ABSTRACT Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between

effector and regulatory populations remains unclear. We found that _Rag1_−/− hosts repopulated with _Bim_−/− conventional CD4+ T cells (Tconv) resulted in a larger induced regulatory T cell

(iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim.

Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 _in vitro_. The iTregs generated following

subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the

apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs. SIMILAR CONTENT BEING VIEWED BY OTHERS TIM-3 DRIVES TEMPORAL DIFFERENCES

IN RESTIMULATION-INDUCED CELL DEATH SENSITIVITY IN EFFECTOR CD8+ T CELLS IN CONJUNCTION WITH CEACAM1 Article Open access 14 April 2021 VENETOCLAX IMPARTS DISTINCT CELL DEATH SENSITIVITY AND

ADAPTIVITY PATTERNS IN T CELLS Article Open access 27 October 2021 CD74 SUPPORTS ACCUMULATION AND FUNCTION OF REGULATORY T CELLS IN TUMORS Article Open access 03 May 2024 MAIN Regulatory T

cells (Tregs) are an important subpopulation of CD4+ T cells that have a critical role in maintaining immune homeostasis and tolerance. Tregs can be divided into natural Tregs (nTreg), that

develop in the thymus, and induced Tregs (iTreg), that develop from conventional CD4+ T cells (Tconv) in the periphery during an immune response. iTregs can be induced in response to either

increased expression of self-antigen or as a consequence of infection. Therefore, their homeostasis must be tightly controlled. Insufficient differentiation of iTregs may result in immune

pathogenesis, whereas prolonged survival of these cells may compromise the establishment of immune memory. Although significant insight has been obtained concerning Treg development and

differentiation,1 little is known about the molecular mechanism(s) controlling nTreg and iTreg survival.2, 3, 4, 5 The BH3-only family member Bim (encoded by _Bcl2l11_) is a pro-apoptotic

molecule that controls the coordinated contraction of the immune T cell response.6, 7, 8, 9 It directly interacts with and deactivates the anti-apoptotic regulators Bcl-2 and Bcl-xl. Thus,

the fine balance between Bim and Bcl-2/Bcl-xl expression levels represents an important checkpoint for cell survival.10, 11 Despite enhanced peripheral T cell expansion, _Bim_−/− mice do not

develop aggressive T cell-mediated autoimmune disease.12 This may be due in part to an increased Treg population in moderately aged _Bim_−/− animals compared with their wild-type (WT)

littermates.13, 14 Interestingly, Bim expression naturally declines in peripheral WT CD4+ T cells during aging,15 which is co-incident with increasing numbers of Tregs. This raises the

possibility that Bim has an important role in Treg homeostasis. As iTregs and nTregs cannot be distinguished in _Bim_−/− animals, the impact that Bim depletion has on either Treg cell

subtype remains unclear. It appears that depletion of Bim may have a limited impact on nTreg development, as the emigration rate of nTregs from the thymus is similar in WT and _Bim_−/−

mice.13 The observation that _Bim_−/− nTregs are more proliferative is, however, contradictory to the observation that _Bim_−/− T cells are hypo-responsive upon TCR activation.16 In this

study, we ask if the balance between Bim and Bcl-2 acts as a critical survival parameter that controls iTreg homeostasis. RESULTS PREFERENTIAL INDUCTION OF ITREGS _IN VIVO_ IN THE ABSENCE OF

BIM To directly investigate the influence of Bim depletion on iTreg homeostasis _in vivo_, we induced inflammatory bowel disease (IBD) in mice by adoptively transferring CD4+CD25−CD45RBhi

Tconv into _Rag1_−/− hosts. In this model, transferred Tconv repopulate the host lymphoid tissue, leading to intestinal inflammation in a certain percentage of Tconv recipients. As only

Tconv are transferred (Supplementary Figure S1a), essentially all Foxp3+ T cells recovered from the recipients are _de novo_-generated iTregs. Although Bim is important for T-cell apoptosis,

we only observed a slight difference between _Bim_−/− and WT Tconv recipients in both percentage and absolute number of CD4+ T cells recovered from the spleens and mesenteric lymph nodes

