*Purpose: Optimizing Regulatory T cells (Tregs) survival is of utmost importance to maximize the efficacy of utilizing them in adoptive cell therapy. We previously described a novel pathway for activated Tregs to undergo self-inflicted damage by granzyme pathways. Work has been underway to manipulate this pathway to preserve Tregs and prolong their lifespan after transfer.

*Methods: Here, we examined the effect of TORC1 inhibition on Tregs homeostasis by both in vitro and humanized mouse model studies.

*Results: We show that in activated human Tregs granzymes (Gr) A and B leak from cytotoxic granules and lead to their apoptosis, despite an increase in endogenous inhibitor proteinase inhibitor 9. Confocal imaging shows GrA and GrB outside of the CD107⁺ granules both in the cytosol and in the nucleus of Tregs. The release of GrB in the cytosol significantly activates its protease activity, leading to multiple substrates cleavage. Gr knockdown in healthy human Tregs with shRNA-expressing lentiviruses protects them from undergoing apoptosis. We hypothesize that decreasing GrB expression in Tregs could protect them from succumbing to apoptosis via granzymes. TORC1 inhibitors have been known to reduce GrB expression in Tregs. In vitro studies show GrB production and self-inflicted damage in activated human Tregs that was reversed by TORC1 inhibition (~2 folds decrease, P*<0.05). We also utilize a humanized mouse model to study the effect of rapamycin treatment on transferred human Tregs overall survival. We observed in the rapamycin-treated group that (57% ±8%) of Tregs expressed GrB compared to 75% of the Tregs in the control group (n=6, p* < 0.05). Subsequently, we also observed that the apoptosis of Tregs from the rapamycin-treated group also decreased to 66% from (80% ±5%) in the control group (n= 6, p* < 0.05). Furthermore, we also observed a variation in GrB expression in Tregs isolated from unidentified human peripheral blood samples. This led us to perform genotypic analysis to identify two known GrB single nucleotide polymorphisms (SNPs), rs714436 and rs8192917, and correlate them with the corresponding GrB expression. However, we found no correlation between the identified SNPs and GrB production (n = 20). By using cytometry by time of flight (CyTOF), we observed an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. Those GrB-expressing Tregs were significantly more apoptotic than non-GrB expressing Tregs (n=20, P*<0.05). We are currently in the process of identifying renal transplant patients on TORC1 inhibitors and performing CyTOF to observe their effects on Tregs homeostasis in these patients.

*Conclusions: These findings might help improve transferred Tregs survival by designing therapies that target their granzymes production.

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