Proapoptotic Protein Bim Is Differentially Required During Thymic Clonal Deletion to Ubiquitous Versus Tissue-Restricted Antigens

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Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens

Alexander Y. W. Suen and Troy A. Baldwin1

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada T6G 2S2

Edited by Michael J. Bevan, University of Washington, Seattle, WA, and approved December 8, 2011 (received for review September 8, 2011) Positive and negative selection of thymocytes in the thymus are positively selected by high-affinity antigen encounter in the thy- critical for the development of a mature and self-tolerant T-cell mus (6). One such example are T-regulatory (Treg) cells, which repertoire. The proapoptotic Bcl-2 family member Bim is important are believed to require high-affinity interactions to develop in the for negative selection by inducing apoptosis in thymocytes receiv- medulla (7). It remains unknown what drives this distinction ing a strong signal through their antigen receptor. However, in the between negative selection and Treg development, although case of ubiquitous self-antigens (UbA), Bim is not required for the secondary signals, such as TGF-β, may play a role in overcoming clonal deletion of self-reactive thymocytes, suggesting the exis- cell death (8). tence of nonapoptotic clonal deletion mechanisms. Unlike UbA, Although it is unclear how the same TCR can transduce a sig- clonal deletion to tissue-restricted antigens (TRAs) requires posi- nal for positive or negative selection, differential mitogen-acti- tive selection and CCR7-mediated migration to the medulla. This vated protein kinase signaling appears to play a role, ultimately led us to hypothesize that Bim is required for the latter. To study leading to the activation of different transcriptional programs (9). the role of Bim in clonal deletion to TRA, we constructed bone Negative selection induces expression of proapoptotic molecules marrow (BM) chimeras using OT-I Bim-deficient or -sufficient do- such as the Bcl-2 homology domain 3 (BH3) only Bcl-2 family nor bone marrow and recipients that express membrane bound member Bim (10, 11), which is essential for the intrinsic pathway

