Decreasing the threshold for thymocyte activation biases CD4+ T cells toward a regulatory (CD4+CD25+) lineage

Authors


Abstract

Thymus-derived CD4+CD25+ regulatory T (Tr) cells play a critical role in suppressing aberrant responses to self in vivo. The factors that influence a CD4+ T cell's decision to commit to an immunoregulatory Tr cell lineage are currently unknown. In the present study, we found that in mice, abundantly expressing a few or one peptide(s) bound to MHC class II molecules, a large portion of conventional CD4+ T cells could be biased towards the commitment to a Tr lineage by reducing the threshold required for thymocyte activation. This occurred in the presence of either an antisense glucocorticoid receptor transgene or a pharmacological inhibitor of glucocorticoid synthesis. These results demonstrate a novel in vivo pathway for the generation of Tr cells, and raise the possibility that therapeutic enhancement of the Tr cell repertoire through pharmacological manipulation of TCR signaling thresholds may provide a feasible means of ameliorating autoimmunity.

Abbreviations:
GR:

Glucocorticoid receptor

GC:

Glucocorticoid

β2m:

β2-Microglobulin

Tr:

CD4+CD25+ regulatory T

Ii:

Invariant chain

1 Introduction

During the process of thymic selection, developing T cells are scrutinized for the ability to recognize peptides bound to host MHC molecules. An important component of this selection process is the elimination of T cells, whose antigen receptors are overtly reactive toward self-derived epitopes 1, 2. However, due to the inherent cross-reactivity of theTCR 3, and a lack of presentation of many tissue-specific antigens in the thymus, a complete purging of the repertoire cannot be accomplished by deletional mechanisms alone. Consequently, among the T cells emigrating from the thymus, some will inevitably be capable of responding to self antigens. However, despite this persistent threat most individuals never develop detectable autoimmune disease.

It has been suggested that the thymus continuously produces a population of CD4 SP (CD4+CD8) T cells capable of suppressing aberrant responses to self-derived antigens in vivo4, 5. Removal of this population, distinguished by expression of the activation marker CD25, leads to the development of fatal autoimmune syndromes inotherwise normal rodents 6. Evidence suggests that this CD4+CD25+ T cell subset is specific for self peptides and, once activated, inhibits the activation of other T cells in an antigen-independent fashion 7, 8. Thus, although thymocyte generation is usually thought of in terms of the generation of effector cells, thymocytes may adopt another fate by becoming regulatory CD4+CD25+ T cells (Tr) 4, 5.

The suggestion that CD4+CD25+ T cells originate from the common double-positive (CD4+CD8+) precursor pool in the thymus raises interesting questions as to how the development of these cells differs from that of effector T cells 4. It has been proposed that the decision to commit to a Tr lineage is determined by the TCR affinity for self-peptide/MHC class II molecules 9, 10. In this model, commitment to a conventional or regulatory fate occurs following low or high affinity interactions, respectively. A testable prediction stemming from this model is that altering the strength of TCR signals during development may potentially influence a CD4 T cell's choice to commit to a Tr lineage.

We sought to determine if increasing a thymocyte's perception of signals arising from TCR engagement might affect the CD4+ versus CD4+CD25+ lineage decision in the context of a polyclonal T cell repertoire. To accomplish this, we looked for mouse models where thymocytes undergoing selection receive higher-than-normal TCR signals. In one such model, decreased glucocorticoid receptor (GR) expression due to the presence of an antisense transgene (TKO mice) has been shown to increase the thymocyte response to TCR occupancy 11, 12. Importantly, because the transgene is targeted to the thymus, the consequences of increasing the perceived avidity of TCR engagement during selection could be examined without subsequent interference with TCR signaling in the periphery 11. One potential caveat to this approach, however, was that increasing TCR-mediated signals might inadvertently result in enhanced negative selection. Indeed, substantial MHC-dependent deletion of CD4+CD8+ thymocytes occurs in TKO mice and in FTOC when glucocorticoid (GC) synthesis is inhibited 11, 13. Thus, it seemed likely that, as a consequence of the reduced threshold required for thymocyte activation, alterations in the commitment of CD4+ T cells might be concealed in this model if many potential Tr were deleted upon cross-reacting with other self-peptide/MHC class II complexes.

