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Peptide Specificity of Thymic Selection of CD4⁺CD25⁺ T Cells

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912

	Abstract

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The CD4⁺CD25⁺ regulatory T cells can be found in the thymus, but their need to undergo positive and negative selection has been questioned. Instead, it has been hypothesized that CD4⁺CD25⁺ cells mature following TCR binding to MHC backbone, to low abundant MHC/peptide complexes, or to class II MHC loaded with peripheral autoantigens. In all these circumstances, processes that are distinct from positive and negative selection would govern the provenance of CD4⁺CD25⁺ cells in the thymus. By comparing the development of CD4⁺CD25^- and CD4⁺CD25⁺ cells in mice expressing class II MHC molecules bound with one or many peptide(s), we show that the CD4⁺CD25⁺ cells appear during natural selection of CD4⁺ T cells. The proportion of CD4⁺CD25⁺ cells in the population of CD4⁺ thymocytes remains constant, and their total number reflects the complexity of selecting class II MHC/peptide complexes. Hence, thymic development of CD4⁺CD25⁺ cells does not exclusively depend on the low-density, high-affinity MHC/peptide complexes or thymic presentation of peripheral self-Ags, but, rather, these cells are selected as a portion of the natural repertoire of CD4⁺ T cells. Furthermore, while resistant to deletion mediated by endogenous superantigen(s), these cells were negatively selected on class II MHC/peptide complexes. We postulate that while the CD4⁺CD25⁺ thymocytes are first detectable in the thymic medulla, their functional commitment occurs in the thymic cortex.

	Introduction

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Thymic selection induces tolerance to self-Ags not only by elimination of potentially self-destructive cells during negative selection, but also by production of naturally suppressive CD4⁺CD25⁺ regulatory T cells (1, 2). While a regulatory capacity of these CD4⁺ T cells has been demonstrated in multiple autoimmunity models, and molecules expressed by these cells (IL-2R, CTLA-4, glucocorticoid-induced TNFR family-related gene, IL-10, and TGF-) have been gradually identified, little is known about how these cells are generated. In particular, it is not known whether TCRs expressed on these cells are scrutinized by thymic selection following the same rules that apply to classical CD4⁺CD25^- cells. In general, the repertoire of TCRs on CD4⁺CD25⁺ T cells is diverse, which contrasts with the canonical or semidiverse repertoires of TCRs expressed on other minor thymic subpopulations of CD4⁺ T cells expressing NK1.1 or selected on CD1, respectively (3, 4). If the development of CD4⁺CD25⁺ T cells depends on self MHC/peptide complexes, as the selection of conventional CD4⁺ T cells does, then TCR diversity and the number of these cells should correspond to the diversity of the peptides bound to class II MHC molecules. Alternatively, it has been postulated that CD4⁺CD25⁺ T cells express TCRs with higher intrinsic affinity to MHC, and a stronger interaction with thymic stromal cells commits them toward the separate lineage of regulatory CD4⁺ T cells (5). The frequency of CD4⁺CD25⁺ cells in mice transgenic for TCR or TCR is reduced, suggesting that some, but not all, TCRs facilitate the development of these cells (3). An exception to these observations was recently described in a TCR-transgenic model where many CD4⁺CD25⁺ cells appear following the exposure to high-affinity, low-avidity class II MHC/peptide ligands (6). Subsequently, the authors proposed that the differentiation of CD4⁺CD25⁺ T cells is independent of classical positive selection and is mediated by high-affinity interactions between TCR and class II MHC/peptide ligands expressed at low abundance in the thymic medulla. The nature of these interactions and their affiliation with particular thymic stromal cells have not been established. Previously, it was suggested that thymocytes that contact cognate self-Ag presented by thymic epithelial cells may become the CD4⁺CD25⁺ T cells. In these experiments thymic epithelium rudiments were grafted into allogenic thymectomized hosts, which resulted in the appearance of recent thymic emigrants that tolerate the residual T cells to graft tissues from the donor strain (7). This result implied involvement of donor-derived Ags in thymic selection of the regulatory cells. However, it also has been reported that contact with cognate peptide is required for maintenance, but not selection, of regulatory CD4⁺ T cells. These latter experiments were performed on athyroid rats in which expansion of the regulatory CD4⁺CD25⁺ T cells required the presence of the relevant autoantigen (8). No matter which of these hypotheses is correct, because CD4⁺CD25⁺ and CD4⁺CD25^- cells differentiate in the thymus, an impact of diversity of self-peptides bound to thymic class II MHC molecules on ontogeny of these cells had to be resolved.

