The Bcl-2 regulated apoptotic pathway is critical for the

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1 The Journal of Immunology Defects in the Bcl-2 Regulated Apoptotic Pathway Lead to Preferential Increase of CD25 low Foxp3 + Anergic CD4 + T Cells Yifan Zhan,*, Yuxia Zhang,* Daniel Gray,* Emma M. Carrington,* Philippe Bouillet,*, Hyun-Ja Ko,* Lorraine O Reilly,* Ian P. Wicks,*, Andreas Strasser,*, and Andrew M. Lew*, Defects in the Bcl-2 regulated apoptotic pathway inhibit the deletion of self-reactive T cells. What is unresolved, however, is the nature and fate of such self-reactive T cells escaping deletion. In this study, we report that mice with such defects contained increased numbers of CD25 low Foxp3 + cells in the thymus and peripheral lymph tissues. The increased CD25 low Foxp3 + population contained a large fraction of cells bearing self-reactive TCRs, evident from a prominent increase in self-superantigen specific Foxp3 + Vb5 + CD4 + T cells in BALB/c Bim 2/2 mice compared with control animals. The survival rate of the expanded CD25 low Foxp3 + cells was similar to that of CD25 high Foxp3 + CD4 T cells in vitro and in vivo. IL-2R stimulation, but not TCR ligation, upregulated CD25 on CD25 low Foxp3 + CD4 + T cells in vitro and in vivo. The expanded CD25 low Foxp3 + CD4 + T cells from Bim 2/2 mice were anergic but also had weaker regulatory function than CD25 high Foxp3 + CD4 + T cells from the same mice. Analysis of Bim 2/2 mice that also lacked Fas showed that the peripheral homeostasis of this expanded population was in part regulated by this death receptor. In conclusion, these results show that self-reactive T cell escapees from thymic deletion in mice defective in the Bcl-2 regulated apoptotic pathway upregulate Foxp3 and become unresponsive upon encountering self-ag without necessarily gaining potent regulatory function. This clonal functional diversion may help to curtail autoaggressiveness of escaped self-reactive CD4 + T cells and thereby safeguard immunological tolerance. The Journal of Immunology, 2011, 187: The Bcl-2 regulated apoptotic pathway is critical for the deletion of self-reactive T cells during thymic development (1). Overexpression of antiapoptotic Bcl-2 (2, 3) or deficiency of proapoptotic Bcl-2 family members, such as the Bcl-2 homology domain 3 (BH3)-only protein Bim (4, 5) or the multi Bcl- 2 homology domain proteins Bax plus Bak (6), cause defects in the killing of self-reactive T cells by apoptosis during their development in the thymus. Accordingly, thymocytes from autoimmune diabetesprone NOD mice have been reported to be defective in TCR stimulation-induced upregulation of Bim (7). Defects in the deletion of high-affinity self-reactive TCRexpressing T cells can lead to rapid destruction of self-ag expressing tissues (8, 9). For example, humans and mice lacking AIRE, which is critical for presentation of autoantigens in the *Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; and Department of Medical Biology, University of Melbourne, Melbourne, Victoria 3010, Australia Received for publication January 5, Accepted for publication June 2, This work was supported by National Health and Medical Research Council of Australia Program Grants and , Project Grants and , National Health and Medical Research Council Australia Fellowship , National Health and Medical Research Council Career Development Awards and , National Health and Medical Research Council Independent Research Institutes Infrastructure Support Scheme Grant , the National Institutes of Health (CA ), the Leukemia and Lymphoma Society (LLS SCOR 7413), Juvenile Diabetes Research Foundation Grant , and a Victorian State Government Operational Infrastructure Support grant. Address correspondence and reprint requests to Dr. Yifan Zhan and Dr. Andrew M. Lew, Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, Parkville, Victoria 3052, Australia. addresses: zhan@wehi.edu.au (Y.Z.) and lew@ wehi.edu.au (A.M.L.) The online version of this article contains supplemental material. Abbreviations used in this article: BH3, Bcl-2 homology domain 3; FasL, Fas ligand; PI, propidium iodide; SP, single-positive; Tg, transgenic; Treg, regulatory T; WT, wild-type. Copyright Ó 2011 by The American Association of Immunologists, Inc /11/$16.00 thymus, have a severe defect in the deletion of autoreactive thymocytes (10) and consequently develop multiorgan autoimmune disease (10, 11). In contrast, Bim 2/2 mice and transgenic (Tg) mice overexpressing Bcl-2 in lymphoid cells do not develop multiorgan T cell-mediated autoimmune diseases, despite a severe defect in deleting autoreactive T (3, 4) and B cells (12). There are at least four possible mechanisms that might restrain autoaggression of self-reactive lymphocytes escaping deletion in mice defective in the Bcl-2 regulated apoptotic pathway. First, escapees from deletion may become functionally unresponsive (anergic) upon encountering self-ags (13, 14). In contrast, such induction of anergy will most likely not occur in AIRE 2/2 mice due to impaired presentation of self-ag. Second, forbidden clones (15) may become subject to regulation by CD25 + Foxp3 + regulatory T (Treg) cells. Third, escapees may undergo peripheral deletion by death mechanisms independent of the Bcl-2 regulated apoptotic pathway, akin to the death receptor-mediated control of mature T cells that had been activated continuously through their TCR by persistent Ags in peripheral lymphoid organs (16 18). Finally, potentially dangerous T cell clones may be diverted to differentiate into an alternate, harmless state to preserve tolerance (19). To explore these possibilities, we studied the fate of self-reactive thymocytes in mice defective in the Bcl-2 regulated apoptotic pathway, including Bim 2/2, Bim 2/2 Puma 2/2, vavbcl-2 Tg, and Bax 2/2 Bak 2/2 mice. We found that a subset of Foxp3 + CD4 T cells was preferentially and substantially increased in these mutant mice and investigated the underlying mechanisms for their selective increase and functional attributes. These studies showed that clonal diversion of differentiation of autoreactive thymocytes can help maintain immunological tolerance, at least when the Bcl- 2 regulated apoptotic pathway within these cells is impaired. Such impairment due to either failure to induce proapoptotic proteins or dysregulation of antiapoptotic regulators can occur in autoimmune NOD mice (7) and in humans (20).