(MLN) (Figures 1a and b). Surprisingly, there was a significant increase in the percentage (∼2–3-fold) and number (∼3–4-fold) of Foxp3+CD4+ iTregs recovered from _Bim_−/− Tconv recipients

compared with WT Tconv recipients in the spleen, MLN and a combined population of intraepithelial lymphocytes and lamina propria lymphocytes (IEL/LPL) isolated from the intestines (Figures

1c and d). This increase of iTregs did not appear to be due to differing levels of Foxp3 protein expression, as iTregs recovered from both _Bim_−/− and WT Tconv recipients exhibited

comparable levels of Foxp3 expression (spleen=1022/958 (WT/_Bim_−/− MFI); MLN=1649/1443; intestine=1272/1258; Figure 1e). Nor was the difference due to an increased ability of _Bim_−/− Tconv

to expand, compared with WT Tconv following transfer into a lymphopenic host, as the percentage of BrdU uptake by CD4+Foxp3− cells was similar in the spleen and surprisingly higher in WT

cells in the MLN (Figure 1f and Supplementary Figure S1b). This difference, however, was minimal in terms of the number of CD4+Foxp3−BrdU+ cells (Supplementary Figure S1c). Interestingly,

there was an increased percentage and number of _Bim_−/− CD4+Foxp3+BrdU+ cells in the spleen (Figure 1f and Supplementary Figure S1c). This may partially explain the larger fold difference

between the number of _Bim_−/− iTregs and WT iTregs in the spleen (∼3-fold difference) compared with MLN (∼2-fold difference). Consistent with these data, mice that received _Bim_−/− Tconv

had an ∼50% reduction in the incidence of IBD (as measured by weight loss greater than 5% initial body weight) than WT Tconv recipients (_P_=7.52 × 10−5; relative risk=0.491; 95% CI

(0.346–0.700); Figure 1g). Taken together, these data suggest that the loss of Bim has a preferential effect on the generation and/or survival of iTregs, compared with the proportion of

transferred Tconv that do not convert to a Foxp3+ population, resulting in a protective effect against IBD. LOSS OF BIM CONFERS A PREFERENTIAL SURVIVAL ADVANTAGE FOR ITREGS The increase in

iTregs observed in the _Bim_−/− Tconv recipients could be a consequence of increased efficiency of the _Bim_−/− iTreg conversion, a selective survival advantage of _Bim_−/− iTregs due to

increased IL-2 secretion by _Bim_−/− Tconv, or an accumulated effect due to an intrinsic survival advantage in iTregs _versus_ Tconv in the absence of Bim. To address the first possibility,

we performed an _in vitro_ iTreg conversion assay. Multiple factors are able to modulate the efficiency of iTreg conversion. TCR signaling is known to have a pivotal effect. Enhanced TCR

activation with higher proliferative capacity leads to more efficient iTreg differentiation _in vitro_ (Supplementary Figures S2a and b) and _in vivo._17 As recent studies have suggested

that _Bim_−/− Tconv are hyporeactive to TCR stimulation,16 one might predict that _Bim_−/− Tconv may not show a differentiation advantage over WT Tconv. Consistent with this notion, _Bim_−/−

Tconv exhibited less efficient iTreg conversion _in vitro_ than WT Tconv (Supplementary Figure S2c), which was likely due to less efficient TCR activation. It is possible that _Bim_−/−

Tconv secrete more IL-2, leading to enhanced conversion and survival of iTregs. However, this did not seem to be likely as WT and _Bim_−/− Tconv activated _in vitro_ secrete similar amounts

of IL-2 (Supplementary Figure S2d). Furthermore, the percentage of CD4+CD25−IL-2+ cells isolated from _Rag1_−/− mice that received either WT or _Bim_−/− Tconv was equivalent (Supplementary

Figure S2e). The third possibility is that Bim depletion has a differential effect on the survival of iTregs _versus_ Tconv. Constitutive expression of the IL-2 receptor (CD25) is a hallmark

for nTregs and it is well known that they are highly dependent on IL-2 for their homeostasis and survival.18, 19 Given the fact that Bim is a principal component in growth factor

deprivation-mediated apoptosis, we compared the survival capacity of WT and _Bim_−/− iTregs, and activated Tconv in the absence of IL-2. The iTregs were generated by activating purified

