Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation

Abstract

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.

Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy

“…Protocol 2 is GMP-compliant and utilizes multiple microbead restimulations to yield the greatest number of expanded Tregs. Unfortunately, the microbeads, which bind more effectively to cells, are difficult to remove from cultures, and, at the time of these experiments, had not yet been validated for clinical release criteria (although the microbeads have since been used in the ThRIL trial 45 ). Protocol 3 utilized GMP-approved materials for expanded T cell products suitable for testing in humans that have been successfully utilized in a phase I trial of APB Tregs 23 …”

Preservation of cell-based immunotherapies for clinical trials

“…In one study, non-expanded Tregs were cryopreserved and, after thawing, cells were washed into culture media and expanded for 7 days for a second infusion of Tregs [22]. In another study, cryopreserved Tregs were found to maintain their phenotype and suppressive function after being stored for 3 months in the vapor phase of LN [23].…”

“…Similar to Tregs, the majority of CAR Tcell therapy clinical studies did not clearly state the freezing and thawing methods. A few studies mentioned using a controlled-rate freezer to freeze cells [23,32] and a 36À38˚C water bath to thaw cryopreserved cells [30,33]. Unlike Tregs, the vast majority of CAR T-cell studies infused the cryopreserved cells immediately upon thawing.…”

IL-33 improves the suppressive capacity of human regulatory T cells

“…Cells were recovered and washed twice with 1X PBS for further huTregs isolation using the CD4+CD25+ T regulatory cells isolation kit from Miltenyi (California, USA). For huTregs culture and expansion we followed a protocol from Safinia et al, 2016. Briefly, 1x10…”

“…The most reported protocols include anti-CD3/anti-CD28-coated beads for T cell stimulation, along with administration of human IL-2 and the immunosuppressor drug Rapamycin [2,3], which favors Tregs growth instead of the proliferation of effector CD4+ T. To date, several clinical studies have been performed, and the data indicates that the administration of huTregs is safe even in high doses [4] and seems to ameliorate the symptoms of Graft versus Host Disease (GvHD) in some cases; however, this has not been accomplished in all patients, thus demonstrating the need for more knowledge and treatment alternatives [5][6][7][8]. One of the limitations of huTregs effectiveness is the potential lost of their functional stability (or plasticity); in other words, huTregs may convert from a suppressive to an inflammatory phenotype [9].…”