Characterization and Expansion of a New Regulatory CD8+CD45RClow T Cell Population in Humans.

Abstract

Background We previously demonstrated that CD8+CD45RClow T cells have a regulatory function in the rat and it has been recently shown a direct correlation between the number of CD8+CD45RChigh T cells before transplantation and an increased risk of acute kidney graft rejection. To date, human CD8+CD45RClow T cells have never been studied for their regulatory function and their potential in immunotherapy. Methods Cell populations were sorted by FACS Aria and the phenotype analyzed by flow cytometry in the blood from PBMCs. CD8+CD45RClow or high T cells were cultured with effector CD4+CD25- T cells from the same donor stimulated by allogeneic APC (T cell-depleted and irradiated PBMC). Cells were expanded for 15 days with cytokines and allogeneic APCs. Results We observed that the CD45RClow subset represents around 70% of CD8+T cells in human PBMC, which does not vary with age or sexe, and that they display distinct phenotype in normal healthy volunteers using several discriminative markers identified in the rat model (GITR, CD40L, HLA-DR). We next proved that CD8+CD45RClow T cells suppressed efficiently effector T cells response to allogeneic APC in a dose dependant manner, while CD45RChigh subset did not. Based on markers differentially expressed by CD45RClow and high subsets, we further defined a regulatory sub-population among CD8+CD45RClow T cells, whose suppression of allo-T cell response was higher than total CD45RClow subset. In addition, preliminary results indicated that Tregs can act at least partially through immunoregulatory cytokines since they were still suppressive when physically separated from responder T cells by transwell membrane, and importantly they were not cytotoxic. Finally, we developed a process to expand human Tregs in short-term culture. Comparing allogeneic and polyclonal stimulations in a two stimulation cycles of 7 days each, we obtained until 900 fold expansion of Tregs with a primary allogeneic and a secondary polyclonal stimulation. Expanded Tregs were significantly more efficient than fresh Tregs to inhibit allo T cell response in vitro. Conclusion We identified and characterized for the first time a new regulatory T cell population with a distinct phenotype and that suppress allo T cell responses in vitro. These Tregs can efficiently be expanded ex-vivo for immunotherapy. In this regard, a pre-clinical evaluation of their suppressive potential in humanized mice transplantation model is under progress.