Tconv from Foxp3GFP reporter mice in the presence of TGF-_β_ and IL-2 for 3 days. Foxp3+ iTregs (GFP+) and activated Foxp3− Tconv (GFP−) were sorted, cultured in the absence of exogenous

IL-2 and their relative survival compared. We found that Foxp3+ iTregs had a two-fold increase in their sensitivity to apoptosis in cultures without the addition of exogenous IL-2, compared

with Foxp3−-activated Tconv (Figures 2a and b). Foxp3+ iTregs were more vulnerable to apoptosis than Foxp3−-activated Tconv over a wide range of limited exogenous IL-2 supplementation (from

10 U/ml to 100 U/ml; Figure 2c). However, these differences in survival capacity between iTregs and activated Tconv were abrogated in both cell populations in the absence of Bim (Figures 2a

and b). These data suggest that iTregs are more sensitive to IL-2 deprivation-mediated apoptosis than activated Tconv. Loss of Bim expression confers a greater survival advantage for Foxp3+

iTregs, nullifying their increased sensitivity to growth factor withdrawal. INTRINSIC VULNERABILITY TO APOPTOSIS DUE TO ALTERED BIM, BCL-2 AND BCL-XL BALANCE IN ITREGS We next investigated

the mechanism responsible for the survival disparity between iTreg and activated Tconv in the presence of limited IL-2. Activated T cells only express IL-2 transiently following TCR

engagement. Blocking IL-2 signaling by using an anti-CD25 antibody had little effect on activated Tconv survival in the absence of exogenous IL-2 addition. Thus, the survival advantage of

activated Tconv over iTregs under limiting IL-2 conditions was likely due to cell intrinsic mechanisms rather than an autocrine IL-2 pathway. As deletion of the pro-apoptotic molecule Bim

was able to mitigate the survival differences between iTregs and activated Tconv, we compared the expression levels of several pro- and anti-apoptotic Bcl-2 family members in iTregs and

activated Tconv before (pre) and after (post) a 2-day culture in the absence of exogenous IL-2 (Figure 3a). The expression of the pro-apoptotic members of the Bcl-2 family, Bim and Bax, was

increased to a similar extent in iTregs and activated Tconv (change between expression pre- and post-IL-2 removal). No change in Bid expression level was observed. Expression of the

anti-apoptotic molecules Bcl-2 and Bcl-xl was slightly higher in iTregs compared with activated Tconv before IL-2 withdrawal (Figure 3a; noted as ‘pre’). However, culturing cells in the

absence of exogenous IL-2 for just 48 h resulted in a much more dramatic decrease of Bcl-2 expression in iTregs compared with activated Tconv, which was accompanied with a larger increase of

Bim expression (Figure 3a; noted as ‘post’). Similar but less pronounced changes were observed with Bcl-xl and Bax. Mcl-1 and Noxa have recently been shown to be important in regulating

T-cell apoptosis during clonal expansion.20 Despite substantial upregulation of Noxa following T-cell activation, there was no difference in the relative expression of Mcl-1 and Noxa mRNA

between Tconv and iTregs (Supplementary Figure S3). As cell survival is dependent on the balance between pro- and anti-apoptotic molecules rather than their absolute expression levels,

analysis of the relative ratios of interactive partners may be an informative parameter to elucidate cellular sensitivity to apoptosis. Minimal differences in the Bim/Bcl-2, Bim/Bcl-xl,

Bax/Bcl-2 and Bax/Bcl-xl expression ratios were observed between freshly sorted iTregs and activated Tconv (Figure 3b; pre), suggesting that their initial sensitivity to apoptosis may be

similar. However, continued culture in the absence of IL-2 resulted in a significant increase in the Bim/Bcl-2 expression ratio in iTregs (∼7-fold – pre _versus_ post) _versus_ activated

Tconv (∼3-fold). There was also an ∼3-fold increase in the Bim/Bcl-xl ratio in iTregs, which was not observed with activated Tconv (Figure 3b). Similar changes were observed with the

