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. Author manuscript; available in PMC: 2009 Jan 1.
Published in final edited form as: Immunity. 2008 Jan;28(1):100–111. doi: 10.1016/j.immuni.2007.11.021

A two-step process for thymic regulatory T cell development

Chan-Wang Joaquim Lio 1, Chyi-Song Hsieh 1,*
PMCID: PMC2248212  NIHMSID: NIHMS38563  PMID: 18199417

Summary

Recognition of self-antigens is required for regulatory T (Treg) cells to exert dominant tolerance. However, the mechanism by which self-reactive developing T cells are diverted into the Treg cell subset is unclear. To address this question, we looked for the immediate precursors to Treg cells within Foxp3−CD4+CD8− thymocytes. Using intrathymic transfer, we found that the CD25hi subset is highly enriched in Treg cell precursors. This was supported by tracking of thymocyte development via analysis of TCR repertoires in a TCRβ transgenic model. Remarkably, these Treg cell precursors exist at a developmental stage where they are poised to express Foxp3 without further TCR engagement, requiring only stimulation by IL-2 or IL-15. Thus, we propose that the selection of self-reactive thymocytes into the Treg cell subset occurs via an instructive rather than stochastic-selective model whereby TCR signals result in the expression of proximal IL-2 signaling components facilitating cytokine-mediated induction of Foxp3.

Introduction

Natural Foxp3+ regulatory T (Treg) cells play an important role in the preservation of self-tolerance and prevention of autoimmunity (Fontenot and Rudensky, 2005; Izcue et al., 2006; Sakaguchi, 2004). Genetic ablation of Treg cell development due to Foxp3-deficiency leads to overwhelming autoimmunity and death at a young age in both humans (Bennett et al., 2001) and mice (Fontenot et al., 2003). Moreover, acute depletion of Foxp3-expressing cells turns a normal resting immune system into one that is hyperactive and uncontrolled, resulting in the death of an adult animal within a mere 10 days (Kim et al., 2007; Lahl et al., 2007). Thus, Treg cells are required throughout life to preserve immune homeostasis.

Foxp3+ T cells have been found in both the CD8 and CD4 subsets, though the latter comprises greater than 90% of Foxp3+ cells in both central and peripheral lymphoid organs (Fontenot et al., 2005c). Although all of these Foxp3+ cells may be considered “regulatory,” here, we use “Treg cell” in the traditionally defined manner to indicate CD4+ Foxp3+, and mostly CD25+, cells.

A characteristic feature of Treg cells is their ability to recognize self-antigens (Apostolou et al., 2002; Jordan et al., 2001; Seddon and Mason, 1999; Taguchi et al., 1994). This is consistent with the finding that the TCR repertoires of Treg and non-Treg cells are different with a small degree of overlap (Hsieh et al., 2004; Hsieh et al., 2006; Pacholczyk et al., 2006; Wong et al., 2007). The ability of many Treg, but not non-Treg, cell TCRs to facilitate in vivo expansion, and the observation of some Treg cell TCRs in Foxp3-deficient autoimmune mice, further support the notion that Treg cell TCRs are self-reactive (Hsieh et al., 2004; Hsieh et al., 2006). Importantly, self-reactivity appears to be required for Treg cells to maintain self-tolerance and prevent autoimmunity in TCR transgenic and non-transgenic models (Samy et al., 2006; Seddon and Mason, 1999; Taguchi et al., 1994; Tang et al., 2004).

Although it is commonly accepted that Treg cell development predominantly occurs in the thymus (Hsieh et al., 2006; Itoh et al., 1999; Pacholczyk et al., 2006; Wong et al., 2007), the mechanisms by which self-reactive thymocytes are selected to become Treg cells remain controversial. Monoclonal TCR transgenic studies suggested that thymic encounter with “self-antigen” expressed as a second transgene facilitates Treg cell development (Apostolou et al., 2002; Jordan et al., 2001). A different study using another αβ-TCR, however, argued that the percentage increase of Treg cells was due to the deletion of non-Treg cells associated with graded increases of cognate antigen controlled via a doxycycline regulated promoter (Van Santen et al., 2004). Thus, it has been proposed that the development of a Treg cell subset which is relatively more self-reactive than the non-Treg cell subset occurs via a stochastic-selective process by which thymocytes expressing self-reactive TCRs are preferentially eliminated in the non-Treg cell subset.

Cytokines, primarily IL-2, have also been suggested to play a role in thymic Treg cell development. IL-2Rβ (CD122)-deficient mice exhibit a decreased number of thymic Treg cells (Burchill et al., 2007; Malek et al., 2002), therefore suggesting a role of IL-2 in thymic Treg cell development (Bayer et al., 2007). However, other studies have argued that IL-2 is not critical for thymic Treg cell development as IL-2 deficient mice have only a 50% decrease in thymic Treg cell numbers (D'Cruz and Klein, 2005; Fontenot et al., 2005b). Instead, they suggested that the principle role of IL-2 is for the survival of Treg cells in the periphery.

Finally, a recent report has suggested that unknown trans-acting factors which precede the expression of TCR in the thymus play an important role in the process of thymic Treg cell development (Pennington et al., 2006). Because these factors appear to act so early in thymic development at the “double negative” CD4− CD8− stage, it remains unclear how these factors would result in the generation of a self-reactive Treg cell subset. This model has recently attracted proponents based on their report that non-self-, rather than self-, antigens represent the cognate antigens of the Treg cell subset (Pacholczyk et al., 2007). Thus, the developmental steps that result in the diversion of thymocytes into the thymic Treg cell subset, and the relationship of those steps to TCR specificity and cytokines, remains ill defined and controversial.

