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Using methods to inhibit the NFkB family member c-Rel, a transcription factor that upon antigen receptor triggering regulates lymphocyte survival and proliferation, we developed a novel strategy to diminish alloactivation of T cells while preserving GVT activity.
We investigated the role of c-Rel during GVHD in MHC mismatched as well as MHC matched, minor antigen mismatched murine HSCT models. c-Rel-/- T cells caused significantly less GVHD than normal T cells, as determined by survival (p<0.001), clinical GVHD score (p<0.01), and histopathology of GVHD target organs. On day 3 post transplant, proliferation and activation of c-Rel-/- T cells was impaired (p<0.001 CFSElow/high ratio; p<0.01 CD25+ donor cells) and pSTAT5 expression was decreased (p<0.05), resulting in less expansion of donor derived effector T cells by day 7 after transplant (p<0.001). Unexpectedly, serum levels of IL-2 were increased on day 7, likely secondary to decreased pSTAT5-mediated negative feed back on IL-2 secretion. In addition, IL-21, a target gene of c-Rel and negative regulator of IL-2, was decreased (p<0.05) in recipients of c-Rel-/- T cells during early GVHD. By day 14 post BMT we observed increased numbers of donor derived Tregs with increased CD25 expression in recipients of c-Rel-/- T cells (p<0.05), suggesting that the increased IL-2 levels primarily benefited the expansion of Tregs. Consistent with this hypothesis, in vivo depletion of donor Tregs in recipients of c-Rel inhibited Foxp3DTR transgenic T cells exacerbated GVHD.
We next evaluated if c-Rel deficient T cells were able to mediate antitumor activity. We challenged allogeneic HSCT recipients with either a liquid tumor (A20) or a solid tumor (RENCA) and found that GVT activity in c-Rel-/- T cells recipients was intact in the absence of GVHD, resulting in significantly improved survival compared to recipients of wildtype T cells (p<0.001 A20; p<0.01 RENCA). Using syngeneic GVT models with EL4 as well as B16 we could demonstrate strong anti-tumor activity of c-Rel deficient polyclonal T cells targeting EL4 as well as melanoma antigen-specific pmel-1 transgenic T cells in the absence of T cell alloactivation, reinforcing the notion of separation of GVHD from GVT activity through inhibition of c-Rel signaling.
The potential for clinical translation of our approach is highlighted by a series of experiments testing the efficacy of a recently developed Pyrimidinetrione-based small molecule c-Rel inhibitor compound. Using T cells that were pre-incubated with this c-Rel inhibitor, we reproduced our above described effects on GVHD and antitumor activity (Figure).
Taken together, our findings identify c-Rel as a promising target for the development of a clinical strategy to prevent GVHD while preserving GVT activity, and to possibly even enable adoptive T cell therapy across MHC barriers that would be incompatible with a conventional transplant approach.
