*Purpose: Tacrolimus (Tac) is an effective anti-rejection drug in kidney transplant recipients (KTR), but its off-target effects make withdrawal desirable. Studies indicate that Tac can be withdrawn in a subset of KTR, but the immune mechanisms that drive successful vs. unsuccessful Tac withdrawal are unknown.

*Methods: We performed microarray analyses of peripheral blood mononuclear cell (PBMC) RNA samples from 18 KTR enrolled in the Clinical Trials in Organ Transplantation (CTOT)-09 study, in which stable patients were randomized to withdraw or continue Tac immunosuppression (Figure 1A). Samples were collected before (6mo post-tx), at the end of Tac withdrawal (9mo post-tx) and 12-18 mo post-tx. We also measured donor-reactive T cells (IFN-g ELISPOT, with and without CD25+ regulatory T cell depletion) and CD4+ and CD8+ T cells for PD1/CD57 expression by flow cytometry as a measure of exhaustion.

*Results: Analysis of differentially expressed genes (DEG) before vs. immediately after Tac withdrawal in subjects who developed acute rejection or donor-specific antibodies (withdrawal rejection, WR, n = 8) revealed upregulation of genes involved in immune activation. DEGs of PBMC RNA from those who successfully weaned Tac (withdrawal stable, WS; n = 6) enriched for downregulation of immune activity and upregulation of apoptosis. CellCODE analyses of gene expression data showed that within the WS subjects, both the downregulation of the T cell activation transcriptional program and upregulation of the apoptotic program following Tac withdrawal were preferentially confined to CD4⁺ and CD8⁺ T cells. Functional analyses of PBMCs suggested stronger donor-reactive T cell responses in WR vs WS without evidence of Tac-induced regulatory T cell dysfunction (Figure 1B-C). Flow cytometry of PBMC from the WS group show higher frequencies of exhaustion marker+ T cells up to 18 mo after Tac withdrawal compared to controls who remained on Tac (no withdrawal, NW; Figure 1D).

*Conclusions: Our data support the hypothesis that successful Tac withdrawal in KTRs can unleash an active, protective T cell program with features of exhaustion.

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