Bax/Bcl-2 or Bax/Bcl-xl ratios. As anticipated, IL-2 supplementation prevented the increase of Bim/Bcl-2 ratio, which correlated with their capacity to promote iTreg survival to a level

comparable to activated Tconv (Supplementary Figure S4). Consistent with this mRNA analysis, we observed a much larger proportion of iTregs that lost intracellular Bcl-2 protein expression

following IL-2 withdrawal, compared with activated Tconv (Figure 3c). Collectively, these data suggest that iTregs are intrinsically less capable of maintaining an appropriate balance

between the pro- (Bim/Bax) and anti- (Bcl-2/Bcl-xl) apoptotic proteins to ensure survival. REDUCED BCL-2 EXPRESSION IN ITREGS _IN VIVO_ To extend our findings _in vivo_, we transferred the

Foxp3GFP Tconv or CD4+CD25−CD45RBhi Tconv from WT mice into _Rag1_−/− recipients and analyzed the expression levels of the Bcl-2 family molecules in iTregs and Tconv recovered 4 weeks

post-transfer. Although expression levels of Bim, Bcl-xl and Bax were fairly comparable between iTreg and Tconv, iTregs consistently expressed four to five times less Bcl-2 than Tconv

recovered from the spleen (Figure 4a). Consequently, the Bim/Bcl-2 and Bax/Bcl-2 mRNA ratios were substantially higher in iTregs than in Tconv (Figures 4b and c). No difference in Bax/Bcl-xl

ratio was observed (Figure 4c). Interestingly, Bcl-2 mRNA expression in iTregs and Tconv recovered from the MLN was similar, which mirrored our observation that there was a somewhat less

profound increase in the number of iTregs in MLN _versus_ the spleen in the absence of Bim (Figures 1c and d). Given that the MLN serves as the primary site of immune response in the IBD

model, it is possible that the levels of growth factors such as IL-2 are more abundant here compared with more distal lymphoid tissues such as the spleen. Intracellular Bcl-2 protein

staining confirmed that iTregs consistently express less Bcl-2 (∼3-fold) compared with Foxp3− Tconv isolated from the spleen, non-draining lymph nodes, MLN and IEL/LPL of repopulated

_Rag1_−/− hosts (Figures 4d and e). This was also observed with cells isolated from _Rag1_−/− hosts repopulated with _Bim_−/− Tconv (Figure 4e). Bcl-2 expression in iTregs was also

consistently higher in the MLN. Collectively, iTregs seem to have a deficiency in Bcl-2 expression, which may render them more susceptible to apoptosis _in vivo_. FOXP3+BCL2LO TREG

POPULATIONS _IN VIVO_ The nTregs are reported to express higher levels Bcl-2 than Tconv.21 If low expression of Bcl-2 is a characteristic feature of iTregs, in comparison with nTregs, we

asked whether it could potentially mark iTregs in WT C57BL/6 mice _in vivo_. Consistent with our hypothesis, there were clearly two populations of Tregs, distinguishable as Bcl-2lo and

Bcl-2hi in the spleen and peripheral lymph nodes, respectively (Figure 5a). Although Bcl-2 expression levels in Tregs did not correlate with Helios expression, a suggested marker of

iTregs,22, 23 about 50% of the Bcl-2lo Tregs were CD69lo and CD44hi, suggesting that this Treg population was antigen-experienced but not recently activated (Figures 5a and b). Therefore,

although we cannot exclude the possibility that a proportion of the Bcl-2lo Tregs were derived from nTregs after recent activation, it is possible that Foxp3+Bcl-2loCD69hiCD44hi cells

represent newly differentiated iTregs, whereas Foxp3+Bcl-2loCD69loCD44hi cells represent a memory iTreg population.24 However, further analysis would be required to determine if nTregs and

iTregs can be distinguished by the level of Bcl-2 expression. DISCUSSION Previous studies have reported increased numbers of Tregs in _Bim_−/− mice, as well as in aged mice.13, 14 However,

the mechanisms that control the homeostasis of Treg populations remain to be fully defined. It has been suggested that increased proliferation of Bimlo Tregs (marked by increased BrdU

incorporation in the Foxp3+Bimlo population) was the basis for preferential Treg expansion.13 However, this model is somewhat controversial as studies have suggested that responses following