We reasoned that studying the process of thymic Treg cell development, like any developmental system, would be greatly simplified by the in vivo identification of an intermediate in this process, i.e. a Treg cell precursor. Although many cells may be considered precursors to Treg cells, including the aforementioned “double negative” cells (Pennington et al., 2006), we were interested in precursors which are closer in the developmental program to expressing Foxp3, the best available marker for Treg cells (Fontenot et al., 2005c). Other studies have examined cells which were induced to express Foxp3, but were unable to make functional protein (Gavin et al., 2007; Lin et al., 2007). While these studies were important in addressing the contribution of Foxp3 to the Treg cell phenotype, they shed little light regarding the mechanisms which actually induce Foxp3 in developing thymocytes. Finally, it has been reported that CD25hi Foxp3− cells in the CD4+ CD8− (CD4SP) thymocytes are observed prior to the appearance of Foxp3+ cells in the neonatal period (Fontenot et al., 2005a). Although it was suggested that these cells may be Treg cell precursors, this was not experimentally demonstrated. Thus, the developmental destination of the CD25hi subset is unknown, as these cells could face alternative fates such as becoming negatively selected (Kishimoto et al., 1995), or exported into the periphery.

Based on the previous study by Rudensky's group (Fontenot et al., 2005a) and our own observations regarding the phenotypic overlap between Foxp3+ and Foxp3− CD4SP subsets, we formally tested whether the CD25hi Foxp3− CD4SP subset was enriched in Treg cell precursors. We found that this subset was highly enriched in cells which express Foxp3 after intrathymic transfer. Surprisingly, there was a population of Treg cell precursors within the CD25hi subset which appeared to be at a pent-ultimate step in Treg cell differentiation, requiring only non-TCR derived signals to express Foxp3 in vivo, and a minimal quantity of IL-2 in vitro. As the ability of IL-2 to induce Foxp3 correlated with the ability to detect proximal IL-2 signaling, we propose that Treg cell development occurs via an instructive, rather than stochastic-selective, model whereby self-reactive thymocytes upregulate components of the proximal IL-2 signaling pathway to compete for cytokine-mediated induction of Foxp3.

Results

Identification of Treg cell precursors within the CD25hi Foxp3− CD4SP subset

To address the mechanism of thymic Treg cell differentiation, we asked whether there are definable intermediates in this developmental program. Since the vast majority of Foxp3+ T cells appear at the CD4+ CD8− stage (Fontenot et al., 2005c), we focused on understanding Treg cell development in this subset. For this report, we have utilized Foxp3gfp reporter mice to measure Foxp3 expression (Fontenot et al., 2005c). Using a variety of phenotypic markers, we were struck by the overlap in GITR and CD25 expression between the Treg cells and a subset of Foxp3− CD4SP thymocytes (Figure 1A). Of note, both GITR and CD25, the IL-2 receptor α chain, have been used as markers for Treg cells. Interestingly, this CD25hi population also arises in the neonatal period immediately prior to the development of Foxp3+ thymocytes (Fontenot et al., 2005a), and is greatly diminished in TCR transgenic x Rag2-deficient mice which harbor few, if any, Treg cells (Apostolou et al., 2002). This CD25hi GITRhi subset represents approximately 3% of CD4SP, and 0.2% of all thymocytes. The low frequency of these cells resulted in a substantial technical challenge in purification, requiring magnetic bead depletion of CD8+ cells followed by an extended period of flow cytometric sorting of an approximately 1% target population. To test whether thymic subsets were enriched for precursors to Treg cells, we performed intrathymic transfer to assess the frequency of cells which upregulated the expression of Foxp3. For these in vivo experiments, we avoided the use of anti-GITR DTA-1 monoclonal antibody due to its potentially activating properties in vivo (Shimizu et al., 2002). Three days after intrathymic transfer, we found that on average, 13.8% of purified CD25hi Foxp3− CD4SP cells acquired Foxp3 expression after 3 days, in contrast to 0.05% of CD25lo cells–a two hundred and eighty-fold enrichment (Figure 1B). This was not a transient increase in Foxp3 expression, as we observed that the percentage of Foxp3+ cells was stable 14 days after intravenous adoptive transfer (Figure 1C). Thus, the CD25hi Foxp3− CD4SP thymocyte subset is enriched for Treg cell precursors.

Figure 1. CD25hi Foxp3− CD4SP thymocytes are enriched for Treg cell precursors.

Figure 1

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(A) Overlap between Foxp3+ and Foxp3− CD4SP thymocytes for the expression of CD25 and GITR. Thymocytes from Foxp3gfp reporter mice were analyzed by flow cytometry. Cells are gated on the CD4SP subset, and are further gated based on Foxp3 (left panel) into the Foxp3− (middle) and Foxp3+ subsets (right). (B) Development of Foxp3+ cells from CD25hi, but not CD25lo, Foxp3− CD4SP cells after intrathymic transfer. FACS purified CD25lo or CD25hi Foxp3− CD45.2+ CD4SP cells (105) were intrathymically injected into congenically marked CD45.1+ recipients. Development of Foxp3+ cells was analyzed by flow cytometry 3 days post transfer. Representative FACS plots for the intrathymically injected CD45.1− CD45.2+ CD4SP cells from two independent experiments are shown on the top, and the percent Foxp3+ cells from individual mice plotted on the bottom. The mean value is indicated by a bar. (C) Maintenance of Foxp3+ cells from CD25hi Foxp3− CD4SP thymic precursors. 105 sorted CD25lo or CD25hi Foxp3− CD4SP thymocytes (Thy1.2+) together with 5x105 MACS purified peripheral CD4+ cells (Thy1.1+) were intravenously transferred into TCRβ−/− mice, and analyzed at 3 and 14 days by flow cytometry of pooled spleen and lymph nodes cells. Representative FACS plots from two independent experiments show cells gated on donor Thy1.2+ Thy1.1− TCRβ+ CD4+ cells. (D) Flow cytometric characterization of CD25hi Foxp3− cells. Thymocytes from Foxp3gfp mice were stained with the indicated antibodies along with CD4, CD8, and CD25; and analyzed on a FACSAria. Histogram plots shown are gated on CD4SP cells.

We then performed an extensive flow cytometric analysis of the CD25hi Foxp3− subset (Figure 1D and Suppl. Figure 1). We found that the CD25hi cells are TCRβhi, suggesting that these cells have undergone positive selection. Consistent with the proposed developmental progression, CD25hi cells appear more immature than Treg cells based on their higher expression of HSA (heat stable antigen). In contrast with CD25 and GITR, other Treg cell markers such as FR4 and CD103 are not elevated in the CD25hi subset, and potentially may represent Foxp3 induced markers. However, CD69 and CD44 are upregulated. Along with the increased expression of CD25 and GITR, these data suggest that CD25hi Foxp3− CD4SP cells represent a thymocyte subset that has recently been activated through the TCR.

