The purpose of this study was to investigate the role of mTOR versus calcineurin inhibition in anti-tumoral CD8⁺ T cell responses in posttransplant malignancy. To precisely study this in an antigen-specific way, we used B6 recipients, cytotoxic T (cT) cells from TCR-transgeneic B6.OTI recipients and B6.OVA-transgeneic donors in skin transplant experiments. OVA-expressing B16F10 melanoma cells were used as targets in tumor experiments. Also, we created CD8-OTI-transgeneic reporter mice by crossbreeding DsRed-expressing B6.Nagy mice with B6.OTI mice to track color-coded OTI cT cells in anti-tumoral and alloresponses in vivo. First, we explored the differentiation and function of alloreactive cT cells in vitro by stimulating cT cells from OTI mice with OVA-transgeneic APCs in the presence of rapamycin (Rapa) or cyclosporine A (CsA). Rapa (1, 10 and 100 ng/mL), but not CsA (50, 100, 500, 1000 ng/mL), induced a dose-dependent phenotypic shift from cT effector-memory (EM) cells to highly tumor-reactive cT central-memory (CM) cells. Importantly, cT memory cells treated with Rapa up-regulated T-bet and Eomes and showed preserved levels of IFNΓ and perforin (FACS and qRT-PCR). In contrast, CsA blocked expression of T-bet, IFNΓ and perforin, indicating that mTOR, but not calcineurin, inhibition might spare functional anti-tumoral cT memory cells in transplant recipients. To test this hypothesis in vivo, we adoptively transferred purified OTI or DsRed⁺OTI cT cells into B6 recipients of either OVA-transgeneic skin transplants or subcutaneous OVA-B16F10 tumors. While OVA-skin allograft survival was equally prolonged in both Rapa- and CsA-treated recipients (15.5±2.6 and 15.1±2.6 vs 11.9±1.9 days in controls), OVA-melanoma growth was enhanced (37%) in CsA-treated mice, but substantially reduced (59%) with Rapa-treatment (vs. controls). Moreover, DsRed⁺OTI cT cells of CM phenotype could be readily recovered from 20 lymphoid organs and tumor infiltrates of Rapa-treated B6 recipients (FACS analysis). Therefore, mTOR, but not calcineurin, inhibition preserves anti-tumoral immune responses in this model, which may be important for understanding and reducing posttransplant malignancy.

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