Fig. 1

Trial Flow chart Part 1 Observational study: Identification of patients eligible for Part 2, based on pattern of IFNg/IL-17 reactivity on FluoroSpot assay. Some patients may have already had these assays performed in 2020 as part of an observational study (REC ref 16/WM/0370). Additional patients (up to n = 100) will be recruited and have blood withdrawn for testing to determine HLA-specific patterns of responsiveness in FluoroSpot assays, to identify those eligible for Treg therapy in Part 2, and to determine the size of the population for our proposed follow-on trial (beyond this study). All patients with either positive IL-17 or IFN gamma anti-HLA reactivity without evidence of regulation by CD25 + cells will be eligible to enter Part 2. The first 9 patients identified as being eligible, from any site, will undergo baseline immune monitoring for two months to inform statistical analysis of primary endpoint. If < 21 patients with Fluorospot patterns eligible for inclusion in Part 2 are identified, the study will terminate early. Part 2 Interventional Study: This is a two stage, adaptive, open labelled, trial based on Simon’s two stage design, with 12 patients treated in Part 2 Stage 1 and 9 treated in Part 2 Stage 2. Because of an expected high withdrawal rate, all patients recruited to Part 1 will consent to inclusion in Part 2. If more than 21 eligible patients are identified, the statisticians will draw up an algorithm at the end of milestone 1 to determine which patients are included in Part 2, to ensure that eligible patients from each of the three sites are included in a ratio proportionate to the number found eligible from each site. For instance, if after 75 patients have been analysed in Part 1, the number found eligible at each site is 15 (site 1), 10 (site 2) and 10 (site 3), then 9 from site 1 will be included, and 6 each from sites 2 and 3. The order of Treg dosing will be determined by the order in which eligibility for Part 2 was determined. Treg dosing: The exact number in Part 2 Stage 2 who receive Tregs will be determined by the results from Part 2 Stage 1, according to Simon’s 2 stage design. If fewer than 2 of the 12 in stage 1 reach primary endpoint (suppression of anti-HLA responses for at least two months post treatment), the trial will be stopped, concluding that Treg therapy has no efficacy. If 6 or more of the 12 in group 1 reach primary endpoint, we will proceed with dosing all 9 in stage 2. In the event that 3, 4 or 5 of the 12 patients in stage 1 respond, the estimates of efficacy are below our hoped for 50% but are not so low as to be regarded as evidence of futility. We will therefore pause the trial and re-estimate the number of patients that we need to recruit, after discussion with the funders. We anticipate withdrawals from both Part 1 and Part 2, for various reasons including transplantation, and so will likely enrol more than 21 patients to Part2