expressed Foxp3 (Fig 2a, c). The CD25+ICOS+Foxp3+ and CD25+ICOS−Foxp3+ TR subsets were also found in the blood (Fig 2e). Although both subsets expressed similar levels of CD28, CD27, CD58, CD54, CD62L and CCR7, the ICOS+Foxp3+ subset expressed relatively higher CTLA-4 and CD38 (Fig 2f). Both subsets expressed little or no CD127 (IL-7Rα (Liu et al., 2006; Seddiki et al., 2006a)), CRTH2 (a marker for TH2 memory cells (Wang et al., 2006)), CD57 (a marker for follicular T helper cells in humans (Rasheed et al., 2006)) and CD103. A quantitative analyses from 6 human thymus and 18 human peripheral blood shows that the percentage of the ICOS+Foxp3+ TR is 4.02% in thymus versus 2.45% in the peripheral blood and the percentage of the ICOS−Foxp3+ TR is 2.56% in the thymus versus 3.59% in the periphery (Fig 2g).

Figure 2.

Figure 2

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Two subsets of Foxp3+ TR defined by ICOS expression in periphery. a,c, Double staining of Foxp3 (red) and ICOS (blue) in the human tonsil (a) and lymph node (c) showing two Foxp3+ T cell populations: Foxp3+ICOS+ T cells and Foxp3+ICOS− T cells at a low magnification (100×). All scale bars, 50 μm. b,d, Immunofluorescence staining of Foxp3 (red) and ICOS (green) performed on the same section of tonsil (b) and lymph node (d) showing two Foxp3+ T-cell populations: nuclear Foxp3+ cytoplasmic ICOS+ TR and nuclear Foxp3+ cytoplasmic ICOS− TR. e, Human peripheral blood CD25high CD4+ TR were separated into ICOS+ and ICOS− subpopulations. Foxp3 expression in each subset was determined by flow cytometry. f, h, Phenotypical analysis of two subsets of TR in adult blood (f) and cord blood (h). Intranuclear Foxp3 and intracytoplasmic CTLA4 and the cell surface markers on sorted blood CD25high ICOS+ CD4+ TR and CD25high ICOS− CD4+ TR were determined by flow cytometry. g, Percentages of two subsets of TR in 6 thymus and 18 adult blood from healthy donors. Percentages of CD25high ICOS+ CD4+ TR and CD25high ICOS− CD4+ TR among CD4+ T cells were determined by flow cytometry. Bars represent the group means. Statistical significance was determined using paired Student’s t-test. Similar results were observed in three (a–d) and five (e) experiments, and at least three experiments for each marker (f, h), and the results of a representative experiment are shown.

Resent studies have shown that there are at least two populations of Foxp3+ TR in human adult blood, one with CD45RA+ naïve phenotype and one with CD45RO+ memory phenotype (Seddiki et al., 2006b; Valmori et al., 2005). The Foxp3+ TR with the CD45RA+ naïve phenotype represent the majority in human cord blood (Takahata et al., 2004). To establish the relationship of the two subsets of Foxp3+ TR classified by ICOS expression and the two subsets of Foxp3+ TR based on the expression of CD45 isoforms, we analyzed the expression of CD45RA and CD45RO by the ICOS+Foxp3+ and ICOS−Foxp3+ TR from both adult blood and cord blood. Now we show that while all the ICOS+Foxp3+ TR expressed CD45RO, the ICOS−Foxp3+ TR contained CD45RO+ and CD45RO− two subpopulations in adult blood (Fig 2f). Although the majority of the cord blood CD25+Foxp3+ TR expressed high CD45RA, the ICOS+Foxp3+ TR contained a fraction of TR with the CD45RO+CD45RAlow memory phenotype (Fig 2h). These data suggest that the ICOS+Foxp3+ TR from both adult and cord blood are more related to the TR with the memory phenotype and the ICOS−Foxp3+ TR are more related to the TR with the naïve phenotype previously reported. In addition, the CD25+Foxp3+ TR did not express the typical naïve T cell phenotype. Another recent study has divided the peripheral Foxp3+ TR into a MHC class II+ subset and a MHC class II− subset (Baecher-Allan et al., 2006). However, the ICOS+Foxp3+ TR and ICOS−Foxp3+ TR did not show significant differences in MHC class II expression (data not shown). In order to establish the relationship between the two subsets of TR in the blood with the Foxp3+ CD31+ recent thymic emigrant (Valmori et al., 2005, Haas et al., 2007, Kimmig et al., 2002), we analyzed the expression of CD31 by flow cytometry (Fig 2f and 2h). We found that about 50% of the ICOS+Foxp3+ TR and all of the ICOS−Foxp3+ Treg in cord blood expressed CD31, therefore suggesting that both subsets of TR contain the recent thymic emigrants. Interestingly fewer TR of both subsets from adult blood express CD31, consisting with the fact that there is a dramatic decrease in the thymic output during adult life.

