Cell Autonomous Role of TGFβ and IL-2 Receptor in the In Vivo Generation of CD4 and CD8 Inducible Regulatory T Cells During Graft-Versus-Host Disease,

Abstract 4015

FoxP3⁺ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from expansion of pre-existing natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor conventional T cells (Tconvs) in the transplant recipient. Although the co-adoptive transfer of nTregs or in vitro -derived iTregs has been shown to prevent the development of GVHD, the relative contribution of these two Treg subsets in protection against GVHD has been unclear. To investigate the contribution of the different FoxP3⁺ Treg subsets, we used a MHC-mismatched mouse model of acute GVHD. Lethally irradiated (500cGy × 2) B6D2F1.SJL (H-2^bxd) host mice were injected with T cell-depleted bone marrow cells and FACS-sorted Tconvs (WT or Foxp3-deficient) with or without FACS-sorted Tregs of C57BL/6 (H-2^b) mouse origin. Weight loss in mice receiving Foxp3-deficient Tconvs alone was significantly more pronounced compared to other groups. The presence of either donor-derived nTregs or iTregs alone protected against GVHD-induced weight loss but was suboptimal compared to the presence of both donor-derived nTregs and iTregs.

Next, we sought to determine how the donor-derived Treg pool was established during acute GVHD and tracked the appearance of Tregs in the secondary lymphoid organs at different time points post transplant. On Day 8 post GVHD induction, ∼5% of the donor-derived CD4⁺ T cells in the spleen were FoxP3⁺. We found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Unexpectedly, we found a significant fraction of CD8⁺FoxP3⁺ T cells (1–3% of all CD8⁺ T cells) in the spleen and in the lymph nodes. These CD8⁺FoxP3⁺ T cells representing ∼70% of the iTreg pool on Day 8 post GVHD induction. These CD8⁺FoxP3⁺ T cells shared phenotypic markers with their CD4⁺ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4⁺ and CD8⁺ Tregs expanded in vivo with IL-2 treatment and required IL-2 and TGFβ receptor expression for their generation.

In summary, we found that donor derived-iTregs are generated during GVHD and contribute to suppression of acute GVHD induced-weight loss. Surprisingly, CD8⁺Foxp3⁺T cells were a major contributor to the donor derived-iTreg pool after transplantation. The generation of CD8⁺ and CD4⁺ iTregs occurred at least in part by a cell autonomous IL-2 and TGFβ receptor-dependent mechanism. Thus, our data suggest that in addition to increasing nTregs, concomitant strategies aimed at enhancing the conversion of donor-derived Tconvs to iTregs for example by engaging the IL-2 and TGFβ signaling pathways might be beneficial for the treatment of GVHD.