*Purpose: Belatacept-based immunosuppression confers fewer off-target toxicities compared to CNI but comes at a cost of increased incidence and severity of acute rejection, potentially due to its deleterious effect on both the number and function of Foxp3⁺ Treg. Here we sought to determine if targeting the TIGIT pathway can enhance the function of Tregs in the presence of costimulation blockade.

*Methods: We assessed the efficacy of a TIGIT agonist in combination with CTLA-4Ig vs. CTLA-4Ig treatment alone in a minor antigen mismatch model of murine skin transplantation. Mice were monitored to assess graft survival or sacrificed 10 days after grafting to assess donor-reactive T cell responses in the graft. In addition, we used novel Foxp3-Cre x TIGIT^fl/fl mice in which TIGIT is conditionally knocked (cKO) out from Treg to determine Treg-specific effects of TIGIT agonism.

*Results: TIGIT agonism combined with CTLA-4Ig significantly prolonged graft survival in mice when compared to animals treated with CTLA-4Ig alone, extending the MST from 25 days to 85 days (p= 0.0007). When TIGIT was conditionally knocked out on Tregs, treatment with CTLA-4Ig+TIGIT agonist resulted in more rapid graft rejection compared to wildtype littermate controls treated with the combination therapy (p=0.0107). Analysis of graft-infiltrating T cells revealed that CTLA-4Ig+TIGIT agonist resulted in a significant decrease in CD8⁺ T cell infiltration compared to CTLA-4Ig alone (p=0.0326). CTLA-4Ig+TIGIT agonist also resulted in a significant increase in the number of Tregs in the allograft compared to CTLA-4Ig alone (p=0.0046). These effects were not detected when Treg cKO mice were treated with CTLA-4Ig+TIGIT agonist. Analysis of RNA sequencing revealed 67 differentially expressed genes (DEG) with an adj. p value <0.05 in TIGIT cKO Treg as compared to WT Treg from draining lymph nodes after allograft challenge. Gene set enrichment analysis revealed that DEG in WT vs. cKO Treg are enriched in a Treg vs. T conventional helper cell gene set. Significantly higher expression of genes including Foxp3, Axl, Dusp4, IL-2ra, and IL-2rb in WT Treg vs. cKO Treg contribute to this profile.

*Conclusions: These data demonstrate that a TIGIT agonist synergizes with CTLA-4Ig to promote prolonged allograft survival in a Treg-dependent manner. Mechanistically, TIGIT agonism functions to promote a favorable ratio of Tregs: effector CD8⁺ T cells within the graft in the context of CTLA-4Ig. Together, these data show that TIGIT agonism promotes Treg stability and survival in the face of costimulation blockade and improves allograft outcomes.

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