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

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Abstract
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.