The expression of CD25 on peripheral T cells does not indicate that these cells have regulatory functions; however, the CD4⁺CD25⁺ thymocytes are consider as solely committed to the regulatory lineage. To determine the significance of peptide diversity in the development of CD4⁺CD25⁺ cells, we compared the selection of CD4⁺ T cells in mice expressing class II MHC molecules occupied with a range of different self-peptides. If CD4⁺CD25⁺ T cells are selected by a special class of autoantigens present in both thymus and periphery, one could again expect that these autoantigens are not sufficiently presented in mice expressing a dominant or covalent peptide bound to class II MHC. On the contrary, the fraction of CD4⁺CD25⁺ thymocytes should be increased in mice expressing limited peptide diversity if their TCRs are biased to recognize the MHC framework, because the reduction of peptide diversity affects the selection of TCRs that depend more on peptide residues then those on those that recognize mostly MHC framework determinants. Collectively, the information gathered allows us to determine whether TCRs expressed on CD4⁺CD25⁺ T cells show unusual preference toward the MHC backbone or rare endogenous peptides. Interestingly, we found that the CD4⁺CD25⁺ cells constituted the same fraction of CD4⁺ thymocytes in mice expressing a different diversity of peptides bound to class II MHC molecules, implying that their selection resembles the selection of conventional CD4⁺ thymocytes. In addition, we report that although CD4⁺CD25⁺ thymocytes can resist apoptosis by endogenous superantigens, these cells remain sensitive to deletion mediated by class II MHC/peptide ligands.

	Materials and Methods

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Animals

C57BL/6 mice (A^b wild type (A^bwt)²) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Mice expressing the transgenic A^bEp complex were generated at the National Jewish Medical and Research Center (Denver, CO) as previously described (9). The same cloning strategy was used to generate transgenic mice expressing the A^bEp63K complex. Both types of mice expressing covalent A^b/peptide complexes were further backcrossed with mice deficient for the invariant chain (Ii) (A^bIi^-; provided by E. Bikoff (Harvard Medical School, Boston, MA) and R. Germain (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD)), endogenous A^b chain (A^b-; provided by D. Mathis, Harvard Medical School), and ₂-microglobulin (₂m) (A^bwt₂m^-) as indicated. The H2-M knockout mice (A^bH2-M^-) were provided by L. van Kaer (Vanderbilt University, Nashville, TN). All mice were housed under specific pathogen-free conditions in the animal care facility at the Medical College of Georgia (Augusta, GA). Mice used in experiments were 8–12 wk old. All mouse strains used for these experiments and their relevant properties are listed in Table I.

Single-cell suspensions were prepared from thymi, lymph nodes (pooled axillary, inguinal, mesenteric, and para-aortic lymph nodes), and spleen by mechanical disruption through nylon mesh. Spleen cell suspensions were additionally incubated with buffered ammonium chloride to remove RBCs. To analyze thymic stromal cells in suspension, thymi were incubated for 30 min at 37°C with collagenase (1 mg/ml; type IV; Sigma-Aldrich, St. Louis, MO) and DNase (0.02 mg/ml; bovine pancreatic DNase I; Sigma-Aldrich), followed by incubation with EDTA (0.01 M) for 5 min, then filtered to remove debris and used for FACS analysis. The following Abs were used for flow cytometric analyses: BP1-PE (anti-Ly-51), anti-CD4-allophycocyanin, anti-CD8-PerCP, anti-CD11c-PE, anti-CD25-FITC (7D4), anti-CD25-biotin (7D4), anti-V2-, anti-V3-, anti-V4-, anti-V5-, anti-V6-, anti-V7-, anti-V8-, anti-V9-, anti-V10-, anti-V11-, anti-V12-, anti-V13-, and anti-V14-FITC (all from BD PharMingen, San Diego, CA), and Y3P-FITC (prepared in our laboratory). Biotinylated Ab was detected with streptavidin-PE (BD PharMingen). Staining was performed on ice in 1x wash buffer (BSS containing 2% FBS and 0.1% NaN₃). All FACS analyses were performed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and CellQuest software (BD Biosciences). Dead cells were excluded by gating of forward and side scatters.