chicken ovalbumin under control of the rat insulin promoter (Rip- of apoptosis (12). It was generally held that Bim-mediated apo- IMMUNOLOGY mOVA). We found that clonal deletion to TRA was completely ptosis was required for negative selection (13). This was supported abrogated in the absence of Bim and large numbers of mature by the fact that Bim-deficient mice develop late onset autoim- OT-I CD8 T cells survived in the periphery. Despite the large num- mune disease and that nonobese diabetic mice, which spontane- bers of autoreactive T cells, the chimeras did not develop diabetes ously develop diabetes, have a defect in the induction of Bim (11, and OT-I Bim-deficient T cells from these chimeras were function- 14, 15). In contrast to this paradigm, recent studies demonstrated ally impaired. Collectively, these data provide unique evidence of that, whereas Bim is required for thymocyte apoptosis, it is not a differential, thymocyte-intrinsic, molecular requirement down- required for clonal deletion to the ubiquitous s-mcy male peptide stream of the T-cell receptor (TCR) for clonal deletion to UbA ver- (16, 17) or superantigen-mediated clonal deletion (18). sus TRA and highlight the profound ability of other tolerance It is clear that negative selection is induced by two different mechanisms to control T-cell autoreactivity in the absence of thy- classes of self-antigens: ubiquitous self-antigens (UbA) and tis- mic clonal deletion. sue-restricted antigens (TRAs). TRA expression is restricted to the medulla with expression of many TRAs controlled by the autoimmunity | thymocyte development AIRE gene (19). After much debate, recent data suggest that negative selection of DP thymocytes to UbA occurs in the cortex uring T-cell development, the T-cell receptor (TCR) is without medullary involvement (20), whereas positive selection Dgenerated by random rearrangements at the TCRα and and CCR7-dependent migration to the medulla are required for TCRβ loci. This allows for a large variety of TCR specificities to negative selection to TRA (4). Once in the medulla, CD4+CD8lo be generated but also requires that T cells be educated. The or CD4loCD8lo thymocytes can interact with mTECs that directly education process ensures that the T cells can respond to antigen present TRA or DCs that can cross-present TRAs derived from in the context of self-major histocompatibility complex (MHC) mTECs (5). Therefore, negative selection to UbA and TRA molecules but cannot respond to self-antigens in the context of differ with respect to the developmental stage of the thymocyte self-MHC. These selection events begin at the CD4+CD8+ and the anatomical location within the thymus. These factors double-positive (DP) stage of thymocyte development within the may result in differential molecular requirements for negative cortex of the thymus. DP thymocytes may undergo one of three selection to UbA versus TRA, although no such thymocyte in- fates depending on the affinity of their TCR for self-peptide trinsic differences downstream of the TCR have been reported. presented on self-MHC (pMHC) on cortical thymic epithelial Using OT-I TCR transgenic bone marrow (BM) and mice cells (1). If the TCR cannot functionally interact with self- expressing membrane bound chicken ovalbumin (mOVA) under pMHC, the thymocytes die by neglect. Thymocytes expressing control of the chicken β-actin promoter (Act-mOVA) (21) or rat a TCR with high affinity for self-pMHC undergo negative se- insulin promoter (RIP-mOVA) (22), we demonstrate that unlike lection, resulting in the elimination of that particular TCR clonal deletion to UbA, Bim is required for clonal deletion to − − specificity from the T-cell repertoire. Low or moderate affinity TRA. Although OT-I Bim / CD8 T cells are abundant in RIP- interactions between the TCR and self-pMHC result in positive selection, leading to thymocyte survival and subsequent differ- + + entiation into CD4 or CD8 single-positive (SP) thymocytes Author contributions: A.Y.W.S. and T.A.B. designed research; A.Y.W.S. performed (1). These positively selected thymocytes then undergo CCR7- research; A.Y.W.S. and T.A.B. analyzed data; and A.Y.W.S. and T.A.B. wrote the paper. dependent migration to the thymic medulla (2). During the 3- to The authors declare no conflict of interest. 