TCR stimulation are defective in _Bim_−/− and Bimlo T cells.15, 16 Our data suggest that decreased Bim expression confers a preferential survival advantage on iTregs. Therefore, depletion

or downregulation of Bim limits the rapid peripheral Treg cell turnover,25 which consequently results in increased Treg numbers. As optimal TCR signaling is required for iTreg

differentiation, and the T-cell population with the highest proliferative capacity tends to differentiate into iTregs more efficiently, higher BrdU incorporation in the Foxp3+Bimlo T-cell

population may represent enhanced iTreg survival due to lower Bim expression _in vivo_ rather than nTreg expansion. In line with our model, overexpression of the anti-apoptotic molecule

Bcl-2 in T cells also results in an increased proportion of iTregs _in vivo_ and reduces the incidence of autoimmune disease.26 The importance of IL-2 in the development and homeostatic

maintenance of nTregs, iTreg and Tconv is well documented.18, 19, 27 IL-2- or IL-2R-deficient mice have a two-fold reduction in the number of Tregs and suffer from a severe

lymphoproliferative disease.2, 28 However, crossing IL-2- or IL-2R-deficient mice with _Bim_−/− mice eliminated this numerical deficiency, supporting our data that demonstrates the level of

vBim expression is important for iTreg survival.28 Nevertheless, restoration of Foxp3+ Treg numbers in _Il2_−/−_Bim_−/− mice did not prevent the lymphoproliferative disease unless exogenous

IL-2 was added, suggesting that IL-2 is also required for the function of Tregs _in vivo_.28 It has been reported that IL-2 and signaling through the IL-2R regulates Bcl-2 expression.29 Our

data supports this observation as IL-2 deprivation resulted in a significant decrease in both Bcl-2 and Bcl-xl expression _in vitro_. Taken together, this strongly suggests an IL-2-dependent

link between Bim and Bcl-2 levels, and supports our conclusion that in the absence of Bim, there is increased survival of iTregs and consequently a higher number of Tregs. How this relates

to a decrease in IBD incidence may be two-fold. First, in the absence of Bim, there may be a reduction in apoptosis resulting in more iTregs. Second, the increased number of _Bim_−/− iTregs

results in a lower ratio of Tconv/iTregs in the tissues (spleen, WT –.86 : 1, _Bim_−/− – 22 : 1; MLN, WT – 31 : 1, _Bim_−/− – 20 : 1; Gut, WT – 303 : 1, _Bim_−/− – 120 : 1), which may

increase the probability that Bim-deficient iTregs outcompete Tconv for the available IL-2 that is required for optimal Treg suppression _in vivo_.28 Although the notion that different

T-cell subpopulations may preferentially use different pro- and anti-apoptotic pathways to regulate homeostasis has been previously proposed,30 no clear examples have been documented. Our

data suggest that balanced expression within the Bim/Bcl-2 axis has a greater impact on the survival of iTregs than activated Tconv _in vitro_ and _in vivo_. It is possible that

lineage-specific transcription factors may preferentially modulate the expression of specific Bcl-2 family apoptotic modulator(s). A previous ChIP–ChIP study revealed that Foxp3 is

constitutively bound to the Bcl-2 locus, but not any other Bcl-2 family members.31 It remains unclear whether such binding promotes or represses Bcl-2 expression. Although depletion of the

Foxp3-recruited transcriptional repressor Eos results in increased Bcl-2 expression, iTregs themselves express more Bcl-2 than activated Tconv.21 A more intriguing hypothesis is that Foxp3

may modulate Bcl-2 expression in a conditional manner. By recruiting different nuclear factors, Foxp3 may promote Bcl-2 expression when IL-2 is in excess, but repress Bcl-2 expression when

IL-2 is limited. This may be particularly relevant in specific diseases such as autoimmune diabetes, in which defective IL-2 production has been documented.32 If Foxp3 is directly modulating