Calculation of total cell number

The number of CD4⁺CD25⁺ cells in spleen and lymph nodes was calculated from the number of cells recovered using flow cytometric analysis. The total number was obtained by doubling the number of CD4⁺CD25⁺ T cells from lymph nodes and adding it to the number of CD4⁺CD25⁺ T cells from spleen.

Complement depletion

Single-cell suspensions from thymi were incubated for 30 min at 4°C with supernatant containing cytotoxic anti-CD8 (HO 2.2) mAb. Cells were rinsed and incubated for 35 min at 37°C with guinea pig complement (Life Technologies, Grand Island, NY). After a single washing, thymocytes depleted of the CD8 population were used for FACS sorting.

AutoMACS sorting of cells

Single-cell suspensions from lymph nodes were incubated with anti-CD4-biotin (BD PharMingen) for 15 min at 4°C, washed, incubated again for 15 min at 4°C with MACS streptavidin beads (Miltenyi Biotec, Bergisch Gladsbach, Germany), and sorted using an AutoMACS cell separator (Miltenyi Biotec). The positive fraction of cells was used for FACS sorting.

FACS sorting of cells

CD4-biotin (BD PharMingen)-labeled lymphocytes or thymocytes were stained with anti-CD25-FITC (7D4) and streptavidin-PE (both from BD PharMingen), and were subjected to cell sorting on FACSVantage flow cytometer (BD Biosciences). The purity of CD4⁺CD25^- and CD4⁺CD25⁺ populations was >99 and >94%, respectively.

In vitro proliferation assay

Lymphocytes (10⁴) or thymocytes (10⁴), sorted as described above, were cocultured with 5 x 10⁵ irradiated (3000 rad) RBC-lysed spleen cells for 3 days in 96-well flat-bottom plates (Costar, Cambridge, MA) at 37°C in MEM (Cellgro, Herndon, VA) supplemented with 10% FBS (Life Technologies), 2 mM L-glutamine, 50 µM 2-ME, nonessential amino acids, penicillin (100 U/ml), streptomycin (100 µg/ml), and human rIL-2 (10 U/ml; Roche, Indianapolis, IN) as indicated. Wells were coated overnight with 10 µg/ml anti-CD3 (145-2C11) where indicated. During the last 12–16 h of the culture, incorporation of [³H]thymidine (0.5 µCi/well) by proliferating lymphocytes was measured.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thymic selection of CD4⁺CD25⁺ thymocytes in mice expressing different numbers of self-peptides bound to class II MHC molecules