To obviate this potential hindrance, we crossed mice that differed in their abilities to present self peptides bound to MHC class II with mice expressing reduced levels of the GR (TKO mice) in thymocytes 11. Our rationale for this approach was based on the observation that when a single peptide bound to MHC class II mediates both positive and negative selection, many CD4+ T cells are selected that would otherwise be deleted in wild-type mice because of their significant cross-reactivity with other self peptides 14, 15. Thus, we examined naïve mice with impaired negative selection due to invariant chain deficiency or the enforced expression of one peptide covalently linked to MHC class II (Ab) molecules in the context of reduced GC signaling. This approach revealed the commitment of a large portion of the selected CD4+ T cell repertoire towards a Tr lineage. Moreover, when neonatal mice expressing a single Ab-bound peptide were administered a pharmacological inhibitor of GC synthesis, a similar increase in regulatory T cells was observed. Our data suggest that increasing the perceived intensity of TCR engagement during thymic selection may provide a novel means of diverting the repertoire of conventional CD4+ T cells towards commitment to a Tr fate.

2 Results

2.1 GC influence CD4+CD25+ lineage commitment

We compared the efficiency of CD4+CD25+ T cell selection in thymocytes and lymph nodes from mice expressing many (Abwt), few (Ii°), one (AbEpIi°), or no self-peptide/Ab complexes (MHC II°) in the context of either normal or impaired GC signaling. Consistent with our prediction, when mice expressed normal levels of a diverse repertoire of peptides (Abwt) and reduced levels of the GR, alterations in lineage commitment were not detectable in CD4 SP thymocytes (Fig. 1Ca) and lymph node T cells (Fig. 1Cb) (Fig. 1Aa vs. Ab, Fig. 1Ac vs. Ad). In lymph nodes of non-TKO mice presenting few MHC class II-bound peptides (Ii°), CD4+CD25+ T cells were somewhat more frequent, although in the thymus their frequency was similar to Abwt-expressing mice (Fig. 1Ca). In contrast, expression of reduced amounts of the GR during T cell development in Ii° (TKO.Ii°) mice led to a significant increase in the percentage of CD4 SP thymocytes (Fig. 1Ca) and CD4+ LN T cells (Fig. 1Cb) expressing CD25 (Fig. 1Ae vs. Af, Fig. 1Ag vs. Ah). When the peptide diversity was further restrained to a single detectable peptide bound to MHC class II molecules, the frequency of CD4 SP CD25+ thymocytes (Fig. 1Ca) and T cells (Fig. 1Cb) in non-TKO AbEpIi° mice was only slightly increased over Abwt mice (Fig. 1Cb). However, similar to TKO.Ii° mice, a substantially higher proportion of CD4 SP thymocytes (Fig. 1Ca) and LN T cells (Fig. 1Cb) from TKO.AbEpIi° mice expressed CD25 (Fig. 1Ai vs. Aj, Fig. 1Ak vs. Al).

Despite their increased frequency, in the absence of the TKO transgene the absolute number of lymph node CD4+CD25+ T cells present in Ii° and AbEpIi° mice were reduced compared to mice presenting a diverse array of MHC-bound peptides (Fig. 1Cd and 16). In contrast, the increased percentage of CD4+CD25+ T cells in both TKO.Ii° and TKO.AbEpIi° mice was also accompanied by a significant increase in the absolute number of CD4+CD25+ T cells (Fig. 1Cd). Remarkably, although wild-type mice had approximately five times more total lymph node CD4+ T cells (Fig. 1Ca) due to more efficient CD4+ T cell selection, both TKO.Ii° and TKO.AbEpIi° mice contained more total lymph node CD4+CD25+ T cells (Fig. 1Cd) and splenocytes (data not shown). Reduced GR expression in thymocytes did not, however, cause a significant increase in the absolute number of CD4+ lymph node T cells in TKO.Ii° or TKO.AbEpIi° mice (Fig. 1Cc).