The ICOS+Foxp3+ TR have the ability to produce the highest levels of IL-10

We investigated whether the two subsets had a similar capacity to produce cytokines. First, we examined the IL-10 and IL-2 production by the total TR and two TR subsets, as well as naïve and memory T cells immediately after activation for 6 hours with phorbol myristate acetate plus ionomycin (FACS analyses) or for 24 hours with anti-CD3 plus anti-CD28 antibodies (enzyme-linked immunosorbent assay; ELISA). We found that the CD25+Foxp+ICOS+ TR contained the highest numbers of IL-10-producing cells, which is is about 4 times more than the CD25+Foxp+ICOS− (Fig 3a). While freshly isolated naïve and memory T cells produced high levels of IL-2, total TR and the two subsets of TR produced very low levels of IL-2 imediately after activation, as analyzed by both flow cytometry and IL-2 ELISA (Fig 3a), indicating a key feature of TR. We found that both subsets of freshly isolated TR produced very low levels of IL-4, IL-5, IL-13, IFN-γ, TNF-α, and mTGF-β without priming (data now shown).

Figure 3.

Figure 3

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Different capacity of two TR subsets to produce cytokines. Blood CD25high ICOS+ TR, CD25high ICOS− TR, and whole CD25high CD4+ TR, as well as CD25−CD45RO− naïve and CD25−CD45RO+ memory T cells were isolated by same donor. a, b, Analysis of IL-10 and IL-2 production by freshly isolated T cell subsets (a) or T cells primed for 5 days with anti-CD3 antibody in the presence of IL-2 on parental L cells (b) by intracellular cytokine staining (left) and ELISA (right). ELISA data are means ± s.e.m. of four independent experiments. c–e, Blood TR and other T-cell subsets were cultured for 5 days with anti-CD3 antibody in the presence of IL-2 on parental L cells or ICOSL-L cells. Cytokine production of primed blood TR subsets were analyzed by flow cytometry (c, d) and ELISA (e). ELISA data are means ± s.e.m. of four independent experiments. Statistical significance was determined using paired Student’s t-test. *: p<0.05, **: p<0.01 (e). f, g, Flow cytometric analysis of CD25, ICOS, CTLA4, and Foxp3 expression with CFSE labeling by CD25−CD45RO+ memory T cells and TR subsets after culturing for 5 days with anti-CD3 antibody in the presence of IL-2. h, Flow cytometric analysis of IL-10 and Foxp3 expression in T-cell subsets after culturing for 5 days with anti-CD3 antibody in the presence of IL-2 on ICOSL-L cells. Similar flow cytometric results were observed in three experiments (a–d, f, g, h), and the results of a representative experiment are shown.

Then we examined the capacity of cytokine production by the two TR subsets after 5 days of prining with anti-CD3 antibody or anti-CD3 antibody plus ICOSL. Although 5 day-primed total TR and the two subsets of TR produced more IL-2 (Fig 3b) than the freshly isolated TR subsets did, they produced much less IL-2 than the 5 day-primed naïve and memory CD4+ T cells as indicated by both FACS and ELISA analyses (Fig 3b). We found that the ICOS+ TR produced a huge amount of IL-10 (47–76% by intracellular IL-10 staining in Fig 3b and 3d, and 4.9–7.9 ng/ml by IL-10 ELISA in Fig 3e). In contrast, the ICOS− TR produced only a moderate amount of IL-10 (8–16% by intracellular IL-10 staining in Fig 3b and 0.8–1.4 ng/ml by IL-10 ELISA in Fig 3e). In addition as indicated by both ELISA and intracellular cytokine staining, the ICOS+ TR produced more IFN-γ but less TNF-α and IL-2 than the ICOS− TR (Fig 3c, d, and e). Both subsets of TR produced low or undetectable levels of IL-4 or IL-13 (Fig 3c and e). After 5 days of activation by anti-CD3 antibody, while the ICOS+Foxp3+ TR maintained their expression of high CD25, ICOS, CTLA4 and Foxp3, the ICOS−Foxp3+ TR acquired the expression of ICOS and CTLA4, and maintained the expression of CD25 and Foxp3 (Fig 3f). However, the ICOS−Foxp3+ TR did not acquire the capacity to produce high IL-10 (Fig 3b, d, and e). Two color flow cytometric analyses further showed that although ICOS−Foxp3+ TR and CD25−CD45RO+ memory T cells rapidly expressed ICOS after activation and divison (Fig 3g), they produced much lower IL-10 than did the in vivo-derived ICOS+ TR (Fig 3h). These data suggest that the two subsets of Foxp3+ TR did not convert to each other following in vitro activation.