Currently, several hypotheses have been proposed to explain the origin of CD4⁺CD25⁺ T cells in the thymus. We wished to examine the impact of the diversity of self-peptides bound to class II MHC A^b on the in vivo development of CD4⁺CD25^- vs CD4⁺CD25⁺ thymocytes. The goal of these experiments was to elucidate whether CD4⁺CD25⁺ thymocytes express TCRs with distinct requirements for self-MHC/peptide ligands that will predispose these cells to positive selection on MHC per se or whether specific low-abundance peptides or peripheral autoantigens are required. To examine thymic ontogeny of CD4⁺CD25⁺ T cells, we followed the development of these cells in mice that differ in the diversity and level of expression of class II MHC/peptide complexes (including thymic stromal cells; Table I and Fig. 1). One set of mice had a high diversity of self-derived peptides presented by either A^bwt or transgenic A^b composed of transgenic A^b chain and endogenous A^b chain (A^bEpIi⁺). The transgenic A^b chain was originally covalently coupled with Ep peptide, but in the presence of Ii, the covalent peptide is cleaved and replaced with many endogenous peptides (10). Despite the significant difference in expression level of A^b between A^bwt and A^bEpIi⁺ mice, the number of CD4⁺ cells was similar (Figs. 1 and 2A). Another set of mice used in this study had a low diversity of class II MHC-bound peptides. H2-M-deficient mice had a high expression of A^b bound to dominant Ii-derived peptide (class II-associated Ii peptide (CLIP)) (11, 12). In contrast, mice transgenic for A^b chain with covalently bound peptide and devoid of Ii and endogenous A^b chain had low expression of A^b/single-peptide complexes (Fig. 1) (13). In this study we used two mouse lines expressing transgenic A^b and bound with either Ep_52–68 peptide (A^bEpIi^- mice) or its analog, Ep63K, with glycine at position 63 changed to lysine (A^bEp63KIi^- mice). A^bEpIi^- and A^bEp63KIi^- mice had the same number of CD4⁺ thymocytes despite a much lower expression of A^b in the latter mice (Figs. 1 and 2A). Two different transgenic lines were used to assure that the lack of diversity of self-peptides and not the unique properties of the arbitrarily chosen peptide changes the outcome of selection of CD4⁺CD25⁺ T cells. The efficiency of thymic selection of conventional CD4⁺CD25^- and regulatory CD4⁺CD25⁺ thymocytes did not depend on the level of class II MHC expression (Figs. 2A and 3A). However, peptide diversity is the main factor that affects the number of selected CD4⁺ thymocytes in both populations. Mice expressing diverse A^b/peptide complexes had roughly three to four times more CD4⁺ thymocytes than mice expressing single A^b/peptide complexes (Fig. 2A). The fraction of regulatory CD4⁺CD25⁺ thymocytes remained the same in mice expressing a different diversity of MHC-bound peptides (Figs. 2A and 3A). These findings suggest that the selection requirements of CD4⁺CD25⁺ and CD4⁺CD25^- thymocytes are similar. In all mice examined, regulatory CD4⁺CD25⁺ thymocytes had low constitutive expression of CTLA-4 (data not shown). We also noticed a decreased level of TCRs on the CD4⁺CD25⁺ thymocytes, the biological significance of which is currently unknown.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Expression of Ab molecules bound with one or many peptides on thymic epithelial cells. Thymic stromal cells from mice with indicated genotypes were stained with mAb BP1-PE (specific for thymic cortical epithelial cells) or CD11c-PE (specific for dendritic cells) and Y3P-FITC (specific for I-Ab complexes). The mean fluorescence for each histogram is shown.

View larger version (60K): [in this window] [in a new window]   FIGURE 2. CD4+CD25+ cells are selected in thymi expressing Ab occupied with many or a single peptide. Thymocytes (A) or lymph node cells (B) were stained with anti-CD4-allophycocyanin, anti-CD8-PerCP, and anti-CD25-FITC. Dot plots show expression of CD4 and CD8 molecules. Histograms show expression of CD25 molecules on CD4+CD8- gated cells. Numbers indicate the percentages of different subpopulations of cells in the analyzed organs.

CD4⁺CD25⁺ thymocytes from mice expressing A^bwt or single peptide-A^b complexes have the same functional properties

To ensure that CD4⁺CD25⁺ thymocytes isolated from thymi expressing covalent A^b/peptide complexes are true precursors of regulatory CD4⁺ T cells, we performed two functional assays. In the first experiment we compared the activation requirement of CD4⁺CD25⁺ thymocytes isolated from A^bwt and A^bEp63KIi^- mice. As reported, regulatory thymocytes from both mice responded to anti-CD3 stimulation only in the presence of exogenous rIL-2 (Fig. 4A) (5). In the second experiment we tested the suppressive properties of CD4⁺CD25⁺ thymocytes from A^bwt vs A^bH2-M^- and A^bEp63KIi^- mice. As shown in Fig. 4B, these cells had equal capacities to inhibit the response of conventional CD4⁺ thymocytes to anti-CD3 stimulation. These experiments prove that the regulatory CD4⁺CD25⁺ cells from mice expressing single class II MHC/peptide complexes have the same properties as the relevant population in wild-type mice.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. CD4+CD25+ thymocytes from single-peptide mice reveal functions similar to their counterparts from Abwt mice. CD4+CD25- or CD4+CD25+ thymocytes from Abwt or AbEp63K Ii- mice were cultured in the presence of anti-CD3 and rIL-2 as indicated along with irradiated autologous splenocytes (A). Purified CD4+CD25- thymocytes (104/well) were cocultured with indicated amounts of CD4+CD25+ thymocytes in the presence of anti-CD3 (B). Results are shown as the arithmetic mean ± SD of triplicate cultures. Data are representative of three independent experiments.

