Developing in vitro expanded CD45RA⁺ regulatory T cells as an adoptive cell therapy for Crohn's disease

Background: and aim Thymus-derived regulatory T cells (T_regs) mediate dominant peripheral tolerance and treat experimental colitis. T_regs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. T_reg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of T_regs expanded from Crohn's blood is unknown. The potential for adoptively transferred T_regs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to T_reg-mediated suppression in active CD. The capacity for expanded T_regs to home to gut and lymphoid tissue is unknown. Methods: To define the optimum population for T_regcell therapy in CD, CD4⁺CD25⁺CD127^loCD45RA⁺ and CD4⁺CD25⁺CD127^loCD45RA^- T_reg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. Results: T_regs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA⁺ T_regs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA^- T_regs. CD45RA⁺ T_regs highly express α₄β₇ integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA⁺ T_regs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA⁺ T_regs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. Conclusions: CD4⁺CD25⁺CD127^loCD45RA⁺ T_regs may be the most appropriate population from which to expand T_regs for autologous T_reg therapy for CD, paving the way for future clinical trials.