Bim is required for thymocyte deletion in a range of settings, however, the absence of organ-specific autoimmunity in Bim-deficient mice suggests that other apoptotic regulators play a role in PTA-mediated deletion. This prediction was borne out by our observations of organ-specific autoimmunity in Rag1•/• mice reconstituted with precursors from Vav-BCL-2 transgenic mice, in which the intrinsic pathway of apoptosis is potently blocked. By reconstituting Rag1•/• mice with hematopoietic precursors from a range of compound mutant mice lacking Bim and other BH3-only proteins, we found that only the combined loss of Puma and Bim provoked a similar pathology in this sensitized assay of autoimmunity. Importantly, aged Bbc3•/•Bcl2l11•/• mice spontaneously developed organ-specific autoimmunity (albeit with a reduced severity), indicating that Puma was unique among BH3-only proteins in synergizing with Bim to impose deletional tolerance of peripheral tissues.
The autoimmunity in Bbc3•/•Bcl2l11•/•
mice was reminiscent of that observed in Aire•/•
mice, but there are important differences in the pathologies and, presumably, the auto-antigens involved. For instance, retinopathy and serum autoantibodies directed against the interphotoreceptor layer develop in Aire•/•
mice (Anderson et al., 2002
) but were not observed in mice deficient in both Puma and Bim. Conversely, the lymphocytic infiltration of the pancreas in Bbc3•/•Bcl2l11•/•
mice (which became destructive in reconstituted Rag1•/•
mice) was not observed in C57BL/6.Aire•/•
mice (Jiang et al., 2005
). These findings suggest that Puma and Bim are not required for deletional tolerance of all Aire-regulated PTA, and conversely, can affect tolerance to Aire-independent PTA.
Defects in T-cell tolerance underpinned the autoimmunity we observed in Bbc3•/•Bcl2l11•/•
mice. Their T-cells showed evidence of increased activation and could transfer autoimmunity to Rag1•/•
recipients. In the thymus, Puma was again unique amongst the BH3-only proteins in preventing further accumulation of SP thymocytes in the absence of Bim. In fact, increased numbers of mature CD69−
SP thymocytes was a prominent, distinguishing feature of Bbc3•/•Bcl2l11•/•
mice in both the polyclonal and TCR transgenic settings. Evidence that this SP defect reflected impaired tolerance came from the TCR transgenic models where compound deficiency in Puma and Bim in the OT-I and OT-II models significantly increased the escape of PTA-reactive thymocytes beyond that in Bcl2l11•/•
mice. Importantly, the increase could not be attributed to an increased proliferative response to the antigen because there was no change in BrdU incorporation by these cells. Rather, our data indicate that Puma is required for normal deletion of semi-mature CD69+
SP thymocytes following high avidity TCR stimulation, extending upon Bim’s function in this process (Villunger et al., 2004
Is the increased production of PTA-reactive T-cells escaping Puma- and Bim-mediated deletion driving the autoimmunity observed in these mice? Defects in thymic deletion cause the autoimmunity observed in Aire•/•
mice (reviewed in (Taniguchi and Anderson, 2011
)), without any requirement for “danger” signals or inflammation (Gray et al., 2007b
). The PTA-mediated deletion of OT-I and OT-II thymocytes was even more severely affected in Bbc3•/•
mice than in Aire•/•
mice (Anderson et al., 2005
; Hubert et al., 2011
), suggesting the lesion to central tolerance caused by crippling the intrinsic pathway of apoptosis would be sufficient to explain the autoimmunity we observed. In addition, there is good evidence that dominant tolerance imposed by Treg
remains intact in Bbc3•/•Bcl2l11•/•
mice (indeed, it may be amplified); CD25+
from mice with deficiencies in the intrinsic pathway of apoptosis function normally and are significantly increased in number (Zhan et al., 2011
), while conventional T-cells from Bbc3•/•Bcl2l11•/•
mice can be efficiently suppressed (Szymczak-Workman et al., 2011
). Therefore, the thymic production of PTA-reactive T-cells in mice deficient in Puma and Bim is likely to be sufficient to provoke autoimmunity, overwhelming peripheral tolerance mechanisms.
By contrast to Aire•/•
mice, thymocytes that avoid deletion in Bbc3•/•Bcl2l11•/•
mice show evidence of antigen encounter in the thymus (increased CD69 and reduced co-receptor expression). This stimulation would presumably increase the scope for the induction of non-deletional negative selection mechanisms, which may explain the finding that Bbc3•/•Bcl2l11•/•
OT-I => RIP-mOVA chimeras did not develop diabetes and exhibited peripheral OT-I T-cells with an anergic phenotype (consistent with the data in Bcl2l11•/•
mice from (Suen and Baldwin, 2012
)). Therefore, among the PTA-reactive clones that escape to the periphery, only some will cause organ-specific autoimmunity, while others may become anergic or be diverted into Treg
. The next challenge is to elucidate the most important synergies between non-deletional pathways and the physiological mediators of deletion we have identified here.
The reason Puma is unique among BH3-only proteins in providing an ancillary function in central tolerance is probably because it can, like Bim, engage in strong interactions with all of the pro-survival Bcl-2 proteins (Chen et al., 2005
; Kuwana et al., 2005
). The increased expression of the anti-apoptotic Bcl-2 family members throughout late thymocyte differentiation would require potent antagonism from BH3-only proteins for efficient induction of apoptosis following high avidity TCR interactions in the medulla. The selective interactions of the other BH3-only proteins (e.g. Noxa, Bmf and Bad) with the prosurvival Bcl-2 family members, or the requirement for death receptor engagement (for Bid activation) might render them ineffective for PTA-mediated thymocyte deletion. It is also possible that the requirement for Puma and Bim in combination might be for direct activation of Bax and Bak (Dewson and Kluck, 2009
). However, we also found that the loss of Noxa, Puma and Bim together was required to phenocopy the Bax•/•Bak•/•
chimeras. Given that the combined loss of Bax and Bak completely cripples the intrinsic apoptotic pathway, this observation indicates that Bim, then Puma, and then Noxa are the major BH3-only proteins involved in the life and death decisions that shape thymocyte differentiation.
Our results also demonstrate that this important pathway is extremely robust. The likelihood that the combination of loss-of-function mutations necessary to completely ablate the intrinsic pathway of apoptosis (i.e. to Noxa, Puma and Bim or Bax and Bak) would manifest in thymocytes is extremely low. It is more probable that partial defects to deletion and anergy or T-cell regulation could cooperate to drive autoimmune disease (e.g. (Teh et al., 2010
)). Understanding the molecular basis to those tolerance mechanisms that synergise with deletion should reveal better ways to deal with autoreactivity and autoimmune disease.