Because the limited diversity of A^b/peptide complexes did not interfere with thymic production of CD4⁺CD25⁺ cells, we examined what percentage of these cells can be selected in mice devoid of ₂m. For that purpose we crossed our single-peptide A^bEpIi^- and A^bEp63KIi^- mice with ₂m-deficient mice. In these mice the class II MHC/peptide complexes remain intact, but the selection of CD4⁺ T cells by nonclassical MHC/peptide complexes such as CD1, Qa, or TL is aborted. Furthermore, in these mice the peripheral CD4⁺ T cells undergo significant peripheral expansion due to the lack of CD8⁺ T cells. As shown in Fig. 5, we found no significant reduction of CD4⁺CD25⁺ thymocytes in A^bwt₂m^-, A^bEpIi^-₂m^-, or A^bEp63KIi^-₂m^- mice (see also Fig. 2A). Therefore, although few CD4⁺ T cells are selected by the nonclassical MHC/peptide complexes, possibly including some that are also CD25⁺, this process has no significant contribution to the generation of the regulatory CD4⁺CD25⁺ T cells. Also, the rapid expansion of CD4⁺CD25^- T cells in the periphery due to the lack of CD8⁺ T cells was not followed by a significant increase in the number of CD4⁺CD25⁺ T cells. This phenomenon has resulted in a changed ratio of CD4⁺CD25^-:CD4⁺CD25⁺ cells, from 5:1 to 9:1. Subsequently, we concluded that the proliferative capacity of CD4⁺CD25⁺ T cells is more restrained than that of conventional CD4⁺ T cells, and that although these cells represent <10% of peripheral cells in the normal repertoire, they may be selected in an excess, because fewer of these cells continue to control increased numbers of CD4⁺CD25^- cells.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. The CD4+CD25+ population is efficiently selected in thymi of mice devoid of 2m-dependent MHC molecules. Thymocytes (A) or lymph node cells (B) from indicated mice were stained and analyzed as described in Fig. 2.

It has been found that the frequency of V and V gene families encoding TCRs of regulatory and conventional thymocytes in mice expressing A^bwt was similar, suggesting that both repertoires are diverse (4). However, it is not known how the restriction of the available MHC/peptide ligands impacts the diversity and encoded specificities of the TCRs expressed on CD4⁺CD25⁺ thymocytes. We have compared the contribution of V gene families to the TCR repertoires in A^bwt, A^bH2-M^-, and A^bEp63KIi^- mice. As shown in Fig. 5, both conventional and regulatory thymocytes have diverse TCR repertoires with similar distributions of TCR V segments. The only irregularities in the frequency of V genes pertained to the V5 and V14 gene families (see below). To compare the range of Ag specificities encoded by TCRs on regulatory and conventional thymocytes, we measured their Ag responses. In mice expressing only single covalent A^b/peptide complexes, thymocytes are not tolerant to self-peptides and therefore respond strongly to syngeneic APCs expressing A^bwt complexes. This response depends on the wide range of bound endogenous peptides (9). In this study we examined the proliferation of CD4⁺CD25⁺ and CD4⁺CD25^- thymocytes cocultured with syngeneic APCs expressing A^b occupied with wild-type peptides, CLIP, or covalently bound Ep peptide (Fig. 6). Both thymocyte populations responded only to wild-type APCs. This result suggests that the Ag specificities of regulatory and conventional thymocytes overlap and that the precursor frequency of TCRs specific to syngeneic APCs in both CD4⁺CD25⁺ and CD4⁺CD25^- cells is similar. Both cell populations responded only to APCs expressing multiple antigenic epitopes, indicating that this response is polyclonal, corroborating flow cytometric data about the diversity of the TCR repertoires.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Regulatory T cells selected by covalent Ab/peptide complex recognize syngeneic Ab molecules occupied with many peptide(s). CD4+CD25- or CD4+CD25+ thymocytes from AbEp63K Ii- mice were stimulated with either a CD3 mAb or different type of APCs. Unstimulated thymocytes cultured in the presence of IL-2 did not proliferate (see Fig. 4). Results are shown as the arithmetic mean ± SD of triplicate cultures. Data are representative of three independent experiments.

