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

Abstract

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.

Foxp3-expressing Regulatory T Cells Expanded With CD28 Superagonist Antibody Can Prevent Rat Cardiac Allograft Rejection

“…The stimulation of CD4 ϩ CD25 Ϫ T cells leads to induction of cell-surface expression of CD25. Therefore, it may be possible that some or all of the CD4 ϩ CD25 ϩ T cells are the result of recent activation, whereas our data, in agreement with previous reports, 6,18 demonstrate that supCD28 MAb has a unique capacity to expand intrinsic constitutive CD25 Ϫ and Foxp3-expressing nTreg cells, which are not activated CD25 ϩ effector T cells (see Figure 5). …”

Superagonistic CD28 stimulation of allogeneic T cells protects from acute graft-versus-host disease

“…Upon in vitro stimulation with a CD28-SA, a significant subpopulation of T conv cells also expresses CD25, 20 which is not the case after in vivo treatment. 27 To be able to directly assess the role of in vitro-expanded T reg cells in the protection from aGVHD in vivo, we substituted the CD90.1 ϩ donor T reg cells with CD90.2 ϩ -congenic T reg cells before starting the LN cell culture.…”

“…[19][20][21] Therefore, we either pretreated donor mice in animal models of aGVHD and GVT responses with a mouse anti-mouse CD28-SA in vivo or added the CD28-SA to total lymph node (LN) cell suspensions in vitro to activate and expand T reg cells before transplantation of purified T cells into allogeneic recipient mice. The latter approach with human peripheral blood mononuclear cells as starting material may bear clinical potential because it avoids the in vivo use of anti-human CD28-SA, for which there is at present no safe protocol.…”

Delivering safer immunotherapies for cancer

“…Crosslinking of CD28 using superagonist antibodies led to proliferation of all subsets T-cells in mouse and rat models [103]. In rodents CD28SAs also trigger the rapid expansion of Tregs, allowing for potential applications in autoimmune disease in addition to cancer [104]. However, when applied in healthy human volunteers, the release of proinflammatory cytokines was so significant that all six patients were hospitalized, indicating major shortcomings in the extensive preclinical research [105].…”