Figure 4.

Figure 4

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Foxp3+ICOS+CD25+ TR have the capacity to produce the highest levels of IL-10. a, Blood CD4+ T cells were separated into ICOS+CD45RO+ memory, ICOS−CD45RO+ memory, and ICOS−CD45RO− naive T cells based on the expression of ICOS and CD45RO. Flow cytometric analysis of intracellular cytokines in each T-cell subset after culturing with anti-CD3 antibody in the presence of IL-2 on parental L cells or ICOSL-L cells for 5 days. b, Flow cytometric analysis of Foxp3 expression by five subsets of CD4+CD45RO+ memory subpopulations: CD25−ICOS−CD45RO+ memory, CD25−ICOS+ follicular TH-like, CD25+ICOS+ TR, CD25+ICOS− TR, and CD25lowCD45RO+CRTH2+ TH2 memory T cells. c, d, Five blood CD4+ memory T-cell subpopulations were cultured with anti-CD3 antibody in the presence of IL-2 on parental L cells or ICOSL-L cells for 5 days. IL-10 production by T-cell subsets were analyzed by flow cytometry (c) and ELISA (d). ELISA data are means ± s.e.m. of four independent experiments. Similar flow cytometric results were observed in four experiments (a–c), and the results of a representative experiment are shown.

Because the ICOS+Foxp3+ TR had the capacity to more IL-10 than the ICOS−Foxp3+ TR after priming, we questioned whether the ICOS+Foxp3+ TR have the capacity to produce more IL-10 than other CD4+ T cell subsets. Peripheral blood CD4+ T cells were separated by cell sorting into CD45RO− naïve T cells, CD45RO+ICOS+ memory T cells and CD45RO+ICOS− memory T cells (Fig 4a). After 5 days of priming with anti-CD3 antibody or anti-CD3 antibody plus ICOSL, the major IL-10-producing cells were found to be in the CD45RO+ICOS+ memory T cells (Fig 4a). We then analyzed the capacity of IL-10 production among all CD45RO+ memory T cell subsets including: CD25−Foxp3− non-regulatory T cells, CRTH2+ TH2 memory cells, CD25−ICOS+ follicular TH-like cells (Rasheed et al., 2006), ICOS+ TR, and ICOS− TR (Fig 4b). After 5 days of culture, the ICOS+ TR were found to produce the highest level of IL-10 (Fig 4c and d). All other subsets produced 5 to 10 times lower amounts of IL-10 (Fig 4c and d). These data suggest that the CD25+Foxp+ICOS+ TR have the ability to produce the highest amounts of IL-10 among the circulating CD4+ T cell pool.

The ICOS−Foxp3+ TR express higher TGF-β than other CD4+ T cells

Because TGF-β has been suggested to be the major molecules used by the Foxp3+ TR for immunosuppression (Fahlen et al., 2005; Green et al., 2003; Nakamura et al., 2001), we analyzed the expression of mTGF-β by the The ICOS+Foxp3+ TR, the ICOS−Foxp3+ TR, and the CD4+CD25−Foxp3− T cells after activation. We found that the ICOS− TR expressed higher levels mTGF-β than the ICOS+ TR did and the CD25−CD45RO+ total memory T cells expressed little mTGF-β (Fig 5a). These findings which were confirmed by quantitative polymerase chain reaction analyses (Fig 5b) suggest that the CD4+CD25+Foxp3+ naturally occurring TR can be divided into an ICOS+ subset that has the capacity to produce massive amounts of IL-10 and express moderate levels of mTGF-β and an ICOS− subset that express higher levels of mTGF-β but produce low amounts of IL-10.

Figure 5.

Figure 5

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Different capacity of two subsets of TR to express TGF-β. a, Flow cytometric analysis of membrane TGF-β on the primed blood TR subsets after culturing with anti-CD3 antibody in the presence of IL-2 on parental L cells or ICOSL-L cells for 5 days. The numbers in the histograms indicate the mean fluorescence intensity, which is calculated by subtraction of mean fluorescence intensity with the isotype control from that with the anti-TGF-β antibody. Similar results were observed in three experiments, and the results of a representative experiment are shown. b, The real-time quantitative RT-PCR analysis for TGFB mRNA expression in blood CD25−CD45RO+CD4+ memory T cells, and two TR subsets after culturing with anti-CD3 antibody in the presence of IL-2 for 5 days. Data are means ± s.e.m. of three independent experiments.