It has been shown that whereas immature CD4⁺CD8⁺ and CD4⁺CD25^- thymocytes are extensively negatively selected upon exposure to endogenous superantigens, the CD4⁺CD25⁺ thymocytes are spared from deletion (18). Different susceptibility of CD4⁺CD25⁺ vs CD25^- thymocytes to negative selection provided the main support for the hypothesis that these cells develop independently of conventional CD4⁺ thymocytes.

To reexamine the resistance of CD4⁺CD25⁺ thymocytes to negative selection, we followed the fate of the polyclonal thymocyte population in the presence of high-affinity ligand encoded by the 3' open reading frame of mouse mammary tumor virus (MMTV)-9 retrovirus or transgenic class II MHC/peptide complex (19). Although A^b molecules do not present endogenously derived superantigens very efficiently, the deletion of thymocytes expressing V5 TCRs mediated by MMTV-9 is evident when the frequency of V5-bearing thymocytes has been compared in preselection and postselection repertoires. The V distribution in the preselected repertoire was estimated by analyzing TCR usage by CD4⁺CD8⁺ thymocytes in mice simultaneously devoid of A^b and ₂m (Fig. 7A). Concordantly with the previously published data (20), there was a visible reduction of CD4⁺CD25^- thymocytes with V5 TCRs, while the CD4⁺CD25⁺ thymocytes bearing V5 TCRs were spared from deletion (Fig. 8A). We have also noticed that if A^b molecules were loaded with Ep peptides or CLIPs, negative selection of both CD25^- and CD25⁺ thymocytes was abolished, implying that the diversity of peptides bound to A^b validates superantigen binding and subsequent deletion of thymocytes (Fig. 8A) (21). Recently, it has been shown that uniform occupancy of A^b molecules with either covalent Ep peptide or dominant CLIP profoundly blocks the deletion of T cells bearing TCRs specific for viral superantigen (vSAG)-7. These same mice had aborted susceptibility to MMTV infection, which was not the case for mice expressing A^b bound with many endogenous peptides (22). To examine whether CD4⁺CD25⁺ T cells are eligible to deletion by conventional A^b/peptide complex, we used mice that express the A^bEp63K complex. The antigenic response to A^bEp63K complex is dominated by T cells expressing V14 genes (17). Subsequently, thymocytes bearing TCRs with this V segment should be prone to negative selection in mice that exclusively express this complex. In fact, analysis of the V repertoire of thymocytes in A^bEp63KIi^- mice showed that V14 TCRs are less frequently represented on CD4⁺ thymocytes compared with wild-type mice (Fig. 7B). Because CD4⁺ thymocytes expressing V14 TCRs were also missing in double-peptide A^bEpA^bEp63KIi^- mice, deletion, not lack of positive selection, was responsible for this phenomenon (Fig. 7B). Subsequently, we were interested to determine whether V14-bearing thymocytes may avoid deletion on A^bEp63K complex by committing to the CD4⁺CD25⁺ lineage. In contrast to the previous marginal deletion of V5⁺ regulatory thymocytes by MMTV-9, both CD4⁺CD25⁺ and CD4⁺CD25^- thymocytes expressing V14 were deleted to similar extents in A^bEp63K mice. Therefore, the CD4⁺CD25⁺ thymocytes have differential sensitivity to antigenic peptide- or superantigen-mediated deletion, which is most likely caused by the expression pattern of MMTV on thymic stromal cells (see Discussion).

