Our immune system protects us from disease by recognizing and mounting a defence against harmful pathogens that enter our bodies. T cells, a type of white blood cell, play a key role in this process. Each T cell has a unique protein called a T cell receptor on its surface that is able to recognize particular pieces of pathogens. Together, the millions of T cells in our bodies, each with its own unique T cell receptor, can initiate an immune response to eliminate a vast array of potential pathogens.

T cells are made in an organ called the thymus. During this production process, immature T cells are generated, including some with T cell receptors that recognize the harmless molecules that make up our bodies. If allowed to enter the bloodstream and left to their own devices, these T cells could trigger an immune response against the body, leading to the development of autoimmune disease.

Normally, many of these ‘auto-reactive’ T cells are instructed to die in the thymus by a process called negative selection. Furthermore, auto-reactive cells that escape into the blood can also be shut down by additional failsafe mechanisms. Given the success of these failsafe mechanisms, notably the effectiveness of a class of T cells called T regulatory cells, some researchers have begun to ask if negative selection is necessary to prevent autoimmunity.

During negative selection T cells die as a result of a process called apoptosis. Multiple proteins have been implicated in T cell apoptosis, including Bim, Puma and the Nur77 family of nuclear receptors. Blocking the function of some these proteins individually can rescue some autoreactive T cells from death: however, this rarely results in the development of autoimmune disease.

Burger et al. have now created a mouse strain with T cells that produce large amounts of a mutant form of the anti-apoptotic protein, Bcl-2, which can block the function of multiple pro-apoptotic proteins, including Bim and Puma. Additionally, it has been proposed that Bcl-2 can be converted to a pro-apoptotic protein by Nur77 proteins, but the Bcl-2 proteins in the mutant strain are able to resist this process.

T cells in the thymus of the mutant mice were highly resistant to apoptosis accompanying negative selection. Moreover, as the mice aged, they accumulated autoreactive T cells in the blood, which led to symptoms of autoimmune disease and early death. While various failsafe mechanisms were engaged, they did not provide sufficient protection. The work of Burger et al. thus provides strong evidence that negative selection in the thymus is crucial for the prevention of autoimmune disease.

Maintenance of T cell self-tolerance is essential for the prevention of autoimmune diseases. A series of mechanisms are thought to be involved in establishing T cell tolerance, the first of which is deletion of potentially self-reactive T cells in the thymus. During T cell development, autoreactive T cells bind self-peptide presented on major histocompatibility complex (MHC) molecules with high affinity and receive strong T cell receptor (TCR) signals that trigger apoptosis and clonal deletion. Some of these cells can also differentiate down an alternative pathway to a harmless lineage, such as T regulatory cells (T_reg), or also CD8αα and invariant NK T cells (Stritesky et al., 2012). The importance of this process, termed negative selection, in establishing self-tolerance and preventing autoimmunity was best demonstrated in studies of mice deficient in the autoimmune regulator (Aire) gene. These mice develop a multi-organ autoimmune disease as the result of defective deletion of auto-reactive T cells specific to tissue-restricted antigens (TRA), which would normally be expressed by medullary thymic epithelial cells under the control of AIRE (Anderson et al., 2002). Humans with loss of AIRE function develop similar disease pathology (Finnish-German APECED Consortium., 1997; Nagamine et al., 1997). However, additional models that implicate a breakdown in negative selection as the driving force behind the development of autoimmune disease are lacking (von Boehmer and Melchers, 2010). This might suggest that under most circumstances, subsequent tolerizing mechanisms in the peripheral compartments such as anergy or T_reg-mediated suppression are sufficient to compensate for defective thymic deletion. Even in the case of AIRE-deficiency, impaired tolerization of CD4⁺ T cells in the periphery by extra-thymic AIRE expressing cells likely contributes to the development of autoimmunity (Gardner et al., 2013). This has cast doubt on the relative importance of negative selection for maintaining immunological self-tolerance.

