Large-scale expansion of rat CD4+ CD25+ Treg cells in the absence of T-cell receptor stimulation

T-cell receptor (TCR) stimulation is both central to homeostatic maintenance of CD4(+) CD25(+) regulatory T cells (T-reg cells) in vivo and a prerequisite for the initiation of suppression by T-reg cells, both in vivo and in vitro. However, TCR-independent stimulation of T-reg 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 T-reg cells in vitro. Interestingly, CD28-SA stimulation plus interleukin (IL)-2 was even superior to conventional costimulation plus IL-2 in promoting T-reg cell growth in vitro. Despite their highly activated phenotype suppression by T-reg 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 T-reg cells. Taken together, we provide a novel protocol for long-term propagation of T-reg cells in vitro and our data are the first to reveal a difference in the signals required for activation and expansion of T-reg cells and those, involving the TCR, necessary for the initiation of suppression.