The paucity of conventional CD4+ T cells present in both Ii° and AbEpIi° mice results in a passive increase in the proportion of CD4+ T cells selected on non-classical MHC molecules 17, 18. The majority of these cells belong to the NK T cell lineage, whose selection requires the β2-microglobulin (β2m)-dependent molecule CD1d. They, like conventional CD4+ T cells, do not constitutively express CD25 unless activated 19. Nevertheless, given their autoreactivity 19, the increased number of CD4+CD25+ T cells present in low peptide diversity TKO mice might have reflected activated cells of this type rather than the Tr cell subset. To exclude this possibility, we compared the frequency of CD4+CD25+ thymocytes and T cells present in TKO and non-TKO AbEpIi° mice devoid of β2m. Removal of β2m in TKO.AbEpIi° mice did not lead to a reduction in the proportion of CD4+CD25+ thymocytes and T cells (Fig. 1Ba vs. Bb, Fig. 1Bc vs. Bd), which remained a substantially higher fraction of the CD4+ population. TKO mice were crossed with MHC class II-deficient mice to further rule out the participation of MHC class II-independent CD4+ T cells. On this background, we found a low frequency of CD4 SP CD25+ thymocytes in both TKO and non-TKO mice (Fig. 1Bi vs. Bj). Furthermore, the frequency of lymph node CD4+ T cells expressing CD25 was similar between TKO and non-TKO animals devoid of MHC class II (Fig. 1Bk vs. Bl), indicating that the enhanced selection of CD4+CD25+ T cells observed in low peptide diversity strains of TKO mice was dependent on MHC class II engagement.

In addition to its effect on peptide diversity, invariant chain deficiency results in an approximately tenfold reduction in surface Ab levels. To discriminate between the effects of low levels of Ab and reduced peptide diversity in the increased selection of CD4+CD25+ T cells, TKO mice were crossed with mice expressing reduced numbers of Ab bound with a diverse array of peptides (AbEpIi+) 20. Despite the reduction in surface Ab levels, we did not find an increase in the proportion of CD25-expressing CD4 SP thymocytes or lymph node CD4+ T cells in TKO.AbEpIi+ mice (Fig. 1Be vs. Bf, Fig. 1Bg vs. Bh), implying that reduced self-peptide diversity, and not reduced expression of Ab, spares CD4+CD25+ T cells in this model.

Figure 1.

Frequency and absolute numbers of CD4+CD25+ T cells present in TKO and non-TKO mice. (A, B) Lymph node or thymocyte suspensions (two inguinal, two axillary) were stained with allophycocyanin-labeled anti-CD4, perCP-labeled anti-CD8, and FITC-labeled anti-CD25. Histograms depicting CD25 expression are gated CD4+ cells. (Ca) Comparison of the percentage of CD4 SP thymocytes expressing CD25. (Cb) Comparison of the percentage of lymph node CD4+ T cells expressing CD25, and (Cc) the total number of lymph node CD4+ T cells. (Cd) The absolute numbers of CD4+CD25+ T cells present in the lymph nodes of TKO and Non-Tg mice of the indicated genotypes. All flow cytometric analyses are representative of three to four independent experiments. Results are represented as means ± SEM. (TKO.AbEpIi°, n=4; AbEpIi°, n=4; TKO.Ii°, n=5; Ii°, n=5; TKO.Abwt, n=5; Abwt, n=4). **p<0.005, *p<0.020

2.2 Phenotypic analysis of CD4+CD25+ T cells from TKO mice

To establish more precisely the relationship between CD4+CD25+ T cells observed in TKO mice and those described previously, a comparison of several genes differentially expressed in the CD4+CD25+ subset was performed 5, 10, 21. Levels of TCR and IL-7Rα were reduced on CD4+CD25+ lymph node T cells from TKO and non-TKO mice compared to the CD25 population (Fig. 2A), consistent with previous studies 21. In contrast, CD44, GITR, and CD5 were all expressed more highly on CD4+CD25+ T cells from both sets of mice (Fig. 2A). A comparison of TCR Vβ chain usage demonstrated that the repertoire of CD4+CD25+ T cells from TKO mice was not strongly biased toward the usage of any particular segment(s), in particular those used predominately by NK T cells (as shown by the Vβ usage of CD4+CD25+ from MHC class II-deficient mice, which is skewed towards usage of Vβ7 and -8), but, rather was diverse like its CD4+CD25 T cell counterpart (Fig. 2B). Furthermore, analysis of cell size by flow cytometry indicated that the CD4+CD25+ T cell subset present in TKO.AbEpIi° mice was not blasting and, therefore, likely not activated (Fig. 2C). This was further supported by cell cycle analysis, which demonstrated a low basal rate of proliferation in both TKO and non-TKO backgrounds (data not shown). These results established that CD4+CD25+ T cells from TKO mice were phenotypically indistinguishable from those found in non-TKO mice.

