Direct Expansion of Human Allospecific FoxP3+CD4+ Regulatory T Cells with Allogeneic B Cells for Therapeutic Application

Abstract

Compelling evidence from animal studies has demonstrated that allospecific FoxP3+CD4+ regulatory T (Treg) cells expanded ex vivo can be used as effective therapeutic tools in the treatment of allograft rejection and graft-vs-host disease. Despite the promising results from animal studies, there remain major barriers to developing Treg cell-based immunotherapy in humans. Currently, no effective approach has been established for selective expansion of human allospecific Treg cells ex vivo. Additionally, the very low frequency of Treg cells present in human peripheral blood could pose a formidable challenge to obtaining a sufficient number of Treg cells from a single donor for ex vivo expansion for therapeutic utilization. Extending our recent finding that mouse B cells preferentially induce expansion of alloreactive Treg cells, we report herein that human Treg cells can be expanded ex vivo with allogeneic B cells. The expanded Treg cells express very high levels of FoxP3, maintain anergic phenotype, and are potent suppressors capable of inhibiting the alloproliferation of third-party responder T cells at very low Treg-to-T effector cell ratio in an alloantigen-specific manner. The alloantigen specificity demonstrated by B cell-expanded Treg cells is not determined by the HLA haplotypes of the Treg cells, but it is induced and determined by the haplotype of the B cells used to expand them. Our findings represent a significant advance in the development of Treg cell-based immunotherapy in humans and raise the possibility of using third-party Treg cells for therapeutic applications.

Constitutive Nuclear Localization of NFAT in Foxp3+ Regulatory T Cells Independent of Calcineurin Activity

“…To isolate Foxp3 + GFP + cells, CD4 + T cells were first isolated from splenocytes of Foxp3EGFP mice (32) by negative selection using a CD4 T Cell Isolation Kit (Miltenyi Biotec), followed by sorting of GFP + cells by FACS (33). All of the isolated cells were rested in culture or treated with cyclosporine A (CsA) for 30 min before being processed for protein extracts.…”

Adenosine production by human B cells and B cell–mediated suppression of activated T cells

“…For example, coculturing of CD19 1 human B cells with CD4 1 CD25 1 alloreactive T cells in the presence of IL-2 and CD28-specific antibody (Ab) was reported to induce a 40-fold expansion of Treg. 12,13 The current hypothesis is that B cells exert dual and potentially opposing effects on T-cell responses. On the one hand, they can promote primary T-cell responses and the generation of memory T helper (Th)1 and Th2 cells through antigen-dependent but Ab-independent mechanisms.…”

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

“…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.…”

“…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.…”

“…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).…”