Clinical Grade Manufacturing of Human Alloantigen-Reactive Regulatory T Cells for Use in Transplantation

Abstract

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

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

“…This finding may also explain the observation that a simultaneous influx of both Ag-specific effector T cells and Tregs is observed after s.c. purified protein derivate injection (69). Expansion of alloantigen-specific Tregs through stimulation with other cell types like PBMCs (18,19) and B cells (10,20) has been documented previously. Our results with PBMCs as allogeneic stimulator cells for nTreg expansion are in line with previous publications, and relatively high stimulator/Treg ratios in combination with IL-2 and IL-15 are needed.…”

“…Allogeneic mature moDC-expanded nTregs are superior in suppressing alloantigen-induced proliferation by effector T cells compared with polyclonal Tregs. This is most likely the result of a skewing in the TCR repertoire imposed by application of an Ag-specific expansion protocol (10,20). This superior alloantigenspecific suppressive capacity, induced by applying an allogeneic stimulus during Treg expansion, was confirmed by several in vitro (18) and in vivo (9,26,66) studies.…”

“…Our results with PBMCs as allogeneic stimulator cells for nTreg expansion are in line with previous publications, and relatively high stimulator/Treg ratios in combination with IL-2 and IL-15 are needed. Using B cells in combination with an anti-CD28 Ab (20) over a period of 2 wk is inferior to those obtained by allogeneic mature moDCs in a period of 10-12 d. More recently, using a blast-promoting cell line, to enhance levels of costimulatory molecules on B cells, significantly increased the potential of these cells to generate large numbers of Tregs (10,70). At a higher ratio of B cells to Tregs, equal numbers of Tregs were obtained within a similar expansion period compared with allogeneic mature moDCs used at a ratio of 1:1.…”

“…Data from transplantation models using animals have also shown the efficacy of Tregs in controlling the alloreactive immune response, thereby preventing or delaying allograft rejection. Moreover, in a humanized mouse skin transplant model, alloantigen-specific Tregs were superior in controlling skin rejection compared with polyclonal-expanded Tregs (9,10).…”

“…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).…”