Figure 2.

(A) Data representative of expression of TCR, IL-7Rα, CD44, and GITR gated on either CD4+CD25+ (solid line) or CD4+CD25 (broken line) T cells from TKO and non-transgenic mice as assessed by flow cytometry. (B) Analysis of Vβ usage by CD4+CD25+ T cells from AbEpIi° and TKO.AbEpIi° mice as determined by flow cytometry. MHC class II-deficient mice were included for reference. (C) Representative dot plots from TKO.AbEpIi° mice of forward scatter (FSC) versus side scatter (SSC) as assessed by flow cytometry. Gated populations are indicated above the respective dot plots. Rightmost FSC/SSC profile of in vitro activated CD4+ T cells is included for reference. All flow cytometric analyses are from at least three independent experiments.

2.3 Purified CD4+CD25+ T cells from TKO mice inhibit T cell responses

A functional assessment of CD4+CD25+ T cells from TKO mice was used to establish their immunoregulatory properties. Co-culture of CD4+CD25+ and CD4+CD25 T cells from TKO mice resulted in a complete suppression of proliferation, similar to that seen in non-TKO strains (Fig. 3A). Using this same experimental setup, CD4+CD25+ T cells from TKO mice failed to proliferate upon CD3ϵ stimulation (Fig. 3B, white bars), unless provided exogenous IL-2 (Fig. 3B, black bars). CD4+CD25+ T cells isolated from each mouse strain did not proliferate in the presence of IL-2 alone (data not shown).

The similar responses of IL-2-supplemented CD4+CD25+ and CD4+CD25 T cells selected on single Ab complexes to APC from Abwt-expressing mice previously implied a degree of overlap in these two repertoires 16, 22. Given that negative selection acts comparably on CD4+ T cells on both CD25+ and CD25 lineages 16, 22, 23, we examined the responses of FACS-purified CD4+CD25+ and CD4+CD25 T cells from non-TKO and TKO.AbEpIi° mice cultured in the presence of IL-2 with splenocytes from either self or β2m-deficient mice. CD4+CD25 T cells from TKO.AbEpIi° mice demonstrated levels of proliferation only slightly above background in response to β2m-deficient APC, while the same population from non-TKO AbEpIi° mice responded measurably (Fig. 3D, white bars). However, in sharp contrast, CD4+CD25+ T cells from TKO.AbEpIi° displayed a greater proliferative response than those from non-TKO AbEpIi° mice in the presence of β2m° splenocytes and IL-2 (Fig. 3D, black bars), supporting the possibility that CD4+ T cells may be biased to follow a regulatory fate when TCR-mediated signals are increased during thymic development.

2.4 Pharmacological inhibition of GC synthesis influences Tr lineage commitment

We next examined if pharmacological inhibition of GC synthesis using metyrapone 13 could induce the generation of thymus-derived Tr cells in vivo. These studies were carried out on newborn AbEpIi° mice, where little CD4+CD25+ T cell development had already taken place, thereby allowing us to easily distinguish alterations in CD4+CD25+ cell numbers. This might have otherwise been difficult in adult mice due to reduced thymic output, and the presence of a substantial preexisting population of CD4+CD25+ T cells. As a control, newborn Abwtβ2m° mice were included as our data suggested that extensive negative selection of CD4+CD25+ T cells on diverse Ab peptide complexes accompanies a decreased TCR signaling threshold. Furthermore, these mice allowed us to exclude the possibility that metyrapone treatment itself induced CD25 up-regulation on peripheral T cells independently of thymic selection processes.

Analysis of lymph nodes and spleens of newborn mice demonstrated a significant increase in the proportion of CD25-expressing CD4 T cells in metyrapone-treated AbEpIi° mice (Fig. 4A, top and 4B, left; p<0.050). The difference was more noticeable in the spleens of treated mice, perhaps due to the reported reduced sensitivity of CD4+CD25+ T cells to lymph node-specific chemokines 21. Consistent with the results from TKO mice, we found a similar percentage of CD25-expressing CD4+ T cells in treated versus untreated Abwtβ2m° mice (Fig. 4A, bottom and 4B, left graph; p>0.050). The total number of CD4+CD25+ T cells was also significantly increased in treated AbEpIi° mice, although no difference was noted in treated Abwtβ2m° mice (Fig. 4B, right graph; p<0.010). Lymphoid organs from AbEpIi° mice treated with metyrapone were pooled, and various numbers of CD4+CD25+ T cells were sorted by FACS into single wells of a 96-well plate. When purified CD4+CD25 T cells from adult (untreated) AbEpIi° mice were co-cultured with metyrapone-induced CD4+CD25+ T cells from AbEpIi° mice and splenocytes from Abwtβ2m° mice, a cell dose-dependent inhibition of CD4+CD25 T cell proliferation was observed, indicating that CD4+CD25+ T cells from treated AbEpIi° mice are fully functional regulatory T cells.