The notion that TCR stimulation may be responsible for the generation of CD25hi cells is consistent with several studies using polyclonal TCR models which have uniformly shown that the Treg cell TCR repertoire is different from, but overlapping with, the non-Treg cell TCR repertoire in the thymus (Hsieh et al., 2006; Pacholczyk et al., 2006; Wong et al., 2007). However, it is unclear whether TCR derived signals played a direct role in the developmental process resulting in the induction of Foxp3, or whether it acted in a circumstantial fashion by favoring the negative selection of self-reactive Foxp3− versus Foxp3+ cells (Van Santen et al., 2004). In fact, it has been argued that TCR specificity to self-antigen plays little to no role in thymic Treg cell development (Pacholczyk et al., 2007), implying that the cell surface phenotype consistent with TCR activation may be a stochastic, and antigen non-specific occurrence. We therefore asked whether the population of Treg cell precursors within the CD25hi CD4SP subset arose due to TCR specificity characteristic of Treg, and not non-Treg, cells. To test this, we utilized a fixed TCRβ model for tracking the developmental fate assigned to individual TCRs (Hsieh et al., 2004; Hsieh et al., 2006). We purified T cells from the Foxp3+ Treg, and Foxp3− CD25hi GITRhi and CD25lo GITRint CD4SP subsets, and sequenced TRAV14 (Vα2) chains as previously described (Hsieh et al., 2006). GITRlo CD4SP cells were excluded as they appear to be immature thymocytes that are transiting to either CD4SP or CD8SP cells (data not shown). We found that the thymic Treg cell TCR repertoire is more similar to the CD25hi GITRhi subset versus the CD25lo GITRint subset (Figure 2A, Suppl. Figure 2), consistent with the notion that the Treg cell precursors in the CD25hi Foxp3− subsets have already been selected based on TCR specificities characteristic of mature Foxp3+ Treg cells. The enrichment of Treg cell TCRs in the CD25hi subset is in agreement with our previous observation that the CD25hi subset is enriched in functional Treg cell precursors (Figure 1B). We did not expect that the CD25hi Foxp3− CD4SP subset would share the exact same TCR repertoire as Foxp3+ cells since a large proportion of the CD25hi subset does not develop into Treg cells (Figure 1B). Similar to our previous results (Hsieh et al., 2006), the TCR repertoire from CD25lo GITRint CD4SP subset was different from that of the Treg cell subset with a small degree of overlap. We also compared the TCR repertoires from the thymic subsets to those from peripheral Foxp3+ Treg, naïve (Foxp3− CD44lo), and memory/activated (Foxp3− CD44hi) CD4+ cells. In accordance with our observations between thymic data sets, we also found that the thymic CD25hi GITRhi, in comparison with the CD25lo GITRint subset, showed greater usage of TCRs found in peripheral Treg cells (Figure 2B). This TCR tracking study therefore suggests that the Treg cell precursors within the CD25hi subset are generated due to TCR specificities characteristic of Treg cells.

Figure 2. CD25hi Foxp3− CD4SP TCR repertoire is enriched for Treg cell TCRs.

Figure 2

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(A) Analysis of shared TCRα sequences among thymic CD4SP populations. Foxp3+, CD25hi GITRhi Foxp3−, or CD25lo GITRint Foxp3−, CD4SP thymocytes were sorted from TCli TCRβ-transgenic x Foxp3gfp x Tcra+/− mice and TRAV14 (Vα2) TCRα sequences obtained as previously described (Hsieh et al., 2004). Data shown is pooled from two independent experiments (Suppl. Figure 2). Each pie graph represents the TCRα sequences in the data set which were also found in at least one other thymic data set. The portion of the TCRα sequences which overlaps another data set is indicated by a different color in the pie graph, and the data set overlapped is denoted by an arrow. The number of TCRα chains which were not found in another subset was similar between the three subsets (73%, 65%, and 67% for CD25lo, CD25hi, and Foxp3+ subsets, respectively). (B) Comparison of TCRα sequences from thymic and peripheral subsets. Unique TCRα sequences from the indicated thymic CD4SP subsets which were also found in the peripheral TCR data sets are plotted on the x-axis. Each vertical bar represents the likelihood that an individual thymic TCR would be found within the peripheral Foxp3+, naïve (Foxp3− CD44lo), or memory/activated (Foxp3− CD44hi) CD4+ T cell data sets. The size of each color in the bar is calculated by the frequency of the TCR in the subset divided by the sum of the frequencies in all three subsets.

A TCR-independent phase of thymic Treg cell development

Although it appeared that TCR specificity is important for the generation of Treg cell precursors, it was still possible that additional TCR signals are required to complete the process of Treg cell differentiation. To directly address this issue, we intravenously transferred CD25hi Foxp3− thymocytes into MHC class II-deficient or MHC class II-sufficient αβ-T cell-deficient hosts. We observed that regardless of MHC class II expression in the host, a similar frequency of adoptively transferred CD25hi CD4SP thymocytes acquired Foxp3 three days after transfer (Figure 3). By contrast, the small frequency of CD25lo cells that became Foxp3+ in an MHC class II-sufficient host was essentially abrogated in an MHC class II-deficient host, supporting the notion that CD25lo thymocytes are at an earlier stage in Treg cell development requiring TCR-derived signals. Taken together, these data suggest that TCR-derived signals are involved prior to, or at the time of, development of Treg cell precursors within the CD25hi Foxp3− subset. Importantly, once that developmental stage is achieved, further development is independent of TCR-derived signals.

Figure 3. A subset of CD25hi CD4SP thymocytes is at a TCR-independent stage of Treg cell development.

Figure 3

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CD25lo or CD25hi Foxp3− CD4SP thymocytes (CD45.1+ Thy1.2+) were purified and 105 cells were intravenously transferred along with 5x105 peripheral MACS-purified CD4+ cells (CD45.2+ Thy1.1+) into either TCRβ−/− or MHC class II−/− mice (CD45.2+ Thy1.2+). Development of Foxp3+ T cells from the thymocyte population was assessed 3 days post transfer by flow cytometry. FACS plots are representative of two independent experiments, and are gated on CD45.1+ CD45.2− CD4+ cells. Percentage of cells which are Foxp3+ are plotted below, and shown as mean ± s.d.

