Purpose: We have shown that CD57+PD1- CD4 T cells are present in higher numbers in the peripheral blood and allograft biopsies of patients who experienced rejection on belatacept-based immunosuppression compared to patients with a stable outcome, but the mechanisms of their costimulation blockade resistance and the means by which they exert a negative impact on graft rejection are unknown. Therefore, to determine the role that CD57+ CD4 T cells play in BRR, we sought to investigate the in vitro functionality of these cells.

Methods: Peripheral blood lymphocytes were obtained from kidney recipients on the day of transplant and prior to drug administration. Cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Brefeldin A was added after 1 hour to inhibit cytokine release. Flow cytometry was used to assay T cell subsets for surface expression of CD57, CD2, LFA1, VLA4 and CD28, along with intracellular staining for inflammatory cytokines IFNg and TNFa as well as granzyme B.

Results: Analysis of surface molecules revealed a significant inverse correlation of CD57 and CD28 expression (p < 0.01). All CD57+ CD4 T cells expressed high levels of adhesion molecules CD2, LFA1 and VLA4. A significant majority of CD57+CD28- CD4 T cells produce both IFNg and TNFa (p < 0.01) following stimulation. Furthermore, all CD57+CD28- CD4 T cells were granzyme B positive in contrast to CD57-CD28+ CD4 T cells that were granzyme B negative.

Discussion: The increased expression of adhesion molecules on CD57+ CD4 T cells coupled with their ability to produce high levels of IFNg, TNFa, and granzyme B licenses them to infiltrate kidney allografts and mediate rejection. Moreover, the lack of CD28 expression on these highly cytotoxic cells indicates a costimulation independent mechanism of action. This may explain the increased risk of rejection associated with belatacept-based immunosuppression in patients with an elevated percentage of CD57+PD1- CD4 T cells. Future studies utilizing sorted cell populations and mixed lymphocyte reactions will investigate the ability of these cells to proliferate and produce cytokines in response to allogeneic stimulation in the setting of costimulation blockade. The ability to define the role that CD57+PD1- CD4 T cells play in rejection yields a potential therapeutic target to decrease the rates of early acute rejection initially observed with belatacept-based immunosuppression.

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