Apoptosis of thymocytes during negative selection is dependent on the mitochondrial apoptosis pathway regulated by the Bcl-2 family of proteins (Tischner et al., 2010). In particular, the pro-apoptotic, Bcl-2 homology domain 3 (BH3) only protein, Bim, which is upregulated in response to strong TCR signals, is essential for this process. Bim and other BH3-only proteins are thought to promote apoptosis by binding and inhibiting anti-apoptotic Bcl-2 proteins (such as Bcl-2, Bcl-X_L and Mcl-1) that directly suppress downstream effectors Bax and Bak, which are responsible for disrupting mitochondrial membrane integrity (Youle and Strasser, 2008). Bim-deficiency has been shown to cause defective thymocyte apoptosis in several TCR transgenic (HY, OTI, OTII) models of negative selection (Bouillet et al., 2002; Moran et al., 2011; Zhan et al., 2011; Suen and Baldwin, 2012). However, this apoptotic defect does not lead to emergence of autoreactive mature T cells except in the case of negative selection to TRA (Hu et al., 2009; Kovalovsky et al., 2010; Suen and Baldwin, 2012). Interestingly, Bim^−/− mice on a mixed 129/Sv X C57BL/6 background develop a systemic lupus erythematosus (SLE)-like autoimmune disease, indicative of a breakdown in tolerance (Bouillet et al., 1999). However, loss of Bim in other immune compartments likely contributes to the disease, especially B cells, which are important mediators of SLE pathology. This phenotype was greatly ameliorated on the autoimmune-resistant C57BL/6 background and is distinct from the multi-organ, T cell dominant disease found in AIRE-deficient mice (Bouillet et al., 2001; Labi et al., 2014). Recently, a study showed that additional loss of Puma (Bbc3), another BH3-only protein, enhanced the Bim^−/− thymic deletion defect and led to the development of immune pathology more similar to that found in AIRE-deficient mice (Gray et al., 2012). T cells from these mice were able to transfer the disease, lending support to it being T cell-driven. However, the role of non-T cells in these mice cannot be excluded due to germline deletion of Puma and Bim. In addition, T-cell specific over-expression of Bcl-2, which can inhibit both Bim and Puma (Chen et al., 2005), does not lead to autoimmunity (Sentman et al., 1991; Linette et al., 1995), suggesting that a defect in T cells alone may be insufficient to cause disease. Alternatively, redundant negative selection pathways involving Bim, Puma and the Nur77 family members (See below) may not allow Bcl-2 over-expression to block all pathways leading to negative selection.

In addition to Bim, the Nur77 family of orphan steroid receptors, which includes Nur77, Nor-1 and Nurr1, has also been implicated in apoptosis accompanying negative selection. Nur77 expression, like that of Bim, is induced by strong TCR signals that result in negative selection. Low expression of both proteins has been correlated with defective clonal deletion in Non-obese Diabetic (NOD) mice (Sohn et al., 2003; Liston et al., 2004). Furthermore, T cell-specific over-expression of Nur77, or Nor-1, results in massive apoptosis of thymocytes (Cheng et al., 1997b). Expression of a dominant negative Nur77 protein that can block all family members results in inhibition of apoptosis in the F5 and HY TCR transgenic models of negative selection (Calnan et al., 1995; Zhou et al., 1996). Deficiency in Nur77 alone (Nr4a1^−/−) results in moderate inhibition of apoptosis accompanying negative selection in the OTII and BDC2.5 TCR transgenic models (Fassett et al., 2012). Interestingly, the Nur77 family has also been shown to transcriptionally regulate Foxp3 expression and deletion of all three Nur77 family members (Nr4a3^−/− X Cd4-Cre⁺Nr4a1^fl/flNr4a2^fl/fl) results in impaired T_reg cell production and development of a quick onset, severe autoimmune disease resembling that of Foxp3-deficient mice (Sekiya et al., 2013). Therefore, Nur77 could contribute to the maintenance of immunological tolerance both by facilitating deletion of autoreactive thymocytes and ensuring production of T_reg cells. The mechanism by which Nur77 and its family members promote thymocyte apoptosis has long been under investigation. We and others showed that strong TCR signals induce the translocation of Nur77 and Nor-1 from the nucleus to the mitochondria in thymocytes, a phenomenon first reported to occur in cancer cells in response to apoptotic stimuli (Lin et al., 2004; Thompson and Winoto, 2008; Wang et al., 2009). At the mitochondria, Nur77 and Nor-1 bind Bcl-2 and cause exposure of Bcl-2's BH3 domain, the ‘death’ domain of the Bcl-2 family of proteins. Bcl-2 BH3 exposure correlates with thymocyte apoptosis in TCR-stimulated thymocytes and HY and F5 TCR transgenic thymocytes undergoing negative selection (Thompson and Winoto, 2008). BH3-exposed Bcl-2 might act similarly to BH3-only protein Bim to bind and inhibit other anti-apoptotic Bcl-2 proteins, such as Bcl-X_L, as was demonstrated in cancer cells (Kolluri et al., 2008). It is thus possible that Nur77 converts Bcl-2 to a pro-apoptotic effector during negative selection, preventing over-expression of Bcl-2 from completely blocking negative selection.

Here, we generated a T cell-specific Bcl2 BH3 mutant transgenic mouse, in which Bcl-2's purported pro-apoptotic BH3 function should be abolished by changing its conserved BH3 residues, GDD, to alanines (Cheng et al., 1997a). We found that over-expression of both wild-type and BH3 mutant Bcl-2 efficiently rescued thymocyte apoptosis in two TCR transgenic models of negative selection. However, the BH3 mutant transgene more effectively blocked TCR-induced thymocyte apoptosis in vitro and better rescued high affinity TCR clones from deletion in polyclonal systems in vivo. Interestingly, BH3 mutant transgenic mice, in contrast to the reported wild-type Bcl2 transgenic mouse phenotype, developed multi-organ autoimmune pathology and died around one year-of-age. Thus, we provide strong evidence that a breakdown in thymocyte apoptosis during negative selection is indeed sufficient to cause autoimmune disease.