The observation that deletion of the common γ-chain of the IL-2 receptor completely inhibited the development of thymic Treg cells (Fontenot et al., 2005b) suggested that cytokines may be TCR-independent factors for Treg cell development. We therefore tested whether freshly isolated thymocytes would upregulate Foxp3 in response to exogenous cytokines. Remarkably, we observed that IL-2 induced Foxp3 in 25.5% (± 4.8 S.D.) of purified CD25hi GITRhi CD4SP cells by 24 hours (Figure 4A and 4B). Interestingly, this in vitro percentage is approximately twice the percentage of Foxp3+ cells observed after in vivo transfer (Figure 1B), suggesting that cytokine levels may be limiting in vivo. By contrast, only 0.6% (± 0.2 S.D.) of purified CD25lo GITRint CD4SP thymocytes upregulated Foxp3 in response to IL-2. Consistent with the notion that this is a TCR-independent phase of Treg cell development, addition of plate bound anti-CD3 did not markedly increase the frequency of Foxp3+ cells in the presence or absence of IL-2 (Figure 4C). CD25hi cells which upregulated Foxp3 were suppressive in vitro, suggesting that they have acquired functional characteristics of Treg cells (Figure 4D). Thus, the administration of a single cytokine to precursors within the CD25hi subset is sufficient for inducing Foxp3, the final step in Treg cell development (Gavin et al., 2007).

Figure 4. Cytokine-mediated induction of Foxp3 in CD25hi Foxp3− CD4SP thymocytes.

Figure 4

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(A) IL-2 is sufficient to induce Foxp3 in Treg cell precursors in vitro. Sorted CD25lo or CD25hi Foxp3− CD4SP cells (5x103) were cultured for 24 hours in 96-well round bottom plates in the presence or absence of human IL-2 (5 U/ml), then analyzed by flow cytometry for induction of Foxp3. This amount of human IL-2 is approximately equivalent to 1 ng/ml or 58.1 pM recombinant murine IL-2 (17.2 kDa, Peprotech) in this assay (Figure 6A and data not shown). This concentration of IL-2 would be expected to saturate approximately 90% of high affinity IL-2Rαβγ receptors. All wells also contained murine IL-7 (5 ng/ml) to promote cell survival (Suppl. Figure 6). The addition of increased amounts of IL-7 (up to 100 ng/ml) in the absence of IL-2 did not increase the percentage of CD25lo or CD25hi cells which became Foxp3+ (data not shown). However, IL-7 was not required to observe the induction of Foxp3 in vitro (Figure 4B). (B) Time course of Foxp3 induction by IL-2. Purified CD25hi Foxp3− CD4SP thymocytes were cultured with indicated combinations of 5 U/ml human IL-2 and 5 ng/ml IL-7 and analyzed for Foxp3 expression by flow cytometry at 12, 24, and 48 hours. (C) TCR stimulation plays little role in the induction of Foxp3 from CD25hi cells. Purified CD25hi Foxp3− CD4SP cells were labeled with DDAO-SE (Invitrogen) and then cultured in the presence of IL-7 (5 ng/ml) and/or human IL-2 (5 U/ml) or plate bound anti-CD3 (10 μg/ml) as indicated. Expression of Foxp3 and dilution of DDAO-SE were assessed by flow cytometry at 24 hours. At 72 hours, division of both Foxp3+ and Foxp3− cells is easily observed (Suppl. Figure 8). (D) Cells induced to express Foxp3 by IL-2 are suppressive in vitro. Purified CD25hi Foxp3− CD4SP thymocytes (Thy1.2+) were cultured with 5 U/ml human IL-2 for 24 hours. Cells which upregulated Foxp3 were purified by FACS (bottom) and co-cultured with freshly FACS purified peripheral CD25− CD4+ effector T cells (Thy1.1+) labeled with CFSE (Invitrogen). CD25lo Foxp3− (middle) and Foxp3+ CD4SP thymocytes (top) cultured 24 hours in vitro with IL-2 were used as controls. Three days after stimulation with 1 μg/ml anti-CD3 and irradiated splenic APCs, effector T cell proliferation was analyzed by CFSE dilution in the Thy1.1+ CD4+ T cell population shown. Numbers shown are the mean ± s.d. from duplicate wells. (E) CD25 expression correlates with the ability for IL-2 to induce Foxp3. The four Foxp3− CD4SP populations (a, b, c, and d) were sorted and cultured with IL-2 as indicated in part (A). Post sort analysis is shown in Suppl. Figure 7.

The enrichment of Treg cell precursors within the CD25hi CD4SP thymocytes subset suggested that the presence of the IL-2 receptor α chain, CD25, may facilitate the induction of Foxp3. We sorted four Foxp3− thymocyte subsets with varying levels of CD25 and GITR expression, three of which (denoted a, b, and c in Figure 4E) were in areas of overlap between the Foxp3+ and Foxp3− subsets. Consistent with the hypothesis that IL-2 signaling plays a role in Foxp3 acquisition, we found that the level of CD25 correlated with the frequency of cells which upregulate Foxp3 in response to IL-2.

An alternative explanation for the above results is that IL-2, instead of directly inducing Foxp3 expression, may simply permit the expansion or survival of thymocytes pre-committed to becoming Treg cells. We first assessed whether IL-2 induced the proliferation of CD25hi cells which become Foxp3+. Since CFSE interfered with the use of Foxp3gfp as a Treg cell marker, we used DDAO-SE (Invitrogen) to track cell proliferation via the dilution of the fluorescent dye. We observed essentially no decrease in DDAO-SE staining during our 24 hour short term IL-2 culture, though the induction of Foxp3 was easily visualized (Figure 5A). Next, we performed real-time quantitative RT-PCR for Foxp3 to evaluate time points that could not be easily visualized by flow cytometry due to the time required for protein folding of GFP for fluorescence (Figure 4B). We observed that IL-2 rapidly induced Foxp3 mRNA by 3 hours (Figure 5B). Importantly, delay of IL-2 administration by 3 hours did not negatively affect the induction of Foxp3 mRNA, arguing that IL-2 is not simply a survival or growth factor allowing the expression of Foxp3 via a pre-programmed path, but that IL-2 directly induces the expression of Foxp3 in Treg cell precursors.