4-d residence in the medulla (3), SP thymocytes are subjected to This article is a PNAS Direct Submission. a further round of negative selection through interactions with 1To whom correspondence should be addressed. E-mail: [email protected]. medullary thymic epithelial cells (mTECs) and medullary den- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dritic cells (DCs) (4, 5). Interestingly, some T-cell subsets are 1073/pnas.1114834109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1114834109 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 mOVA chimeras, they do not cause diabetes, but rather are Bim Is Required for Clonal Deletion to TRA. Despite the lack of re- impaired in their response to antigen. quirement for Bim in clonal deletion in the OT-I model and other models, the fact remains that Bim deficiency leads to au- Results toimmune disorders (14). Whereas the previous models exam- Bim Is Not Required for Clonal Deletion to UbA. Recently, it has ined clonal deletion to UbA or superantigen they did not address become clear that Bim is not required for clonal deletion in clonal deletion to TRA, which differs with respect to the de- a number of physiological model systems (16, 17). However, in velopmental stage of the thymocyte and anatomical location these models, the high-affinity antigen was expressed ubiqui- within the thymus. We hypothesized that these distinctions may tously and therefore the role of Bim in clonal deletion to TRA is result in a difference in the requirement for Bim in clonal de- still unclear. As these models are not easily manipulated to ex- letion to TRA. amine clonal deletion to TRA, we sought to use the OT-I TCR To generate a model of clonal deletion to TRA, we grafted −/− transgenic system. OT-I thymocytes express a TCR specific for OT-I or OT-I Bim BM to RIP-mOVA recipients. The rat the SIINFEKL peptide of OVA in the context of H-2Kb and can insulin promoter drives expression of mOVA in the pancreas, the be detected using an anti-Vα2 mAb. Furthermore, mice that kidney, and the medulla of the thymus. In this model, OT-I fi express membrane OVA ubiquitously or in a tissue-restricted thymocytes will rst undergo positive selection in the cortex fashion are available. To ensure that clonal deletion of OT-I before encountering their cognate antigen in the medulla. The fi α + → thymocytes to UbA was Bim independent, we generated BM CD4/CD8 thymic pro les of V 2 thymocytes from OT-I B6 −/−→ chimeras using Act-mOVA recipients and either OT-I or OT-I and OT-I Bim B6 chimeras were not drastically altered by − − fi A Bim / BM donors. We found that Bim deficiency did not affect Bim de ciency (Fig. 2 ). In contrast and as previously demon- fi α + strated (23), the OT-I→RIP-mOVA chimeras displayed a signif- the CD4/CD8 thymic pro les of V 2 thymocytes in the OT-I + −/− icant reduction in the proportion of Vα2 CD8SP thymocytes Bim →B6 chimeras compared with the OT-I→B6 chimeras + int with an increase in the DN compartment (Fig. 2A). Contrary to (Fig. 1A). In both cases, DP, CD4 CD8 , and CD8SP thymo- − − the OT-I→RIP-mOVA chimeras, the OT-I Bim / →RIP-mOVA cyte percentages were similar (Fig. 1A). In both Act-mOVA − − chimeras showed no evidence of clonal deletion compared with chimeras, the majority of thymocytes were CD4 CD8 double −/−→ + int the OT-I Bim B6 chimeras. Interestingly, CD8 expression negative (DN) with reductions in DP, CD4 CD8 , and CD8SP −/−→ A was slightly down-regulated in the OT-I Bim RIP-mOVA populations, indicating deletion of OT-I thymocytes (Fig. 1 ). The chimeras and may be indicative of high-affinity antigen encounter increased percentage of DN thymocytes was more pronounced in A α + −/−→ (Fig. 2 ). With the exception of the V 2 CD8SP compartment, the OT-I Bim Act-mOVA chimeras. Cell numbers in the the number of Vα2+ thymocytes in the other compartments was various compartments further supported Bim-independent clonal similar (Table 1). We also examined CD24 expression on Vα2+ α + deletion to UbA. There was a similar reduction in V 2 DP and CD8SP thymocytes to assess the maturity of the thymocytes. α + → − − V 2 CD8SP cell numbers in OT-I Act-mOVA compared with Compared with either OT-I or OT-I Bim / →B6, OT-I→RIP- → −/−→ OT-I B6 and in OT-I Bim Act-mOVA compared with OT-I mOVA chimeras showed a considerable reduction in the pro- −/−→ B Bim B6 chimeras (Fig. 1 ). Furthermore, cleaved caspase 3 portion of mature, CD24lo,Vα2+ CD8SP thymocytes, whereas − − staining showed significant caspase 3 activity in the OT-I→Act- the proportion of mature OT-I thymocytes in OT-I Bim / →RIP- mOVA chimeras that was dramatically reduced in the OT-I mOVA chimeras was equivalent (Fig. 2B). Cell numbers rein- −/− − − Bim →Act-mOVA chimeras (Fig. S1), indicating that Bim- forced these observations with OT-I and OT-I Bim / →B6 and − − independent mechanisms of clonal deletion are functioning. OT-I Bim / →RIP-mOVA displaying similar numbers of mature These data suggest that OT-I thymocytes are efficiently deleted OT-I CD8SP thymocytes while the number of these cells was in the thymus in response to ubiquitously expressed OVA re- reduced approximately 50-fold in OT-I→RIP-mOVA chimeras gardless of Bim expression. These results recapitulate those compared with OT-I→B6 chimeras (Fig. 2C). Collectively, this observed in previous studies (16–18), providing further support rescue of the mature OT-I CD8SP population by Bim deficiency that Bim is not required for clonal deletion to UbA and dem- suggests that clonal deletion of TRA-specific thymocytes is onstrate that the OT-I model is comparable to other models. completely abrograted in the absence of Bim.