Bcl-2 family proteins and the Nur77 family of orphan steroid receptors are critical mediators of thymocyte apoptosis during negative selection. However, whether thymocyte deletion is necessary to maintain immunological self-tolerance remains under debate. Here, we find that T cell-specific over-expression of a Bcl2 BH3 mutant transgene results in T cell-driven multi-organ autoimmunity, strongly suggesting that a defect in thymic deletion is indeed sufficient to cause a breakdown in tolerance. Consistent with previous observations, we found that T cell-specific over-expression of wild-type Bcl-2 did not cause significant autoimmune pathology (Linette et al., 1995). However, our results show that Bcl-2 Tg mice may develop autoimmunity at low penetrance, as some mice exhibited a modest accumulation of activated T cells and autoantibodies. In contrast, expression of a Bcl2 BH3 mutant transgene significantly increased the severity of the disease and resulted in greater than 70% lethality by one year-of-age. A significant accumulation of activated T cells was observed starting at 20 to 30 weeks-of-age and ultimately resulted in massive lymphoid hyperplasia, inflammation, lymphocyte infiltration into non-lymphoid organs and organ-targeted auto-antibody accumulation. This phenotype of low ANA antibody titers, activated T cells and organ-targeted autoantibodies is suggestive of a primarily T cell-mediated disease.

Our data in the Nur77^GFP and superantigen mouse models indicate that the Bcl-2 BH3 mutant is better able to rescue autoreactive thymocytes from negative selection than wild-type Bcl-2. Abolishing the Nur77-dependent pro-apoptotic function of Bcl-2 by BH3 mutation might therefore enhance the anti-apoptotic effects of Bcl-2 over-expression. Alternatively, it is also possible that slightly higher BH3 transgene expression levels in DP and CD4 SP cells could be responsible for this phenotype, although the levels of transgene expression do not necessarily correlate with GFP expression as demonstrated by the higher GFP M.F.I. in CD4⁺ T cells of Bcl-2 over BH3 Tg mice (Figure 5A). Cause set aside, a greater defect in negative selection could allow a threshold to be crossed in which other mechanisms of maintaining tolerance are no longer sufficient to prevent disease. This would be consistent with the recent study comparing the severity of disease in Bim^−/− vs Bim^−/−Bbc3^−/− mice. Bim^−/− mice on the C57BL/6 background were reported to have attenuated autoimmunity and 80% of them still survive at 70 weeks-of-age (Bouillet et al., 2001; Labi et al., 2014). Additional loss of Puma (Bbc3), however, enhanced the thymic deletion defect of Bim^−/− mice and resulted in more severe, T cell-driven multi-organ autoimmune pathology (Gray et al., 2012). It remains to be seen if loss of Bim and Puma in T cells alone would result in the same phenotype. Our observations in a T cell-specific model provide strong evidence that a severe thymic clonal deletion defect is sufficient to cause autoimmune pathology. Arguably, a defect in peripheral T cell deletion due to increased T cell survival may also contribute to the BH3 Tg phenotype. Although we cannot completely rule out this possibility, the fact that Bcl-2 Tg mice do not develop autoimmunity would suggest a minimal role for defective peripheral tolerance in this model.

Despite the large disparity in autoimmune phenotype, we found that both BH3 and Bcl-2 Tg thymocytes were significantly protected from apoptosis in TCR transgenic models of negative selection. Previously, Bcl-2 over-expression was directly compared with Bim-deficiency in the HY model and Bcl-2 over-expression fell significantly short of Bim-deficiency in rescuing thymocytes from deletion (Bouillet et al., 2002). However, in our thymic slice assay of HY TCR-mediated deletion, we find that Bcl-2 over-expression almost completely blocks thymocyte death. Similarly, over-expression of Bcl-2 in F5 TCR transgenic mice dramatically blocked thymocyte deletion by thymic slice assay and in vivo peptide injection. One possible explanation for the discrepancy between our results and previously published observations might be the negative selection models used. Apoptosis of HY TCR transgenic mice is notoriously difficult to block due to accelerated T cell development and early expression of the transgenic TCR (Baldwin et al., 2005). Indeed, using thymic organ culture, another group has previously reported a significant inhibition of negative selection by Bcl-2 in the F5 model (Williams et al., 1998). Regardless, however, we have demonstrated that Bcl-2 over-expression has a potent anti-apoptotic effect on negative selection in a normal, polyclonal system. Mirroring observations in Bim^−/−/Nur77^GFP mice (Stritesky et al., 2013), we observed a striking increase in GFP-high post-selection thymocytes in Bcl-2/Nur77^GFP mice, indicating a significant rescue of high affinity TCR clones from deletion. Here, over-expression of the BH3 mutant transgene resulted in substantially greater rescue of GFP-high DP and CD4 SP thymocytes compared to over-expression of wild-type Bcl2. BH3 transgene expression also enhanced rescue of vβ5 and vβ6 DP and CD4 SP thymocytes from deletion in a polyclonal superantigen model of negative selection. Increased rescue of these cells in the thymus might lead to the development of a more self-reactive T cell repertoire, accelerating the expansion of activated T cells in BH3 Tg mice.

