The agonistic anti-human CD3ε antibody , OKT-3, has been used to control acute transplant rejection. The in vivo administration of OKT-3 was previously shown to induce the partial depletion of T cells and anergy in the remaining CD4+ T cells. However, this therapy is also associated with the systemic release of several cytokines, which leads to a series of adverse side effects. We established a novel anti-human CD3ε Ab, 20-2b2 (#1 abs), which recognized a close, but different determinant on the CD3ε molecule from that recognized by OKT3. 20-2b2 was non-mitogenic for human CD4+ T cells, could inhibit the activation of T cells in vitro, and induced T cell anergy in in vivo experiments using humanized mice. Cytokine release in humanized mice induced by the administration of 20-2b2 was significantly less than that induced by OKT-3. Our results indicated that the CD3ε molecule is still an attractive, effective, and useful target for the modulation of T cell responses. The establishment of other Abs that recognize CD3ε, even though the determinant recognized by those Abs may be close to or different from that recognized by OKT-3, may represent a novel approach for the development of safer Ab therapies using anti-CD3 Abs, in addition to the modification of OKT-3 in terms of the induction of cytokine production. Overall design: We examined whether a novel anti-human CD3 antibody (#1 abs: 20-2b2) could suppress T-cell activation in vitro, and we would liken to find which genes could suppress in the cells. #1 abs was incubated with human peripheral blood mononuclear cells (PBMC) from a healthy volunteer in cell culture plate for 2 days (48 hr). In addition, we also used Rat IgG (a negative control) & OKT-3 abs (an experimental control) in the cultures. We then extracted total RNA of abs-treated PBMC (n=3).