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DNA methyltransferase 3a (DNMT3a) is an important part of the epigenetic machinery that stabilizes patterns of activated T cell responses. We hypothesized that donor T cell DNMT3a regulates alloreactivity after allogeneic blood and marrow transplantation (allo-BMT). T cell conditional Dnmt3a KO mice were used as donors in allo-BMT models. Mice receiving allo-BMT from KO donors developed severe acute graft-versus-host disease (aGVHD), with increases in inflammatory cytokine levels and organ histopathology scores. KO T cells migrated and proliferated in secondary lymphoid organs earlier and demonstrated an advantage in trafficking to the small intestine. Donor T cell subsets were purified after BMT for whole-genome bisulfite sequencing (WGBS) and RNA-Seq. KO T cells had global methylation similar to that of WT cells, with distinct, localized areas of hypomethylation. Using a highly sensitive computational method, we produced a comprehensive profile of the altered epigenome landscape. Hypomethylation corresponded with changes in gene expression in several pathways of T cell signaling and differentiation. Additionally, Dnmt3a-KO T cells resulted in superior graft-versus-tumor activity. Our findings demonstrate a critical role for DNMT3a in regulating T cell alloreactivity and reveal pathways that control T cell tolerance. These results also provide a platform for deciphering clinical data that associate donor DNMT3a mutations with increased GVHD, decreased relapse, and improved survival.

Background: Although severe haemoglobinopathies can be cured with allogeneic blood or bone marrow transplantation, availability of matched donors and toxic effects can be problematic. We previously found that non-myeloablative haploidentical related bone marrow transplantation with post-transplantation cyclophosphamide expanded the donor pool while limiting graft-versus-host disease (GVHD). However, graft failure-albeit with full host haemopoietic recovery-occurred in 50% of patients. In this study, we investigated whether increasing total body irradiation from 200 cGy to 400 cGy would improve engraftment while maintaining the safety profile., Methods: This study was done at Johns Hopkins Hospital (Baltimore, MD, USA). Patients aged 2-70 years receiving their first bone marrow transplant were eligible for inclusion in the study. Patients received rabbit-derived intravenous anti-thymocyte globulin 0·5 mg/kg on day -9 and 2 mg/kg on days -8 and -7, intravenous fludarabine 30 mg/m 2 on days -6 to -2, intravenous cyclophosphamide 14·5 mg/kg on days -6 and -5, and total body irradiation 400 cGy administered as a single fraction on day -1. We collected unmanipulated bone marrow and infused on day 0. GVHD prophylaxis comprised intravenous cyclophosphamide 50 mg/kg per day on days 3 and 4 after transplantation, oral mycophenolate mofetil 15 mg/kg per dose (maximum 1 g) every 8 h on days 5 to 35, and oral sirolimus to maintain a level of 5-15 ng/dL for at least 1 year starting on day 5. The original planned primary objectives of this phase 2 clinical trial were transplant-related mortality and progression-free survival. However, the coverage decision by the Centers for Medicare and Medicaid Services to only provide payment for allogeneic bone marrow transplantation for patients with sickle cell disease on a clinical trial that had a comparison arm with patients not receiving bone marrow transplantation prompted the closure of this trial to accrual in 2017. Therefore, as we were unable to perform our planned statistical analysis, the primary objective was modified to evaluate engraftment, assessed by chimerism. This trial is registered with ClinicalTrials.gov, number NCT00489281. The study is closed to new participants and this is the primary analysis., Findings: Between Sept 24, 2014, and Aug 1, 2017, we enrolled 17 consecutive patients: 12 (71%) with sickle cell disease and 5 (29%) with β-thalassaemia major. The median patient age was 16 years (range 6-31, IQR 7·7-27·5). One (6%) of 17 patients had primary graft failure with recovery of host haemopoiesis. 13 (76%) of 17 patients achieved full donor chimerism and three (18%) had mixed donor-host chimerism. Five (29%) of 17 patients developed grade 2-4 acute GVHD, including four (24%) with maximal grade 2 GVHD and one (6%) with grade 3 GVHD. Chronic GVHD developed in three (18%) patients. As of their last follow-up visit, GVHD had resolved in all patients and no patients were receiving systemic GVHD therapy. All patients remained alive as of Aug 4, 2019, and the median follow-up duration was 705 days (range 355-1294; IQR 398-943). Only one (6%) of the 16 engrafted patients remained transfusion dependent, and 14 (88%) discontinued immunosuppression., Interpretation: Increasing total body irradiation to 400 cGy substantially reduced graft failure while maintaining the safety of haploidentical bone marrow transplantation with post-transplantation cyclophosphamide. These results suggest that engraftment after haploidentical bone marrow transplantation for haemoglobinopathies is possible, and primary graft failure-the main problem previously reported-might be addressed by this strategy. Therefore, this curative approach should no longer be restricted to patients with HLA-matched donors., Funding: Maryland Stem Cell Research Fund and US National Institutes of Health., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

High-dose cyclophosphamide given after HLA-matched related and unrelated allogeneic bone marrow transplantation (BMT) for patients with hematologic malignancies is effective single-agent graft-versus-host disease (GVHD) prophylaxis in adults. Data describing outcomes for pediatric and young adult patients have not been reported. Between the years 2007 and 2013, 29 pediatric and young adult patients ages ≤21 years of age treated at our institution for high-risk hematologic malignancies underwent myeloablative HLA-matched related T cell-replete BMT. Eleven patients received post-transplantation cyclophosphamide (PTCy) as single-agent GVHD prophylaxis and were followed prospectively. Eighteen patients received calcineurin inhibitor (CNI)-based standard GVHD prophylaxis and were studied retrospectively as a control group. No acute GVHD (aGVHD) developed in patients receiving PTCy, whereas patients receiving CNI-based GVHD prophylaxis had cumulative incidences of grades II to IV and grades III and IV aGVHD of 27% and 5%, respectively. No patients receiving PTCy developed chronic GHVD, compared to 1 in the control group. Two-year overall survival was similar between the 2 groups (54% PTCy versus 58% CNI-based prophylaxis), as was event-free survival (42% PTCy versus 47% CNI-based). The 5-year cumulative incidence of relapse was 58% for PTCy and 42% for CNI-based GVHD prophylaxis (P = .45). These results suggest that PTCy is a safe and efficacious method of GVHD prophylaxis after an HLA-matched related BMT in the pediatric and young adult population that affords patients to be off all post-transplantation immunosuppression on day +5., (Copyright © 2016 American Society for Blood and Marrow Transplantation. All rights reserved.)