ICOS+Foxp3+ TR and ICOS−Foxp3+ TR use different molecular mechanisms for suppression

An important question is whether the ICOS+ and ICOS− TR have different functions. CD4+CD45RO−CD25− naïve T cells underwent strong proliferation in culture with allogeneic myeloid dendritic cells (DCs), which was strongly inhibited by activated ICOS+ TR and ICOS− TR (Fig 6a). Neutralizing antibody to IL-10 or TGF-β inhibitor partially blocked the inhibitory function of ICOS+ TR, and anti-IL-10 antibody plus TGF-β inhibitor led to a complete blockage, as indicated by [3H]thymidine incorporation (Fig 6b) and CFSE-labeling experiments (Fig 6c). However, only TGF-β inhibitor but not anti-IL-10 antibody blocked the function of ICOS− TR (Fig 6b and c). The ICOS− TR–mediated suppression through mTGF-β was dependent on the cell-cell contact because a Transwell system completely block the function of ICOS− TR, whereas ICOS+ TR–mediated suppression was only partially blocked by the Transwell (Fig 6d). This is consistent with the fact that the ICOS+ TR used both mTGF-β and soluble IL-10 suppression mechanisms. We found that CD86 expression on DCs was suppressed by the coculture with ICOS+ TR but not by the ICOS+ TR and this suppression was restored by anti-IL-10 antibody (Fig 6e), indicating that ICOS+ TR use IL-10 to inhibit DC maturation. Freshly isolated ICOS+ TR and ICOS− TR show similar functions when compared with the primed ICOS+ TR and ICOS− TR in the above experimental systems (Supplementary Fig 1)

Figure 6.

Figure 6

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Suppressive function of two subsets of TR. CD4+ naive T cells and autologous CD25high ICOS+ CD4+ TR or CD25high ICOS− CD4+ TR were sorted from human peripheral blood. CD4+ naive T cells were used as responder, and primed TR after culturing with anti-CD3 antibody in the presence of IL-2 on ICOSL-L cells for 5 days were used as suppressor. Mixtures of T-cell populations were cultured for 5 days with allogeneic monocyte-derived DCs as stimulator. The results using primed TR as suppressor were shown here, and that using freshly isolated TR were shown in Supplementary Figure 1. a, Ratios of TR to naive T cells are shown in the figure. Proliferation was assessed by [3H]thymidine incorporation and error bars represent the s.e.m. of triplicate wells. b, c, Ratio of TR to naive T cells is 1 to 2. Neutralizing anti-IL-10 plus IL-10 receptor antibodies and/or TGF-β receptor type I (TβRI) inhibitor were added into T-cell culture. Proliferation was assessed by [3H]thymidine incorporation and error bars represent the s.e.m. of triplicate wells. Statistical significance was determined using Student’s t-test. **: p<0.01 (b). CD4+ naive T cells were labeled with CFSE before culture, and cells after culturing were analyzed by flow cytometry, gated on CD4+CFSE+ cells (c). d, Indicated T-cell populations were cultured in Transwell at 1:2 ratio of TR to naive T cells with allogeneic monocyte-derived DCs. Proliferation was assessed by [3H]thymidine incorporation and error bars represent the s.e.m. of triplicate wells. e, Primed TR, naive T cells, and allogeneic monocyte-derived DCs were cultured at 1:1:1 ratio. After 4 days of culture, CD86 expression on DCs were analyzed by flow cytometry, gated on CD11c+ cells. Filled histogram indicates CD86 expression on DCs in coculture with naive T alone, and line histogram indicates CD86 expression on DCs in coculture with naive T and TR. Dot histogram indicates isotype control on DCs. Similar results were observed in three experiments (a–e), and the results of a representative experiment are shown.

Survival and proliferation of ICOS+Foxp3+ TR and ICOS−Foxp3+ TR are differentially regulated

Another key question is whether the survival and expansion of the peripheral ICOS+ TR and ICOS− TR were differentially regulated. We found that the ICOS+ TR but not ICOS− TR underwent a massive apoptosis in culture without IL-2, unless signaling ICOS (Fig 7a, and b). In the presence of IL-2, ICOSL strongly promoted the proliferation of anti-CD3-activated ICOS+ TR (Fig 7c). By contrast, anti-CD28 antibody strongly inhibited the proliferation of ICOS+ TR induced by anti-CD3 antibody plus ICOSL. However, both ICOSL and anti-CD28 antibody promoted the proliferation of ICOS− TR and CD4+ naïve T cells induced by anti-CD3 antibody and IL-2 (Fig 7c). These suggest that the survival and homeostatic proliferation of the ICOS+ TR and ICOS− TR are regulated by different costimulatory molecules.

Figure 7.