Bcl-2 expression in Tregs, it is unclear whether nTregs, like iTregs, are also hypersensitive to apoptosis. A decreased Treg/Tconv cell ratio has been implicated in the onset of autoimmune

diabetes.32 Indeed, consistent with our finding that iTregs have a greater apoptotic tendency _in vitro_ and _in vivo_, profound Treg apoptosis has been shown to occur during diabetes

onset.32 Interestingly, our study suggests that there may be two subpopulations of Tregs expressing different levels of Bcl-2, and it is the Bcl-2hi population that is preferentially lost

during progression towards diabetes. The majority of the detectable Tregs in islets are phenotypically CD4+Foxp3+Bcl-2loKi67+, which may represent newly differentiated iTregs at the site of

inflammation. In summary, the balance between Tregs and Tconv has a central role in maintaining immune tolerance and promoting immune response. Perturbation of the Bim/Bcl-2 balance may

represent a new therapeutic approach to modulate the immune environment under diverse disease conditions. Modulation of Bcl-2 expression could be used as an approach to promote survival of

iTregs generated for therapeutic application. MATERIALS AND METHODS MICE, T-CELL PREPARATION AND ANTIBODIES C57BL/6 (WT) and _Rag1_−/− mice were obtained from The Jackson Laboratory (Bar

Harbor, ME, USA). The _Bim_−/− mice were provided by Andreas Strasser (The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia).12 The _Bim_−/− mice were

crossed with Foxp3GFP reporter mice2 to generate _Bim_−/−Foxp3GFP mice. All mice were bred and maintained at St. Jude Children's Research Hospital. All animal experiments were performed

in the American Association for the Accreditation of Laboratory Animal Care-accredited, specific pathogen-, Helicobacter- and MNV-free facilities, following national, state and

institutional guidelines. Animal protocols were approved by the St. Jude Animal Care and Use Committee. For IEL/LPL purification, intestines were removed, cut into 0.5 cm pieces and shaken

at 37°C for 20 min in 45 ml HBSS with 1 mM DTT. Suspension was decanted and filtered through a 70 _μ_m cell strainer into a 50 ml conical tube, and tissue was pelleted by centrifugation for

5 min at 1200 r.p.m. Supernatant was discarded and pellet was reserved on ice. This process was repeated two more times with the strained tissue, pelleting the suspension in the same 50 ml

conical tube. The remaining intestinal tissue was finely chopped and incubated in 25 ml RPMI supplemented with 10% FBS and 8000 U of Collagenase Type IV (Worthington Biochemical, Lakewood,

NJ, USA), rotating at 37°C. Cell suspension was strained and pooled with the previous pelleted collection, centrifuged and supernatant discarded. This was repeated with the strained

intestinal tissue. The final cell pellet was washed in RPMI/10% FBS, centrifuged and supernatant discarded. The cell pellet was resuspended in 4 ml of 40 × percoll (4.0 ml of 100 ×

percoll+6.0 ml RPMI/10% FBS; GE Healthcare, Piscataway, NJ, USA) and strained through a 70 _μ_m cell strainer. Cell suspension was gently layered over 5.0 ml of 100 × percoll in a 15 ml

conical tube and centrifuged at 2500 r.p.m. with no brake for 20 min at room temperature. Media overlying the band of IEL/LPL at the interface between the two percoll layers was carefully

removed. The band of cells was transferred by pipette to a new 15 ml conical tube to which RPMI/10% FBS was added to a final volume of 10 ml. The tube was shaken and centrifuged 10 min at

1500 r.p.m. to pellet the cells. For Tconv and Treg preparation, spleens and lymph nodes from mice were processed and stained with fluorochrome-conjugated antibodies and purified by

single-cell sorting using anti-CD4 (clone GK 1.5), anti-CD25 (clone PC61) and anti-CD45RB (clone C363-16A). Tconv used in this study were defined as CD4+CD45RBhiCD25−, or if the Foxp3GFP

reporter mice were used, CD4+GFP−. All antibodies used were from Biolegend (San Diego, CA, USA) or eBiosciences (San Diego, CA, USA). Bcl-2 expression was determined using clone 10C4, and

staining performed with Foxp3 fixation/permeablization reagents from eBioscience. IBD MODEL Induction of IBD was performed as described previously.33 Briefly, 0.4 × 106 FACS-purified Tconv