Differential expression of the BH3 vs wild-type Bcl2 transgenes in the CD8 T cell compartment might also contribute to the development of autoimmunity in BH3 Tg, but not Bcl-2 Tg mice. Cd4-driven transgene expression in the BH3 transgenic mice preferentially enhances CD4 T cell seeding of the peripheral lymphoid organs. BH3 Tg mice have about a 3:1 ratio of CD4 to CD8 T cells in the lymph nodes compared to a 2:1 ratio in wild-type mice. In Bcl-2 Tg mice, the proportions are skewed in the opposite direction, resulting in a 1:1 ratio. This is likely due to a combination of transgene-biased rescue of cells from negative selection and increased survival of transgene-expressing T cells. BH3 Tg mice are therefore expected to have increased numbers of autoreactive CD4 T cells, which as key mediators of autoimmunity, could be responsible for the acceleration of disease in these mice (Palmer and Weaver, 2010). Alternatively, as Bcl-2 over-expression reduces the ability of T cells to proliferate (Linette et al., 1996; Cheng et al., 2004), downregulation of the BH3 transgene in CD8 T cells might enhance their functional capacity over that of Bcl-2 Tg CD8 T cells. As CD8 T cells play a well-documented role in T cell-mediated, tissue-specific autoimmune diseases (i.e. multiple sclerosis and diabetes), more functionally competent autoreactive CD8 T cells in the BH3 Tg mice might contribute to the development of disease (Gravano and Hoyer, 2013).

In response to defective clonal deletion in the thymus, we observed compensatory induction of alternative tolerizing mechanisms that might prevent earlier onset of autoimmunity in BH3 Tg mice. An increase in the proportion and number of T_reg cells was noted in both the thymus and lymph nodes of young mice. Interestingly, CD25⁻ T_reg cells accounted for most of the increase in total T_reg numbers and these cells have been reported to have reduced suppressive function (Zhan et al., 2011). These CD25⁻ T_reg cells are thought to be derived from diverted, autoreactive cells that were not deleted in the thymus. Consistent with this, CD25⁻ T_reg cells in the thymus expressed GFP at a higher level than CD25⁺ T_reg cells and at a similar level to the most GFP-high naïve CD4 SP cells in the BH3/Nur77^GFP mice. Hence, diverting a portion of these GFP-high autoreactive cells to this lineage might be the more effective tolerizing strategy. Notably, our observation that CD25⁺ T_reg proportions were not increased in the BH3 Tg mice suggests that T cells with self-reactive TCRs are not automatically committed to the T_reg lineage and that there is likely a saturated niche for CD25⁺ T_reg development determined by availability of other factors, such as IL-2 and IL-15 (Stritesky et al., 2012). In addition to increased T_reg cells, we observed a 2-fold increase in the proportion of anergic CD4 T cells in the BH3 and Bcl-2 Tg mice. This was similarly observed in Bim^−/− mice (Stritesky et al., 2013). Induction of anergy could cooperate with diversion to the T_reg lineage/T_reg suppression to inhibit T cell-mediated promotion of autoimmunity and prevent early onset of disease. However, differential induction of these tolerizing mechanisms was not observed in BH3 Tg vs Bcl-2 Tg mice and therefore cannot account for the eventual development of autoimmunity in BH3 Tg, but not Bcl-2 Tg mice. Interestingly, in aged BH3 Tg mice, T_reg cells made up almost 50% of the CD4 T cell pool in mice displaying autoimmune pathology. However, this protective response was still apparently insufficient to suppress autoimmunity.

