Graft versus host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of non-human primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3+ T cells from five cohorts: allogeneic transplant recipients receiving 1) no immunoprophylaxis (No Rx); 2) sirolimus monotherapy (Siro); 3) tacrolimus-methotrexate (Tac-Mtx); as well as 4) autologous transplant recipients (Auto) and 5) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for allo-reactive T cells and determine the impact of both mTOR and calcineurin inhibition on GVHD-mediated pathway dysregulation. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function and cytokine synthesis. Within these pathways we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD. This transcriptome enables a systems-based approach to the identification of targets for disease control, many of which are immediately amenable for clinical evaluation. The first such target identified, AURKA should now be considered a lead target for prevention of GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD.

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Although there is evidence linking hematopoietic chimerism-induction and solid organ transplant tolerance, the mechanistic requirements for chimerism-induced tolerance are not clearly elucidated. To address this, we used an MHC-defined primate model to determine the impact of impermanent, T cell-poor, mixed-chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow + renal transplant (“BMT/renal”) and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40-directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole-blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p= 0. 46), with histopathology documenting T-cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28-/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28-negative Tem and costimulation blockade-resistant rejection. These results suggest that in some settings, transient T cell-poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.

The integrin avb6 activates latent transforming growth factor-b (TGF-b) within the kidney and may be a target for the prevention of chronic allograft fibrosis after kidney transplantation. However, TGF-b also has known immunosuppressive properties that are exploited by calcineurin inhibitors (CNIs); thus, the net benefit of avb6 inhibition remains undetermined. To assess the acute impact of interference with avb6 on acute rejection, we tested a humanized avb6-specific monoclonal antibody (STX-100) in a randomized, double-blinded, placebo-controlled nonhuman primate renal transplantation study to evaluate whether avb6 blockade alters the risk of acute rejection during CNI-based immunosuppression. Rhesus monkeys underwent renal allotransplantation under standard CNI-based maintenance immunosuppression; 10 biopsy-confirmed rejection-free animals were randomized to receive weekly STX-100 or placebo. Animals treated with STX-100 experienced significantly decreased rejection-free survival compared to placebo animals (p ¼ 0.049). Immunohistochemical analysis confirmed avb6 ligand presence, and avb6 staining intensity was lower in STX-100-treated animals (p ¼ 0.055), indicating an apparent blockade effect of STX-100. LAP, LTBP-1 and TGF-b were all decreased in animals that rejected on STX-100 compared to those that rejected on standard immunosuppression alone, suggesting a relevant effect of avb6 blockade on local TGF-b. These data caution against the use of avb6 blockade to achieve TGF-b inhibition in kidney transplantation.