The safety and efficacy of adoptive transfer of regulatory T cells (Tregs) are being extensively investigated in ongoing clinical trials. However, these trials have been hindered by the short lifespan of transferred Tregs. We show that activated human Tregs undergo self-inflicted damage due to leakage of granzymes (Gr) A and B from cytotoxic granules, despite an increase in endogenous inhibitor Protease inhibitor 9 (PI9). Confocal microscopy with Z stack shows GrA and GrB outside of the CD107+ granules both in the cytosol and in the nucleus of Tregs. While GrB is inactive at the acidic pH of the granules, we show that its release in the cytosol significantly activates its protease activity, leading to multiple substrates cleavage. We used flow cytometry (GranToxiLux assay) and immunoblotting to probe for known Gr substrates at 0.5, 1, 2, and 3 days after in vitro activation of healthy donor Tregs (3 separate donors/experiments, P< 0.01). 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) were first seen to decline 2 days after stimulation. Most declined further by day 3, while levels of the loading control β-actin remained unchanged. We also observed a Gr mediated disruption of the mitochondrial transmembrane potential leading to apoptosis (3 separate donors/experiments, P<0.05). Gr Knockdown in healthy human Tregs using shRNA-expressing lentiviruses lead to protection from self-inflicted damage. Furthermore, we identified TORC1 pathway as key pathway leading to GrB production and self-inflicted damage that was reversed by TORC1 inhibition (~2 folds decrease, P<0.05). By using cytometry by time of flight (CyTOF), 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 increased expression of chemokine receptors that mediate trafficking to sites of inflammation (n=20, P< 0.05). However, those GrB-expressing Tregs were significantly more apoptotic than non–GrB expressing Tregs (n=20, P<0.05). This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.

CITATION INFORMATION: Al Dulaijan B., Eskandari S., Karnyski J., Assaker J., Cai S., Mansouri A., Mohamed M., Azzi J. TORC1 Inhibition Protects Activated Human Regulatory T Cells from Self-Inflicted Damage via Granzyme B Am J Transplant. 2017;17 (suppl 3).

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