2 The Journal of Immunology 1567 Materials and Methods Mice C57BL/6 Bim 2/2 (line 266), Puma 2/2, Bim 2/2 Puma 2/2,andOT-II-Bim 2/2 mice were described previously (21, 22). BALB/c Bim 2/2 mice were generated by backcrossing C57BL/6 Bim 2/2 mice for 10 generations onto a BALB/c background. Foxp3GFP KI /Bim 2/2 mice and Foxp3GFP KI / vavbcl-2 Tg mice were generated by mating Foxp3GFP KI mice (backcrossed for at least six generations with C57BL/6 mice) (23) with C57BL/6 Bim 2/2 mice. C57BL/6-Ly5.1 Rag-1 2/2 mice reconstituted with a Bax 2/2 Bak 2/2 hemopoietic system (using Bax 2/2 Bak 2/2 fetal liver cells as donors) after lethal irradiation ( Gy, 4 h apart). vavbcl-2 Tg mice and FAS lpr/lpr mutant mice were maintained at the Walter and Eliza Hall Institute animal facilities under specific pathogen-free conditions. All experiments with mice were conducted in accordance with the rules of the Walter and Eliza Hall Institute s Animal Ethics Committee. Flow cytometry Abs used in this study were all purchased from BD Biosciences (San Jose, CA) unless stated otherwise. PE-Cy7 or APC-conjugated anti-mouse CD4 (clone RM4-5), allophycocyanin-cy7 conjugated anti-mouse CD8 (clone ), and FITC- or PE-conjugated anti-mouse CD25 (clone PC61) Abs were used for surface staining. FITC-conjugated Abs specific for individual TCR Vb-chains were supplied as a kit by BD Biosciences. To stain cell-surface proteins, cells were incubated with optimally diluted Abs for 30 min on ice. Viable cells, identified by propidium iodide (PI) exclusion, were analyzed using an FACSAria (BD Biosciences) and Cell- Quest software (CellQuest, San Carlos, CA). Intracellular Foxp3 protein was detected using a staining kit from ebioscience (San Diego, CA). Cells from the thymus, spleen, and lymph node were stained for surface markers CD4, CD8, and CD25, and sorted in an FACSAria (BD Biosciences). Cell survival assays Cell survival assays were performed as previously reported (24). Purified cell subsets (.95% pure) were cultured at in 200 ml medium in U- bottom 96-well plates in the presence or absence of 5 mg/ml anti-cd3 and 2 mg/ml anti-cd28 Abs for 2 d. Cell survival was measured by flow cytometry with PE-conjugated FACS calibration beads (CaliBRITE; BD Biosciences) and PI to determine numbers of viable cells. Recombinant human IL-7 (2 ng/ml; PeproTech, Rocky Hill, NJ) or human IL-2 (20 U/ ml; National Cancer Institute Biometric Research Branch Preclinical Repository, Rockville, MD) was used in some cultures. In vivo tracking of transferred CD4 T cell subsets CD4 T cell subsets were sorted. Each population was labeled with CellTrace violet dye according to the manufacturer s instructions (Invitrogen, Carlsbad, CA). Labeled cells ( ) were injected i.v. into C57BL/6- Ly5.1 mice. Days 3 and 7 posttransfer, spleens were harvested, and singlecell suspensions were prepared. Spleen cells were then stained for Ly5.2, CD4, CD8, and CD25. Transferred cells were identified as Ly5.2 + Cell- Trace violet + CD4 + cells. The total number of transferred cells was calculated by multiplying the total spleen cellularity with the percentage of transferred cells within the total spleen cell population. Quantitative real-time PCR RNA was extracted from pellets of purified cell populations using an RNeasy MiniKit (Qiagen, Melbourne, Victoria, Australia) as per the manufacturer s instructions. A total of 250 ng RNA was treated with DNase I recombinant, RNase-free (Roche, Mannheim, Germany) and reverse-transcribed to cdna using random primers and the Superscript III First-Strand Synthesis System RT-PCR kit (Invitrogen). Quantitative realtime PCR was performed to determine the expression of Foxp3, CD25, and CTLA4 in CD4 + T cell subsets. The reactions consisted of GoTaq qpcr Master Mix (Promega, Madison, WI), 0.5 mm specific primers, and 2 ml cdna, and were carried out on a LightCycler (Roche). Specific primers (Sigma-Aldrich, Castle Hill, New South Wales, Australia) for quantitative real-time PCR were as follows: b-actin: F, 59-GATCTGGCACCACA- CCTTCT-39; R, 59-GGGGTGTTGAAGGTCTCAAA-39; Foxp3: F, 59- ATGTTCGCCTACTTCAGAAACC-39, R, 59-CAAATTCATCTACGGT- CCACAC-39; CD25: F, 59-TTCCGAAGACTAAAGGAATTGG-39; R, 59- TCTGTTGTGGTTTGTTGCTCTT-39; and CTLA4: F, 59-AGTTTCCT- GGTCACTGCTGTTT-39; R:59-TTTTCACATTCTGGCTCTGTTG-39. An initial activation step was carried out at 95 C for 15 min. Amplification was then carried out for 35 cycles with denaturation at 95 C for 15 s, annealing at 55 C for 30 s, and extension at 72 C for 30 s, followed by melting point analysis. Data analysis was performed with SDS 2.2 software (Applied Biosystems). The expression level for each gene was determined using a standard curve prepared from 1 to 1026 pg of specific DNA fragment, then expressed as a ratio to the levels of b-actin mrna. Assays to measure T cell proliferation and Treg cell activity CD25 2 Foxp3 2, CD25 low Foxp3 +, and CD25 high Foxp3 + CD4 + T cell subpopulations were purified by FACS sorting from the thymi, spleens, or lymph nodes of GFP KI /vavbcl-2 Tg mice, Foxp3-GFP KI /Bim 2/2 mice, and Foxp3-GFP KI mice for the following functional assays. For cell proliferation assays, subsets of T cells ( cells/well) were cultured in 96-well V-bottom plates with 5 mg/ml anti-cd3 Ab together with gamma-irradiated and T cell-depleted spleen cells as APCs. For Treg cell functional assays, CFSE-labeled CD25 2 CD4 + T cells from C57BL/6-Ly5.1 mice used as effector cells were cultured either with CD25 low Foxp3 + or CD25 + Foxp3 + CD4 + T (from the mice listed above) in the presence of APCs. Cells were stimulated for h with 5 mg/ml soluble anti-cd3 Ab plus 2 mg/ml anti-cd28 Ab. Proliferation of effector cells was measured by flow cytometric analysis of CFSE dilution. PEconjugated calibration beads (BD Biosciences) were included to determine total cell numbers. In some experiments, unlabeled CD25 2 CD4 + T cells from wild-type (WT) mice were used as effector cells. Cultures were pulsed with 1 mci/well [ 3 H]thymidine for the final 8 h of culture, harvested with a Tomtec Harvester96 (Tomtec, Hamden, CT), and cell incorporated radioactivity measured in a Packard TopCounter (Packard Instruments, Sterling, VA). Cytokine production assays Supernatants from cultured T cell subsets were investigated for cytokine content using the 23-plex on Bio-plex 2200 system according to the manufacturer s protocol (Bio-Rad, Hercules, CA). Fas ligand-induced apoptosis assays Spleen cells ( /well) from vavbcl-2 Tg/Foxp3GFP KI were cultured in RPMI 1640 plus 5% FCS and treated with serial dilutions of Fas ligand (FasL)-Fc (harvested from stable 293 cells expressing FasL-Fc [ , clone H10; kind gift from Drs P. Schneider and J. Tschopp, University of Lausanne, Lausanne, Switzerland]) in 96-well flat-bottom plates for 16 h. Cells were then stained for CD4, CD8, and CD25. The numbers of live cells were calculated by determining the ratio between PI-negative cells and FITC-coupled FACS calibration beads, which were added prior to FACS analysis. Results Defects in the Bcl-2 regulated apoptotic pathway cause a preferential