Ex Vivo Generation of Human Alloantigen-Specific Regulatory T Cells from CD4posCD25high T Cells for Immunotherapy

Abstract

BackgroundRegulatory T cell (Treg) based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy.Methodology/Principal FindingsHere, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4posCD25high Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent.Conclusions/SignificanceThe ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.

Large-scale in vitro expansion of human regulatory T cells with potent xenoantigen-specific suppression

“…5b). Taken together, our results suggest that multiple expansion cycles, regardless of the mode of stimulation, may lead to reduced suppressive capacity due to cell exhaustion and subsequent cell death (Peters et al 2008). The xenoantigen specificity in suppression of xenogeneic response shown by xenoantigen stimulated Treg was not detected when Treg expanded with one cycle of xenoantigen stimulation were assessed in xenoantigen-stimulated MLR, as indicated by a similar suppressive potency revealed by both Pc-and Xn-Treg at all ratios tested (Fig.…”

“…Selection and use of antigen-specific Treg has been proposed to improve Treg therapeutic efficiency and lower the risk for unwanted nonspecific immune suppression caused by transferring large numbers of polyclonal Treg (Koenecke et al 2009;Hall et al 2009;Peters et al 2008;Veerapathran et al 2011). Recent studies have shown that ex vivo expanded alloantigen specific Treg obtained enhanced suppressive capacity in allogeneic responses in vitro (Peters et al 2008;Golshayan et al 2007) and were more potent than polyclonal Treg in protecting against alloimmune-mediated injury of human skin grafts in a humanized mouse model (Yamano et al 2011;Sagoo et al 2011). However, strategies for large scale expansion of xenoantigen-specific human Treg remain to be developed.…”

“…Thus, these findings indicate that the protocol applying a combination of one initial polyclonal expansion cycle and two subsequent cycles of xenoantigen stimulation would become a practical strategy to obtain high numbers of porcine xenoantigen-specific human Treg. Indeed, the use of two expansion cycles has been reported to be more efficient than any other cycles for expansion of highly suppressive alloantigen-specific human Treg in large numbers (Peters et al 2008;Fan et al 2012). …”

“…However, the infused polyclonal Treg may also deliver panimmunosuppressive effects, such as opportunistic infections and tumor-growth. Studies with transplantation animal models and immune monitoring of transplant patients indicate that these side effects could be avoided by specific suppression of the expansion of donor-alloreactive effector T cells after transplantation, which can be achieved by using donor allospecific Treg that are more effective at preventing allograft and improving clinical outcome than polyclonal Treg (Masteller et al 2006;Peters et al 2008;Sagoo et al 2011).…”

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15

“…Addition of rapamycin to this cell culture is used to diminish outgrowth of the contaminating effector T cells (16,17). Alloantigen-specific Tregs with a much greater specificity can be generated by expansion in the presence of allogeneic PBMCs (18,19) or B cells (10,20). The potential benefit of these alloantigen-specific Tregs is targeted suppression rather than general immunosuppression and increased suppressive potency (9,(21)(22)(23)(24)(25)(26).…”