Finally, the enhanced ability of the Bcl2 BH3 mutant transgene to rescue autoreactive thymocytes from apoptosis compared to wild-type Bcl2 could suggest that the Bcl-2 BH3 domain might indeed have some pro-apoptotic function. This finding may provide in vivo support for the notion that Nur77 promotes thymocyte apoptosis through conversion of Bcl-2 to a pro-apoptotic effector. However, this still needs to be further confirmed in a knock-in model. Additionally, analysis of a Bcl-2 protein that cannot interact with Nur77 would also be required to confirm that Bcl-2 pro-apoptotic function is Nur77-dependent. Intriguingly, it has previously been suggested that Nur77 and Bim might function in the same pathway, as Bim-deficiency was unable to synergize with Nur77-deficiency to inhibit thymocyte apoptosis in the OTII TCR transgenic model of negative selection (Fassett et al., 2012). If Bcl-2 conversion is indeed critical for Nur77-mediated thymocyte apoptosis, this would support the idea that Nur77 and Bcl-2 family members come together at the mitochondria to cooperatively regulate cell death during negative selection.

1. 1. Adlam M
   2. Siu G

   (2003) Hierarchical interactions control CD4 gene expression during thymocyte development

   Immunity 18:173–184.

   https://doi.org/10.1016/S1074-7613(03)00021-9

   • Google Scholar
2. 1. Anderson MS
   2. Venanzi ES
   3. Klein L
   4. Chen Z
   5. Berzins SP
   6. Turley SJ
   7. von Boehmer H
   8. Bronson R
   9. Dierich A
   10. Benoist C
   11. Mathis D

   (2002) Projection of an immunological self shadow within the thymus by the aire protein

   Science 298:1395–1401.

   https://doi.org/10.1126/science.1075958

   • Google Scholar
3. 1. Baldwin TA
   2. Sandau MM
   3. Jameson SC
   4. Hogquist KA

   (2005) The timing of TCR alpha expression critically influences T cell development and selection

   The Journal of Experimental Medicine 202:111–121.

   https://doi.org/10.1084/jem.20050359

   • Google Scholar
4. 1. Bouillet P
   2. Cory S
   3. Zhang LC
   4. Strasser A
   5. Adams JM

   (2001) Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim

   Developmental Cell 1:645–653.

   https://doi.org/10.1016/S1534-5807(01)00083-1

   • Google Scholar
5. 1. Bouillet P
   2. Metcalf D
   3. Huang DC
   4. Tarlinton DM
   5. Kay TW
   6. Kontgen F
   7. Adams JM
   8. Strasser A

   (1999) Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity

   Science 286:1735–1738.

   https://doi.org/10.1126/science.286.5445.1735

   • Google Scholar
6. 1. Bouillet P
   2. Purton JF
   3. Godfrey DI
   4. Zhang LC
   5. Coultas L
   6. Puthalakath H
   7. Pellegrini M
   8. Cory S
   9. Adams JM
   10. Strasser A

   (2002) BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes

   Nature 415:922–926.

   https://doi.org/10.1038/415922a

   • Google Scholar
7. 1. Calnan B
   2. Szychowski S
   3. Chan FKM
   4. Cado D
   5. Winoto A

   (1995) A role of the orphan steroid receptor Nur77 in apoptosis accompaying antigen-induced negative selection

   Immunity 3:273–282.

   https://doi.org/10.1016/1074-7613(95)90113-2

   • Google Scholar
8. 1. Chen L
   2. Willis SN
   3. Wei A
   4. Smith BJ
   5. Fletcher JI
   6. Hinds MG
   7. Colman PM
   8. Day CL
   9. Adams JM
   10. Huang DC

   (2005) Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function

   Molecular Cell 17:393–403.

   https://doi.org/10.1016/j.molcel.2004.12.030

   • Google Scholar
9. 1. Cheng EH
   2. Kirsch DG
   3. Clem RJ
   4. Ravi R
   5. Kastan MB
   6. Bedi A
   7. Ueno K
   8. Hardwick JM

   (1997a) Conversion of Bcl-2 to a Bax-like death effector by caspases

   Science 278:1966–1968.

   https://doi.org/10.1126/science.278.5345.1966

   • Google Scholar
10. 1. Cheng LE
    2. Chan FK
    3. Cado D
    4. Winoto A

    (1997b) Functional redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell apoptosis

    The EMBO Journal 16:1865–1875.

    https://doi.org/10.1093/emboj/16.8.1865

    • Google Scholar
11. 1. Cheng N
    2. Janumyan YM
    3. Didion L
    4. Van Hofwegen C
    5. Yang E
    6. Knudson CM

    (2004) Bcl-2 inhibition of T-cell proliferation is related to prolonged T-cell survival

    Oncogene 23:3770–3780.

    https://doi.org/10.1038/sj.onc.1207478

    • Google Scholar
12. 1. Dzhagalov IL
    2. Chen KG
    3. Herzmark P
    4. Robey EA

    (2013) Elimination of self-reactive T cells in the thymus: a timeline for negative selection

    PLOS Biology 11:e1001566.

    https://doi.org/10.1371/journal.pbio.1001566

    • Google Scholar
13. 1. Dzhagalov IL
    2. Melichar HJ
    3. Ross JO
    4. Herzmark P
    5. Robey EA

    (2012)

    Two-photon imaging of the immune system

    Current Protocols in Cytometry / Editorial Board, J Paul Robinson, Managing Editor... [et al.], Chapter 12 Unit12 26, 10.1002/0471142956.cy1226s60.