Fig. 1. Bim is not required for negative selection to UbA. (A) CD4 by CD8 proﬁle of Vα2+ thymocytes from OT-I→B6 (n = 7), OT-I→Act-mOVA (n = 3), OT-I − − − − Bim / →B6 (n = 8), and OT-I Bim / →Act-mOVA (n =3).(B) Number of Vα2+ DP and Vα2+ CD8+ thymocytes recovered from thymi of indicated strains. Data depict means and are representative of the indicated number of samples. *P < 0.05, **P ≤ 0.01.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114834109 Suen and Baldwin Downloaded by guest on September 26, 2021 within the Vα2+ population compared with the OT-I→B6 and − − OT-I Bim / →B6 chimeras (Fig. 3B). Similar to the OT-I→B6 and − − OT-I Bim / →B6 chimeras, the Vα2+ splenocytes from OT-I − − Bim / →RIP-mOVA chimeras were largely CD8+.Asinthe thymus, we observed down-regulation of the CD8 coreceptor, suggesting antigen encounter in the periphery (Fig. 3 B and C). − − OT-I→B6 and OT-I Bim / →B6 chimeras had similar numbers of Vα2+ CD8+ T cells, demonstrating that Bim does not signiﬁcantly alter mature peripheral T-cell numbers (Fig. 3B). As expected, Vα2+ CD8+ T-cell numbers were signiﬁcantly reduced in the OT-I→RIP-mOVA chimeras, demonstrating effective deletion of these autoreactive T cells through central and peripheral tolerance − − mechanisms (Fig. 3B). However, in the OT-I Bim / →RIP- − − mOVA chimeras, there was an abundance of OT-I Bim / T cells in the periphery (Fig. 3B), further suggesting that not only was thymic clonal deletion abrogated but peripheral clonal deletion was impaired as well. The few OT-I T cells that did enter the periphery in OT-I→RIP-mOVA chimeras expressed high levels of CD44 (Fig. 3C). It is unclear whether CD44 induction occurred in response to antigen or lymphopenia. Interestingly, Vα2+ CD8+ T − − cells from the OT-I Bim / →RIP-mOVA chimeras had a CD44lo phenotype, suggesting that they were not overtly activated in the periphery, even though cognate antigen was present and CD8 dulling was apparent (Fig. 3C). It has been previously shown that adoptive transfer of 5 × 106 OT-I T cells into a RIP-mOVA mouse will result in rapid onset of diabetes (24). However, blood glucose readings obtained for up to 14 wk post-BM transplant demon-

