Forced Revision Can Still Inform—Lessons and Questions From the “Original” TWO Study

Kidney transplantation is the preferred treatment for patients with end-stage kidney disease, but the life-long immunosuppression required remains a source of long-term complications. One approach to minimize or eliminate immunosuppression that has gained traction recently is the use of cellular therapies. The ONE Study, a multicenter phase 1 trial, demonstrated the safety of CD4+Foxp3+ regulatory T cells (Treg) in the setting of kidney transplantation.1 However, the specifics of which cells to use, how to isolate them, and how to administer them are active areas at several international centers. In the accompanying article, Brook et al2 described results from a limited study with lymphodepletion and delayed administration of ex vivo expanded polyclonal Treg. The TWO study (Transplantation Without Overimmunosuppression)3 was originally designed as a phase 2b randomized control trial of delayed administration of autologous Treg therapy in living donor kidney transplant recipients. Study participants underwent induction with alemtuzumab (anti-CD52) lymphodepletion therapy followed by maintenance immunosuppression with mycophenolate mofetil (MMF) and tacrolimus. Participants in the cell therapy arm had progressive MMF reduction starting at 12 wk posttransplant with full discontinuation of MMF at 6 mo at which time they also received 5–10 × 106 Treg/kg; control participants were maintained on both drugs and received no cell therapy. The provided protocol biopsies performed at 38 wk posttransplant showed no acute rejection, and the dose of tacrolimus was reduced from a goal of 5–10 to 4–6 ng/dL in the cell therapy arm. Participants were followed up to 18 mo posttransplant with clinical assessment and immune monitoring performed longitudinally. Unfortunately, the COVID-19 pandemic led authorities in the United Kingdom to suspend the use of alemtuzumab limiting enrollment to a total of only 9 participants, 4 in the cell therapy arm, 3 in the control arm, and 2 were withdrawn due to COVID-19-related delays in transplantation. The TWO study was subsequently substantially modified,3 so these 7 represent a unique cohort with no additional participants. Overall, lymphodepletion followed by delayed administration of CD4+Foxp3+ Treg was well tolerated and there were no safety concerns. All 3 patients were weaned to single-drug immunosuppression with "low-dose" tacrolimus without evidence of acute rejection. However, despite the lower tacrolimus target postinfusion in the cell therapy arm, drug levels were not statistically different from those in control participants. Protocol biopsies performed at 9 mo post-transplant/12 wk post-infusion demonstrated a lack of cellular infiltrate and minimal tubulointerstitial fibrosis. Notably, 1 patient in each group developed mild clinical cytomegalovirus (CMV) disease that was treated with 3 wk of valganciclovir, leading to full resolution; neither group had any participants with evidence of BK virus nephropathy. Immune monitoring studies showed a trend toward an increase in Treg number and frequency, but only during the first 1–2 wk postinfusion. Although there was transient depletion of B cells following alemtuzumab, naive and marginal-zone B-cell numbers were similar between the 2 groups at the time of cell infusion and the 72-wk endpoint of the study. The overall findings of safety and a lack of acute rejection are reassuring. An earlier phase 1 study by the Northwestern group showed that Treg could be safely given 2 mo after alemtuzumab,4 but those patients remained on MMF, whereas the current study with Treg infusion delayed until 6 mo posttransplant suggests that minimization to tacrolimus monotherapy can be achieved using this approach. Similarly, the STEADFAST study (NCT04817774) is a phase 1/2a trial of Chimeric Antigen Receptor (CAR)-Treg with delayed administration at 3 mo following rabbit anti-thymocyte globulin induction.5 Based on data publicly presented but not yet published, this study has had no significant safety concerns at this time. Combined with the current study, there is ever-increasing evidence that autologous Treg can be safely administered after lymphodepletion. Regarding infectious complications, unlike The ONE Study where the cell therapy arm had a noticeable absence of CMV viremia,1 1 of 4 patients in the cell therapy arm had CMV reactivation; similar to the frequency of 1 of 3 control participants. Although not a safety signal and only a small number of participants, these data may decrease the optimism surrounding the correlation between cellular therapy and protection from viral reactivation. From a mechanistic stance, the authors provide data for major T- and B-cell populations including longitudinal Treg frequencies, with no statistical differences between the cell therapy and control groups. Unfortunately, the authors were not able to quantify changes in donor-reactive T cells after the infusion because of technical challenges. With the biopsy tissue obtained as part of the protocol and the group's growing expertise in spatial profiling, it will be interesting to see high-dimensional data from the tissues. Will there be differences in innate or adaptive cell infiltration or signals that could point toward markers consistent with successful immunosuppression tapering? These types of studies, combined with planned gene expression studies as well as cytokine and metabolic profiling (per the original protocol provided in the Supplement), are sure to provide the community with invaluable mechanistic insights. Overall, the conclusions that can be drawn from this study are limited because of the numbers enrolled; the authors should be acknowledged and applauded for disseminating the data on this unique set of patients as it has the potential to guide the planning of new trials involving lymphodepletion and delayed administration of Treg post–kidney transplantation.