Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells

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Abstract
CD4+CD25+ regulatory T (Treg) cells are pivotal for the maintenance of self-tolerance, and their adoptive transfer gives protection from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4+CD25+ Treg cells display phenotypic and functional characteristics similar to those of murine CD4+CD25+ Treg cells: namely, hyporesponsiveness to T-cell receptor (TCR) stimulation and suppression of CD25- T cells. Thus far, the detailed characterization and potential clinical application of human CD4+CD25+ Treg cells have been hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to 40 000-fold expansion of highly purified human CD4+CD25high T cells in vitro through the use of artificial antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose interleukin 2 (IL-2). Expanded CD4+CD25high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4+CD25high T cells, and maintained expression of the lymph node homing receptors L-selectin (CD62L) and CCR7. The ability to rapidly expand human CD4+CD25high Treg cells on a large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell–mediated diseases and transplantation medicine.