Figure 7

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Survival and expansion of two TR subsets were differentially regulated. a, b, Sorted blood CD25high ICOS+ CD4+ TR and CD25high ICOS− CD4+ TR were cultured in the absence of IL-2 on the idicated condtions for 5 days. Flow cytometric analysis of annexin V staining on each TR subset after culturing. The numbers in the histograms indicate the percentage of cells in the gate (a). The number of viable T cells counted by trypan-blue exclusion is shown in the bar graph (b). c, 5 × 104 TR subsets and CD4+ naive T cells were cultured in the presence of IL-2 on the indicated conditions for 5 days. The viable cell number of each TR subset counted by trypan-blue exclusion is shown. Bars represent means of four experiments. d, 5 × 104 TR subsets were cultured with blood pDCs or mDCs (DC/T cell ratio of 1:2) for 4 days in the presence of neutralizing anti-ICOSL mAb or neutralizing anti-CD80/CD86 mAbs. The viable cell number of each TR subset counted by trypan-blue exclusion is shown. Similar results were observed in three (a) and four (b, d) experiments, and the results of a representative experiment are shown.

Plasmacytoid DCs but not myeloid DCs promote the proliferation of ICOS+ TR through ICOSL

We and other have recently shown that while plasmacytoid DCs (pDCs) preferentially express ICOSL, myeloid DCs (mDCs) preferentially express CD80/86 following activation (Ito et al., 2007; Janke et al., 2006). pDCs but not mDCs have unique ability to prime naïve CD4+ T cells to differentiate into IL-10-producing cells (Kadowaki and Liu, 2002; Rissoan et al., 1999). Therefore we further investigated the pDCs and mDCs may differentially regulate the proliferation of the ICOS+ TR versus the ICOS− TR. We found that pDCs but not mDCs selectively promoted the proliferation of the autologous ICOS+ TR (Fig 7d). The ability of pDCs to promote the proliferation of the ICOS+ TR was dependent on ICOSL but not on CD80 and CD86 (Fig 7d). By contrast, mDC preferentially promoted the proliferation of the autologous ICOS−TR through CD80/CD86-dependent mechanism (Fig 7d). These data suggest that while the homeostasis of the ICOS+ TR is preferentially maintained by the pDCs through ICOSL, homeostasis of the ICOS− TR is preferentially maintained by the mDCs through CD80/CD86.

DISCUSSION

In this study, we reported the identification of two subsets of natural Foxp3+ TR in human thymus, and peripheral blood and secondary lymphoid tissues. The ICOS+ TR subset uses two mechanisms for immunosuppression, including IL-10-mediated suppression of antigen-presenting cell function and mTGF-β-mediated T cell-T cell contact-dependent suppression. The ICOS− TR subset uses predominantly the mTGF-β-mediated T cell-T cell contact-dependent suppression. The ICOS+ TR display a striking propensity to undergo rapid apoptosis in culture, unless signaled by ICOSL. In addition, ICOS and CD28 costimulation have opposing effects on the ICOS+ TR; while ICOSL costimulates their proliferation, anti-CD28 signaling inhibits their proliferation. By contrast, the ICOS− TR do not display such propensity for apoptosis and anti-CD28 signaling strongly promotes their proliferation. We provided further experimental data showing that while activated pDCs preferentially promote the proliferation of the autologous ICOS+ TR through ICOSL, activated mDCs preferentially promote the proliferation of the autologous ICOS− TR through B7 signaling.

The question is whether the ICOS+ TR are really generated in the thymus and whether ICOS+ TR are simply derived from the ICOS− TR in the periphery following activation. Although it is possible that the ICOS− TR can be converted into the ICOS+ TR in the periphery under similar circumstances that induce the generation of IL-10-producing Tr1 cells from naïve T cells, the following lines of evidence support the concept that thymus not only plays a critical role in the selection of the Foxp3+ naturally occurring TR but also have the ability to imprint the two TR subsets that have the potential to produce different cytokines in the periphery upon activation. First, both newborn thymus and cord blood already contains the ICOS+Foxp3+ TR, and furthermore thymus appears to contain more ICOS+Foxp3+ TR than the adult peripheral blood. Second, although ICOS is rapidly upregulated on ICOS− non-regulatory T cells and ICOS−Foxp3+ TR, these in vitro activated T cells acquires only ICOS expression, but do not acquire the ability to produce high IL-10 as the in vivo-derived ICOS+ TR. Third, the dramatic differences between the ICOS+Foxp3+ TR and ICOS−Foxp3+ TR in terms of prone to apoptosis and differential molecular regulation of survival and proliferation suggest that the two subsets have undergone very different differentiation programs in the thymus, and that one does not simply represent the other one at a transient activation state. Fourth, we found that all the ICOS−Foxp3+ TR and about 50% of the ICOS+Foxp3+TR from cord blood express CD31, a marker for the recent thymic emigrants (Valmori et al., 2005, Haas et al., 2007, Kimmig et al., 2002), suggesting that the two subsets could be derived directly from the thymus.