increase in Foxp3 + CD4 single-positive thymocytes To explore the nature and fate of self-reactive T cells that escape thymic deletion due to a defect in the Bcl-2 regulated apoptotic pathway, we first examined Bim 2/2 mice. Bim 2/2 mice had higher numbers of CD4 single-positive (SP) thymocytes (mean 6 SEM: million) than that of WT mice (mean 6 SEM: million; p, 0.001) (Fig. 1Ai). Furthermore, Bim 2/2 mice had a striking increase in Foxp3 + CD4 SP thymocytes ( 14-fold) ( million in Bim 2/2 mice versus million in WT mice; p, ). However, these cells did not express as high levels of CD25 as the corresponding cells from WT mice (Fig. 1Aii, iii). The CD25 high Foxp3 + CD4 SP thymocytes were also increased in Bim 2/2 mice, albeit to a more moderate extent (2 6- fold) ( million in Bim 2/2 mice versus million in WT mice; p, 0.04). Like Bim, Puma is also a potently proapoptotic BH3-only protein that binds with high affinity to all antiapoptotic Bcl-2 family members (25). We observed an even greater increase in CD25 low Foxp3 + thymocytes in Puma 2/2 Bim 2/2 mice compared with WT mice (.40-fold) ( million in Puma 2/2 Bim 2/2 mice versus million in WT mice; p, 0.01), although loss of Puma alone did not cause a significant increase in such cells (Fig. 1Aiv). Next, we examined vavbcl-2 Tg mice that overexpress antiapoptotic Bcl-2 in all hematopoietic cells (26). Similar to Bim 2/2

3 1568 APOPTOSIS AND DIFFERENTIATION OF Foxp3 + T CELLS FIGURE 1. Inhibition of the Bcl-2 regulated apoptotic pathway causes a marked increase in CD25 low Foxp3 + CD4 + SP thymocytes. Thymi were harvested from 6 10-wk-old mice. Thymocytes were first surface stained for CD4, CD8, and CD25 and then for intracellular Foxp3. A, Comparison between Bim 2/2 and WT mice: CD4 and CD8 expression on total thymocytes (i). ii, Expression of CD25 and Foxp3 gated on CD4 SP thymocytes. iii, Bar graphs show the mean numbers 6 SD of the indicated thymic subpopulations. Nine independent experiments were performed with similar results. iv, Bar graphs show the mean numbers 6 SD of the indicated thymic subpopulations from the indicated genotypes of mice. Two independent experiments were performed with similar results. B, Comparison between vavbcl-2 Tg and WT mice: CD4 and CD8 expression on total thymocytes (i). ii, Expression of CD25 and Foxp3 gated on CD4 SP thymocytes. iii, Bar graphs show the mean numbers 6 SD of the indicated thymic subpopulations. Three independent experiments were performed. C, Comparison between Bax 2/2 Bak 2/2 and control (WT) reconstituted mice: CD4 and CD8 expression on total thymocytes from RAG-1 2/2 mice reconstituted with fetal liver cells from WTor Bax 2/2 Bak 2/2 mice(i). ii, Expression of CD25 and Foxp3 gated on CD4 SP thymocytes. iii, Bar graphs show the mean numbers 6 SD of the indicated thymic subpopulations. Three independent experiments were performed. Numbers in dot plots show the percentages of corresponding populations. Numbers in bar graphs indicate the fold increase of a given population in the indicated mice compared with that in control WT animals. mice, the proportion and numbers of CD25 low Foxp3 + cells within the CD4 SP thymocytes from vavbcl-2 Tg mice were profoundly increased ( 15-fold) compared with WT mice ( million in vavbcl-2 Tg mice versus million in WT mice; p, 0.01) (Fig. 1B). The increase in CD25 high Foxp3 + CD4 SP thymocytes was again less pronounced but significant ( 2- fold) ( million in vavbcl-2 Tg mice versus million in WT mice; p, 0.01). Finally, we examined C57BL/6-Ly5.1 Rag 2/2 mice reconstituted with Bax 2/2 Bak 2/2 fetal liver cells, this approach being taken because most Bax 2/2 Bak 2/2 mice die soon after birth (27). Bax and Bak activation is required for all Bcl-2 regulated apoptotic responses, and their combined loss causes severe defects in intrathymic T cell development (6). Accordingly, the cell subset composition of Bax 2/2 Bak 2/2 thymocytes (CD ) was similar to that of Bim 2/2 and Puma 2/2 Bim 2/2 thymocytes. There was a great increase (.40-fold) in CD25 low Foxp3 + CD4 SP thymocytes in Bax 2/2 Bak 2/2 reconstituted mice compared with WT reconstituted mice ( million of Bax 2/2 Bak 2/2 thymocytes versus million in WT thymocytes; p, 0.01) (Fig. 1C). The numbers of total CD4 SP thymocytes and CD25 high Foxp3 + CD4 SP thymocytes in Bax 2/2 Bak 2/2 -reconstituted hosts were also increased compared with control WT-reconstituted animals, albeit to a comparatively smaller extent ( 4-fold) (mean 6 SEM: million of Bax 2/2 Bak 2/2 thymocytes versus million in WT mice; p, 0.01). In addition to using intracellular staining of Foxp3 protein to characterize thymocytes from mice defective in the Bcl-2 regulated apoptotic pathway, we also crossed Bim 2/2 mice and vavbcl-2 Tg mice with Foxp3GFP KI reporter mice, in which one Foxp3 allele has been replaced by GFP (23). Thus, live cells expressing Foxp3 can be tracked and their function analyzed. Compared to control Foxp3GFP KI mice, Foxp3GFP KI /Bim 2/2 mice had similar numbers of total thymocytes, but 5-fold increase in total CD4 SP thymocytes (p, 0.05) and greatly increased percentages and numbers of Foxp3 + (GFP + ) CD4 + SP thymocytes ( 15-fold; p, 0.05) (Fig. 2A). Similar to our observations in Bim 2/2 mice, the percentages of CD25 low Foxp3 + cells were higher in Foxp3GFP KI /Bim 2/2 mice compared with control Foxp3GFP KI mice ( % in Bim 2/2 mice versus % in WT mice; p, 0.05), whereas the percentages of CD25 high Foxp3 + cells were only moderately increased in Foxp3GFP KI /Bim 2/2 mice ( % in Bim 2/2 mice versus % in WT mice; p = 0.293) (Fig. 2A). Correspondingly, the increase in the numbers of CD25 low Foxp3 + thymocytes in Foxp3GFP KI /Bim 2/2 mice was more profound ( 40-fold; p,

4 The Journal of Immunology ) compared with the increase in the numbers of CD25 high Foxp3 + thymocytes ( 6-fold; p, 0.05). A disproportionate increase in CD25 low Foxp3-GFP + CD4 SP thymocytes was also observed in Foxp3GFP KI /vavbcl-2 Tg mice (Fig. 2B). The increase in CD25 low Foxp3 + CD4 + T cells was also observed in secondary lymphoid organs (e.g., spleen and lymph nodes; Supplemental Fig. 1) of mice with defective Bcl-2 regulated apoptosis, suggesting that these cells are exported from the thymus. The increase in CD25 high Foxp3 + CD4 + T cells in these mice was statistically significant but lesser in extent than the increase in the CD25 low Foxp3 + T cells. Collectively, these results show that inhibition of the Bcl-2 regulated apoptotic pathway causes a preferential expansion of Foxp3 + CD4 + T cells, particularly the CD25 low Foxp3 + population. T cells bearing autoreactive TCRs contribute preferentially to the increase in Foxp3 + CD4 thymocytes in mice with defects in the Bcl-2 regulated apoptotic pathway Next, we examined whether the increase in Foxp3 + CD4 thymocytes in mice with defects in the Bcl-2 regulated apoptotic pathway was linked to impaired deletion of self-reactive thymocytes. FIGURE 2. Inhibition of the Bcl-2 regulated apoptotic pathway causes a marked increase in CD25 low Foxp3 GFP+ CD4 + thymocytes. A, Thymocytes