International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

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_reg cell 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.

Adoptive transfer of ex vivo expanded CD4⁺CD25⁺FOXP3⁺ regulatory T cells is a successful therapy for autoimmune diseases and transplant rejection in experimental models. In man, equivalent manipulations in bone marrow transplant recipients appear safe, but questions regarding the stability of the transferred regulatory T cells during inflammation remain unresolved. In this study, protocols for the expansion of clinically useful numbers of functionally suppressive and stable human regulatory T cells were investigated. Regulatory T cells were expanded in vitro with rapamycin and/or all-trans retinoic acid and then characterized under inflammatory conditions in vitro and in vivo in a humanized mouse model of graft-versus-host disease. Addition of rapamycin to regulatory T-cell cultures confirms the generation of high numbers of suppressive regulatory T cells. Their stability was demonstrated in vitro and substantiated in vivo. In contrast, all-trans retinoic acid treatment generates regulatory T cells that retain the capacity to secrete IL-17. However, combined use of rapamycin and all-trans retinoic acid abolishes IL-17 production and confers a specific chemokine receptor homing profile upon regulatory T cells. The use of purified regulatory T-cell subpopulations provided direct evidence that rapamycin can confer an early selective advantage to CD45RA⁺ regulatory T cells, while all-trans retinoic acid favors CD45RA⁻ regulatory T-cell subset. Expansion of regulatory T cells using rapamycin and all-trans retinoic acid drug combinations provides a new and refined approach for large-scale generation of functionally potent and phenotypically stable human regulatory T cells, rendering them safe for clinical use in settings associated with inflammation.

The concept of regulatory T cell (Treg)-based immunotherapy has enormous potential for facilitating tolerance in autoimmunity and transplantation. Clinical translation of Treg cell therapy requires production processes that satisfy the rigors of Good Manufacturing Practice (GMP) standards. In this regard, we report our findings on the implementation of a robust GMP compliant process for the ex vivo expansion of clinical grade Tregs, demonstrating the feasibility of this developed process for the manufacture of a final product for clinical application. This Treg isolation procedure ensured the selection of a pure Treg population that underwent a 300-fold expansion after 36 days of culture, while maintaining a purity of more than 75% CD4+CD25+FOXP3+ cells and a suppressive function of above 80%. Furthermore, we report the successful cryopreservation of the final product, demonstrating the maintenance of phenotype and function. The process outlined in this manuscript has been implemented in the ONE study, a multicenter phase I/IIa clinical trial in which cellular therapy is investigated in renal transplantation.

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell‐based clinical therapy. However, human Treg cells are “plastic”, and are able to produce IL‐17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL‐17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL‐17 in vitro when activated in the presence of IL‐1β, but not IL‐6. “IL‐17 potential” is restricted to population III (CD4⁺CD25^hiCD127^loCD45RA⁻) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL‐17 induction. Importantly, we find that IL‐17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL‐17. Finally, we show that CD161⁺ population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL‐17‐producing Treg‐cell population at these sites. As IL‐17 production from this Treg‐cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.