CD4+CD25+ T cells have been identified as a population of immunoregulatory T cells, which mediate suppression of CD4+CD25− T cells by cell–cell contact and not secretion of suppressor cytokines. In this study, we demonstrated that CD4+CD25+ T cells do produce high levels of transforming growth factor (TGF)-β1 and interleukin (IL)-10 compared with CD4+CD25− T cells when stimulated by plate-bound anti-CD3 and soluble anti-CD28 and/or IL-2, and secretion of TGF-β1 (but not other cytokines), is further enhanced by costimulation via cytotoxic T lymphocyte–associated antigen (CTLA)-4. As in prior studies, we found that CD4+CD25+ T cells suppress proliferation of CD4+CD25− T cells; however, we observed here that such suppression is abolished by the presence of anti–TGF-β. In addition, we found that CD4+CD25+ T cells suppress B cell immunoglobulin production and that anti–TGF-β again abolishes such suppression. Finally, we found that stimulated CD4+CD25+ T cells but not CD4+CD25− T cells express high and persistent levels of TGF-β1 on the cell surface. This, plus the fact that we could find no evidence that a soluble factor mediates suppression, strongly suggests that CD4+CD25+ T cells exert immunosuppression by a cell–cell interaction involving cell surface TGF-β1.

In previous studies, we have shown that murine CD4+CD25+ regulatory T cells produce high levels of TGF-β1 in a cell surface and/or secreted form, and blockade of such TGF-β1 by anti-TGF-β curtails the ability of these cells to suppress CD25− T cell proliferation and B cell Ig production in in vitro suppressor assays. In further support for the role of TGF-β1 in suppression by CD4+CD25+ T cells, we show in this study that another TGF-β1-blocking molecule, recombinant latency-associated peptide of TGF-β1 (rLAP), also reverses suppression by mouse CD4+CD25+ T cells as well as their human counterparts, CD4+CD25high T cells. In addition, we show that CD25− T cells exposed to CD4+CD25+ T cells in vitro manifest activation of Smad-2 and induction of CD103, the latter a TGF-β-inducible surface integrin. In further studies, we show that while CD4+CD25+ T cells from TGF-β1-deficient mice can suppress CD25− T cell proliferation in vitro, these cells do not protect recipient mice from colitis in the SCID transfer model in vivo, and, in addition, CD4+LAP+, but not CD4+LAP− T cells from normal mice protect recipient mice from colitis in this model. Together, these studies demonstrate that TGF-β1 produced by CD4+CD25+ T cells is involved in the suppressor activity of these cells, particularly in their ability to regulate intestinal inflammation.