from 6 8-wk-old Foxp3GFP KI or Foxp3GFP KI /Bim 2/2 mice were surface stained for CD4, CD8, TCRb, and CD25. TCRb expression on total thymocytes (i); CD4 and CD8 expression gated on TCRb + thymocytes (ii); and CD25 and Foxp3 GFP expression gated on TCRb + CD4 + SP thymocytes (iii). Bar graphs show cell numbers 6 SD of the indicated thymic cell populations from three mice. Four independent experiments were performed. B, Thymocytes from 10-wk-old Foxp3GFP KI or Foxp3GFP KI / vavbcl2 Tg mice were stained as in A. Dot plots show expression of CD25 versus Foxp3 on gated CD4 SP thymocytes. Three independent experiments yielded similar results. *p, 0.05, **p, 0.01 compared with WT. To address this question, we analyzed the selective deletion of thymocytes bearing specific TCR components (e.g., Vb5) that confer specificity to endogenous retroviral superantigens (a self- Ag) presented by I-E d MHC molecules in BALB/c mice (28). Analysis of thymocytes revealed that the percentage of Vb5 + CD4 SP thymocytes, but not Vb6 + or Vb8.1/8.2 + CD4 SP thymocytes (which do not confer reactivity to self-superantigens), was substantially increased in BALB/c Bim 2/2 mice (.10-fold; p, 0.01 compared with WT; Fig. 3A). Furthermore, the percentage of Foxp3 + cells was much higher among Vb5 + CD4 SP thymocytes from BALB/c Bim 2/2 mice compared with the Vb6 + thymocytes in the same mice (Fig. 3A). In absolute numbers, Foxp3 + Vb5 + CD4 SP thymocytes were increased.250-fold, whereas the increase in Foxp3 + cells bearing TCRVb6 or TCRVb8.2 was,5- fold in BALB/c Bim 2/2 mice compared with WT mice (Fig. 3B). Furthermore, when CD4 + T cells from Bim 2/2 mice were separated into Foxp3 + and Foxp3 2 populations, Vb5 + cells were much more abundant in Foxp3 + fraction (Fig. 3C). Although C57BL/6 mice do not express MHC I-E molecules, Vb5 CD4 + T cells can still be reactive to endogenous retroviral Ags and are partially deleted via I-A (6). When Vb5 CD4 + T cells were compared between C57BL/6 and C57BL/6 Bim 2/2 mice, Vb5 CD4 + T cells from WT mice contained a much larger proportion of Foxp3 + cells (30%), and Vb5 CD4 + T cells from C57BL/6 Bim 2/2 mice had an even higher proportion of Foxp3 + cells (.40%) (Supplemental Fig. 2A). In contrast, cells bearing non negative-selecting Vb6 contained 10% Foxp3 + cells in WT and 16% in C57BL/6 Bim 2/2 mice (Supplemental Fig. 2A). Thus, accumulation of Foxp3 + CD4 + T cells in Bim 2/2 mice is more prominent in cells expressing TCRs reactive to self-ags. A profound increase in CD25 low Foxp3 + thymocytes was also observed in normal (i.e., Bim +/+ ) TCR Tg mice in the presence of cognate selecting ligands (membrane-bound OVA) (Supplemental Fig. 2B). Thymic expression of membrane-bound OVA depleted most of the high-affinity DO11 TCR-expressing thymocytes. However, for those remaining self-ag specific TCR-expressing cells, a significant proportion became CD25 low Foxp3 +. Thus, the increase in CD25 low Foxp3 + thymocytes reflects self-reactivity rather than a peculiarity of mice with defects in the Bcl-2 regulated apoptotic pathway. CD25 low Foxp3 + CD4 + T cells and CD25 high Foxp3 + have a similar survival rate The preferential accumulation of CD25 low Foxp3 + CD4 + cells in Bim 2/2 or Bcl-2 Tg mice described above is consistent with clonal diversion of self-reactive T lymphoid cells escaping deletion. However, the expansion could also reflect a survival advantage of CD25 low Foxp3 + CD4 + cells over the other CD4 + T cell subsets. To examine this, sorted CD4 T cell subpopulations from vavbcl- 2 Tg/Foxp3 KI mice were labeled with CellTrace violet dye (Invitrogen) and adoptively transferred into C57BL/6-Ly5.1 recipient mice. Transferred cells (identified by CellTrace dye [Invitrogen] and CD ) were analyzed 3 and 7 d after transfer. Several observations were made: 1) the phenotype of the transferred cells remained stable. Most of the CD25 low Foxp3 + CD4 + T cells remained CD25 low (Fig. 4A); 2) recovery of CD25 low and CD25 high Foxp3 + CD4 + T cells was similar, suggesting that they had similar survival capacity, although both populations did not survive as well as Foxp3 2 CD4 + T cells (Fig. 4B); and 3) all three CD4 + T cell subpopulations did not divide substantially, at least within the first 7 d after transfer (Fig. 4C). These in vivo data with transferred cells from vavbcl-2 Tg mice were also supported by in vitro survival analysis of CD4 + T cell subpopulations from Bim 2/2 mice. CD25 2 Foxp3 2, CD25 low

5 1570 APOPTOSIS AND DIFFERENTIATION OF Foxp3 + T CELLS FIGURE 3. Loss of Bim causes a selective increase in self-superantigen specific TCR Vb5 + Foxp3 + CD4 + thymocytes on the BALB/c background. Thymocytes from three individual 6- to 7-wk-old mice of the indicated genotypes were first surface stained for CD4, CD8, CD25, and individual TCR Vb-chains and then fixed, permeabilized, and finally stained for intracellular Foxp3. A, Percentages of subpopulations of CD4 SP thymocytes: mean 6 SEM of the percentages of CD4 SP thymocytes bearing the indicated TCR Vb-chain within total thymocytes (left panel). Mean 6 SEM of the percentages of Foxp3 + CD4 + cells within CD4 + SP thymocytes bearing the indicated TCR Vb-chain (right panel). B, Numbers of cells within the different subpopulations of CD4 SP thymocytes: mean 6 SD of the numbers of total thymocytes (left panel) and mean 6 SEM of Foxp3 + CD4 SP thymocytes bearing the indicated TCRVb-chain (right panel). C, Usage of different Vb TCRs by Foxp3 + and Foxp3 2 CD4 SP thymocytes from Bim 2/2 mice. Bar graph shows mean 6 SEM of the percentages of Foxp3 + and Foxp3 2 CD4 SP thymocytes carrying different TCRVb-chains from three individual Bim 2/2 mice. Three experiments were performed with similar results. *p, 0.05, ***p, compared with WT mice. Foxp3 +, and CD25 high Foxp3 + from lymph nodes of Bim 2/2 mice survived better than the corresponding cells from WT mice. However, CD25 low Foxp3 + cells and CD25 high Foxp3 + cells from the same Bim 2/2 mice had a similar survival rate. With the same input number, the recovered number of viable CD25 low Foxp3 + cells was fewer than that of CD25 2 Foxp3 2 cells and comparable to CD25 high Foxp3 + cells. Provision of IL-2 greatly enhanced the survival of Foxp3 + T cells from WT mice, whereas IL-7 enhanced the survival of Foxp3 2 T cells from WT mice. Enhancement of survival of Bim 2/2 T cells by IL-2 and IL-7 was modest (because they already survived so well in their absence). Stimulation by anti-cd3 plus anti-cd28 Abs also did not significantly alter the pattern of survival of these cell populations (data not shown). FIGURE 4. Survival of CD25 low Foxp3 + CD4 + T cells is similar to that of CD25 high Foxp3 + CD4 + T cells in vivo after adoptive transfer. Total of CellTrace-labeled (Invitrogen) CD4 + T cells were injected i.v. into C57BL/6-Ly5.1 recipient mice. Three and 7 d after transfer, spleens were recovered. Transferred cells were identified as Ly5.2 + CellTrace + cells. A, Dot plots show the expression of CD25 and Foxp3-GFP by CD4 + T cell subpopulations before and after transfer. B, Bar graphs show the numbers of transferred cells (mean of two to three mice per group). C, Histograms show CellTrace intensity (Invitrogen) of CD4 + T cell subpopulations. Two independent experiments were performed with similar results. Overall, these results show that the selective increase in the CD25 low Foxp3 + CD4 + subset is unlikely to be a consequence of preferential survival of this cell subset. CD25 low Foxp3 + CD4 T cells are hyporesponsive to TCR ligation Functional analysis of Foxp3 + CD4 + T cells relies on the use of reporter GFP because Foxp3 is an intracellular transcription factor. GFP expression in these reporter mice faithfully reflects Foxp3