Figure 5. Rapid induction of Foxp3 mRNA by IL-2.

Figure 5

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(A) Induction of Foxp3 is not associated with cell proliferation. Purified CD25lo or CD25hi Foxp3−, and Foxp3+ CD4SP cells were labeled with DDAO-SE and cultured in the presence of 5 U/ml human IL-2 and 5 ng/ml IL-7 for 24 hours. Dilution of DDAO-SE and acquisition of Foxp3 was assessed by flow cytometry. DDAO-SE staining after proliferation is shown in Suppl. Figure 8. (B) Analysis of Foxp3 mRNA induction by real time RT-PCR. Purified CD25hi Foxp3− CD4SP cells were cultured with or without 5 U/ml human IL-2 for either 3 hours, or for the last 3 hours of a 6 hour culture, and assessed for Foxp3 mRNA expression using real time RT-PCR normalized to HPRT expression (Hori et al., 2003). For reference, freshly isolated Foxp3+ thymocytes express 3.6 normalized amount of Foxp3 mRNA.

Since a deficiency in the common γ-chain, and not IL-2, results in a virtual absence of thymic Treg cells (Fontenot et al., 2005b), we also tested the ability of other common γ-chain cytokines to induce Foxp3 in CD25hi CD4SP thymocytes (Figure 6A). Of the cytokines tested, only IL-2 and IL-15 were effective in inducing Foxp3 expression in a high proportion of CD25hi cells. However, the in vitro potency of these two cytokines for inducing Foxp3 expression differed by at least 50-fold (Figure 6B), although in trans presentation may enhance the potency of IL-15 in vivo (Burkett et al., 2004). It should be noted that these data may underestimate the true sensitivity of the Treg cell precursors to IL-2 induction due to our inability to purify the CD25hi subset without the use of monoclonal antibodies to CD25 which inhibit IL-2 signaling. None-the-less, the dose of IL-2 required to achieve 15% induction of Foxp3 expression, the level observed after intrathymic transfer, is a mere 75 pg/ml. This represents a molar concentration of 4.4 pM, which is in the 10 pM Kd range of the high affinity receptor IL-2Rαβγ (Sharon et al., 1986). In vitro blockade of IL-2 did not reveal a role for endogenous production of IL-2 (Suppl. Figure 3). Although TGFβ has been suggested to be important for the development of Foxp3+ cells from peripheral T cells in vitro (Chen et al., 2003), we did not observe a role for TGFβ in our in vitro thymocyte assay (Suppl. Figure 4), consistent with in vivo observations from conditional T cell knockouts of TGFβRII (Li et al., 2006; Marie et al., 2006). From these data, we conclude that IL-2 and IL-15 are the predominant common γ-chain cytokines responsible for induction of Foxp3 in CD25hi Treg cell precursors.

Figure 6. IL-2 and IL-15 are the predominant common γ-chain cytokines which induce Foxp3.

Figure 6

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(A) Analysis of common γ-chain cytokines for their ability to induce Foxp3. Sorted CD25hi Foxp3− CD4SP cells were assayed with the indicated murine cytokines (100 ng/ml) and IL-7 (5 ng/ml) as described in Figure 4A. Titration of cytokines down to 1 ng/ml did not enhance the percentage of Foxp3+ cells observed (data not shown). (B) IL-2 is more potent than IL-15 for the induction of Foxp3. Purified CD25hi Foxp3− CD4SP thymocytes were cultured with IL-7 (5 ng/ml) and indicated concentrations of murine IL-2 or IL-15 and analyzed for Foxp3 expression by flow cytometry at 24 hours. Data is representative of two independent experiments. The difference in potency between IL-2 and IL-15 was calculated using the concentration at half maximal Foxp3 induction.

Regulation of proximal IL-2 signaling during the process of Treg cell development

The ability of cytokines to induce Foxp3 in CD25hi Treg cell precursors but not CD25lo cells could be due to a differential ability of those cells to respond at the level of proximal signaling. As Janus kinase (Jak) 1/3–Stat5 is the main signaling pathway that is activated by IL-2, we sought out to determine whether Jak kinase activity is required for the induction of Foxp3. Indeed, pharmacologic inhibition of Jak kinase activity prevented the expression of Foxp3 (Figure 7A). Thus, Jak kinase activation, presumably via Stat5, is required for the induction of Foxp3 in Treg cell precursors.

Figure 7. Upregulation of proximal IL-2 signaling correlates with the ability to induce Foxp3.

Figure 7

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(A) Foxp3 induction by IL-2 is Jak kinase dependent. Purified CD25hi Foxp3− CD4SP cells were cultured with or without 5 U/ml human IL-2 in the presence of Jak inhibitor I or the carrier (DMSO) only. Foxp3 expression was assessed by flow cytometry at 24 hours. (B) CD25hi, but not CD25lo, Foxp3− CD4SP thymocytes are capable of generating proximal IL-2 signals. Purified cells were incubated with serum free DMEM for 10 minutes, stimulated with 1000 U/ml human IL-2 or 100 ng/ml IL-7 at a concentration of 5x105 cells/ml for 20 minutes, then fixed and stained for phospho-Stat5 (pStat5) as previously described (Van De Wiele et al., 2004), and analyzed by flow cytometry. FACS plots are representative from three independent experiments. Shaded area and solid line represent unstimulated and stimulated cells, respectively.

