Bim Regulates the Survival and Suppressive Capability of CD8+ Foxp3+ Regulatory T Cells during Graft Versus Host Disease

Graft versus host disease (GVHD) is a proinflammatory syndrome that is the major complication of allogeneic hematopoietic stem cell transplantation and can lead to significant pathological damage. The failure to reconstitute an effective regulatory T cell compartment is a defining characteristic of this disease and has been the genesis for strategies designed to augment reconstitution of regulatory T cells (Tregs) by either in vivo expansion or adoptive transfer of this population. While CD4⁺ Foxp3⁺ T cells have been the most carefully examined Treg population, CD8⁺ Foxp3⁺ Tregs also emerge early during GVHD, have suppressive function, and can attenuate disease severity. The precise role of these cells and the extent to which CD8⁺ Tregs possess functional and ontological similarities to CD4⁺ Tregs, however, have not been well delineated. Using established murine models of GVHD, we observed that adoptively transferred alloantigen-specific CD8⁺ Tregs had reduced in vivo survival in all target organs and were significantly less potent than CD4⁺ Tregs for the prevention of lethal GVHD. To define the mechanistic basis for their reduced efficacy, we first examined the T cell receptor (TCR) repertoire of in vivo-derived CD4⁺ and CD8⁺ Tregs to determine whether a restricted repertoire was a potential explanation for the diminished ability of CD8⁺ Tregs to prevent GVHD. We employed GVHD models in which both polyclonal CD8⁺ [B6→Balb/c] and alloantigen-specific [B6→Balb.B] CD8⁺ H60⁺ Treg populations could be assessed and used the Shannon entropy index to define TCR repertoire diversity which is critical for the maintenance of self-tolerance by Tregs. Amplicon sequencing of TCR-β rearrangements to analyze the TCR repertoires of in vivo-induced CD8⁺ and CD4⁺ Tregs, along with their conventional CD8⁺ and CD4⁺ T cell counterparts, revealed that polyclonal CD8⁺ and alloantigen-specific CD8⁺ H60⁺ Tregs had repertoire diversity that was similar to that of conventional CD8⁺ T cells, indicating that a more restricted repertoire was not the proximate cause of decreased suppressive capability. Subsequent transcriptional profiling and principal component analysis revealed that CD8⁺ Tregs arising during GVHD possessed a canonical Treg transcriptional signature that was similar to that observed in CD4⁺ Tregs, yet distinct from conventional CD8⁺ T cells. Gene set enrichment and Ingenuity pathway analyses, however, both demonstrated that CD8⁺ Tregs had differential gene expression in pathways involved in cell death and survival when compared to CD4⁺ Tregs. This was further confirmed by gene expression analysis and protein expression studies which revealed that CD8⁺ Tregs had increased expression of the pro-apoptotic proteins Bim, cleaved caspase 3, and cleaved caspase 7, as well as a reduction in the anti-apoptotic protein, Mcl-1. To determine whether Bim was functionally important for CD8⁺ Treg survival, we comparatively examined in vivo-derived CD4⁺ and CD8⁺ Treg induction using wild type Foxp3^EGFP and Bim^-/-Foxp3^EGFP animals as donors in transplantation studies. The absence of Bim in the CD8⁺ Treg compartment resulted in a significant increase in absolute CD8⁺ Treg numbers in the liver, lung, and colon when examined on days 7, 14, and 21 post transplantation. Given that the absence of Bim augmented in vivo-generated CD8⁺ Treg survival, we then examined whether Bim also regulated the survival of adoptively transferred, alloantigen-specific CD8⁺ Tregs which we had observed were inferior to CD4⁺ Tregs for the suppression of GVHD. We noted an increased percentage and absolute number of adoptively transferred CD8⁺ Foxp3⁺ Bim^-/- Tregs relative to wild type CD8⁺ Tregs in all tissue sites, indicating that the absence of Bim resulted in enhanced stabilization of Foxp3 expression and augmented in vivo survival. Finally, animals that were transplanted with in vitro-derived, alloantigen-specific CD8⁺ Tregs from Bim^-/- Foxp3^EGFP mice had significantly prolonged survival relative to mice reconstituted with wild type CD8⁺ Foxp3^EGFP Tregs, demonstrating that the prolonged in vivo survival conferred by the absence of Bim resulted in an increased ability to prevent GVHD. Collectively, these studies demonstrate that Bim plays a critical role in the regulation of CD8⁺ Treg survival which may have clinical implications for the use of regulatory T cell therapy in the treatment and prevention of GVHD.