Administration of a humanized monoclonal anti-CD3 antibody (mAb) to patients with type 1 diabetes (T1D) increases their C-peptide responses and the CD8/CD4 ratio. Incubation of human peripheral blood mononuclear cells (PBMC) with mAb in vitro has been shown to induce CD8(+) regulatory T cells (Tregs) capable of inhibiting proliferation of CD4(+) T cells. We hypothesized that CD8(+) Tregs function through secretion of cytokines. To test that possibility, we generated CD8(+) Tregs, sorted them by FACS, incubated them with syngeneic CD8-depleted PBMC in the presence of staphylococcal enterotoxin B (SEB), and measured proliferation of T cells and cytokines. Using neutralizing anti-cytokine mAbs, we show that the inhibitory effect of CD8(+) Tregs could be partially alleviated by anti-CCL-4, anti-TNF, and to a lesser extent anti-IL2, suggesting that these cytokines contribute to CD8(+) Treg function.