Expansion and Characterization of Human Double Negative Regulatory T Cells

A subset of αβ-T cell receptor (TCR) positive, NK lineage marker negative, CD4−CD8− double negative regulatory T cells (DN Tregs) comprise only ~1% of peripheral blood T lymphocytes. Clinical applications of DN Tregs in humans are limited by their scarce number and the lack of effective expansion method. Herein is described a protocol for large-scale ex vivo expansion of functional and pure human DN Tregs. In vitro, expanded DN Tregs induce a cell-contact dependent immunosuppression of autologous T cells and B cells, and are cytotoxic towards various lung cancer, and leukemic cells. In vivo, infusion of DN Tregs delays an onset of xenogeneic graft-versus-host disease (GVHD) in humanized mouse model. Furthermore, short treatment of expanded DN Tregs with rapamycin augments their suppressive function. Taken together, these results indicate a dual function of ex vivo expanded DN Tregs and suggest their therapeutic potential in suppression of allograft rejection and treatment of malignancies.M.Sc