View larger version (22K): [in this window] [in a new window]   FIGURE 7. In mice with single Ab/peptide complexes, CD4+CD25+ thymocytes express a diverse set of TCRs. Thymocytes from AbEp63K Ii- (A), AbH2-M- (B), or Abwt (C) mice were stained with Abs against CD4, CD8, CD25, and different Vs. Percentages of populations as a fractions of CD4+CD8- thymocytes bearing TCRs with a particular V are shown as arithmetic mean ± SD of five mice of the same phenotype.

View larger version (21K): [in this window] [in a new window]   FIGURE 8. CD4+CD25+ thymocytes are resistant to clonal deletion by endogenous superantigen vSAG-9 but are susceptible to negative selection by specific class II MHC/peptide complexes. Thymocytes from the indicated mice were stained with Abs against CD4, CD8, CD25, and V5 (A) or V14 (B). Percentages of V-bearing populations as a fraction of single-positive CD4+ thymocytes are shown ± SD.

	Discussion

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To examine the impact of peptide diversity on thymic development of CD4⁺CD25⁺ T cells, we have followed their thymic ontogeny in mice that express one, few, or many peptides bound to class II MHC. We have also determined how the CD4⁺CD25⁺ T cells develop depending on the expression level of the selecting class II MHC/peptide complexes. Because the properties and diversity of selecting ligands recognized by CD4⁺CD25⁺ cells are not known, the goal of this investigation was also to resolve whether the TCR repertoire of these cells resembles the repertoire of conventional CD4⁺ T cells. First, we found that the total number of selected CD4⁺CD25⁺ T cells is proportional to the diversity of self-peptides presented in the context of A^b on thymic epithelium. The population of regulatory CD4⁺ thymocytes comprised a constant proportion (4–6%) of all the CD4⁺ thymocytes in mice expressing one or many peptides bound to A^b. Therefore, it is most likely that the complexity of peptides bound to class II MHC controls the selection of both regulatory and conventional T cells. Secondly, there was no evidence that TCRs used by CD4⁺CD25⁺ thymocytes preferentially bind the MHC framework due to their inherent bias to MHC. If that were the case, we would expect to find more CD4⁺CD25⁺ T cells in A^bEpIi^- mice, where the development of conventional CD4⁺ T cells is restrained. Instead, the total number of CD4⁺CD25⁺ thymocytes was reduced in mice expressing one or few peptides bound to A^b and mirrored the reduced number of CD4⁺CD25^- thymocytes. Lastly, because the development and peripheral persistence of CD4⁺CD25⁺ T cells was normal in two types of mice exclusively expressing covalent class II MHC/peptide complexes, the hypothesis that these cells depend on the presentation of specific autoantigens in the thymus and in the periphery is likely to be incorrect (7).

CD4⁺ T cells expressing activation markers are found in mice with ablated expression of class II MHC (27). It was also reported that the CD4⁺ T cells with regulatory functions may be selected on nonclassical MHC molecules (28). To examine whether CD4⁺CD25⁺ T cells are selected on MHC complexes other than class II MHC, we examined the development of these cells in mice devoid of ₂m. Our results show that the selection of regulatory CD4⁺ T cells remains normal in the absence of nonclassical MHC molecules. These experiments also revealed that these cells have reduced capacity to expand in the absence of CD8⁺ T cells.

It has been reported that CD4⁺CD25⁺ T cells appeared resistant to apoptosis mediated by endogenously encoded superantigens (18). This finding implies that regulatory T cells acquire their properties by avoiding negative selection and thus express TCRs prone to recognize self-Ags. To examine the sensitivity of CD4⁺CD25⁺ T cells to negative selection, we have compared the extent of deletion of V5- or V14-bearing thymocytes exposed to MMTV-9 or transgenic A^bEp63K complex, respectively. Consistently with the previous reports, the fraction of V5-expressing thymocytes in A^bwt mice was higher in the preselection repertoire on CD4⁺CD8⁺ cells than in CD4⁺CD25^- cells, indicating clonal negative selection (20). In contrast, when self-Ag was encoded by covalent A^b/peptide complex, both regulatory and conventional thymocytes bearing V14⁺ TCRs were negatively selected. The A^bEp63K complexes are expressed in both thymic cortex and medulla, while MMTV-9-encoded vSAG was not found in thymic cortex (29). It is known that MMTV-mediated clonal deletion occurs late in T cell ontogeny and affects mature, single-positive, medullary thymocytes (30). Therefore, low susceptibility of CD4⁺CD25⁺ T cells to superantigen-mediated deletion implies that commitment to the CD25⁺ lineage, manifested by resistance to TCR-mediated deletion, occurred in thymic cortex before interaction with the A^b/superantigen in the medulla. In conclusion, we favor the hypothesis that some CD4⁺CD8⁺ thymocytes with increased affinity to self-MHC/peptide complexes are positively selected toward regulatory CD4⁺CD25⁺ T cells.