(CD4+CD45RBhiCD25−) were injected via the tail vein into _Rag1_−/− mice. The Tconv recipients were monitored and the clinical sign of disease was defined as 5–10% body-weight loss. Spleen,

lymph node and IEL/LPL cellularity was analyzed approximately 4 weeks post-Tconv transfer. For _ex vivo_ analysis of gene expression pattern, iTregs and Tconv were sorted 4 weeks

post-transfer. ANALYSIS OF IL-2 BY ELISA AND INTRACELLULAR STAINING WT and _Bim_−/− Tconv were sorted and plated in 96-well round-bottom plates (1 × 105 per well) with anti-CD3/CD28-coated

M-450 tosylactivated dynabeads (Invitrogen, Grand Island, NY, USA) at a ratio of 2.5 cells per bead. After 72 h stimulation, supernatants were collected and analyzed by ELISA. Supernatants

were incubated on plates pre-coated with 1 _μ_g/ml purified anti-IL2 (clone JES6-1A12, Biolegend) for 2 h at room temperature. Then, IL-2 was detected with 1 _μ_g/ml biotinylated anti-IL-2

(clone JES6-5H4 biotin, Biolegend), followed by streptavidin-HRP (Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA) and developed with TMB substrate (Thermo Fisher Scientific,

Pittsburgh, PA, USA). For intracellular staining, spleen and MLN of _Rag1_−/− that had received WT or _Bim_−/− Tconv 4 weeks prior were processed, and the cells were stimulated for 12 h with

PMA/ionomycin (100 ng/ml and 500 ng/ml, respectively) in the presence of Brefeldin A (1 : 1000, Biolegend). Following stimulation, cells were stained for surface CD4 and CD25, fixed and

stained with antibodies to IL-2 (clone JES5-5H4) or an IgG isotype control, using Cytofix/Cytoperm kit following the manufacturer's instruction (BD Biosciences, San Diego, CA, USA), and

analyzed by flow cytometry. INTRACELLULAR STAINING FOR BCL-2 AND FOXP3 Purified Tconv (CD4+CD45RBhiCD25−) were injected i.v. via the tail vein into _Rag1_−/− mice. After 4 weeks, spleen and

MLN were processed and cells were stained for surface CD4, and then fixed, permeabilized and stained with antibodies to Bcl2 (clone BCL/10C4), Foxp3 (clone 150D) and an IgG1 isotype

control, using the Foxp3 Fixation/Permeabilization buffer set from EBiosciences according to manufacturer's instructions, and analyzed by flow cytometry. BRDU ANALYSIS Purified Tconv

were injected via the tail vein into _Rag1_−/− mice. At 4 weeks post-transfer, mice were injected i.p. with 200 _μ_l of BrdU (10 mg/ml) in PBS. After 16 h, cells from spleen and MLN were

harvested, stained for CD4, Foxp3 and BrdU, using the BrdU-APC flow kit (BD Biosciences) following manufacturer's instructions, and analyzed by flow cytometry. INDUCED REGULATORY T-CELL

DIFFERENTIATION AND SURVIVAL ANALYSIS Tconv were isolated from WT or _Bim_−/− Foxp3GFP reporter mice and activated with either plate-bound anti-CD3 (clone 2C11) and soluble anti-CD28 (clone

37.51) or pre-coated CD3/CD28 beads in the presence of 5 ng/ml TGF-_β_ and 100 U/ml IL-2 for 3 days. GFP+ iTreg and activated GFP− Tconv were sorted by FACS. Either cell type was then

cultured with or without exogenous IL-2 at a cell density of 5 × 104 per well in 96-well round-bottom plates for the times indicated. Samples were collected and stained with eFluor-670

(eBiosciences), AnnexinV (BD Biosciences) and PI (Sigma-Aldrich, St. Louis, MO, USA). Live cells were defined as AnnexinV−PI−. QUANTITATIVE REAL-TIME PCR RNA was purified using Qiagen