Figure 3.

(A) CD4+CD25+ T cells purified from TKO mice suppress CD4+CD25 T cell proliferation. Lymphocyte suspensions from the indicated mice were stained with anti-CD4-PE and CD25-FITC and sorted into 96-well round-bottom plates. In the cases of Ii° and AbEpIi° mice, lymphocyte suspensions were prepared by pooling lymph nodes with CD4+ T cells pre-enriched by MACS sorting from splenocyte suspensions. Cells were cultured for 72 h in the presence of anti-CD3ϵ with splenocytes from MHC (I×II)° mice. [3H]Thymidine (1 μCi/well) was added to the cultures during the last 12 h. Ordinate values are in cpm. The suppressor to responder ratio is indicated in the top left. Representative data from two such experiments with each type of mouse are shown. (B) Cells were cultured as described in (A) either with or without exogenously added rIL-2. (C) CD4+CD25 T cells from Non-Tg AbEpIi° were sorted in 96-well round-bottom plates (1×104) with or without the FACS-purified CD4+CD25+ from TKO.AbEpIi° mice. Cultures were carried out for 72 h as described above with the indicated suppressor to responder ratios shown on the × axis. (C) FACS-purified CD4+CD25+ (white bars) or CD4+CD25 (black bars) (1×104) were sorted into 96-well plates and cultured for 72 h with IL-2 in the presence of γ-irradiated splenocytes (5×104) from Abwtβ2m° mice. Cultures were pulsed with [3H]thymidine (1 μCi/well) during the last 12 h. Ordinate values are in counts per million where indicated (cpm). All results are representative of two independent experiments. In all of the above graphs, bars represent the means ± SEM calculated from duplicate or triplicate cultures.

Figure 4.

(A) Representative flow cytometry analysis of expression of CD25 gated on CD4+ cells from lymph nodes (left) and spleen (right) of 16-day-old AbEpIi° and Abwtβ2m° mice after metyrapone treatment regime. (B) Bar graphs represent the means ± SEM of percentages (left) or absolute cell numbers (right) calculated from flow cytometric analysis. (C) Metyrapone-induced CD4+CD25+ T cells suppress the proliferation of CD4+CD25 T cells in vitro. The suppression assay was performed as described above. (D) The percent maximal response was calculated based on the proliferation of CD4+CD25 AbEpIi° cells alone. Data is from two independent experiments (metyrapone-injected AbEpIi° and Abwtβ2m° mice, n=6 total for each group; 5% EtOH+PBS-injected AbEpIi° and Abwtβ2m° mice, n=4 total for each group). *p<0.050, **p<0.010.

3 Discussion

Here, we show that a substantial portion of a polyclonal repertoire of CD4+ T cells, which might otherwise play a conventional role in mediating adaptive immunity, instead committed towards a Tr cell fate when GR signaling was impaired. This was revealed in both TKO and metyrapone-treated mice with a limited capacity to present self peptides to developing CD4+ T cells. Previous reports suggested that reduced GC exposure during thymocyte development led to two distinct outcomes: positive selection of T cells that usually perish due to neglect, and an elimination of T cells that otherwise would be positively selected. Our data supports those original findings, while offering an alternative to those two choices: by modulating the strength of the signal perceived by immature thymocytes upon TCR engagement, GC may also regulate the generation of regulatory T cells.

