Regulatory T cells (Tregs) are a lymphocyte subset with intrinsic immunosuppressive properties that can be expanded in large numbers ex vivo and have been shown to prevent allograft rejection and promote tolerance in animal models. To investigate the safety, applicability, and biological activity of autologous Treg adoptive transfer in humans, we conducted an open‐label, dose‐escalation, Phase I clinical trial in liver transplantation. Patients were enrolled while awaiting liver transplantation or 6‐12 months posttransplant. Circulating Tregs were isolated from blood or leukapheresis, expanded under good manufacturing practices (GMP) conditions, and administered intravenously at either 0.5‐1 million Tregs/kg or 3‐4.5 million Tregs/kg. The primary endpoint was the rate of dose‐ limiting toxicities occurring within 4 weeks of infusion. The applicability of the clinical protocol was poor unless patient recruitment was deferred until 6‐12 months posttransplant. Thus, only 3 of the 17 patients who consented while awaiting liver transplantation were dosed. In contrast, all six patients who consented 6‐12 months posttransplant received the cell infusion. Treg transfer was safe, transiently increased the pool of circulating Tregs and reduced anti‐donor T cell responses. Our study opens the door to employing Treg immunotherapy to facilitate the reduction or complete discontinuation of immunosuppression following liver transplantation.

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial, ThRIL, recently commenced at King's College London, proposes to use Treg cell therapy to induce tolerance in liver transplant recipients, the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads, IL-2 and rapamycin. We report the enrichment of a pure, stable population of Tregs (>95% CD4+CD25+FOXP3+), reaching adequate numbers for their clinical application. Our protocol proved successful in, influencing the expansion of superior functional Tregs, as compared to freshly isolated cells, whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF), a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL, will undoubtedly inform the improved management of the liver transplant recipient.

Regulatory T cells (Tregs) are an essential component of the cellular immune response, occupying a key role in maintaining immunological tolerance and present an attractive therapeutic target in a range of immunopathologies. Comprehensive analysis of the human Treg compartment has been restricted due to technical limitations. The advent of mass cytometry enables simultaneous assessment of vastly increased phenotypic parameters at single-cell resolution. In this study, we used mass cytometry to examine the complexity of human Tregs using an extensive panel of surface markers associated with Treg function and phenotype. We applied unsupervised clustering analysis, revealing 22 distinct subpopulations of Tregs, representing previously identified and novel subpopulations. Our data represent the most in-depth phenotypic description of the human Treg compartment at single-cell resolution and show a hitherto unrecognized degree of phenotypic complexity among cells of the regulatory lineage.