Similar to GITR, CTLA4, and CD25, ICOS is a T cell activation maker, which can all be upregulated on naïve T cells upon activation. Although CD25 can be readily rapidly induced on peripheral non-regulatory T cells upon activation by anti-CD3 stimulation, however these in vitro activated CD25+ T cells do not became regulatory T cells. We believe that the expression of ICOS during Foxp3+ TR selection reflect a dramatic functional changes on the Foxp3+ TR in terms of cytokine production potential (such as IL-10 and TGF-β expression), and molecular regulation of cell survival and proliferation. Our study highlights an important principle in the developmental immunology that a signal upregulating ICOS or CD25 on T cells during their development in thymus have more dramatic impact on their function than a similar event that effects the mature T cells in the periphery. In another word, early education in thymus is more important.

Currently, three types of TR have been reported, including Foxp3+ naturally occurring TR, inflammation-induced IL-10-producing Tr1 cells, and TGF-β-expressing TH3 cells (O’Garra and Vieira, 2004; Roncarolo and Levings, 2000; Thompson and Powrie, 2004). IL-10-producing Tr1 cells were originally isolated from patients with severe combined immunodeficiency who had undergone successful HLA-mismatched bone marrow transplantation (Bacchetta et al., 1994; Groux et al., 1997). Subsequently, IL-10-producing Tr1 cells were generated from naïve CD4+ T cells during antigen-driven T cell immune responses (Asseman and Powrie, 1998; McGuirk et al., 2002). It was recently shown that IL-10-producing Tr1 cells generated in vitro from naïve CD4+ T cells in the presence of dexamethasone and the active form of vitamin D3 did not express Foxp3 (Vieira et al., 2004). Our finding that the Foxp3+ICOS+ naturally occurring TR produced the highest levels of IL-10 after activation among all CD4+ T cell subsets, suggests that Foxp3+ICOS+ naturally occurring TR are the major precursors of circulating IL-10-producing regulatory T cells, and may play a complementary function with the IL-10-producing Tr1 cells that are generated during inflammation in the tissues. The TGF-β-expressing TH3 cells were originally identified in mice after oral tolerance induction to MBP (Chen et al., 1994; Weiner, 2001). TH3 cells suppress the function of MBP-specific TH1 effector cells in a TGF-β-dependent fashion in vivo and in vitro (Chen et al., 1994). The identification of the Foxp3+ICOS− TR subset that mainly use mTGF-β but not IL-10 to directly inhibit T cell proliferation suggests that the CD25+Foxp3+ICOS− TR may represent major precursors of circulating TGF-β-expressing regulatory T cells, and may play a complementary function with the TGF-β-expressing TH3 that are generated during inflammation in the tissues. Therefore, the two subsets of naturally occurring Foxp3+ TR may functionally mirror the two subsets of peripheral-induced TR.

In vivo studies demonstrated that the function of Foxp3+ TR depends on IL-10 and/or TGF-β (Asseman et al., 1999; Belkaid et al., 2002; Fahlen et al., 2005; Green et al., 2003; Nakamura et al., 2001). However studies using cultured and cloned CD4+CD25+ TR in vitro have generated conflicting data as to whether the TR function depends on IL-10 or TGF-β (Jonuleit et al., 2001; Piccirillo et al., 2002; Taams et al., 2001; Thornton and Shevach, 1998). Recent studies showed that the cultured or cloned human CD4+CD25+ TR expressed TGF-β but not IL-10 and these cells depend on TGF-β for immune regulation and were distinct from the IL-10-producing Tr1 cells (Annunziato et al., 2000; Levings et al., 2002). Our current study suggests that these studies may be over looked the function of the ICOS+Foxp3+ TR subset. This is because that the ICOS+Foxp3+ TR subset that has the ability to produce a huge amount of IL-10 is more prone to undergoing apoptosis in cultures than the ICOS−Foxp3+ TR subset is. In addition, IL-10 does not directly inhibit T-cell proliferation induced by anti-CD3 and anti-CD28 antibodies, it only inhibits T-cell proliferation by blocking the function of antigen-presenting cells, such as DCs. However, many studies on TR function use a T-cell proliferation assay induced by anti-CD3 and anti-CD28 antibodies. Recent studies show the presence of Foxp3+IL-10+ TR preferentially colonize the lamina propria of colon (Kamanaka et al., 2006; Uhlig et al., 2006). Our study suggests that the ICOS+Foxp3+ TR and ICOS−Foxp3+ TR may have preferences in homing to different tissues.

