CD4+CD25+ regulatory T cells (T reg cells) play a key role in controlling autoimmunity and inflammation. Therefore, therapeutic agents that are capable of elevating numbers or increasing effector functions of this T cell subset are highly desirable. In a previous report we showed that a superagonistic monoclonal antibody specific for rat CD28 (JJ316) expands and activates T reg cells in vivo and upon short-term in vitro culture. Here we demonstrate that application of very low dosages of the CD28 superagonist into normal Lewis rats is sufficient to induce T reg cell expansion in vivo without the generalized lymphocytosis observed with high dosages of JJ316. Single i.v. administration of a low dose of the CD28 superagonist into Dark Agouti (DA) rats or Lewis rats that suffered from experimental autoimmune encephalomyelitis (EAE) proved to be highly and equally efficacious as high-dose treatment. Finally, we show that T reg cells that were isolated from CD28-treated animals displayed enhanced suppressive activity toward myelin basic protein–specific T cells in vitro, and, upon adoptive transfer, protected recipients from EAE. Our data indicate that this class of CD28-specific monoclonal antibodies targets CD4+CD25+ T reg cells and provides a novel means for the effective treatment of multiple sclerosis and other autoimmune diseases.

Summary T‐cell receptor (TCR) stimulation is both central to homeostatic maintenance of CD4+ CD25+ regulatory T cells (Treg cells) in vivo and a prerequisite for the initiation of suppression by Treg cells, both in vivo and in vitro. However, TCR‐independent stimulation of Treg cells, e.g. with superagonistic CD28‐specific monoclonal antibodies (CD28‐SA), not only expands these cells in vivo but, as we show here, also mediates large‐scale expansion of rat Treg cells in vitro. Interestingly, CD28‐SA stimulation plus interleukin (IL)‐2 was even superior to conventional costimulation plus IL‐2 in promoting Treg cell growth in vitro. Despite their highly activated phenotype suppression by Treg cells expanded in the absence of TCR stimulation remained fully dependent on TCR‐triggering for initiation and cell contact was required to exert suppression. With regard to the regulation of suppression by CD28 stimulation we observed that neither the presence of a conventional anti‐CD28 monoclonal antibody nor a CD28‐SA generally rendered conventional T cells resistant to suppression by preactivated Treg cells. Taken together, we provide a novel protocol for long‐term propagation of Treg cells in vitro and our data are the first to reveal a difference in the signals required for activation and expansion of Treg cells and those, involving the TCR, necessary for the initiation of suppression.