A critical question raised in this study pertains as to why we observe that thymic deletion particularly targets the regulatory population, or conversely, preferentially spares conventional CD4+ T cells in TKO mice presenting a diverse array of self peptides. To explain this apparent discrepancy, it is perhaps noteworthy that in AbEpIi° mice, where a single peptide mediates both positive and negative selection, many CD4+ T cells are selected to mature that would be deleted in wild-type mice because of their strong cross-reactivity with other self peptides 14, 15. This observation led to the hypothesis that peptide recognition during positive selection of CD4+ T cells is rather promiscuous 14, as a single peptide can select a relatively diverse CD4+ T cell repertoire, while negative selection of T cells appears inherently peptide-specific 2. Therefore, in mice expressing diverse arrays of self-peptide/MHC class II complexes, many potential Tr cells in TKO mice are likely deleted by even higher avidity interactions upon encounter with bone marrow-derived APC presenting other self peptides bound to MHC class II molecules. Thus, our results suggest that a disproportionate number of cells that would otherwise become Tr cells are eliminated in TKO.Abwt mice, due to their intrinsically high avidity towards self peptide/MHC. A loss of an appropriate amount of GC-mediated blunting of TCR signals likely renders them especially susceptible to clonal elimination during development.

Evidence suggests that the repertoire of CD4+CD25+ T cells selected on single Ep or CLIP complexes is diverse, implying that promiscuous recognition of MHC class II peptides during positive selection is a feature of both conventional (CD4+CD25) and Tr cells 16, 22. This observation seems somewhat at odds with the idea that positive selection of Tr cells is an intrinsically peptide-specific process 9, 10, 24. If, however, the selection of Tr cells were strictly mediated by recognition of agonist peptides presented during positive selection, mice expressing a single peptide should contain a repertoire of regulatory cells dominated by cells specific for the positively selecting ligand, which is inconsistent with other reports 16, 22.

Although we interpreted the reduced proliferative responses of CD4+CD25 T cells from TKO.AbEpIi ° mice to syngeneic Abwt APC as evidence that GC hyporesponsiveness diverts CD4+ T cells towards a regulatory fate, other explanations might account for this observation. Our results could be interpreted to reflect an enhanced deletion of the cohort of T cells, which normally respond to other self peptides bound to Ab, or perhaps an overall decrease in the functional avidities of the selected T cells, or both in TKO.AbEpIi° mice. It certainly seems plausible that an enhanced purging of Ab-reactive precursors from the repertoire of TKO.AbEpIi° mice could be a consequence of an overall lowered thresholdrequired for negative selection, which occurs in the absence of GC-mediated signals. However, it seems unlikely that a reduction in GC signaling would altogether alter the specificity of a particular TCR such that the selecting peptide, which may not necessarily resemble the potential agonist peptide, now deletes this receptor. Furthermore, this possibility is entirely incongruent with the fact thatthe repertoire of CD4+CD25+ T cells in TKO.AbEpIi° mice is fully capable of recognizing endogenous peptides presented in the context of Ab.

Two previous reports also demonstrated an absence of antigen-specific precursors in TKO mice. In one study, mice bred to express the Ek MHC class II haplotype in the presence of the TKO transgene failed to mount the normal stereotyped response, usually dominated by Vβ3+Vα11+-bearing CD4+ T cells, after immunization with MCC peptide. In a secondreport, inhibition of GR expression in the presence of the TKO transgene abrogated the development of the lupus-like syndrome that spontaneously develops in the MRLlpr/lpr strain of mice. Both studies concluded that a contraction of the conventional CD4+ T cell repertoire occurred because of hyporesponsiveness to GC during development in the thymus. Those studies, however, could not have distinguished between deletional and non-deletional mechanisms of tolerance induction, which was only revealed in the present study when the diversity of peptides presented during thymic selection was limited.

Although our current results are consistent with those published previously, in light of the current data, some reinterpretation of those results is now possible. One report suggested that at least some of the immunoregulatory defects observed in the MRLlpr/lpr strain could be due to an impaired functionality or development of the Tr cell subset 25. In support of this latter possibility, MRLlpr/lpr mice expressing a functional Fas gene specifically in the thymus have substantially reduced symptoms of autoimmune disease 26. Together, these results imply that at least part of the autoimmune syndrome that occurs in the MRLlpr/lpr mouse may be attributed to functional defects in a population of regulatory Tcells. Although speculative, it is plausible that in addition to repertoire contraction, GC hyporesponsiveness in TKO.MRLlpr/lpr mice led to a conversion of some of those self-reactive T cell precursors into self-specific regulatory T cells, which functioned to stave off disease.