    • Google Scholar
14. 1. Fassett MS
    2. Jiang W
    3. D'Alise AM
    4. Mathis D
    5. Benoist C

    (2012) Nuclear receptor Nr4a1 modulates both regulatory T-cell (Treg) differentiation and clonal deletion

    Proceedings of the National Academy of Sciences of USA 109:3891–3896.

    https://doi.org/10.1073/pnas.1200090109

    • Google Scholar
15. 1. Finnish-German APECED Consortium

    (1997) An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains

    Nature Genetics 17:399–403.

    https://doi.org/10.1038/ng1297-399

    • Google Scholar
16. 1. Gardner JM
    2. Metzger TC
    3. McMahon EJ
    4. Au-Yeung BB
    5. Krawisz AK
    6. Lu W
    7. Price JD
    8. Johannes KP
    9. Satpathy AT
    10. Murphy KM
    11. Tarbell KV
    12. Weiss A
    13. Anderson MS

    (2013) Extrathymic Aire-expressing cells are a distinct bone marrow-derived population that induce functional inactivation of CD4(+) T cells

    Immunity 39:560–572.

    https://doi.org/10.1016/j.immuni.2013.08.005

    • Google Scholar
17. 1. Gravano DM
    2. Hoyer KK

    (2013) Promotion and prevention of autoimmune disease by CD8+ T cells

    Journal of Autoimmunity 45:68–79.

    https://doi.org/10.1016/j.jaut.2013.06.004

    • Google Scholar
18. 1. Gray DH
    2. Kupresanin F
    3. Berzins SP
    4. Herold MJ
    5. O'Reilly LA
    6. Bouillet P
    7. Strasser A

    (2012) The BH3-only proteins Bim and Puma cooperate to impose deletional tolerance of organ-specific antigens

    Immunity 37:451–462.

    https://doi.org/10.1016/j.immuni.2012.05.030

    • Google Scholar
19. 1. Hu Q
    2. Sader A
    3. Parkman JC
    4. Baldwin TA

    (2009) Bim-mediated apoptosis is not necessary for thymic negative selection to ubiquitous self-antigens

    Journal of Immunology 183:7761–7767.

    https://doi.org/10.4049/jimmunol.0902181

    • Google Scholar
20. 1. Kolluri SK
    2. Zhu X
    3. Zhou X
    4. Lin B
    5. Chen Y
    6. Sun K
    7. Tian X
    8. Town J
    9. Cao X
    10. Lin F
    11. Zhai D
    12. Kitada S
    13. Luciano F
    14. O'Donnell E
    15. Cao Y
    16. He F
    17. Lin J
    18. Reed JC
    19. Satterthwait AC
    20. Zhang XK

    (2008) A short Nur77-derived peptide converts Bcl-2 from a protector to a killer

    Cancer Cell 14:285–298.

    https://doi.org/10.1016/j.ccr.2008.09.002

    • Google Scholar
21. 1. Kovalovsky D
    2. Pezzano M
    3. Ortiz BD
    4. Sant'Angelo DB

    (2010) A novel TCR transgenic model reveals that negative selection involves an immediate, Bim-dependent pathway and a delayed, Bim-independent pathway

    PLOS ONE 5:e8675.

    https://doi.org/10.1371/journal.pone.0008675

    • Google Scholar
22. 1. Labi V
    2. Woess C
    3. Tuzlak S
    4. Erlacher M
    5. Bouillet P
    6. Strasser A
    7. Tzankov A
    8. Villunger A

    (2014) Deregulated cell death and lymphocyte homeostasis cause premature lethality in mice lacking the BH3-only proteins Bim and Bmf

    Blood 123:2652–2662.

    https://doi.org/10.1182/blood-2013-11-537217

    • Google Scholar
23. 1. Lin B
    2. Kolluri SK
    3. Lin F
    4. Liu W
    5. Han YH
    6. Cao X
    7. Dawson MI
    8. Reed JC
    9. Zhang XK

    (2004) Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3

    Cell 116:527–540.

    https://doi.org/10.1016/S0092-8674(04)00162-X

    • Google Scholar
24. 1. Linette GP
    2. Hess JL
    3. Sentman CL
    4. Korsmeyer SJ

    (1995)

    Peripheral T-cell lymphoma in lckpr-bcl-2 transgenic mice

    Blood 86:1255–1260.

