Comparison of the suppressive function of alloantigen-reactive Treg cells and non-stimulated Treg cells in allogeneic skin transplantation

Abstract

Abstract Adoptive cellular therapy with Treg cells is being evaluated in numerous clinical trials in GvHD and organ transplantation. Cellular biotherapy with Treg cells requires ex vivo expansion. While alloantigen stimulated Treg cells have been used in several studies, very few studies have directly compared the efficacy of alloantigen-reactive with non-stimulated Treg cells in vitro and no studies have compared their efficacy in vivo in organ transplantation. In this study, we obtained enriched population of alloantigen-reactive Treg by stimulating proliferation dye-labeled C57BL/6 CD4+Foxp3+CD25+ Treg cells with allogeneic H-2bm12 dendritic cells in the presence of IL-2. After 5~7 days, the alloantigen-reactive Treg cells and resting Treg cells were separated by FACS. Treg cells that had proliferated in response to alloantigen stimulation were more potent suppressors of the reactivity of C57BL/6 CD4+Foxp3− T cells to H-2bm12 stimulators than non-reactive Treg cells. In contrast, both Treg populations equally suppressed the response of C57BL/6 CD4+Foxp3− T cells to 3rd party BALB/c dendritic cells. Both alloantigen-reactive and non-reactive Treg cells were co-transferred with C57BL/6 CD4+Foxp3− T cells (1:2 ratio) to immunodeficient recipients followed by transplantation of allogeneic H-2bm12 and 3rd party BALB/c skin. Recipients of alloantigen-reactive Treg cells demonstrated delayed rejection (26 days -> 49 days) of H-2bm12 skin, but normal rejection of 3rd party skin. Taken together, this protocol offers a simple, rapid method for enrichment of alloantigen-reactive Treg with enhanced antigen-specific suppressive function in vitro and in vivo. This research was supported by the Intramural Research Program of NIAID, NIH.