*Purpose: Despite a significantly reduced toxicity profile and improved long-term graft function versus calcineurin inhibitors, widespread adaptation of belatacept (CTLA-4 Ig) has been limited due to acute rejection. Deeper understanding of relevant effector T cell populations that mediate acute rejection will lead to more targeted strategies to prevent it. Following priming, expression of numerous surface receptors has been used to describe effector CD8⁺ T cell populations in different models. However, the phenotype of CD8⁺ T cell effectors after transplantation is not well understood. We sought to characterize the phenotype of effector CD8⁺ T cells that are elicited following grafting in order to identify directed therapeutic approaches to inhibit them.

*Methods: We used a fully allogeneic Balb/c H-2(d) into C57BL/6 H-2(b) mice skin graft model. We utilized an MHC Class I tetramer, H-2L(d)/QL9 peptide, in order to identify antigen-specific CD8⁺ T cells in the spleen and draining lymph nodes following transplantation. CD8⁺ T cells were assessed using multiparameter flow cytometry with both FlowSOM clustering and conventional gating.

*Results: Skin grafting resulted in the expansion of CD44⁺ L(d)/QL9-specific CD8⁺ T cells, with a peak in absolute number and Ki67⁺ at day 10 post-graft. FlowSOM identified a cluster of CD8⁺ effector T cells expressing activated CD43 receptor (clone 1B11) that was elevated at day 10 and decreased at memory timepoints (Day 0 vs Day 10 p=0.0020). CD43⁺ CD8⁺ T cells expressed high levels of Ki67 and Granzyme B relative to CD43^– CD8⁺ T cells (Ki67 p<0.0001, Granzyme B p=0.0035). When stimulated with H-2(d) antigen, the CD43⁺ CD8⁺ T cells population expressed a higher frequency of IFN-gamma (CD43^– 22.82 ± 9.61% vs CD43⁺ 73.82 ± 9.99%, p=0.0480). CD43⁺ CD8⁺ T cells were also formed following CTLA-4 Ig treatment, indicating resistance to CD28 blockade. On day 10, CD43⁺ CD8⁺ T cells expressed high levels of ICOS in the presence of CTLA-4 Ig, suggesting that ICOS is a potential therapeutic target. Treatment with both CTLA4-Ig and ICOS antibody resulted in prolonged Balb/c skin graft survival versus CTLA-4 Ig alone (p=0.0094).

*Conclusions: Using MHC Class I tetramers, we identified a population of CD43⁺ CD8⁺ T cells which are potent effectors. This CD43⁺ population also expresses high levels of ICOS, even in the presence of CTLA-4 Ig. Future experiments will seek to understand the dynamics of CD43⁺ effector function in vivo and identify strategies to inhibit their function.

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