Introduction. Cell therapy using CD8⁺ Tregs has been shown to be effective in preclinical models of allogeneic rejection of human skin graft and xenogeneic GvHD in humanized NSG mice. However, their potential has never been evaluated in a clinical trial. Thus, we have translated our findings from the bench to the bedside with the establishment of a GMP protocol and the design of a phase 1/2a safety clinical trial of cell therapy using autologous polyclonal CD8⁺ Tregs to treat kidney transplant patients from living donors.

Method. CD8⁺ cells were isolated from peripheral blood by Clinimacs system, then CD8⁺CD4^-CD45RC^low/-CD56^- cells were sorted by MACSQuant Tyto cell sorter. Cells were stimulated weekly with anti-CD3 and CD28 mAbs and cultured for 3 weeks in X vivo 15 supplemented with human serum, rapamycin, IL-2 and IL-15. Cytotoxicity against allogeneic PBMCs was assessed by Annexin V/DAPI staining and suppressive ability was assessed in vitro on proliferation of syngeneic CD4⁺T cells in response to allogeneic APCs and in vivo in NSG mice irradiated at 1.5 Gy and co-injected with human PBMCs.

Results. First, we set up a method for the isolation of CD8⁺Tregs from peripheral blood of healthy individuals using positive magnetic selection and flow cytometry in a safe closed system. We compared clinical-grade culture media, stimulation methods, and cytokine doses to determine optimal culture conditions that preserve high proliferation rate, baseline phenotypic profile, and cell suppressive function in vitro and in vivo in a model of acute GVHD in NSG mice. We verified that they were not cytotoxic in vitro and in vivo, analyzed their persistence and their efficacy in an NSG mouse model, verified their resistance to immunosuppressive drug treatments which will be administered to the patient. Then, we validated the method on cells from patients with renal insufficiency. Finally, we transferred the manufacturing process in the GMP facility and performed 3 validation runs. We validated their phenotypic and functional stability upon conditioning, determining the time allowed for drug release; determined quality controls and release criteria.

The study was designed with physicians at Nantes Hospital as an one-arm, open-label, prospective trial in patients with end-stage chronic renal failure requiring primary kidney transplantation, with 3 escalating doses of CD8⁺ Tregs administered to the patient the day before the transplant without induction treatment and associated with classic maintenance immunosuppression. Patients will be monitored for long-term safety, immunosuppression burden and occurrences of infections. Blood, biopsy, and urine samples will be taken to monitor donor-directed reactivity of T cells, and persistence, transcriptomic profile, and migration of Tregs to the graft.

Conclusion. We designed a clinically compatible manufacturing process of CD8⁺Tregs and the first-in-human clinical trial evaluating the safety and hints of efficacy of CD8⁺Treg cell therapy in kidney transplant patients.