In vitro induction of regulatory T cells by anti-CD3 antibody in humans

Abstract Therapy with anti-CD3 antibody is effective in controlling models of autoimmune diseases and can reverse or prevent rejection of grafts. We studied the in vitro immunomodulatory effect of anti-CD3 treated human T cells. CD4 + T cells were stimulated with plate-bound anti-CD3 and cultured for 12 days after which they were cultured with autologous peripheral blood mononuclear cells (PBMCs) and stimulated with soluble anti-CD3. We found that CD4 + T cells that were stimulated with anti-CD3 (T αCD3 ) markedly suppressed the proliferation and cytokine production of autologous PBMCs. These regulatory T cells were not induced by incubation with isotype control (T control ) antibody or when anti-CD3 was combined with high doses of anti-CD28 (T αCD3/CD28 ). T αCD3 regulatory cells were anergic and produced lower levels of IFN-γ, TNF-α and IL-2, and higher levels of TGF-β than T control or T αCD3/CD28 . There were no differences in the expression of CD25 or CTLA4 on T αCD3 as compared to T control or T αCD3/CD28 , and CD4 + CD25 − T αCD3 cells were identical to CD4 + CD25 + T αCD3 cells in their in vitro suppressive properties. Recombinant IL-2 in vitro abrogated the suppressive effect of T αCD3 . The suppressive effect was not related to apoptosis, was independent of HLA since T αCD3 also suppressed allogeneic PBMCs, and was not related to soluble factors. Finally, no suppression was observed when non-T cells were removed from culture or when cultures were stimulated with plate-bound anti-CD3, consistent with the ability of T αCD3 to downregulate CD80 on dendritic cells in co-culture experiments. Thus, we have identified human T cells with strong in vitro regulatory properties induced in vitro by anti-CD3 which appear to act in a non-HLA restricted fashion by affecting antigen presenting cells.