*Purpose: The use of Regulatory T cells (Tregs) in adoptive cell therapy has been hindered by their short survival following their adoptive transfer. We have previously described a novel pathway in which activated Tregs undergo self -induced damage by the cytosolic leakage of their granzymes. Multiple works are initiated to manipulate this pathway in order to prolong Tregs lifespan.

*Methods: Flow sorted human Tregs from healthy donors were stimulated in vitro and adoptively transferred into a humanized skin mouse model in vivo.

*Results: We have shown that Tregs undergo self-inflicted damage due to the leakage of Granzymes (Gr) A and B from their cytosolic granules. GrB is inactive in the acidic milieu of the granules, but becomes activated in the neutral pH of the cytosol. Confocal microscopy with Z stacks shows GrA and GrB on the outside of the CD107+ granules, in both the cytoplasm and the nucleus of Tregs. Flow cytometric analysis, using the GranToxiLux assay, and immunoblotting to probe for well-known Gr substrates after in vitro activation of healthy donor Tregs, has revealed that levels of full-length caspase 3; shared GrB, GrA, and caspase 3 substrates (Parp1, Lamin A/C); shared GrB and caspase 3 substrates (bid, α-tubulin); and a unique GrA substrate (SET) significantly decline by day 3 (n=3, P< 0.01), while levels of β-actin (loading control) remained unchanged. Using shRNA-expressing lentiviral particles,human Tregs lacking Gr were significantly protected from self-inflicted damage. We then identified the TORC1 pathway as the main pathway leading to Gr production and that its inhibition in human Tregs lead to a 6-fold decrease in GrB expression (P ≤ 0.0001) and a 2-fold decrease in self-inflicted damage (P ≤ 0.0001). By using cytometry by time of flight, we show an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed a Th1 and Th17 like phenotype with a higher rate of apoptosis.Using a humanized mouse model, we studied the effect of TORC1 inhibition on transferred human Tregs survival. We found that, in the rapamycin treated group, Tregs reduced GrB expression compared to vehicle treated group (n=6, p* < 0.0001) along with decrease in Tregs apoptosis (n= 6, p* < 0.05). By using western blotting and flow cytometry, we also show that T_regs actives the phosphoinositide 3-kinase (PI3K) Akt, mTORC1 that phosphorylates S6 kinase 1 (S6K), and c-JUN. Phosphorylated p-c-Jun then translocates to the nucleus to dimerize with p-c-Fos, creating the transcription factor Activator Protein-1 (AP-1). AP-1 then binds onto the transcription promotor region of GrB, termed the AP-1 binding site, and increases transcription of GrB.

*Conclusions: These data suggests that activated Tregs undergo faster rates of apoptosis due to GrB leakage from their granules that is controlled by the TORC1 pathway.

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