Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/- Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice.

Séverine Bézie,Ignacio Anegon,Elodie Autrusseau,Eric Charpentier,Dimitri Meistermann,Johanna Zoppi,Franck Duteille,Laurent David,Audrey Donnart,Frédérique Bellier-Waast,Laetitia Boucault,Stéphanie Kilens,Carole Guillonneau,Véronique Nerrière-Daguin

doi:10.3389/fimmu.2017.02014

Abstract

Both CD4+ and CD8+ Tregs play a critical role in the control of immune responses and immune tolerance; however, our understanding of CD8+ Tregs is limited while they are particularly promising for therapeutic application. We report here existence of highly suppressive human CD8+CD45RClow/− Tregs expressing Foxp3 and producing IFNγ, IL-10, IL-34, and TGFβ to mediate their suppressive activity. We demonstrate that total CD8+CD45RClow/− Tregs can be efficiently expanded in the presence of anti-CD3/28 mAbs, high-dose IL-2 and IL-15 and that such expanded Tregs efficiently delay GVHD and human skin transplantation rejection in immune humanized mice. Robustly expanded CD8+ Tregs displayed a specific gene signature, upregulated cytokines and expansion in the presence of rapamycin greatly improved proliferation and suppression. We show that CD8+CD45RClow/− Tregs are equivalent to canonical CD4+CD25highCD127low/− Tregs for suppression of allogeneic immune responses in vitro. Altogether, our results open new perspectives to tolerogenic strategies in human solid organ transplantation and GVHD.

Highlights

Immunosuppressive regimens have significantly improved long-term graft survival in the last decades but they still cannot prevent the allograft from chronic graft dysfunction and they remain a significant obstacle for the welfare of transplanted patients, in the last years, improvement of allograft survival has stagnated [1]
As in the rat we previously demonstrated the preferential interaction of CD8+CD45RClow/− Tregs with Plasmacytoid dendritic cells (pDCs) for an optimal suppressive activity [39], we tested the suppressive potential of CD8+CD45RClow/− Tregs in the presence of conventional dendritic cells (cDCs) (CD3−CD19− CD1c+Nrp-1−) and pDCs (CD3−CD19−CD1c−Nrp-1+) sorted simultaneously, in comparison to total MHC class II+ APCs as stimulator cells (Figure 1D)
In a model of cardiac allotransplantation, we have observed that fresh CD8+CD45RClow/− Tregs expressed ~2% of Foxp3 [8] and that Foxp3 was not upregulated following activation [28], the absence of Foxp3-deficient rat models remains a limitation for characterization of the role of Foxp3 in this model of transplantation

Summary

Introduction

Immunosuppressive regimens have significantly improved long-term graft survival in the last decades but they still cannot prevent the allograft from chronic graft dysfunction and they remain a significant obstacle for the welfare of transplanted patients, in the last years, improvement of allograft survival has stagnated [1]. Phase I studies in GVHD and solid organ transplantation have started with regulatory cells from different types (different CD4+ Tregs, macrophages, and DCs) without apparent toxicity [5,6,7], but to date, there are no clinical trials using CD8+ Tregs despite abundant literature in animals models [8,9,10]. Others have shown in mice that adoptive transfer of antigen-specific CD8+ Tregs were potent suppressors of fully MHC mismatch skin allograft and islet allograft [21, 22]. Our own studies have shown in a rat model of CD40-CD40L blockade-induced allograft tolerance the critical role of a CD8+ Tregs population expressing low/no level of CD45RC [8, 25]. Treatment with anti-CD45RC depleted CD45RChigh cells, preserved CD45RClow/− CD8+ and CD4+ Tregs and resulted in inhibition of solid organ rejection and of human GVHD in immune humanized mice [25]. We highlighted the biological role of IFNγ, IL-34, and Fibroleukin-2 (FGL-2) in the suppression exerted by CD8+CD45RClow/− Tregs [8, 17, 26,27,28]

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