It has previously been reported that Stat5 is required for the development of Treg cells, as Stat5a/b-deficient mice are greatly diminished in Treg cell numbers (Burchill et al., 2007; Yao et al., 2007). We therefore analyzed the activation status of Stat5 via intracellular staining of phosphorylated Stat5 (Figure 7B). Interestingly, the response of CD25hi cells to IL-2 is biphasic as approximately half of the cells induced phospho-Stat5 after IL-2 stimulation. By contrast, CD25lo cells which contain few Treg cell precursors uniformly generated little, if any, phospho-Stat5 in response to IL-2. The ability of IL-2 to induce phospho-Stat5 may be a feature of Treg cell development, as Foxp3+ cells are broadly capable of inducing phospho-Stat5. Curiously, addition of IL-7 results in an appreciable induction of phospho-Stat5, but not Foxp3, in CD25lo cells (Figure 7B and 4A), suggesting that activation of Stat5 alone is insufficient to facilitate Foxp3 expression and that additional factors are required for the development of cytokine-responsive Treg cell precursors. The ability to respond to IL-7 is down-regulated in the CD25hi and Treg subsets, implying a greater role for other cytokines such as IL-2 for the generation of phospho-Stat5 during Treg cell development. Since intracellular staining precludes further analysis of the thymocytes, we cannot directly prove that the cells capable of inducing phospho-Stat5 do upregulate Foxp3. Nevertheless, the relative absence of detectable proximal IL-2 signaling in CD25lo cells and in some of the CD25hi cells suggests that differential regulation of the proximal IL-2 signaling machinery plays an important role in the thymic development of Foxp3+ cells from Treg cell precursors.

Since our analysis has thus far addressed only the expression of CD25, the α chain of the trimeric high affinity IL-2Rαβγ, it was possible that other proximal IL-2 components may be missing in the CD25hi cells unable to induce phospho-Stat5. As the common-γ chain is considered to be constitutively expressed, we assessed the expression of CD122 (IL-2Rβ) on the CD25hi Foxp3− subset (Figure 8A). Since only one-third of CD25hi cells expressed high levels of CD122, it was possible that CD122 expression correlated with the ability to induce phospho-Stat5 in response to IL-2. We found that almost all sorted CD25hi CD122hi cells were capable of inducing phospho-Stat5, whereas only 30% of CD25hi CD122lo cells did so (Figure 8B). The ability to induce phospho-Stat5 correlated with the ability to induce Foxp3 after 24 hours of culture (Figure 8C). Since 90% of CD25hi CD122hi cells could signal via Stat5, but only half were capable of inducing Foxp3 expression, this lends further support to the notion that other factors are required in concert with IL-2 derived signals to induce Foxp3. One intriguing possibility is that cytokine signaling itself may be involved in generating this unknown factor, as IL-2 can still induce Foxp3 in some of the CD25hi Foxp3− cells which did not upregulate Foxp3 after the first 24 hour IL-2 stimulation (Suppl. Figure 5). Thus, we propose that the generation of the cytokine-responsive Treg cell precursor requires the induction of an unknown factor which permits Foxp3 expression, in concert with the upregulation of the proximal IL-2 signaling machinery.

Figure 8. Ability to signal via IL-2 does not define the cytokine responsive Treg cell precursor.

Figure 8

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(A) Biphasic expression of CD122 on CD25hi Foxp3− CD4SP thymocytes. Data shown are gated on CD4SP cells. The bottom histogram represents the sort gating for the CD25hi Foxp3− cells of the top and bottom quartile based on CD122 expression. (B) Almost all CD122hi CD25hi Foxp3− cells have the capacity to signal via the IL-2 receptor. Analysis of phospho-Stat5 induced by IL-2 on the indicated cell subsets was performed as described in Figure 7B. (C) Induction of Foxp3 does not correlate with the ability to signal via the IL-2 receptor. Sorted cells used in (B) were also stimulated with 50 U/ml human IL-2 and 5 ng/ml IL-7 to induce Foxp3 expression, which was analyzed by flow cytometry at 24 hours. (D) Fractionation of the CD25hi subset using CD127 (IL-7Rα) and CD122 (IL-2Rβ). Flow cytometric data shown are gated on the top labels and on CD4SP cells. (E) Correlation of CD127 and CD122 with Treg cell precursor frequency in the Foxp3− CD25hi subset. CD25hi Foxp3− cells were purified on the basis of CD127 and CD122 shown in (D) and stimulated with 5 ng /ml IL-7 and 50 U/ml human IL-2 for 24 hours.

Although not absolute, the clear shift from IL-7 to IL-2 signaling between CD25lo and CD25hi Foxp3− cells suggested that CD127 (IL-7Rα) may be downregulated during the process of thymic Treg cell development. Indeed, the upregulation of CD122 in CD25hi cells is associated with a decrease in CD127 levels (Figure 8D). We then sorted subsets of CD25hi cells based on CD122 and CD127 expression, and assessed the ability of IL-2 to induce Foxp3. We found that the expression of CD127 marked a subpopulation of CD25hi cells which have a low frequency of cytokine-responsive Treg cell precursors (Figure 8E). Although further studies are required, these data suggest that Treg cell development within the CD25hi subset may be phenotypically described by the down-regulation of CD127 followed by the upregulation of CD122.

Discussion

The question as to how the immune system selects a portion of self-reactive thymocytes to “police” the periphery as Treg cells is central to understanding an important aspect of immune tolerance. Our observation that a subset of CD25hi Foxp3− CD4SP thymocytes contains precursors for natural Treg cells has enabled us to generate a framework for understanding the process of thymic Treg cell development and potentially explain previous findings which were disparate or controversial.

Temporally, our data suggest that thymic Treg cell development can be divided into at least two distinct stages based on the requirement for TCR-derived signals. The TCR-dependent generation of Treg cell precursors in the CD25hi subset is based on the observation that this subset is enriched in TCRs normally found in the Treg cell subset (Figure 2). Alterations in the TCR repertoire in the CD25hi subset could result from stochastic-selective selection for Treg cell TCRs (Van Santen et al., 2004), or from instructive TCR derived signals. We believe the latter is more likely since TCR transgenic mice on a Rag-deficient background which do not develop Treg cells are also deficient in this CD25hi population based on published (Apostolou et al., 2002) and unpublished results (C.W.L. and C.S.H). Furthermore, upregulation of CD25 has long been associated with TCR activation in the thymus (Kishimoto and Sprent, 1997), favoring the notion that generation of CD25hi cells is a TCR-instructive event.