Despite the fact that GC have long been used clinically for immunosuppression, the primary targets of their action remain obscure. A mechanistic understanding of GC action is complicated by the fact that their activities are largely dictated by the context in which they are administered. Seemingly contradictory to the data presented herein, other reports indicating a role for GC and other steroid hormones in generation of Tr cells from peripheral cells are intriguing 27. Although it is not clear to what extent the cells generated in those studies are related to the naturally occurring thymus-derived regulatory T cell population, they nevertheless support the idea that manipulating GC responsiveness may be an important means of indirectly influencing CD4+ T cells, and ultimately the orchestration of the immune response.

Large-scale gene expression profiling has also implicated CD4+CD25+ T cells themselves as potential producers of GC 28. Thus, it is intriguing to speculatethat CD4+CD25+ T cells may mediate at least part of their function on responder T cells and APC through the local secretion of GC, thereby inhibiting the production of a number ofcytokines, most importantly IL-2. In addition to regulating cytokine genes, GC have recently been shown to induce the expression of molecules known to play important roles in T cell-mediated immuneresponses during inflammation 29. When these two results are taken together, one can envisage a scenario whereby GC produced locally by activated Tr cells might participate in the establishment of an immunosuppressive milieu, which could help to either prevent the induction of or quell an established immune response. Indeed, it is also interesting that another downstream target of GR signaling, GITR (glucocorticoid-induced TNF-like receptor), has recently been shown to play an important role in Tr cells.

A considerable body of evidence suggests that GC affect thymocyte development 1113, 3034. One study has reported that thymocyte development is grossly normal in mice deficient in GR 35. However, the extrapolation of that observation to the present study is problematic. First, the avidity requirements for the selection of the Tr lineage was not analyzed in those mice and, therefore, those data do not bear upon the present analysis of regulatory T cell development. Second, the same authors have recently reported that the animals are not true knockout mice, but express a truncated GR lacking exon 2-encoded residues (the N-terminal half of the molecule), but containing the C-terminal half of the molecule, which includes DNA-binding, ligand-binding, and transactivation domains 36. These truncated GR are able to bind GC with the same affinity as wild-type mice, and cDNA microarray analysis of GC-treated thymocytes has shown that this receptor is capable of modulating the expression of a large number of genes. (Jonathan D. Ashwell, personal communication). Therefore, one cannot conclude that the GR is dispensable for any aspects of thymocyte development based on the analysis of these animals.

In contrast, it is very difficult to reconcile how regulatory T cell development was altered in both TKO mice and in the presence of a specific inhibitor of GC synthesis, given their distinct mechanisms of action (metyrapone acts by inhibiting production of GC by thymic epithelial cells while TKO mice express a specific antisense transgene). The possibility that enhanced/altered commitment to a Tr lineage was caused by non-specific effects of metyrapone treatment was eliminated because the inhibitor had no observable effect on the frequency of Tr lineage cells in syngeneic wild-type mice relative to placebo-injected controls.

It is not clear to what extent the results of this study are ultimately applicable to a more natural setting, as we did not observe any significant difference in the abundance of Tr cells in wild-type animals, regardless of their responsiveness to GC. We consider, however, that perhaps more significant than the increased proportions and numbers of CD4+CD25+ T cells observed in the low peptide diversity strains of mice, is the implication that in a GC-reduced environment, a particular portion of CD4+ T cells with measurable self reactivity to abundant self peptides may be functionally removed from the antigen-responsive repertoire either passively by deletional mechanisms or, alternatively, by being diverted into a Tr cell lineage.

In summary, we show that a substantial portion of a polyclonal repertoire of CD4+ T cells committed towards a Tr cell fate when TCR signaling was increased during development. This was revealed in both TKO and metyrapone-treated mice with a limited capacity to present self peptides to developing CD4+ T cells. We found that primary CD4+CD25 T cells do not respond substantially to Abwt splenocytes, in contrast to CD4+CD25 T cells from non-TKO AbEpIi° mice. This was not due to any intrinsic defect in TCR signaling, as CD4+CD25 T cells from TKO.AbEpIi° mice proliferate normally in response to anti-CD3 cross-linking. Surprisingly, in the present study we found that CD4+CD25+ T cells from TKO.AbEpIi° mice possessed a greater proliferative capacity when cultured with Abwt splenocytes than similar cells isolated from non-TKO AbEpIi° mice. Thus, our results support the possibility that increasing the perceived strength of TCR signals during development diverts CD4+ T cells, which would otherwise play a conventional rolein mediating adaptive immunity, into a regulatory lineage.