6 The Journal of Immunology 1571 transcription (Supplemental Fig. 3). Three populations of CD4 + T cells were purified according to CD25 and GFP expression by FACS sorting. Transcription of CD25 and Foxp3 by these three populations of CD4 + T cells was then analyzed by real-time quantitative PCR. Not surprisingly, transcript levels of CD25 and Foxp3 were lower in CD25 low Foxp3 + cells compared with CD25 high Foxp3 + cells (Supplemental Fig. 3). The expression of CTLA4, a molecule that is critical for the function of Treg cells (29), was also lower in CD25 low Foxp3 + cells compared with CD25 high Foxp3 + cells (Supplemental Fig. 3). To examine the functional status of CD25 low Foxp3 + cells from mice with defects in the Bcl-2 regulated apoptotic pathway, we measured the proliferative response and cytokine production of CD4 + T cell subpopulations to in vitro TCR (anti-cd3 plus anti- CD28 Abs) stimulation comparing Foxp3-GFP KI /vavbcl-2 Tg mice and control Foxp3-GFP KI mice. CD4 + T cells were separated into three subpopulations based on Foxp3-GFP and CD25 expression. Compared to CD25 2 Foxp3 2 CD4 + cells, all Foxp3 + CD4 + cells (either CD25 low or CD25 high ) from both WT as well as vavbcl-2 Tg mice showed only poor proliferation in response to TCR ligation (Fig. 5A). In addition, compared with CD25 2 Foxp3 2 CD4 + T cells, CD25 low Foxp3 + and CD25 high Foxp3 + CD4 + T cells failed to produce appreciable amounts of IL-2, IL-4, and IL-17 (Fig. 5B) as well as several other cytokines (e.g., IL-10 and IFN-g; data not shown). Similarly, CD25 low Foxp3 + as well as CD25 high Foxp3 + CD4 + T cells from Foxp3-GFP KI /Bim 2/2 mice also had a much lower proliferative response to TCR stimulation compared with the corresponding cell subsets from control Foxp3-GFP KI mice (data not shown). Comparable results were obtained with the corresponding populations of T cells from either secondary lymphoid organs (spleen and lymph nodes) or thymus, although thymic T cells generally produced weaker responses (data not shown). CD25 low Foxp3 + CD4 + T cells have reduced regulatory potency compared with CD25 high Foxp3 + CD4 + T cells Because Foxp3 expression is a hallmark of Treg cells, we tested the regulatory capacity of the expanded CD25 low Foxp3 + CD4 + cells from Foxp3-GFP KI /vavbcl-2 Tg mice to suppress CD25 2 CD4 + T cell proliferation in a CFSE dilution-based proliferation assay. We found that CD25 high Foxp3 + CD4 + cells from either vavbcl-2 Tg or WT mice readily suppressed the anti-cd3 Ab-induced proliferation of effector T cells, whereas CD25 low Foxp3 + CD4 + T cells from vavbcl-2 Tg were less potent (Fig. 6). Cytokine production from cocultures of effector cells with CD25 high Foxp3 +, CD25 low Foxp3 + CD4 + T cells, or Foxp3 2 CD4 + T cells from vavbcl-2 Tg mice were also measured. In accordance with the measurements of cell proliferation, the CD25 low Foxp3 + CD4 + T cells from vavbcl-2 Tg mice were also less potent than CD25 high Foxp3 + CD4 + T cells in suppressing IL-2 and IL-17 production (Fig. 7). The two cell subsets were comparable in their ability to suppress IL-4 and IFN-g production (Fig. 7). CD25 high Foxp3 + CD4 + cells from both WT mice as well as CD25 low Foxp3 + CD4 + cells (though very few in number) from WT mice suppressed the production of IL-2 and IL-17. These results show that CD25 low Foxp3 + CD4 + T cells were generally less efficacious than CD25 high Foxp3 + CD4 + T cells in inhibiting proliferation and cytokine production by conventional CD4 effector T cells. CD25 low Foxp3 + CD4 + T cells cannot be readily converted into CD25 high Foxp3 + CD4 + T cells by TCR stimulation Next, we investigated whether CD25 low Foxp3 + CD4 + T cells were the precursors for CD25 high Foxp3 + CD4 + T cells. For this, CD4 + T cells from Foxp3-GFP KI /vavbcl-2 Tg mice and Foxp3-GFP KI / vavbcl-2 Tg mice were sorted into three subpopulations: CD25 high Foxp3 +,CD25 low Foxp3 +,andcd25 2 Foxp3 2 CD4 + Tcells from Foxp3-GFP KI /vavbcl-2 Tg mice. Cells were cultured with or without IL-2 and anti-cd3 plus anti-cd28 Abs. Expression of Foxp3 (reflected by expression of GFP) and CD25 by the three populations was measured after 36 h. In the presence of IL-2, we observed that only 20% of the CD25 low subset upregulated their CD25 expression (Fig. 8) compared with 63% within the CD25 high subset. In response to TCR stimulation, CD25 was upregulated on a proportion of CD25 2 Foxp3 2 cells, but notably there was little change in the expression of CD25 in CD25 low Foxp3 + cells (Fig. 8A). We also observed that IL-2 treatment resulted in a similar change in CD25 expression when CD25 low Foxp3 + cells from Foxp3-GFP KI /vavbcl-2 Tg mice were transferred into B6 mice (Fig. 8B). Changes in CD25 expression did not associate with cell expansion. Overall, these results indicate that TCR stimulation or culture in simple medium does not readily convert the CD25 low Foxp3 + cells into CD25 high Foxp3 + cells but that IL-2 can convert a fraction of the CD25 low Foxp3 + cells into CD25 high cells. The death receptor Fas has only a minor role in regulating thymic differentiation of Foxp3 + cells but is critical for peripheral homeostasis of Foxp3 + CD4 + T cells Although we focused our study on the Bcl-2 regulated apoptotic pathway, we also extended our study to Fas death receptorinduced apoptosis. In contrast to mice with defects in the Bcl-2 regulated apoptotic pathway, Fas-deficient FAS lpr/lpr mutant mice had no significant increase in CD4 SP thymocytes and had only a minor increase in Foxp3 + CD4 + SP thymocytes (Supplemental Fig. 4A). The increase in Foxp3 + CD4 + thymocytes (both CD25 low and CD25 high ) in mice that are defective in both Bim and Fas (Bim 2/2 FAS lpr/lpr ) was only marginally larger than the increase seen in Bim 2/2 mice (both in percentage and absolute cell number) (Supplemental Fig. 4A). However, in mice that are defective in both Bim and Fas (Bim 2/2 FAS lpr/lpr ), we found that the accumulation of CD25 low Foxp3 CD4 + T cells in secondary lymphoid organs (spleen and lymph nodes) was substantially accelerated and increased compared with either Bim 2/2 or FAS lpr/lpr single mutant mice. This increase was a result of both an increased frequency of Foxp3 + cells within the CD4 + T cell subset (Supplemental Fig. 4B) and increased total cellularity of these lymphoid organs (Supplemental Fig. 4B). Although increased accumulation