Efficient generation of human alloantigen-specific CD4₊ regulatory T cells from naive precursors by CD40-activated B cells

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigenspecific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4⁺CD25^- T cells could be expanded 8-fold into alloantigenspecific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO⁺CCR7^- memory cells, and had a CD4^high, CD25⁺, Foxp3⁺, and CD62L (L-selectin)⁺ phenotype. Although these CD4^hiughCD25⁺ Foxp3⁺ alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.