The direct involvement of TCR signaling in this model is in clear contrast with a recent report suggesting that Treg cells do not display increased avidity towards self-antigens (Pacholczyk et al., 2007). Their observation that Treg cell TCRs do not show strong activation to self-antigens on splenic APCs is consistent with our previously published report, where we suggested that the avidity of the majority of Treg TCRs for self ligands is below the range characteristic of conventional T cell responses (Hsieh et al., 2004). It is unclear whether hybridoma cell lines generated without prior immunization would be sufficiently sensitive to test this possibility. Their suggestion that the overlap between Treg and non-Treg cell TCRs is greater when assessed in the absence of frequency information does not, in our opinion, imply that TCR specificity is not important, but rather that control of Treg development by TCR specificity is not absolute, but probabilistic (Hsieh et al., 2004). When the frequency of each TCR is taken into account, their data, like ours and others (Hsieh et al., 2004; Hsieh et al., 2006; Wong et al., 2007), clearly demonstrate that the TCR repertoires of Treg and non-Treg cells differ, as many TCRs are preferentially found in one or the other subset. Although we cannot formally exclude a non-TCR-dependent event that selectively upregulates CD25 on a subset of T cells which express a different (but overlapping) set of TCRs, we believe that the simplest and most likely interpretation is that the CD25hi subset is generated due to moderate avidity TCR interaction with self antigens below or around the threshold for negative selection.

Other molecules may also play a role in the TCR-dependent phase for generating Treg cell precursors. The TCR co-stimulator CD28 has been suggested to play a role in thymic Treg cell development, as CD28 deficient mice show a diminished number of Treg cells (Salomon et al., 2000). Further studies have suggested that CD28 provides a cell-intrinsic signal independent of its ability to promote cell-extrinsic signals such as IL-2 (Tai et al., 2005). It therefore seems likely that CD28 would act in the TCR-dependent phase of Treg cell development, but may also participate in a cell-intrinsic or -extrinsic fashion via production of IL-2 in the TCR-independent phase. Cytokines may also be involved in this TCR-dependent stage, since mice deficient in the common γ-chain show a several fold decrease in CD25hi Foxp3− cells (Fontenot et al., 2005b). However, the proportion of those CD25hi cells which are Treg cell precursors is unknown, and additional studies will be required to address these issues.

After a period of TCR-dependent maturation, Treg cell development enters a TCR-independent phase. Cells within this phase are characterized by their ability to rapidly express Foxp3 in vitro after exposure to IL-2 or IL-15 in the complete absence of antigen presenting cells (Figure 4), suggesting that this is a purely instructive stage of Treg cell development. The ability to upregulate Foxp3 correlates with the ability of the cells at a population level to induce phospho-Stat5 upon IL-2 stimulation, implying that a principle component of the earlier TCR-dependent step is the upregulation of the proximal IL-2 signaling pathway. However, it is likely that other factors may also be upregulated by TCR or other signals which render the Foxp3 promoter permissive to transcription after IL-2 derived signals, since the ability to induce phospho-Stat5 does not imply the ability to induce Foxp3, whether by IL-7 in CD25lo Foxp3− cells (Figures 7 and 4A), or by IL-2 in CD25hi CD122hi cells (Figure 8A-C). Potentially, it may be cytokines themselves which assist in the generation of this unknown factor (Suppl. Figure 5). In concert with this putative unknown factor, it seems likely that the molecular mechanism by which IL-2 and IL-15 induce Foxp3 is Stat5, which has binding sites within the Foxp3 promoter (Burchill et al., 2007; Yao et al., 2007).

Our in vitro data suggest that IL-2 plays a more prominent role in Treg cell development than IL-15 (Figure 6). This may be related to differences in receptor efficiencies, as CD25, but not IL-15Rα, is markedly upregulated on Treg cell precursors (data not shown). Although in trans presentation of IL-15 would enhance its signaling in vivo, the notion that IL-2 plays a greater role than IL-15 in Treg cell development is consistent with previous reports. For example, IL-2 or CD25 knockout mice show impairment in thymic Treg cell development, with approximately 50% of normal Treg cell numbers in CD4SP thymocytes (Fontenot et al., 2005b), and develop lymphoproliferative disease, whereas IL-15 knockout mice show only a modest defect in Treg cell numbers and have no obvious autoimmunity (Burchill et al., 2007). However, mice deficient in both IL-2 and IL-15 exhibit marked deficiency in thymic Treg cell development (Burchill et al., 2007). The most compelling evidence for the hierarchy of IL-2 over IL-15, though, is demonstrated by the observation that thymocytes unable to express CD25 are 5-fold less competitive versus wild-type thymocytes in developing Foxp3+ Treg cells in mixed bone marrow chimeras (Fontenot et al., 2005b). These data suggest that IL-15 or other cytokines compensate poorly for IL-2 in a more natural environment than a full knockout animal. Taken together, these data strongly suggest that IL-15 is not required for thymic Treg cell development when IL-2 is present, but that IL-15 can partially compensate when IL-2 is completely absent.

It appears that the source of IL-2 is likely to be T cells themselves, as bone marrow chimeras with IL-2-deficient donors into wild-type hosts were phenotypically similar to that of IL-2-knockout mice (Tai et al., 2005). Furthermore, this group demonstrated that IL-2 can exert its effect in a paracrine manner as IL-2-deficient thymocytes showed equal expression of CD25 in comparison with IL-2-sufficient thymocytes in a mixed bone marrow chimera. Although the spontaneous production of IL-2 is difficult to observe in situ, experimental induction of TCR activation in vivo suggested that thymocytes undergoing apoptosis can release IL-2 (Bassiri and Carding, 2001).

Our data suggest that only a portion of CD25hi cells are cytokine responsive precursors to Treg cells. Based on our adoptive transfer studies, it appears that many of the cells in the CD25hi subset can survive as CD25lo non-Treg cells in the periphery (Figure 1C). The TCR tracking studies suggest that many TCRs thought to be “non-Treg” cell associated can end up in the CD25hi subset (Figure 2). Potentially, this could be due to weaker TCR-derived signals which are sufficient to upregulate CD25, but not the rest of the proximal IL-2 signaling machinery or other factors required to facilitate Foxp3 expression. Additionally, other unknown factors may be necessary along with TCR signals to generate Treg cell precursors. We have attempted to further specify the populations of Treg cell precursors within the CD25hi subset. Based on preliminary Affymetrix gene chip data from the CD25hi (a, b) and CD25lo (d) Foxp3− CD4SP populations in Figure 4E, we tested a number of cell surface markers in conjunction with CD25. Addition of OX40, CD83, ICOS, PD1, or 4-1BB did not improve our ability to identify cytokine-dependent Treg precursor cells in comparison with CD25 staining alone, either because the staining of the additional antibody was weak, or paralleled that of CD25 (Suppl. Figure 1). Although we are continuing to screen for other surface molecules, it may be an intracellular marker which reveals that a particular cell is cytokine responsive for Foxp3 induction.