of Foxp3 + CD4 + cells was also observed in older FAS lpr/lpr mice, the majority of these cells were CD25 high Foxp3 +. These results show that Fas-mediated apoptosis contributes to the regulation of the numbers of Foxp3 + CD4 + T cells in peripheral lymphoid organs, although Treg cells are thought to be resistant to TCR stimulation-induced apoptosis (activation-induced cell death) (30), which is mediated by FasL/ Fas signaling. As expected, Foxp3 + CD4 + T cells were more sensitive to FasL-mediated killing compared with Foxp3 2 CD4 + T cells in vitro (Supplemental Fig. 4C). Discussion In this study, we found that blocking deletion of autoreactive thymocytes by inhibiting the Bcl-2 regulated apoptotic pathway caused a large increase in Foxp3 + CD4 + T cells in both the thymus and peripheral lymphoid organs. The increase was particularly striking within the CD25 low Foxp3 + CD4 + population. The increase of this population is unlikely due to their preferential survival and is also unlikely due to their enhanced proliferation. Based on the finding that cells bearing self-ag reactive TCR are highly

7 1572 APOPTOSIS AND DIFFERENTIATION OF Foxp3 + T CELLS FIGURE 5. CD25 low Foxp3 + cells are hyporesponsive to TCR stimulation. A, T cell proliferation: distinct CD4 + T cell subpopulations from lymph nodes of Foxp3GFP KI or Foxp3GFP KI /vavbcl-2 Tg mice were sorted according to the levels of Foxp3-GFP and CD25 expression. These T cell subsets ( cells/well) were cultured in 96-well round-bottom plates with 5 mg/ml anti-cd3 plus 2 mg/ml anti-cd28 Abs in the presence of gamma-irradiated and T cell-depleted spleen cells (used as APCs). The mean cpm 6 SD from three to five replicates per culture condition is shown. Three experiments were performed with similar results. B, Cytokine production: supernatants from the above cultures harvested after 3 d of incubation were examined for cytokine content. Data represent the mean 6 SEM from three replicate cultures per cell type and genotype. Three independent experiments were performed and produced similar results. **p, 0.01 compared with Foxp3 2 CD25 2 CD4 + lymph node cells. overrepresented in the population, we suggest that these cells represent a population that has undergone clonal diversion in the differentiation program toward a Foxp3 + T cell fate. Induction of Foxp3, without high expression of CD25, might curtail the autoaggressiveness of abnormally surviving self-reactive CD4 + T cells, but may also bestow upon them reduced potency as Treg cells. Signaling from IL-2R but not from the TCR allowed conversion of a fraction of these CD25 low cells into CD25 + cells. Interestingly, Fas death receptor-mediated apoptosis was found to contribute to the regulation of the peripheral pool of CD25 low Foxp3 + CD4 + T cells and thereby provides an additional checkpoint for cells that escaped from thymic deletion due to a defect in the Bcl-2 regulated apoptotic pathway. In this study, we investigated CD4 + T cell subsets in mice that are defective in different components of the Bcl-2 regulated apoptotic pathway. The increase in CD25 low Foxp3 + CD4 + T cells was most prominent in mice reconstituted with Bax 2/2 /Bak 2/2 fetal liver cells. Deficiency in the BH3-only protein Bim causes a significant increase in Foxp3 + CD4 + T cells in the thymus and peripheral lymphoid tissue. Bim 2/2 /Puma 2/2 mice showed a further increase in Foxp3 + CD4 + T cells, although the loss of Puma alone has no appreciable impact on total thymic cellularity as well as the numbers of Foxp3 + CD4 + cells. Like Bim, Puma is also a BH3- only protein that can bind with high affinity to all prosurvival Bcl- 2 like proteins (25) (31). Puma has a role in regulating apoptosis of Ag-stimulated T cells (32). Furthermore, Puma cooperates with Bim to regulate lymphocyte apoptosis (33). Findings from our study suggest that Bim and Puma have overlapping actions in controlling the pool of Foxp3 + CD4 + T cells. Consistent with the findings from mice lacking proapoptotic proteins, Tg mice

8 The Journal of Immunology 1573 FIGURE 6. CD25 low Foxp3 + CD4 + T cells are less potent suppressors of effector T cell activation compared with CD25 high Foxp3 + CD4 + T cells. Suppression of effector T cell proliferation: CD25 low Foxp3 + CD4 + and CD25 high Foxp3 + CD4 + cells ( cells) from lymph nodes of mice of the indicated genotypes were cultured with CFSE-labeled C57BL/ 6-Ly5.1 CD25 2 CD4 + T cells as effectors in 200 ml medium and left untreated or stimulated with 5 mg/ml anti-cd3 Abs in the presence of spleen cells (used as APCs) for 3 d. After 3 d of culture, supernatants were harvested for measurement of cytokine content (Fig. 8). Recovered cells were stained for Ly5.1, CD4, and CD25. Viable cells were enumerated by FACS analysis using PI exclusion and calibrating PE beads. A, FACS profile of CFSE dilution. Numbers in the plots indicate the percentages of proliferating cells. B, Numbers of proliferating effector T cells. The p values between samples that were compared are indicated. Three independent experiments were performed with similar results. overexpressing antiapoptotic Bcl-2 also showed a profound increase in Foxp3 + CD4 + T cells. However, this increase was lower compared with mice with a Bax/Bak doubly deficient hematopoietic system. Currently, the role of other antiapoptotic molecules, such as Mcl-1 and A1, in regulating the Foxp3 + CD4 + T cell pool remains unclear. Given that an increase in the cellularity of overall T cells including Foxp3 + T cells in mice either lacking proapoptotic Bcl-2 family members or overexpressing antiapoptotic Bcl-2 like proteins may not be surprising and has been documented (24), why is the increase in Foxp3 + CD4 + T cells, particularly the CD25 low Foxp3 + CD4 + subset, much more pronounced than the increase in conventional CD4 + T cells in these mice? Conceivably, there are several potential mechanisms leading to a preferential increase in Foxp3 + CD4 + cells. Firstly, the survival rate of CD25 low Foxp3 + CD4 + T cells might be higher compared with Foxp3 2 CD4 + and CD25 high Foxp3 + CD4 + T cells. However, CD25 low Foxp3 + CD4 + T cells from vavbcl-2 Tg mice and Bim 2/2 mice survived similarly to CD25 high Foxp3 + cells in culture and did not survive as well as Foxp3 2 CD4 + T cells from the same apoptosis-defective mice. Thus, the preferential increase in the CD25 low Foxp3 + CD4 + population in mice with a defect in the Bcl-2 regulated apoptotic pathway is unlikely due to preferential survival, although enhanced survival by T cells in these mice must contribute to the FIGURE 7. CD25 low Foxp3 + CD4 + T cells are less capable of suppressing IL-17 production compared with CD25 high Foxp3 + CD4 + T cells. Cultures were set up as described for Fig. 6. Supernatants from 3-d cultures were harvested to measure the content of the indicated cytokines. Data show the mean concentrations of triplicate cultures for selected cytokines. Two independent experiments produced similar results. *p, 0.05, **p, 0.01 compared with cultures with CD25 22 CD4 + T cells.