Currently, both thymic epithelial cells and DCs have been suggested to select TR in thymus (Aschenbrenner et al., 2007; Bensinger et al., 2001; Goldschneider and Cone, 2003; Liu, 2006; Watanabe et al., 2005). In the periphery, DCs were shown to play key roles in the induction and maintenance of Foxp3+ TR (Kretschmer et al., 2005; Tarbell et al., 2006). A subsets of intestinal DC/macrophage subsets were recently shown to selectively induce the generation of Foxp3+ TR through retinoid acids (Coombes et al., 2007; Sun et al., 2007). These together with our current findings that the pDCs and mDCs play a different role in regulating the proliferation of the ICOS+Foxp3+ TR and ICOS−Foxp3+ TR, respectively, suggest that the ICOS+Foxp3+ TR and the ICOS−Foxp3+ TR are selected and educated by different population of antigen-presenting cells within the thymus.

Experimental Procedures

Isolation of TR subsets

The institutional review board for human research at the M. D. Anderson Cancer Center approved this study. Human thymuses from fetuses (19–23 weeks of gestation), newborns, and children (2 days to 2 years old) were obtained from Advanced Bioscience Resources and the Texas Children’s Hospital, respectively. Cord bloods were obtained from M. D. Anderson Cancer Center (Department of Bone Marrow Transplantation). Adult blood buffy coats from healthy donors were obtained from the Gulf Coast Regional Blood Center, Texas. The following antibodies were used for cell sorting on a FACSAria (BD Biosciences) to reach >99% purity; phycoerythrin (PE)-Cy5.5- or allophycocyanin (APC)-Cy7-conjugated anti-CD4 (S3.5), APC-conjugated anti-CD8 (SK1), fluorescein isothiocyanate (FITC)-, PE-, or PE-Cy7-conjugated anti-CD25 (M-A251), and biotinylated anti-ICOS (ISA-3) antibodies followed by PE- or APC-streptavidin. For the isolation of T-cell lineage thymocytes, thymuses were digested and mononuclear cells were separated by Ficoll centrifugation. CD4+T-cell lineage thymocytes were obtained by negative depletion using a mixture of mouse monoclonal antibodies against markers CD8, CD11c, CD14, CD15, CD20, CD56 and CD235a. This was followed by incubation with goat anti-mouse IgG-coated magnetic beads (M-450, Dynal). CD4+CD8−CD25+ICOS+ and CD4+CD8−CD25+ICOS− thymocytes were isolated by cell sorting. For the isolation of cord blood and adult blood CD4+ TR subsets, CD4+CD25+ T cells were enriched by a CD4+CD25+ regulatory T-cell Isolation Kit (Miltenyi Biotec). CD4+CD25high+ICOS+ and CD4+CD25high+ICOS− TR subsets were isolated by cell sorting. For detection of intranuclear Foxp3, anti-Foxp3 (PCH101; eBioscience) staining kit was used. For detection of membrane-bound TGF-β1 on the surface, cells were stained with anti-TGF-β1 (27232; R&D Systems) antibody (Nakamura et al., 2001), followed by staining with PE- or APC-conjugated anti-mouse IgG1 antibodies (BD Biosciences). The labeling of T cells with CFSE (Molecular Probes) was performed, as described (Watanabe et al., 2005). Isolation of the naive, memory, or other T cell subsets and the other antibodies used are described in the Supplementary Methods online.

T-cell culture

Sorted blood T-cell subsets were cultured for 5 days on irradiated CD32-expressing parental L cells or CD32/ICOSL-expressing L cells (ICOSL-L cells) (described in supplementary Methods) precoated with anti-CD3 antibody (OKT3; 0.2 μg/ml) in the presence of 50 U/ml of IL-2 (eBioscience) in 48-well culture plates (T cell-to-L cell ratio 2.5:1). In some experiments, soluble anti-CD28 antibody (28.2; 1 μg/ml) or isotype-matched control was added in the culture. In another experiment, autologous 5×104 TR subsets were cultured with blood pDCs or mDCs (DC/T cell ratio of 1:2) in round-bottomed 96-well culture plates for 4 days in the presence of 50 μg/ml anti-ICOSL mAb (eBioscience) and a combination of 5 μg/ml anti-CD80 and 10 μ g/ml anti-CD86 mAbs (R&D Systems). pDCs and mDCs were isolated from the buffy coat of healthy adult volunteers as previously described (Ito et al., 2007; Watanabe et al., 2005). Sorted CD4+CD8−CD25+ICOS+ and CD4+CD8−CD25+ICOS− thymocytes were cultured for two rounds of 5 day-stimulation on parental L cells or ICOSL-L cells precoated with anti-CD3 antibody (0.2 μg/ml) in the presence of 50 U/ml of IL-2 and 20 ng/ml of IL-7 (R&D Systems). RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin G, and streptomycin was used for cell cultures. The generation of transfected L cells is described in the Supplementary Methods online.

