The etiology of costimulatory blockade-resistant allograft rejection remains unclear. Therefore, the purpose of this study was to evaluate the responsiveness of distinct CD4+ and CD8+ T cell subsets to costimulatory blockade in mice.

To study T cell subsets in costimulatory blockade resistant-alloresponses, we used mice genetically deficient for hallmark T cell transcription factors such as B6.RORγt knockout (KO) mice, B6.T-bet KO mice, and B6.RORγt-T-bet double-KO (DKO) mice. Purified T cells from these mice and wt controls were adoptively transferred into B6.Rag-common-γc DKO recipients of fully mismatched Balb/c skin allografts (STx) ± treatment with CTLA4Ig and MR-1.

First, we found that untreated control animals from all mouse strains promptly reject their allografts with similar kinetics, confirming the stringency of our skin Tx model. RORγt KO T cell recipients showed a Th1-mediated allograft rejection (55% IFN-γ+ T cells) while T-bet KO T cell recipients featured a Th17/Th2-driven rejection (15% IL-17+, 28% Gata-3+ T cells). Importantly, DKO T cell recipients were characterized by Th2-mediated allograft rejection (42% Gata-3+ T cells) as confirmed by eosinophilic allograft infiltration. Next, we tested the hypothesis that each T cell subset responds differently to double costimulatory blockade. Importantly, we found that RORγt KO recipients had prolonged allograft survival (MST 76d) upon treatment, whereas T-bet KO and DKO STx recipients rejected promptly like untreated controls (22d, 27d, respectively). Critically, Th1, but not Th17 alloresponses were significantly suppressed by costimulatory blockade as indicated by flow cytometry and ELISA. Suprisingly, we found that DKO T cell recipients failed to mount an alloreactive Th2 response under costimulatory blockade conditions. Instead, we detected significantly higher numbers of Eomes+CD8+ T cells in the rejecting DKO recipients when compared to RORγt or T-bet single KO mice, indicating that Eomes+CD8+ T cells emerge as key effector cells under costimulatory blockade conditions in the absence of RORγt and T-bet.

We conclude that Th1 and Th2 cell-mediated alloresponses may be responsive to costimulatory blockade, whereas Th17 and Eomes+Tc cell-mediated alloresponses drive costimulatory blockade-resistant allograft rejection in mice.

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