− − IMMUNOLOGY strated that these OT-I Bim / →RIP-mOVA chimeras did not develop diabetes despite that, on average, there were 6.5 × 107 OT-I T cells present in the spleen. These data suggest that al- − − though OT-I Bim / thymocytes escaped deletion in the thymus of RIP-mOVA mice and survived in the periphery, they were unable A fi Fig. 2. Bim is required for negative selection to TRA. ( ) CD4 by CD8 pro le to cause diabetes. of Vα2+ thymocytes from OT-I→B6 (n =7),OT-I→RIP-mOVA (n = 8), OT-I Bim−/−→B6 (n =8),andOT-IBim−/−→RIP-mOVA (n = 7) and CD8 expression + + TRA-Specific T Cells Are Functionally Impaired in the Periphery of OT-I of Vα2 CD8 thymocytes from indicated strains. (B)CD24byVα2profile of −/− C Bim →RIPmOVA Chimeras. Recently, it was demonstrated that CD8 thymocytes from indicated strains. ( ) Cell numbers of indicated pop- −/− + ulations from indicated mice. Data are representative of the indicated Bim CD4 T cells were unable to produce cytokines following −/− number of samples over four independent experiments. ****P ≤ 0.0001. NS, activation (25). Therefore, if OT-I Bim T cells were func- − − no significance. tionally impaired, it would explain why the OT-I Bim / →RIP- mOVA chimeras did not develop diabetes. However, pathogen − − challenge experiments with OT-I Bim / T cells showed no such TRA-Specific T Cells Persist in the Periphery in the Absence of Bim. To −/− impairment in effector function (26). To further study the ef- determine the fate of the OT-I Bim CD8SP thymocytes that fector capacity of peripheral Vα2+ CD8+ T cells from the OT-I − − escaped clonal deletion in the thymus, we examined the periphery Bim / →RIP-mOVA chimeras, we stimulated carboxy fluores- α + of the chimeras. The proportion of V 2 splenocytes was similar cein succinimidyl ester (CFSE)-labeled bulk splenocytes from the −/− in the OT-I→B6 chimeras and the OT-I Bim →B6 chimeras. In chimeras with either SIINFEKL-pulsed congenic splenocytes or + the OT-I→RIP-mOVA chimeras, the proportion of Vα2 sple- plate-bound anti-CD3/28 antibodies and examined their ability to − − nocytes was significantly reduced compared with the OT-I→B6 proliferate and produce cytokines. While OT-I and OT-I Bim / −/− control chimera, whereas in the OT-I Bim →RIP-mOVA chi- splenocytes from the B6 chimeras proliferated equally on days + meras, the proportion of Vα2 cells was similar to what was seen 2 and 3 poststimulation, only a minority of splenocytes from − − − − in the OT-I Bim / →B6 chimeras (Fig. 3A). Further examination the OT-I Bim / →RIP-mOVA chimeras proliferated in response of the Vα2+ splenocytes for CD4 and CD8 expression revealed to SIINFEKL-pulsed splenocytes (Fig. 4A). Interestingly, pro- − − a similar proportion of CD8+ T cells in the OT-I→B6 and OT-I liferation of CD8 T cells from OT-I Bim / →RIPmOVA chi- − − Bim / →B6 chimeras, although the CD4+ T-cell population was meras was comparable to B6 controls upon stimulation with − − larger in the OT-I Bim / →B6 mice (Fig. 3B). OT-I→RIP-mOVA plate-bound anti-CD3/28 (Fig. 4A). At 4 h poststimulation with chimeras had a dramatically reduced proportion of CD8+ T cells SIINFEKL-pulsed splenocytes, a minor fraction of Vα2+ CD8+

