Freezing Medium Containing 5% DMSO Enhances the Cell Viability and Recovery Rate After Cryopreservation of Regulatory T Cell Products ex vivo and in vivo.

Cell therapies have significant therapeutic potential in diverse fields including regenerative medicine, transplantation tolerance, and autoimmunity. Within these fields, regulatory T cells (Treg) have been deployed to ameliorate aberrant immune responses with great success. However, translation of the cryopreservation strategies employed for other cell therapy products, such as effector T cell therapies, to Treg therapies has been challenging. The lack of an optimized cryopreservation strategy for Treg products presents a substantial obstacle to their broader application, particularly as administration of fresh cells limits the window available for sterility and functional assessment. In this study, we aimed to develop an optimized cryopreservation strategy for our CD4+CD25+Foxp3+ Treg clinical product. We investigate the effect of synthetic or organic cryoprotectants including different concentrations of DMSO on Treg recovery, viability, phenotype, cytokine production, suppressive capacity, and in vivo survival following GMP-compliant manufacture. We additionally assess the effect of adding the extracellular cryoprotectant polyethylene glycol (PEG), or priming cellular expression of heat shock proteins as strategies to improve viability. We find that cryopreservation in serum-free freezing medium supplemented with 10% human serum albumin and 5% DMSO facilitates improved Treg recovery and functionality and supports a reduced DMSO concentration in Treg cryopreservation protocols. This strategy may be easily incorporated into clinical manufacture protocols for future studies.

Regulatory T cells for minimising immune suppression in kidney transplantation: phase I/IIa clinical trial.

OBJECTIVE: To assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model. DESIGN: Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13). SETTING: Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union). PARTICIPANTS: Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9). INTERVENTIONS: CD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×106 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48. MAIN OUTCOME MEASURES: The primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio. RESULTS: For all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire. CONCLUSIONS: The application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies. TRIAL REGISTRATION: NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

Urinary Biomarkers α-GST and π-GST for Evaluation and Monitoring in Living and Deceased Donor Kidney Grafts.

The aim of this study was to analyze the value of urine α- and π-GST in monitoring and predicting kidney graft function following transplantation. In addition, urine samples from corresponding organ donors was analyzed and compared with graft function after organ donation from brain-dead and living donors. Urine samples from brain-dead (n = 30) and living related (n = 50) donors and their corresponding recipients were analyzed before and after kidney transplantation. Urine α- and π-GST values were measured. Kidney recipients were grouped into patients with acute graft rejection (AGR), calcineurin inhibitor toxicity (CNI), and delayed graft function (DGF), and compared to those with unimpaired graft function. Urinary π-GST revealed significant differences in deceased kidney donor recipients with episodes of AGR or DGF at day one after transplantation (p = 0.0023 and p = 0.036, respectively). High π-GST values at postoperative day 1 (cutoff: >21.4 ng/mg urine creatinine (uCrea) or >18.3 ng/mg uCrea for AGR or DGF, respectively) distinguished between rejection and no rejection (sensitivity, 100%; specificity, 66.6%) as well as between DGF and normal-functioning grafts (sensitivity, 100%; specificity, 62.6%). In living donor recipients, urine levels of α- and π-GST were about 10 times lower than in deceased donor recipients. In deceased donors with impaired graft function in corresponding recipients, urinary α- and π-GST were elevated. α-GST values >33.97 ng/mg uCrea were indicative of AGR with a sensitivity and specificity of 77.7% and 100%, respectively. In deceased donor kidney transplantation, evaluation of urinary α- and π-GST seems to predict different events that deteriorate graft function. To elucidate the potential advantages of such biomarkers, further analysis is warranted.

Comprehensive Characterization of a Next-Generation Antiviral T-Cell Product and Feasibility for Application in Immunosuppressed Transplant Patients.

Viral infections have a major impact on morbidity and mortality of immunosuppressed solid organ transplant (SOT) patients because of missing or failure of adequate pharmacologic antiviral treatment. Adoptive antiviral T-cell therapy (AVTT), regenerating disturbed endogenous T-cell immunity, emerged as an attractive alternative approach to combat severe viral complications in immunocompromised patients. AVTT is successful in patients after hematopoietic stem cell transplantation where T-cell products (TCPs) are manufactured from healthy donors. In contrast, in the SOT setting TCPs are derived from/applied back to immunosuppressed patients. We and others demonstrated feasibility of TCP generation from SOT patients and first clinical proof-of-concept trials revealing promising data. However, the initial efficacy is frequently lost long-term, because of limited survival of transferred short-lived T-cells indicating a need for next-generation TCPs. Our recent data suggest that Rapamycin treatment during TCP manufacture, conferring partial inhibition of mTOR, might improve its composition. The aim of this study was to confirm these promising observations in a setting closer to clinical challenges and to deeply characterize the next-generation TCPs. Using cytomegalovirus (CMV) as model, our next-generation Rapamycin-treated (Rapa-)TCP showed consistently increased proportions of CD4+ T-cells as well as CD4+ and CD8+ central-memory T-cells (TCM). In addition, Rapamycin sustained T-cell function despite withdrawal of Rapamycin, showed superior T-cell viability and resistance to apoptosis, stable metabolism upon activation, preferential expansion of TCM, partial conversion of other memory T-cell subsets to TCM and increased clonal diversity. On transcriptome level, we observed a gene expression profile denoting long-lived early memory T-cells with potent effector functions. Furthermore, we successfully applied the novel protocol for the generation of Rapa-TCPs to 19/19 SOT patients in a comparative study, irrespective of their history of CMV reactivation. Moreover, comparison of paired TCPs generated before/after transplantation did not reveal inferiority of the latter despite exposition to maintenance immunosuppression post-SOT. Our data imply that the Rapa-TCPs, exhibiting longevity and sustained T-cell memory, are a reasonable treatment option for SOT patients. Based on our success to manufacture Rapa-TCPs from SOT patients under maintenance immunosuppression, now, we seek ultimate clinical proof of efficacy in a clinical study.