In this study, we show for the first time the key role of a protease inhibitor PI9 in human regulatory T cells (Tregs) homeostasis. Recent clinical trials showed a loss of FoxP3 expression during repeated stimulation of Tregs in vitro and disappearance of transferred Tregs in vivo within 2 weeks. Similarly, our data show that GFP FoxP3 sorted C57BL/6 mice Tregs with very high purity expanded ex vivo lose more than 50% of FoxP3 expression while undergoing apoptosis in vitro but also in vivo. We found similar results when we expanded human Tregs isolated by MACS techniques and stimulated ex vivo 2μg anti-CD3/CD28 and IL2 (250IU). Using flow cytometry, Western blot assays, immunoprecipitation, and confocal microscopy, we showed that mice and human Tregs stimulated in vitro with 2μg anti-CD3/CD28 and IL2 (250IU) increase Granzyme B (GrB) production. Using the GrantoxiLux assay, and z stack confocal microscopy, we observed intracellular leaking of GrB from granules of stimulated Tregs in vitro. Our data was confirmed with subcellular fractionation and GrB activity measurement on the cytoplasm and granules of Tregs. However, human and mice Tregs expressed a protease inhibitor (Spi6 in mice, PI9 in human) that partially inactivated GrB activity as shown by flow cytometry, western blot and immunoprecipitation of the GrB/PI9 complex. Loss of the protease inhibitor Spi6 in mice increased apoptosis of Tregs in vitro and in vivo. In this study, we were able to increase Spi6 expression in mice Tregs and PI9 in human Tregs more than 10 fold using lentivirus techniques, which resulted in significant reduction of apoptosis. Interestingly, screening for small molecules agonist of the PI9 gene promoter, we were able to identify an estrogen receptor agonist. ERA increased protease inhibitor expression and reduced apoptosis. FoxP3 expression during repeated Tregs stimulation in vitro was maintained. This novel discovery enhances our knowledge on human Tregs homeostasis and could help pave the way for better ex vivo Treg expansion and better Treg survival in clinical of Treg cell therapy.

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