Table 1. Bim is required for clonal deletion to TRA Chimeras OT-I→B6 OT-I→RIP-mOVA OT-I Bim−/−→B6 OT-I Bim−/−→RIP-mOVA

Vα2+DPBright 6.38 ± 4.00 4.44 ± 2.52 13.4 ± 6.26 4.90 ± 3.15 Vα2+CD4+CD8int 4.81 ± 2.15 2.32 ± 0.672 13.1 ± 3.04 5.71 ± 3.41 Vα2+DPDull 5.81 ± 3.20 4.61 ± 1.52 9.26 ± 4.12 7.81 ± 5.97 Vα2+CD8SP 4.69 ± 1.94 0.129 ± 0.045 12.4 ± 4.44 8.43 ± 3.17

Absolute number of Vα2+ thymocytes from the indicated strains. Data are compiled from seven to eight mice (mean ± SD × 106).

Suen and Baldwin PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 −/− splenocytes from the B6 control chimeras were producing low levels from OT-I→B6 and OT-I Bim →B6 chimeras, we examined − − of TNFα, whereas the splenocytes from the OT-I Bim / →RIP- CD69 surface expression 1 d poststimulation. The percentage of − − mOVA chimera were not producing any cytokines (Fig. 4B). To OT-I T cells that up-regulated CD69 in the OT-I Bim / →RIP- −/− mOVA chimeras was similar to those from the OT-I→B6 and further examine the effector function of the OT-I and OT-I Bim −/− T cells, following 3 d of stimulation with SIINFEKL-pulsed sple- OT-I Bim →B6 chimeras. However, the median fluorescence nocytes, we rested splenocytes for 2 d in the presence of IL-2 before intensity of CD69 was lower on the OT-I T cells from the OT-I −/−→ → restimulating them with SIINFEKL-pulsed splenocytes for another Bim RIPmOVA chimeras compared with the OT-I B6 and −/−→ → 4 h. After restimulation, the majority of Vα2+ CD8+ splenocytes OT-I Bim B6 chimeras. OT-I T cells from OT-I RIPmOVA → −/−→ chimeras did not up-regulate CD69 following antigen challenge from both OT-I B6 and OT-I Bim B6 chimeras produced −/− − − (Fig. S2A). Collectively, these data indicate that the OT-I Bim cytokines. In contrast, fewer of the OT-I Bim / splenocytes from −/− T cells from RIP-mOVA chimeras are functionally impaired. As the OT-I Bim →RIP-mOVA chimeras produced cytokines −/− the OT-I Bim T cells from B6 chimeras are unaffected, this (Fig. 4B). Interestingly, in contrast to the proliferation data, anti- C impaired function is not due to the absence of Bim but rather CD3/28 stimulation did not rescue cytokine production (Fig. 4 ). suggests that recognition of high-affinity self-antigen in the thy- Furthermore, it was apparent that the subset of cells undergoing mus or periphery induces nonresponsiveness. It is currently un- division was responsible for the cytokine production we observed clear whether the lack of responsiveness is due to dominant B −/−→ (Fig. S2 ). To ensure that OT-I T cells from OT-I Bim RIP- suppression by Treg or recessive anergy, but these data are con- mOVA chimeras were able to interact with antigen as well as those sistent with recent data showing that defects in the Bcl-2 regulated apoptotic pathway leads to an increase in CD25loFoxp3+ anergic CD4+ T cells (27). Discussion It was previously thought that Bim-mediated apoptosis was required for clonal deletion of thymocytes; however, recent data demonstrate that in the case of clonal deletion to UbA, Bim is not required (16–18). Our data from the Act-mOVA chimeras supports the findings of these previous reports, where Bim is essential for thymocyte apoptosis but not clonal deletion to UbA, suggesting Bim-independent clonal deletion mechanisms exist. One such candidate protein that may mediate Bim-independent clonal deletion is the orphan nuclear receptor Nur77 or its family member Nor1; however, the mechanism underlying this form of clonal deletion has not yet been determined. Although in the absence of Bim clonal deletion to UbA appears to be intact, the fact remains that Bim-deficient mice develop autoimmunity (14). In the present study, we demonstrated that unlike clonal deletion to UbA, Bim deficiency completely abrogates clonal deletion to TRA. Whereas defective antigen encounter may explain these data, we find this unlikely as CD8 dulling in the − − thymic Vα2+ CD8SP compartment in OT-I Bim / →RIP-mOVA chimeras suggests antigen encounter. Furthermore, recent work by the Hogquist group determined that Nur77, a gene induced by TCR stimulation, is up-regulated in OT-I CD8SP thymocytes from − − OT-I Bim / →RIP-mOVA chimeras, which indicated there is TCR stimulation and activation of a transcriptional program (28). Finally, it is unlikely that the abrogated clonal deletion resulted from the killing of mOVA expressing APC in the thymus (29) − − because we did not find CD44hi OT-I Bim / cells in the thymus of − − Rip-mOVA recipients and the OT-I Bim / cells from Rip-mOVA recipients had impaired effector function and did not induce diabetes. Collectively, these data demonstrate that, unlike clonal deletion to UbA, thymic clonal deletion to TRA is impaired in the absence of Bim. Importantly, this is a unique report of differential thymocyte intrinsic molecular requirements for clonal deletion to UbA versus TRA downstream of the TCR. These data also demonstrate that other mediators of clonal deletion, such as Nur77 or Nor1, cannot compensate for the absence of Bim in clonal deletion to TRA. Although we have demonstrated that the absence of Bim allows escape of TRA-specific T cells from clonal deletion, we were un- Fig. 3. TRA-specific T cells persist in the periphery in the absence of Bim. (A) able to replicate autoimmune disorders associated with Bim de- SSC by Vα2profile of splenocytes from indicated strains. OT-I→B6 (n =7), fi − − − − ciency. Again, CD8 coreceptor dulling on peripheral OT-I T cells OT-I→RIP-mOVA (n = 8), OT-I Bim / →B6 (n = 8), and OT-I Bim / →RIP-mOVA −/−→ + from OT-I Bim RIP-mOVA chimeras suggested antigen en- (n = 7) and percentage of Vα2 splenocytes from indicated strains. (B) CD4 by fi CD8 profile of Vα2+ splenocytes from indicated strains and Vα2+ CD8+ cell counter in the periphery and this nding is supported by recent numbers from indicated strains. (C) CD8 and CD44 expression of Vα2+ CD8+ data from Moran et al. that demonstrated elevated Nur77 ex- α + + −/−→ T cells from indicated strains. Data are representative of at least five mice pression in V 2 CD8 T cells from OT-I Bim RIP-mOVA from each strain over four independent experiments. ****P ≤ 0.0001. NS, no chimeras (28). This lack of autoimmunity suggests that in the ab- significance. sence of thymic clonal deletion, either a dominant or recessive

