*Purpose: Costimulation blockade with belatacept (CTLA-4 Ig) offers improved renal function and reduced toxicity following transplantation compared to calcineurin inhibitors. Nonetheless, episodes of acute T cell-mediated rejection remain a significant clinical concern. While memory CD8⁺ T cells are recognized to be relatively resistant to costimulation blockade, evaluation of the impact of CTLA-4 Ig on CD8⁺ T cell differentiation has been limited by the absence of defined allogeneic CD8⁺ T cell epitopes in mouse and human, precluding the analysis of endogenous antigen-specific populations using MHC tetramers.

*Methods: We evaluated the C57BL/6 H-2(b)-restricted endogenous CD8⁺ T cell response to an allogeneic H-2L(d)-restricted epitope (termed “QL9”) using an MHC class I tetramer and a magnetic enrichment method.

*Results: We found that the QL9/L(d) tetramer specifically stained naïve CD8⁺ T cells in a peptide-dependent manner. The naïve CD8⁺ T cell compartment of C57BL/6 H-2(b) mice contained approximately 4×10⁴ QL9/L(d) specific T cells per spleen, corresponding to a precursor frequency of approximately 10-fold greater than previously reported precursor frequencies of T cells specific for microbe-derived epitopes. Following allogeneic sensitization with L(d)-expressing BALB/c cells, numbers of QL9/L(d)-specific CD44^hi CD8⁺ T cells expanded approximately 5-fold relative to naïve mice. The QL9/L(d)-specific effector CD8⁺ T cell pool was comprised of predominantly CD62L^loCD45RB^lo effector cells. Treatment with CTLA-4 Ig during allogeneic sensitization greatly inhibited the differentiation of endogenous allo-specific effector CD8⁺ T cells overall, and skewed the effector CD8⁺ T cell population towards a CD62L^hiCD45RB^hi phenotype. Evaluation of endogenous memory QL9/L(d) CD8⁺ T cells 35 days after sensitization revealed that untreated and CTLA-4 Ig-treated mice contained similar frequencies of endogenous allo-specific CD62L^hi central memory cells.

*Conclusions: Future studies will evaluate the functional capacities of these distinct populations of effector CD8⁺ T cells. Together, these data demonstrate that CTLA-4 Ig inhibits a specific population of endogenous, alloreactive CD62L^loCD45RB^lo effector cells, and that endogenous alloreactive CD62L^hiCD45RB^hi effector cells are relatively resistant to CTLA-4 Ig. This novel approach to tracking endogenous allogeneic CD8⁺ T cells has facilitated new insight into the relative susceptibilities of endogenous CD8⁺ T cells to immunosuppression in vivo.

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