9 1574 APOPTOSIS AND DIFFERENTIATION OF Foxp3 + T CELLS FIGURE 8. IL-2R but not TCR stimulation can convert CD25 low Foxp3 + CD4 + T cells into CD25 high Foxp3 + CD4 + T cells. A, In vitro assay. T cells from Foxp3GFP KI /vavbcl-2 Tg mice were sorted into three subpopulations according to the levels of CD25 and Foxp3 expression. Cells were cultured at a starting density of cells/well in 200 ml medium in roundbottom 96-well plates in the presence or absence of IL-2 (20 U/ml) or anti- CD3 plus anti-cd28 Abs. Cells were harvested after 36 h and stained for CD25. Dot plots show expression of Foxp3 and CD25 on freshly sorted viable cells before culture and on viable cells after 36-h culture. B, In vivo assay: B6 mice were i.v. injected with 10 5 CellTrace-labeled (Invitrogen) CD25 low Foxp3 + CD4 + T cells from Foxp3GFP KI /vavbcl-2 Tg mice. Half of the mice were injected i.p. with two daily doses of IL-2 (1000 U/dose). Mice were killed 60 h after the first dose of IL-2. Spleen cells from IL-2 treated and untreated mice were surface stained for CD4, CD8, and CD25. Contour plots show CD25 and Foxp3 expression on CellTrace-labeled (Invitrogen) CD4 + T cells. Histograms show for CellTrace violet intensity. Similar results were obtained from two independent experiments. overall increase in all T cell subsets. Secondly, proliferation of CD25 low Foxp3 + CD4 + T cells might be higher compared with Foxp3 2 CD4 + and CD25 high Foxp3 + CD4 T cells. However, several results from our study do not support this possibility. Isolated CD25 low Foxp3 + CD4 + T cells when adoptively transferred did not show cell division rates above the levels of Foxp3 2 CD4 + or CD25 high Foxp3 + CD4 + T cells. BrdU incorporation was also similar between Foxp3 + CD4 + and the Foxp3 2 CD4 + T cell subsets (D. Gray, unpublished observations); cell cycle analysis of the different CD4 + T cell subsets also did not show a higher percentage of cycling cells within CD25 low Foxp3 + CD4 + T cells compared with the other subsets (Y. Zhan, unpublished observations). Apart from increased cell survival and increased cell proliferation, we also exclude a major contribution of differential thymic location and thymic re-entry of Foxp3 + CD4 + cells from the periphery as a cause of the preferential increase in Foxp3 + CD4 + T cells in these mice (Y. Zhan, unpublished observations). Thus, we favor a view that a preferential increase in CD25 low Foxp3 + CD4 T cells is the consequence of increased differentiation/diversion, most likely originating from cells that are normally deleted by Bcl-2 regulated apoptosis after stimulation of their TCRs by self-ags. In support of the notion that the preferentially expanded population represents the self-reactive cells that escaped thymic deletion and have been diverted in their differentiation, we found an up to 500-fold increase in TCR Vb5-bearing Foxp3 + CD4 + T cells in BALB/c Bim 2/2 mice compared WT BALB/c mice. These T cells bear a TCR that is specific to endogenous superantigens presented by I-E d MHC molecules and are normally deleted on a BALB/c background (28), but loss of Bim allows their escape. Moreover, analysis of Foxp3 expression by CD4 + T cells bearing different TCR Vb-chains in BALB/c Bim 2/2 mice revealed that a much higher proportion of TCRVb5-bearing CD4 + T cells express Foxp3 compared with cells bearing non self-reactive TCRs, such as those containing TCR Vb6. In fact, the percentage of Vb6-bearing Foxp3 + CD4 + T cells was comparable between WT and Bim 2/2 mice on a BALB/c background. When CD4 + T cells are separated into Foxp3 + and Foxp3 2 fractions, Vb5-bearing cells are greatly overrepresented within the Foxp3 + fraction. We therefore conclude that the increased Foxp3 + CD4 + cells in mice with a defect in the Bcl-2 regulated apoptotic pathway are derived from forbidden clones (self-reactive) that are normally deleted in the thymus. A question raised by this study is whether a diversion into CD25 low Foxp3 + CD4 + cells occurs in mice without a profound defect in the Bcl-2 regulated apoptotic pathway. Importantly, we found that although in mice Tg for both an OVA-specific TCR (KJ DO11) and the cognate Ag (OVA) the majority of KJ CD4 + T cells were deleted, most of the residual KJ CD4 + thymocytes in these animals were Foxp3 + but CD25 low. Hence, even in mice with an intact Bcl-2 regulated apoptotic pathway, autoreactive thymocytes that have escaped deletion can be clonally diverted toward a CD25 low Foxp3 + state. There are two unresolved issues relating to the increased differentiation of CD25 low Foxp3 + CD4 + T cells. Firstly, which factors determine the differentiation of Foxp3 + CD4 + T cells? Particularly, what signal differences exist between negative selection and positive selection of Foxp3 + CD4 + cells? What has been revealed is that the strength of TCR engagement plays a critical role in determining Foxp3 + CD4 + cell differentiation. Several elegant and detailed studies concluded that a TCR/MHC plus peptide avidity range somewhere between positive and negative is ideal for Treg differentiation, because very weak TCR interaction as well as very strong TCR stimulation impedes generation of Treg cells (34 36). Notably, in in vitro as well as in vivo studies, not all CD4 + T cells became Foxp3 + even when all T cells expressed the same Treg fate promoting (Tg) TCR (37). This could be due to the fact that not all T cells experience the same dose of TCR stimulation. Alternatively, these findings might imply that the same signals emanating from TCR MHC interaction might result in different cell fates, probably due to differences in intracellular wiring or the fact that different cells may experience different auxiliary stimuli from their microenvironment (38). It remains a challenge to delineate the quantitative or/and qualitative signals directing Foxp3 transcription.