Analysis of intracellular T-cell cytokine production

For detection of intracellular cytokine production, the T cells were restimulated with 50 ng/mlof phorbol myristate acetate plus 2 μg/ml of ionomycin for 6 h. 10 μg/mlbrefeldin A was added during the last 2 h. The cells were stained with PE-anti-Foxp3 (PCH101), PE-anti-IL-4 (8D4-8), or PE-anti-TNF-α (MAb11), PE- or FITC-anti-IL-2 (MQ1-17H12) or FITC-anti-IFN-γ (B27), and APC-anti-IL-10 (JES3-19F1) antibodies using the Foxp3 staining kit (eBioscience) or Caltag FIX and PERM kit.

T-cell cytokine production by ELISA

The blood T-cell subsets were collected, washed, and then restimulated with plate-bound 5 μg/ml anti-CD3 and 2 μg/ml soluble anti-CD28 antibodies at a concentration of 106 cells/ml for 24 h. The levels of IL-2, IL-4, IL-10, IL-13, TNF-α, and IFN-γ in the supernatants were measured by ELISA (R&D Systems). Detail of real time PCR methods is described in the Supplementary Methods online.

Suppressive function assay

Sorted 4×104 CD4+CD45RO−CD25− naive T cells as responder and different numbers of autologous CD4+CD25+ICOS+ TR or CD4+CD25+ICOS− TR as suppressor, these cell type and their mixtures (at TR-to-naive T ratio 1:2 or at different ratios) were stimulated in round-bottom 96-well plates for 5 days by culturing with irradiated 2×104 allogeneic monocyte-derived DCs as stimulator which were generated from isolated CD14+ monocytes by CD14-microbeads (Miltenyi Biotec) by 5 days of culturing with 200 ng/ml GM-CSF and 100 ng/ml IL-4 (R&D Systems). In some experiments, 4×104 CD4+CD25− blood T cells as responder and 2×104 thymic CD4+CD8−CD25+ICOS+ TR or CD4+CD8−CD25+ICOS− TR after culturing with anti-CD3 antibody in the presence of IL-2 and IL-7 on ICOSL-L cells for 5 days as suppressor, these cell type and their mixtures were examined in response to irradiated 4×104 anti-CD3 MACS microbeads-T cell-depleted peripheral blood mononuclear cells as stimulator. Mixture of neutralizing anti-IL-10 plus IL-10 receptor (R&D Systems) antibodies and/or TGF-β receptor type I kinase (ALK5) inhibitor II (Calbiochem) were used in culture at a concentration of 200 ng/ml, 10 ng/ml, and 1 μM, respectively. Cellular proliferation was assessed by [3H]thymidine incorporation, as described (Wang et al., 2006; Watanabe et al., 2005) and analyzed by flow cytometry (CFSE). Transwell experiments were performed in 24-well plates (Corning Costar), as described (Vieira et al., 2004). Briefly, 4×105 naive T cells were stimulated with 2×105 monocyte-derived DCs for 5 days, and 2×105 TR was added into same well or separated by semipermeable membrane. In another experiment, naive T cells, autologous CD25+ICOS+ TR or CD25+ICOS− TR, and allogeneic immature DCs (1:1:1 ratio) were cultured for 4 days, and then the cells were stained with PE-anti-CD11c, FITC-anti-CD86 or FITC-mouse IgG1 antibodies (BD Biosciences) and analyzed by flow cytometry.

T-cell proliferation and viability assay

After 5 days of culture under the several conditions, TR were collected and resuspended in an EDTA-containing medium to dissociate the clusters. Viable cells were counted by Trypan-blue exclusion of the dead cells or by annexin V staining (BD Biosciences).

Immunohistochemistry

Human lymph nodes were obtained from tissue bank of M. D. Anderson Cancer Center with polices established by the institutional committees for human research. Human tonsils were obtained from West Virginia Hospital. Immunohistological and immunoflurescence staining was performed by using anti-human antibodies; mouse anti-ICOS (ANC6C6-A3) and Foxp3 (PCH101) antibodies, as described (Wang et al., 2006; Watanabe et al., 2005).

Supplementary Material

01

Acknowledgments

We thank Karen Ramirez, Zhiwei He, and Eric Wieder for cell sorting and support. Drs MJ Fingold and K. Sternberg for tissue materials. This project is supported by M. D. Anderson Cancer Center Foundation.

Footnotes

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NIHMS55824-supplement-01.pdf (171.4KB, pdf)
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