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1114834109 Suen and Baldwin Downloaded by guest on September 26, 2021 mechanism of tolerance was established to control these TRA specific CD8 T cells that underwent lymphopenia-induced pro- − − reactive T cells in the OT-I Bim / →RIP-mOVA chimeras. There liferation remained CD44lo (31). It is also possible that anergy was − − are several mechanisms by which OT-I Bim / T cells may be induced in the thymus but maintained by continuous cognate an- controlled in the periphery of RIP-mOVA mice. First, the OT-I tigen encounter in the periphery. Because we did not observe PD-1 − − − − Bim / T cells could be rendered anergic upon self-antigen en- to be expressed on thymocytes or peripheral OT-I Bim / T cells counter either in the thymus or periphery. It is currently unclear from RIP-mOVA chimeras, this state of anergy is unlikely to result − − whether the OT-I Bim / thymocytes that encountered OVA as from T-cell exhaustion (Fig. S3B). However, we cannot rule out a TRA in the thymus, but were not deleted, are functional. Be- the expression of other inhibitory cell surface receptors implicated cause thymic anergy is considered a form of negative selection, we in T-cell exhaustion. −/− cannot discount the thymus as the location of anergy induction. Second, OT-I Bim T cells from RIP-mOVA chimeras could Previous work has demonstrated that transfer of Bim-deficient become subject to regulation by Treg. We did not detect any ap- + + DO11.10 T cells into mice that express a soluble form of OVA are preciable increase in CD25 CD4 T cells or thymocytes in the −/−→ A anergized (30). However, in this situation, the anergic DO11.10 OT-I Bim RIP-mOVA chimeras (Fig. S3 ). However, it has − − fi Bim / T cells proliferated and up-regulated CD44 following been shown that high-af nity antigen encounter can result in dif- − − + + + adoptive transfer. Because the peripheral OT-I Bim / T cells ferentiation into CD4 Foxp3 CD25 Treg cells. Therefore, it is fi were CD44lo,wefind it unlikely that they underwent antigen- possible that survival of high-af nity antigen encounter in the driven proliferation, and therefore the mechanism of tolerance thymus could lead to the generation of an unknown CD8 regula- induction may be different. We cannot discount lymphopenia-in- tory population from the OT-I thymocytes. The Santamaria group fi fi + duced proliferation because it was recently demonstrated that self- has recently identi ed low-af nity autoregulatory CD8 Tcells that can potently suppress diabetogenic T cells (32). The possibility exists that either a traditional CD4+ Treg or a CD8+ regulatory cell − − population was induced in the OT-I Bim / →RIP-mOVA chimeras. Experiments designed to test for the presence of a dominant regulatory population are required to address this possibility. − − Finally, the OT-I Bim / T cells may be signaled to further differentiate into another inactive subset (27). This possibility has recently been demonstrated for CD4+ T cells (27). However, further experiments are necessary to examine this possibility. IMMUNOLOGY As plate-bound anti-CD3/28 stimulation rescued proliferation but not cytokine production compared with splenocyte stim- ulations, our data suggest that proliferation may be inhibited by regulatory populations or coinhibitory receptors, whereas intrinsic mechanisms may inhibit cytokine production. This finding again reinforces the fact that the immune system has developed “fail-safe” mechanisms to control autoreactive T cells and limit the potential for autoimmunity. Collectively, our data demonstrate that whereas the proapoptotic molecule Bim is not required for clonal deletion to UbA, it is required for clonal deletion to TRA. This is the first demonstration of differences in the molecular requirements of clonal deletion to TRA versus UbA downstream of the TCR. Although clonal deletion to TRA is abrograted in the absence of Bim, the OT-I T cells remained tolerant. Although the exact nature of this tolerance is unknown, understanding the regulatory mechanisms at play in this situation should provide insight into understanding how autoimmunity progresses beyond impaired clonal deletion. Materials and Methods Mice. C57BL/6 mice were purchased from the National Cancer Institute and The Jackson Laboratory. C57BL/6-Tg(Ins2-TFRC/OVA)296Wehi/WehiJ (RIP- mOVA) mice (22), C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) mice (33), and C57BL/6-Tg(TcraTcrb)1100Mjb/J (Act-mOVA) mice (21) were purchased from The Jackson Laboratory. OT-I Bim−/− mice were kindly provided by Dr. Maureen McGargill (St. Jude Children’s Research Hospital, Memphis, TN). All mice, except Act-mOVA, were bred and maintained in our colony at the University of Alberta, treated in accordance with protocols approved by the University of Alberta Animal Care and Use Committee.

Bone Marrow Chimeras. Donor mice were injected i.p. with 100 μg of puriﬁed anti-CD8 Ab (clone 2.43) on days −2 and −1 before BM harvest to deplete CD8 T cells. BM from the femur, tibia, and humerus was harvested in EasySep media (PBS, 2% FCS, 2 mM EDTA) and was passed through 70-μm nylon cell Fig. 4. CD8 T cells from OT-I Bim−/−→RIP-mOVA chimeras are functionally strainers (Fisherbrand). BM was depleted of T cells using the EasySep Mouse impaired. (A) CFSE stain dilution of Vα2+ CD8+ splenocytes from indicated FITC Selection kit (Stemcell Technologies) and FITC conjugated anti-Thy1.2 Ab strains 2 and 3 d after stimulation. (B and C) IFNγ by TNFα staining of in- (eBiosciences). Between 5 and 10 × 106 BM cells were injected into the tail dicated strains at indicated time points after splenocyte or plate-bound vein of lethally irradiated (1,000 Gy) recipient mice. Mice were provided with α-CD3/28 stimulation, respectively. Data are representative of at least three antibiotic water (40 mg neomycin, 15 mg polymyxin per 1 L) for 4 wk post- mice per strain over three independent experiments. injection. Mice were allowed to reconstitute for at least 8 wk before analysis.