(Valencia, CA, USA) RNeasy Plus Mini or RNeasy Micro kit following the manufacturer's instructions. The expression level of Bim, Bcl-2, Bcl-xl, Bax, Bid, Mcl1 and Noxa were quantified

by real-time PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR green. Taqman probe-based quantitative PCR was used to record actin expression

level for normalization. Primer sets were as follows: bcl2-F: 5′-ACTTCGCAGAGATGTCCAGTCA-3′, bcl2-R: 5′-TGGCAAAGCGTCCCCTC-3′; bcl-xl-F: 5′-GAATGGAGCCACTGGCCA-3′, bcl-xl-R:

5′-GCTGCCATGGGAATCACCT-3′; bax-F: 5′-GCCTCCTCTCCTACTTCGGG-3′, bax-R: 5′-TGAGGACTCCAGCCACAAAGA-3′; Bim-F: 5′-TGCGCCCGGAGATACG-3′, Bim-R: 5′-TTCGTTGAACTCGTCTCCGA-3′; Bid-F:

5′-GCTGTCTCCCTATTTCCAGGTG-3′, Bid-R: 5′-AGCTGAACGCAGAGATGTCCA-3′; Noxa-F: 5′- GTCGCAAAAGAGCAGGATGAG-3′, Noxa-R: 5′-TTGAGCACACTCGTCCTTCAA-3′; Mcl-1F: 5′-AACGGGACTGGCTTGTCAAA-3′, Mcl-1R:

5′-CTAGGTCCTGTACGTGGAAGAACTC-3′. STATISTICS Data were analyzed using paired, unpaired _t_-test or Mann–Whitney test, depending upon the data set, and is described in the figure legends. For

analysis of disease incidence in Figure 1f, a 2 × 2 _χ_2-analysis was used. _P_-values were categorized into three levels: *_P_<0.05, **_P_<0.01 and ***_P_<0.005, unless stated

otherwise. ABBREVIATIONS * Tconv: conventional T cell * nTreg: natural regulatory T cell * iTreg: induced regulatory T cell * WT: wild type * IBD: inflammatory bowel disease * MLN:

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intestinal inflammation. _Immunol Rev_ 2006; 212: 256–271. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We would like to thank Andreas Strasser for the knockout mice.

We are also very grateful to Richard Cross, Greig Lennon, Stephanie Morgan and Jennifer Rogers for FACS; Karen Forbes, Ashley Castellaw and Amy Krause-McKenna for maintenance, breeding and

genotyping of mouse colonies; Fabien Llambi for helpful discussions; and the staff of the St. Jude Animal Resource Center for the animal husbandry. This work was supported by the NIH

(AI39480, AI091977), the St. Jude Cancer Center Support CORE Grant (CA21765) and the American Lebanese Syrian Associated Charities (ALSAC). AUTHOR INFORMATION Author notes * X Wang Present

address: 3Current address: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA, * X Wang, A L Szymczak-Workman and D M Gravano: These authors contributed

equally to this work. AUTHORS AND AFFILIATIONS * Department of Immunology, St. Jude Children's Research Hospital, Memphis, 38105, TN, USA X Wang, A L Szymczak-Workman, D M Gravano, C J

Workman, D R Green & D A A Vignali Authors * X Wang View author publications You can also search for this author inPubMed Google Scholar * A L Szymczak-Workman View author publications

You can also search for this author inPubMed Google Scholar * D M Gravano View author publications You can also search for this author inPubMed Google Scholar * C J Workman View author

publications You can also search for this author inPubMed Google Scholar * D R Green View author publications You can also search for this author inPubMed Google Scholar * D A A Vignali View

author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to D A A Vignali. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no conflict of interest. ADDITIONAL INFORMATION Edited by P Salomoni Supplementary Information accompanies the paper on Cell Death and Disease website SUPPLEMENTARY INFORMATION

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al._ Preferential control of induced regulatory T cell homeostasis via a Bim/Bcl-2 axis. _Cell Death Dis_ 3, e270 (2012). https://doi.org/10.1038/cddis.2012.9 Download citation * Received:

05 August 2011 * Revised: 29 December 2011 * Accepted: 09 January 2012 * Published: 09 February 2012 * Issue Date: February 2012 * DOI: https://doi.org/10.1038/cddis.2012.9 SHARE THIS

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Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * iTreg * apoptosis * Bim * Bcl-2