An intriguing observation is that the number of cytokine-responsive CD25hi Treg cell precursor appears quite sizable, representing approximately 25% of Treg cells (1/4 of CD25hi cells are cytokine responsive; the Foxp3− CD25hi and Foxp3+ CD4SP subsets are about the same frequency, Figures 1A and 4A). Although the rates of Treg cell development and thymic export are unknown, the presence of a large precursor population hints at an in vivo bottleneck in this final phase of Treg cell development. This is supported by the observation that only half of the cells capable of expressing Foxp3 as assessed in vitro (Figure 4A) does so after in vivo transfer (Figure 1B). The simplest explanation for this bottleneck is that a limiting cytokine environment exists in vivo. Thus, competition for cytokines may promote the preferential selection of Treg cells from thymocytes that express the highest level of CD25 (Figure 4E). If the level of CD25 correlates with the degree of TCR activation, and thus self-reactivity, this competition for IL-2 would promote the selection of a thymic Treg cell repertoire that is highly self-reactive. This is consistent with the previously discussed results from mixed bone marrow chimeras in which the CD25-deficient thymocytes were much less competitive than CD25-sufficient cells (Fontenot et al., 2005b). Further support for this hypothesis comes from the observation that the provision of tonic IL-2 signal during thymic development via a transgenic constitutively active Stat5b skews thymic Treg cell development to include many more T cells which are normally destined to be non-Treg cells (Burchill, M et al, manuscript submitted). CD25-mediated IL-2 signaling is therefore an important process for selecting the self-reactive Treg cell TCR repertoire, as bypassing it results in an expansion of the Treg cell TCR avidity window for self-antigens into the realm normally reserved for positive selection of non-Treg cells. We hypothesize that diminishing the difference in self-reactivity between the Treg and non-Treg TCR repertoires would promote autoimmunity due to an imbalance resulting from decreased TCR-dependent Treg cell suppression relative to non-Treg cell-mediated autoimmune inflammation.

The observation that TGFβ appears to be important for peripheral, but not thymic, Treg cell development suggests that the two processes may be distinct (Chen et al., 2003). However, we have found in preliminary studies that an analogous population of cytokine-responsive Treg cell precursors exists in the peripheral lymphoid organs, albeit at a lower frequency. The peripheral CD25hi Foxp3− subset in normal mice may comprise both Treg cell precursors as well as recently activated non-Treg effector cells. These initial results suggest that although peripheral and thymic Treg differentiation may require different initial signals, the latter stages may utilize a common developmental pathway.

In summary, our observations favor an instructive model for thymic Treg cell development that occurs in at least two sequential stages. We propose that TCR signaling results in the upregulation of CD25 as well as other components of the proximal IL-2 signaling pathway, and potentially a putative permissive factor for Foxp3 expression. The expression of CD25 would offer competitive fitness for acquisition of IL-2, which would directly induce Foxp3 expression and finalize Treg cell development (Gavin et al., 2007). The time required to complete this two-step process to generate a functional Treg cell compartment may explain the historic observation that cells at the CD4SP stage can spend more than a week in thymus. Thus, the relationship between TCR activation, IL-2R expression, and induction of Foxp3 by IL-2, may explain the apparent bias towards self-reactivity within the Treg cell subset, enabling thymic Treg cell emigrants to enforce dominant tolerance upon self-recognition in the periphery.

Methods

Mice

Foxp3gfp reporter knockin and TCli TCRβ-transgenic mice were kindly provided by Dr. Alexander Rudensky (U. Washington, Seattle, WA). MHC class II, Tcrb, and Tcra knockout animals were obtained from the Jackson Laboratory, and B6.SJL (CD45.1) were obtained from the National Cancer Institute. Five to eight week old Foxp3gfp mice were used in all instances where Foxp3 protein expression is evaluated. Animals were housed in a specific-pathogen-free animal facility at Washington University and were used following protocols approved by the Institutional Animal Care and Use Committee.

Reagents

Monoclonal antibodies were purchased from eBioscience (San Diego, CA) and Becton Dickenson (San Jose, CA). IL-2, IL-4, IL-7, IL-9, IL-15, IL-21 were obtained from Peprotech (Rocky Hill, NJ). JAK inhibitor I was purchased from Calbiochem (San Diego, CA)

Cell purification

CD8+ thymocytes from Foxp3gfp mice were labeled with biotinylated anti-CD8 antibody (eBioscience) and anti-biotin microbeads (Miltenyi Biotech, Auburn, CA), and then depleted using autoMACS magnetic bead separation (Miltenyi). Remaining cells were stained with directly conjugated fluorescent antibodies for CD4, CD8, CD25, GITR (if indicated) and sorted by flow cytometry using a FACSAria (Becton Dickenson). Other protocols, such as magnetic bead selection for CD25hi thymocytes, did not increase the efficiency of this process. Typical yield from one thymus was on the order of 5x104 cells.

Supplementary Material

01

Acknowledgments

We would like to thank Nicole Bogacki and Pam Simmons for expert technical assistance; Dominic Pham for his crucial role in the analysis of TCR repertoires; Stephanie Lathrop, Jason Fontenot (Biogen Idec), James Scott-Brown (U. of Colorado), Wayne Yokoyama, Paul Allen, and Jhoanne Bautista for helpful discussions and critical review of the manuscript; and Alexander Rudensky for kindly providing the Foxp3gfp reporter and TCli TCRβ transgenic mice. Supported by the Arthritis Foundation, Burroughs Wellcome Fund, and National Institutes of Health (C.S.H.), and the Tertiary Education Services Office, Macau S.A.R. (C.W.L.). The authors have no conflicts of interest to report.

Footnotes

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