Suen and Baldwin PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 Antibodies and Flow Cytometry. All ﬂuorochrome-conjugated and bio- Cytokine Assay. Effectors and stimulators were treated as above, but without tinylated antibodies (Ab) were purchased from eBiosciences except for anti- CFSE labeling, and mixed at a ratio of 3:1 in RP10. On day 3 poststimulation, cells active caspase 3 (Asp-175), which was purchased from Cell Signaling Tech- were washed and resuspended in RP10 plus 1 ng/mL IL-2. On day 5, effectors and nology. Cells were stained with Ab mixtures in FACS buffer (PBS, 1% FCS, freshly pulsed stimulators were mixed at a 3:1 ratio. A total of 3 μg of Brefeldin A 0.02% sodium azide) for 30 min on ice. Cells were washed twice with FACS per 1 mL of culture volume was added to each culture 4 h before harvest and buffer between primary and secondary staining mixtures. Cells were treated anaylsis. Cytokines where detected by internal staining and ﬂow cytometry. with the BD Fix/Perm kit (BD Biosciences) for intracellular staining for active caspase 3 or intracellular cytokines. Cell events were collected on a FACSCanto Blood Glucose Levels. Blood glucose levels were determined using a One- II (BD Biosciences) and analyzed by FlowJo software (Tree Star). Touch UltraMini system with OneTouch Ultra Test Strips. Blood samples (∼5 μL) were obtained by tail vein bleeding. Readings were taken once per week Proliferation Assay. Stimulator splenocytes were harvested from B6 Ly5.1/5.2 and mice were considered diabetic when blood glucose levels exceeded mice, washed, and resuspended at 20 × 106 cells/mL in pure FCS and pulsed 300 mg/dL for 2 consecutive weeks. Blood glucose levels were measured for with 100 nM SIINFEKL peptide at 37 °C for 1 h with gentle shaking every 15– 8–14 wk after BM graft. 20 min. Stimulators were washed three times with 2% FCS–PBS and resusp- ened in RP10 (RPMI, 10% FCS, 5 mM Hepes, 50 units (mg)/mL penicillin/ Statistical Analysis. Mean, SD, and P values were calculated using Prism streptocycin, 2 mM L-glutamine, 50 mM 2-mercaptoethanol, 50 mg/mL gen- tamicin sulfate) at 20 × 106 cells/mL. Plates were prepared with 10 μg/mL of software (GraphPad). α-CD3 and 5 μg/mL of α-CD28 for 1 h at 37 °C. Plates were blocked with 2% BSA in PBS. Effectors were harvested from indicated mice, washed, and ACKNOWLEDGMENTS. The authors thank Qian Hu and Dr. Kristin Hogquist resuspended in sterile PBS at 10 × 106 cells/mL. A total of 1 μL of a 1.25-mM for helpful suggestions and critical review of the manuscript, Dr. Maureen McGargill for the OT-I Bim−/− mice, and Mr. Bing Zhang for excellent tech- solution of CFSE in DMSO was added per 10 × 106 cells, which were then in- nical assistance. This work was supported by Canadian Institutes of Health cubated for 10 min at 37 °C with regular mixing. Staining was quenched with Research Grant MOP-86595 (to T.A.B). T.A.B. is supported by Alberta Heri- RP10 and cells were washed once in RP10 and the cells were resuspended in tage Foundation for Medical Research Scholar and Canadian Institutes for 6 RP10 to 10 × 10 cells/mL. Effectors were mixed with stimulators at a ratio of Health Research New Investigator Awards; and A.S. was supported by a 3:1 and allowed to incubate at 37 °C for the indicated time points. Canadian Institutes of Health Post Graduate Scholarship Masters Award.

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