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Donor Antigen-Specific Human CD8+CD45R Clow Tregs Secreting IFNg, IL-10 and IL-34 Efficiently Inhibits Transplant Rejection

Bézie, Séverine1,2; Boucault, Laetitia1,2; Austrusseau, Elodie1,2; Daguin, Veronique1,2; Ménoret, Séverine1,2; Anegon, Ignacio1,2; Guillonneau, Carole1,2

doi: 10.1097/

1U1064, INSERM, Nantes, France; 2 ITUN, CHU, Nantes, France.

Introduction: We previously reported the suppressive properties of rat CD8 + CD45RClow T cells and characterized the donor-derived antigens recognized by them to stimulate their expansion and function. To date, human CD8 + CD45RClow T cells have never been characterized for their relevance as regulatory cells and potential in transplantation.

Materials and Methods: Cell populations were sorted from PBMCs from healthy volunteers by FACS Aria. Cytokine secretion assay detection kits were used to sort IFNg and/or IL10 secreting Treg subpopulations. Tregs were cultured in vitro with syngeneic CD4 + CD25-T cells and allogeneic APCs to analyze their suppressive function. 9 to 16 mer peptides were designed with 4 overlapping aa along the HLA-DRB1*15:01 sequence and assessed individually on CD8+CD45RClowTregs activation in presence of syngeneic HLA-A2+ pDCs at 4:1 Tregs:pDCs ratio. CD8+CD45RClowTregs were expanded for 14 days with cytokines and allogeneic stimulation. PBMCs with or without expanded Tregs were infused into NSG mice models of GVHD and allograft rejection.

Results: Compared to CD45RChigh subset, CD45RClow cells expressed higher levels of Foxp3, PD1, CD122, GITR and HLA-DR, as well as IL-34, IL-10, TGFb1 and IFNg. We demonstrated that IFNg + IL10+ co-secreting CD8+CD45RClow T cells inhibited more efficiently the allogeneic response in vitro than classical CD4+CD25hiCD127- Tregs. We confirmed the involvement of IL-10, IFNg and IL-34 cytokines in Tregs suppressive function by adding blocking antibodies to the co-culture assay. We identified their mechanisms of action as mediated by IL-2 deprivation, and preferential contact with pDCs, but not cytotoxicity. Screening of MHC class II donor-derived peptides revealed the presentation and recognition of two peptides of 16 aa and one peptide of 9 aa, activating, expanding and increasing their suppressive function. Finally, we observed that these Tregs can be efficiently expanded, until 1055 fold in 14 days. Following expansion, Tregs were enriched in Foxp3+ IL34+ IL10+ and IFNg+cells and possessed a strong suppressive function. Indeed, transfer of expanded Tregs significantly delayed in a dose dependant manner GVHD development and allogeneic skin graft rejection in humanized mice infused with human PBMCs.

Conclusions: We identified and characterized a new natural regulatory T cell population efficiently inhibiting anti-donor immune response and characterized the antigens recognized by them.

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