*Purpose: Costimulation blockade therapeutics for immunosuppression in transplant are less toxic than calcineurin inhibitors offering improved graft longevity and patient quality of life, but belatacept-resistant rejection (BRR) has slowed the wide-spread adoption of costimulation blockade therapeutics in the clinic. To overcome BRR we need to target other costimulatory pathways to enhance immunosuppression by belatacept. The purpose of this study is to determine in mice if agonizing TIGIT, a T cell immune receptor with Ig and ITIM domains, will suppress activated or memory T cells responding to allograft and additionally increase the suppressive capacity of Tregs to work in concert with CTLA-4Ig.

*Methods: To assess the efficacy of an agonistic anti-TIGIT antibody, we used a minor antigen mismatch model of skin graft with the model antigen OVA in addition to full allogenic mismatch with Balb/C to B6 skin grafts and evaluated graft survival and T cell responses in the graft, draining lymph nodes, and spleens of recipient mice. Mice were given 1e⁶ OT-I and OT-II T cells by adoptive transfer or 10e⁶ donor splenocytes prior to bilateral skin graft and received immunosuppression (or vehicle control) on days 0, 2, 4, and 6 after grafting. Animals were sacrificed at day 10 or monitored long-term for graft survival.

*Results: Combination therapy of the TIGIT agonist with CTLA-4Ig improved graft survival compared to individual therapy with either CTLA-4Ig or TIGIT agonist with an MST beyond 80 days, compared to 25 days with CTLA-4Ig alone (p=0.0007). Monitoring OVA-specific cells revealed that this increased survival with dual therapy is accompanied by a reduction of antigen-specific CD8 T cells compared to CTLA-4Ig treatment alone (p= 0.0367 by unpaired t-test). This reduction of activated CD8 T cells was recapitulated in a full allograft model of skin graft with a 16% reduction in central memory CD8+ T cells in combination therapy versus CTLA-4Ig alone (p value = 0.0238). Further we show that the reduction in graft-specific T cells in the presence of CTLA-4Ig + TIGIT agonist compared to the individual therapies is due to an increase in cell death by measuring caspase 3/7 activity, and not changes in proliferative capacity of cytotoxic T cells by measurement of Cell Trace Violet dilutions. Using conditional knock out mice where TIGIT is specifically knocked out only in Foxp3 expressing cells, we show that the decrease in graft-specific CD8 T cell responses is dependent on TIGIT expressing Tregs.

*Conclusions: Together, these data show that TIGIT agonism, when combined with CTLA-4Ig, can promote the death of CD8 T cells involved in allograft rejection in a Treg-dependent manner, improving skin graft survival outcomes in mice.

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