Because the peptide specificities of TCRs expressed by CD4⁺CD25⁺ T cells are not known, we have examined the capacity of these TCRs to recognize multiple peptides bound to syngeneic class II MHC molecules. As mentioned, CD4⁺CD25⁺ T cells selected in mice expressing only covalent A^b/peptide complex are uniquely suited for this purpose. More than two-thirds of conventional CD4⁺ T cells isolated from single-peptide mice express TCRs capable of recognizing A^b molecules loaded with endogenously processed self-peptides (9). Here we report that in the presence of exogenously added IL-2, the CD4⁺CD25⁺ and CD4⁺CD25^- T cells from the single A^b/peptide mice proliferate to a similar extent when incubated in vivo with APCs from A^bwt mice. In contrast, the same subpopulations of CD4⁺ T cells proliferated poorly when incubated with APCs expressing A^b bound with single peptide. These results imply that the repertoire of TCRs expressed on CD4⁺CD25⁺ T cells is restricted to recognize peptides in the context of self class II MHC molecules, and that these cells have been selected by A^b/peptide complexes expressed on thymic cortical cells. The magnitude of the response to A^b bound with multiple, but not single, peptide shows that the specificities of TCRs expressed on CD4⁺CD25⁺ T cells are diverse. Our results agree with the results obtained by analyzing the development of CD4⁺CD25⁺ T cells in mice expressing class II MHC only on thymic cortical epithelium (T. M. Laufer, personal communication). In that report the authors concluded that the selection of this population of CD4⁺ T cells is facilitated by cortical, not modularly thymic, epithelial cells, and that these cells are generated during the natural processes of thymic selection. Furthermore, the CD4⁺CD25⁺ T cells that mature in these mice could recognize syngeneic A^b molecules loaded with endogenous peptides, implying that these cells will be sensitive to natural negative selection.

In vivo deletion of CD4⁺CD25⁺ T cells in wild-type mice leads to autoimmune disease. What the outcome would be of the similar experiment performed in mice in which the expression of endogenous and exogenous peptides is suppressed by covalently attached peptide is not yet known. Nevertheless, in these latter mice the proportion of CD4⁺CD25⁺ T cells increases faster with age than in wild-type mice, implying that these cells play an active role in maintaining tolerance to self. Another function these cells may play in mice expressing one class II MHC/peptide complex is to control the size of the pool of naive CD4⁺ T cells. Regardless of the available space, the number of naive CD4⁺ T cells in mice expressing a single class II MHC/peptide complex remains constant over the life span, whereas, as already mentioned, the proportion of regulatory CD25⁺ T cells increases gradually. This observation agrees with the hypothesis that this subpopulation of CD4⁺ T cells not only controls the function of other T cells but also regulates the pools of naive vs effector T cells in the body.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Selection of CD4+CD25+ regulatory T cells depends on the complexity of peptides bound to Ab. A and B, Percentages of CD4+CD25+ thymocytes and peripheral T cells in indicated types of mice, respectively. C, Total number of peripheral CD4+CD25+ T cells in analyzed mice.

	Footnotes

² Abbreviations used in this paper: A^bwt, A^b wild type; ₂m, ₂-microglobulin; Ii, invariant chain; CLIP, class II-associated Ii peptide; MMTV, mouse mammary tumor virus; vSAG, viral superantigen.

Received for publication August 13, 2001. Accepted for publication November 2, 2001.

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