Introduction: CD8⁺CD45RC^low/-T cells have been described by us and others as highly suppressive cells in rodent and human. Importantly, we highlighted their therapeutic potential in transplantation models of human skin graft allogeneic rejection and human PBMCs-induced GVHD in NSG mice (Bézie et al., Frontiers Immunol., 2018). More recently, we have shown that conferring specificity toward a HLA mismatch in transplantation by using the CAR technology significantly improved their therapeutic efficiency (Bézie et al., Blood Advances, 2019). To date, there has been no clinical trial using CD8⁺Tregs. Thus, our goal was to develop a GMP-compatible protocol to launch a FIH clinical trial using CD8⁺Treg cell therapy in transplantation as a part of the H2020 ReSHAPE program.
Materials and Methods: CD8⁺Tregs were FACS-sorted based on CD3⁺CD8⁺CD45RC^low/- phenotype from blood of healthy volunteers. Clinically compatible culture media from Clinisciences, Miltenyi Biotec, Lonza, ThermoFisher Scientific, Takara, Biotechne, Irvine, CellGenix and Stem cell were compared, when supplemented or not with human AB serum (BioIVT Seralab), human albumin (Vialebex), or CTS Immune cell SR (ThermoFisher Scientific), and with a range of IL-2 (Proleukin) and rapamycin (Pfizer) doses. Anti-CD3/CD28 purified antibodies, coated beads (Miltenyi Biotec), or tetramers (Stem Cell) were used for Treg stimulation at days 0 and 7 of 14 days culture. Suppressive activity of Tregs was assessed in vitro on responder T cell proliferation stimulated by allogeneic APCs and in vivo in a PBMC-humanized NSG mouse model of GVHD.
Results: We sat up a process to isolate and expand circulating CD8⁺Tregs in GMP conditions. Based on cell expansion yield, Treg-associated marker expression and suppressive function, we selected a clinically applicable culture medium combined with serum, cytokines and chemical supplements. In addition, IL-2 and rapamycin doses and stimulation methods were determined and critical to preserve their function. In vivo, we observed that CD8⁺Tregs persist for more than 80 days in NSG mice lacking human cytokines. Finally, we demonstrated in vitro and in vivo that allogeneic off the shelf CD8⁺Tregs can be used in cell therapy.
Conclusion: At the dawn of the FIH clinical trial using CD8⁺Treg cell therapy, we are already working on the next generation of CD8⁺Tregs for transplanted patients with genetic modifications to secure function, stability, high persistence and specificity.