4 Materials and methods

4.1 Animals

Mice engineered to express transgenic AbEp complexes were generated at the National Jewish Medical and Research Center (Denver, CO) as previously described 14. Miceexpressing AbEp complexes were further crossed with mice deficient in invariant chain (Ii°), endogenous Abβ (Abβ °), and β2-microglobulin (β2m°) where indicated to homozygosity. TKO mice that express a fragment of the 3′-untranslated region of the rat GR under the control of the proximal lck promoter were described elsewhere 11. Some TKO mice were backcrossed with AbEp Abβ ° Ii β2m to obtain homozygous TKO mice deficient in Ii, endogenous Aβb, β2m, and transgenic forAbEp. TKO mice singly deficient for invariant chain, Aβb, or β2m resulted from the crosses mentioned above. TKO mice expressing only a single peptide were screened as previously described 14. All mice used in experiments were between 6 and 8 weeks old, on the H-2b (C57BL/6) genetic background and were housed in specific pathogen-free conditions at the animal facility at the Medical College of Georgia. Mice deficient for β2m, which were also on the C57BL/6 genetic background, were purchased from the Jackson Laboratory (Bar Harbor, ME).

4.2 Preparation of cells and flow cytometric analysis

Tissues were harvested and dissociated into single cell suspensions. The following antibodies were used for flow cytometric analysis: allophycocyanin-anti-CD4 (GK1.5), perCP-anti-CD8α (53-6.7), FITC-anti-CD25 antibody (7D4) biotin-anti-IL-7Rα (B12–1), phycoerytherin-anti-CD44 (IM7), FITC-anti-CD5 (53–7.3), (purchased from Pharmingen, San Diego, California). FITC-anti-TCR (57–597)was produced in-house. The anti-GITR mAb (clone Ba42) was kindly provided by Dr. Shimon Sakaguchi. Samples were stained in BSS wash buffer (BSS containing 2% FCS and 0.1% NaN3) after pre-incubation for 5 min with anti-FcγII/III (clone 2.4G2) antibody. Samples were acquired using a dual laser FACSCaliburTM (Becton Dickinson Immunocytometry Systems, Mountain View, CA) withCellQuest software (Becton Dickinson). Analysis of flow cytometry data was carried out using WinMDITM (Joseph Trotter, La Jolla, CA) or WinListTM (Verity Software House, Topsham, ME) software. Dead cells were excluded from analysis by gating appropriate forward and side scatter.

4.3 Cell sorting

Spleen and lymph node cell suspensions prepared from various strains of mice were stained with FITC-conjugated anti-CD25 antibody (7D4) and PE-conjugated anti-CD4 antibody (GK1.5), and sorted using a MoFlo cell sorter (Cytomation), as previously described 16. Purity of the CD25+CD4+ and CD25CD4+ populations was >98%. In some experiments, CD4+ T cells were first enriched from spleen and lymph node cells by removing B cells, CD8+ T cells, and adherent cells by negative selection using immunomagnetic MACS beads and columns. Cells were subsequently stained with FITC–anti-CD25 antibody along antibody with PE–streptavidin as the secondary reagent.

4.4 In vitro proliferation assays

Lymphocytes (1.0×104), sorted as described above, and red blood cell-lysed, γ-irradiated (20 Gy) spleen cells (5×104) were used as APC from the various strains of mice. Anti-CD3 antibody (145–2C11) was added at a final concentration of 10 μg/ml and cells were cultured for 3 days in 96-well round-bottom plates (Costar). Incorporation of [3H]thymidine (1 μCi/well) by proliferating lymphocytes during the last 12 h of the culture was measured.

4.5 Metyrapone treatments

Mice were injected every 2 days with either 35 μg of metyrapone (diluted from fresh stock solution dissolved in EtOH) in PBS or 5% EtOH diluted in PBS as a control. Injections were given i.p. beginning on post-natal day 2.

4.6 Statistical analysis

Statistics were calculated using the Mann-Whitney U test in conjunction with NCSS statistical analysis software.

Acknowledgements

We thank Dr. Jonathan D. Ashwell for the TKO mice, critical review of the manuscript, and helpful discussions. We also thank Mrs. Jeanene Pihkala for assistance in cell sorting and Dr. Shimon Sakaguchi for kindly providing the GITR mAb (clone Ba42).

Footnotes

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