10 The Journal of Immunology 1575 Secondly, unlike Foxp3 + CD4 + T cells from WT mice, a large fraction of Foxp3 + CD4 + T cells from mice with defects in the Bcl-2 regulated apoptotic pathway are CD25 low. This raises the question of what controls CD25 expression in Foxp3 + CD4 + T cells. In conventional CD4 + T cells, expression of CD25 is mainly regulated at the transcriptional level (39, 40), and three major transcription factors have been implicated: NFAT (41), NFkB (42), and AP-1 (41). In the Treg lineage, the transcription factor Foxp3 has been shown to cooperate with NFAT to enhance CD25 expression while suppressing IL-2 transcription (43). Dissection of gene elements critical for CD25 expression revealed that TCR and IL-2 activate different elements of the CD25 gene to enhance transcription (39, 40, 44). In our study, we found that TCR ligation was not sufficient to upregulate CD25 expression on CD25 low Foxp3 + CD4 + T cells even though this stimulus could efficiently increase CD25 expression on Foxp3 2 CD4 + T cells (Fig. 8). Conversely, provision of IL-2 was able to induce CD25 upregulation on a proportion of CD25 low Foxp3 + CD4 + T cells (Fig. 8). This differential effect on CD25 regulation by TCR and IL-2R signaling in CD25 low Foxp3 + CD4 + T cells may reflect specific defects in TCR signaling in these cells. We predict that IL-2/IL-2R activation of Stat5 may proceed normally in these cells, whereas the TCR-activated pathway may somehow be impaired. In vitro, IL-2 can partially convert CD25 low Foxp3 + CD4 + to CD25 high Foxp3 + CD4 + T cells. Using an adoptive transfer system, we found that injection of IL-2 can indeed convert a fraction of CD25 low CD4 + cells into CD25 high CD4 + cells in vivo. Whether these converted CD25 high Foxp3 + CD4 + cells would gain potent regulatory function as bona fide CD25 high Foxp3 + CD4 + cells warrants further investigation. This conversion in CD25 expression levels elicited by IL-2 may have implications for immune regulation. When a strong T cell response is induced, the resultant high-level IL-2 milieu may convert CD25 low Foxp3 + CD4 + T cells into CD25 high Foxp3 + CD4 + T cells to reinforce immune regulation. When there is no such need for immune regulation (and IL-2 levels are low) during steady state, CD25 low Foxp3 + CD4 + T cells may stay stable and serve as a reserve for potential future use. Although we observed that an increase in CD25 low Foxp3 + CD4 + T cells occurs both in the thymus and secondary lymphoid organs of mice with defective Bcl-2 regulated apoptosis, the relationship between the two pools of CD25 low Foxp3 + CD4 + T cells warrants further investigation. There are many examples in which selfreactive T cells can convert from a Foxp3 2 CD4 + into a Foxp3 + CD4 + state in the periphery, particularly under the influence of TGFb (45, 46). Furthermore, TGF-b can also cause an expansion of Foxp3 + CD4 + T cells (47). Thymus-derived Foxp3 + CD4 + cells and TGF-b induced Foxp3 + CD4 + cells differ in several requirements such as TCR signal strength and costimulation for their differentiation (48). The nature of the increased Foxp3 + CD4 + population in secondary lymphoid organs (cf. thymus) of mice with defective Bcl- 2 regulated apoptosis might be dissected based on the aforementioned differences. The abnormally increased CD25 low Foxp3 + CD4 + cells from Bim 2/2 and Bcl-2 Tg mice were found to be hyporesponsive to TCR stimulation (anergic) in culture. This was not surprising given that hyporesponsiveness to TCR stimulation in vitro is a common feature of Foxp3 + CD4 + T cells. Indeed, expression of the transcription factor Foxp3 was shown to be sufficient to render T cells hyporesponsive to TCR stimulation (49, 50), although there are also Foxp3-independent mechanisms for rendering T cells anergic (14, 51, 52). It is presently unclear whether Foxp3- dependent and/or -independent pathways contribute to the anergic status of CD25 low Foxp3 + CD4 + cells. Interestingly, the expanded population of CD25 low Foxp3 + CD4 + T cells from vavbcl-2 Tg and Bim 2/2 mice had a lower capacity to suppress effector T cell proliferation than CD25 high Foxp3 + CD4 + Treg cells. Why do these CD25 low Foxp3 + CD4 + cells lack potent suppressive function? First, it has been reported that only CD4 + T cells with high levels of Foxp3 possess regulatory function (53, 54). We noticed that the CD25 low Foxp3 + CD4 T cells in mice with defects in the Bcl-2 regulated apoptotic pathway express lower levels of Foxp3. Consistent with this finding, CD25 low Foxp3 + CD4 + cells from Bim 2/2 /Foxp3GFP KI mice exhibited greatly reduced expression of GFP during in vitro culture (without exogenous stimuli), whereas the reduction in GFP intensity on CD25 high Foxp3 + CD4 + cells was only moderate, implying less stable Foxp3 expression by CD25 low CD4 + cells. Second, one of the proposed mechanisms by which Treg cells influence effector T cell function is via sequestration of IL-2 and possibly other cytokines (55). Because CD25 (IL-2Ra-chain) functions as a component of the IL-2R, CD25 low Foxp3 + CD4 + cells would be expected to be less potent at binding IL-2 and thereby less potent at depriving effector T cells of this cytokine. Third, Foxp3 alone is unable to induce the full range of Treg transcriptional and functional features (56). For example, although all T cells from Foxp3 Tg mice show reduced TCR stimulation-induced IL-2 production and proliferation (49), they have less regulatory potency compared with naturally occurring CD25 high CD4 + T cells (49). Moreover, we found that CD25 low Foxp3 + CD4 + cells had lower expression of CTLA4 (Supplemental Fig. 3), a molecule critical for regulatory function (29). It appears that CD25 low Foxp3 + CD4 + T cells do not efficiently suppress IL-17 production by Foxp3 2 CD4 + effector cells. However, it is possible that CD25 low Foxp3 + CD4 T cells themselves contribute to the IL-17 production detected in cocultures. It has previously been shown that reducing the levels of Foxp3 in cells committed to become Foxp3 + CD4 + T cells increases their ability to produce proinflammatory and disease-inducing cytokines, including IL-17 (53, 54). Given that CD25 low Foxp3 + CD4 + T cells from mice with defective Bcl-2 regulated apoptosis have lower levels of Foxp3 transcription (Supplemental Fig. 3), it could be argued that CD25 low Foxp3 + CD4 + T cells can gain the ability to produce IL-17, at least under certain conditions (e.g., coculture with effector cells). This issue may be resolved by using retinoic acid-related orphan receptor gt effector cells to distinguish the contribution of Bcl-2 Tg CD25 low Foxp3 + CD4 + T cells to IL-17 production. Although induction of Foxp3 blunts the autoaggressiveness of self-reactive CD4 + T cells, additional tolerance mechanisms may be required to safeguard immune tolerance. One of these mechanisms is deleting self-reactive T cells escaping the censorship by Bcl-2 regulated apoptosis by the death receptor apoptotic pathway. Although Fas had only a very minor role in the differentiation of Foxp3 + CD4 + T cells in the thymus in our studies (Supplemental Fig. 4A), this death receptor exerted a prominent role in controlling the peripheral pool of Foxp3 + CD4 + (particularly CD25 low ) T cells. There was a marked increase in the numbers of CD25 low Foxp3 + CD4 + T cells in mice doubly deficient for Bim and Fas compared with WT or single mutant mice. It has previously been reported that human CD25 + FOXP3 + CD4 + T cells (57) and mouse CD25 + Foxp3 + CD4 + T cells (58) are more sensitive to FasL/Fas-induced apoptosis than effector CD4 + T cells. We also noticed that Foxp3 + CD4 + T cells from mice with defective Bcl-2 regulated apoptosis were more sensitive to FasL-induced apoptosis in vitro than Foxp3 2 CD4 + T cells (Supplemental Fig. 4C). Accordingly, Foxp3 + CD4 + T cells (either CD25 low or CD25 high ) had higher levels of Fas than Foxp3 2 CD4 + T cells (data not shown). Taken together, these data show that Fas-mediated apoptosis can cooperate with the Bcl-2 regulated apoptotic pathway to control self-reactive T cells in a manner