    • Google Scholar
25. 1. Linette GP
    2. Li Y
    3. Roth K
    4. Korsmeyer SJ

    (1996) Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation

    Proceedings of the National Academy of Sciences of USA 93:9545–9552.

    https://doi.org/10.1073/pnas.93.18.9545

    • Google Scholar
26. 1. Liston A
    2. Lesage S
    3. Gray DH
    4. O'Reilly LA
    5. Strasser A
    6. Fahrer AM
    7. Boyd RL
    8. Wilson J
    9. Baxter AG
    10. Gallo EM
    11. Crabtree GR
    12. Peng K
    13. Wilson SR
    14. Goodnow CC

    (2004) Generalized resistance to thymic deletion in the NOD mouse; a polygenic trait characterized by defective induction of Bim

    Immunity 21:817–830.

    https://doi.org/10.1016/j.immuni.2004.10.014

    • Google Scholar
27. 1. Mamalaki C
    2. Elliott J
    3. Norton T
    4. Yannoutsos N
    5. Townsend AR
    6. Chandler P
    7. Simpson E
    8. Kioussis D

    (1993) Positive and negative selection in transgenic mice expressing a T-cell receptor specific for influenza nucleoprotein and endogenous superantigen

    Developmental Immunology 3:159–174.

    https://doi.org/10.1155/1993/98015

    • Google Scholar
28. 1. Mamalaki C
    2. Norton T
    3. Tanaka Y
    4. Townsend AR
    5. Chandler P
    6. Simpson E
    7. Kioussis D

    (1992) Thymic depletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice

    Proceedings of the National Academy of Sciences of USA 89:11342–11346.

    https://doi.org/10.1073/pnas.89.23.11342

    • Google Scholar
29. 1. Martinez RJ
    2. Zhang N
    3. Thomas SR
    4. Nandiwada SL
    5. Jenkins MK
    6. Binstadt BA
    7. Mueller DL

    (2012) Arthritogenic self-reactive CD4+ T cells acquire an FR4hiCD73hi anergic state in the presence of Foxp3+ regulatory T cells

    Journal of Immunology 188:170–181.

    https://doi.org/10.4049/jimmunol.1101311

    • Google Scholar
30. 1. Moran AE
    2. Holzapfel KL
    3. Xing Y
    4. Cunningham NR
    5. Maltzman JS
    6. Punt J
    7. Hogquist KA

    (2011) T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse

    The Journal of Experimental Medicine 208:1279–1289.

    https://doi.org/10.1084/jem.20110308

    • Google Scholar
31. 1. Nagamine K
    2. Peterson P
    3. Scott HS
    4. Kudoh J
    5. Minoshima S
    6. Heino M
    7. Krohn KJ
    8. Lalioti MD
    9. Mullis PE
    10. Antonarakis SE
    11. Kawasaki K
    12. Asakawa S
    13. Ito F
    14. Shimizu N

    (1997) Positional cloning of the APECED gene

    Nature Genetics 17:393–398.

    https://doi.org/10.1038/ng1297-393

    • Google Scholar
32. 1. Palmer MT
    2. Weaver CT

    (2010) Autoimmunity: increasing suspects in the CD4+ T cell lineup

    Nature Immunology 11:36–40.

    https://doi.org/10.1038/ni.1802

    • Google Scholar
33. 1. Scherer MT
    2. Ignatowicz L
    3. Winslow GM
    4. Kappler JW
    5. Marrack P

    (1993) Superantigens: bacterial and viral proteins that manipulate the immune system

    Annual Review of Cell Biology 9:101–128.

    https://doi.org/10.1146/annurev.cb.09.110193.000533

    • Google Scholar
34. 1. Sekiya T
    2. Kashiwagi I
    3. Yoshida R
    4. Fukaya T
    5. Morita R
    6. Kimura A
    7. Ichinose H
    8. Metzger D
    9. Chambon P
    10. Yoshimura A

    (2013) Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis

    Nature Immunology 14:230–237.

    https://doi.org/10.1038/ni.2520

    • Google Scholar
35. 1. Sentman CL
    2. Shutter JR
    3. Hockenbery D
    4. Kanagawa O
    5. Korsmeyer SJ

    (1991) bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes

    Cell 67:879–888.

    https://doi.org/10.1016/0092-8674(91)90361-2

    • Google Scholar
36. 1. Siegel RM
    2. Katsumata M
    3. Miyashita T
    4. Louie DC
    5. Greene MI
    6. Reed JC

    (1992) Inhibition of thymocyte apoptosis and negative antigenic selection in bcl-2 transgenic mice

    Proceedings of the National Academy of Sciences of USA 89:7003–7007.

    https://doi.org/10.1073/pnas.89.15.7003

    • Google Scholar
37. 1. Sohn SJ
    2. Rajpal A
    3. Winoto A

    (2003) Apoptosis during lymphoid development

    Current Opinion in Immunology 15:209–216.

    https://doi.org/10.1016/S0952-7915(03)00004-9

    • Google Scholar
38. 1. Strasser A
    2. Harris AW
    3. Cory S

    (1991) bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship

    Cell 67:889–899.

    https://doi.org/10.1016/0092-8674(91)90362-3

    • Google Scholar
39. 1. Stritesky GL
    2. Jameson SC
    3. Hogquist KA

    (2012) Selection of self-reactive T cells in the thymus

    Annual Review of Immunology 30:95–114.

    https://doi.org/10.1146/annurev-immunol-020711-075035

    • Google Scholar
40. 1. Stritesky GL
    2. Xing Y
    3. Erickson JR
    4. Kalekar LA
    5. Wang X
    6. Mueller DL
    7. Jameson SC
    8. Hogquist KA

    (2013) Murine thymic selection quantified using a unique method to capture deleted T cells

    Proceedings of the National Academy of Sciences of USA 110:4679–4684.

    https://doi.org/10.1073/pnas.1217532110

    • Google Scholar
41. 1. Suen AY
    2. Baldwin TA

    (2012) Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens

    Proceedings of the National Academy of Sciences of USA 109:893–898.

    https://doi.org/10.1073/pnas.1114834109

    • Google Scholar
42. 1. Teh HS
    2. Kishi H
    3. Scott B
    4. Borgulya P
    5. von Boehmer H
    6. Kisielow P

    (1990) Early deletion and late positive selection of T cells expressing a male-specific receptor in T-cell receptor transgenic mice

    Developmental Immunology 1:1–10.

    https://doi.org/10.1155/1990/18208

    • Google Scholar
43. 1. Thompson J
    2. Winoto A

    (2008) During negative selection, Nur77 family proteins translocate to mitochondria where they associate with Bcl-2 and expose its proapoptotic BH3 domain

    Journal of Experimental Medicine 205:1029–1036.

    https://doi.org/10.1084/Jem.20080101

    • Google Scholar
44. 1. Tischner D
    2. Woess C
    3. Ottina E
    4. Villunger A

    (2010) Bcl-2-regulated cell death signalling in the prevention of autoimmunity

    Cell Death & Disease 1:e48.

    https://doi.org/10.1038/cddis.2010.27

    • Google Scholar
45. 1. von Boehmer H
    2. Melchers F

    (2010) Checkpoints in lymphocyte development and autoimmune disease

    Nature Immunology 11:14–20.

    https://doi.org/10.1038/ni.1794

    • Google Scholar
46. 1. Wang A
    2. Rud J
    3. Olson CM Jnr.
    4. Anguita J
    5. Osborne BA

    (2009) Phosphorylation of Nur77 by the MEK-ERK-RSK cascade induces mitochondrial translocation and apoptosis in T cells

    Journal of Immunology 183:3268–3277.

    https://doi.org/10.4049/jimmunol.0900894

    • Google Scholar
47. 1. Williams O
    2. Norton T
    3. Halligey M
    4. Kioussis D
    5. Brady HJ

    (1998) The action of Bax and bcl-2 on T cell selection

    The Journal of Experimental Medicine 188:1125–1133.

    https://doi.org/10.1084/jem.188.6.1125

    • Google Scholar
48. 1. Xue L
    2. Sun Y
    3. Chiang L
    4. He B
    5. Kang C
    6. Nolla H
    7. Winoto A

    (2010) Coupling of the cell cycle and apoptotic machineries in developing T cells

    The Journal of Biological Chemistry 285:7556–7565.

    https://doi.org/10.1074/jbc.M109.035535

    • Google Scholar
49. 1. Youle RJ
    2. Strasser A

    (2008) The BCL-2 protein family: opposing activities that mediate cell death

    Nature Reviews Molecular Cell Biology 9:47–59.

    https://doi.org/10.1038/nrm2308

    • Google Scholar
50. 1. Zhan Y
    2. Zhang Y
    3. Gray D
    4. Carrington EM
    5. Bouillet P
    6. Ko HJ
    7. O'Reilly L
    8. Wicks IP
    9. Strasser A
    10. Lew AM

    (2011) Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells

    Journal of Immunology 187:1566–1577.

    https://doi.org/10.4049/jimmunol.1100027

    • Google Scholar
51. 1. Zhou T
    2. Cheng J
    3. Yang P
    4. Wang Z
    5. Liu C
    6. Su X
    7. Bluethmann H
    8. Mountz JD

    (1996) Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells

    The Journal of Experimental Medicine 183:1879–1892.

    https://doi.org/10.1084/jem.183.4.1879

    • Google Scholar

Author details

1. Megan L Burger

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   MLB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Kenneth K Leung

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   KKL, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Margaux J Bennett

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   MJB, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Astar Winoto

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   AW, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   winoto@berkeley.edu

   Competing interests

   The authors declare that no competing interests exist.

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