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An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4 + /CD8 + double-positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develops from antigen-stimulated CD8 T cells, which become transcriptionally, metabolically, and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when retransplanted into naïve mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.

Relapsed/refractory T cell acute lymphoblastic leukemia (T-ALL) is difficult to salvage especially in heavily pretreated patients, thus novel targeted agents are sorely needed. Hyperactivated JAK/STAT and BCL2 overexpression promote increased T-ALL proliferation and survival, and targeting these pathways with ruxolitinib and venetoclax may provide an alternative approach to achieve clinical remissions. Ruxolitinib and venetoclax show a dose-dependent effect individually, but combination treatment synergistically reduces survival and proliferation of Jurkat and Loucy cells in vitro. Using a xenograft CXCR4+ Jurkat model, the combination treatment fails to improve survival, with death from hind limb paralysis. Despite on-target inhibition by the drugs, histopathology demonstrates increased leukemic infiltration into the central nervous system (CNS), which expresses CXCL12, as compared to liver or bone marrow. Liquid chromatography-tandem mass spectroscopy shows that neither ruxolitinib nor venetoclax can effectively cross the blood-brain barrier, limiting efficacy against CNS T-ALL. Deletion of CXCR4 on Jurkat cells by CRISPR/Cas9 results in prolonged survival and a reduction in overall and neurologic clinical scores. While combination therapy with ruxolitinib and venetoclax shows promise for treating T-ALL, additional inhibition of the CXCR4-CXCL12 axis will be needed to eliminate both systemic and CNS T-ALL burden and maximize the possibility of complete remission.

The expansion and persistence of chimeric antigen receptor (CAR) T-cells in patients are associated with response, toxicity, and long-term efficacy. As such, the tools used to detect CAR T-cells following infusion are fundamental for optimizing this therapeutic approach. Nevertheless, despite the critical value of this essential biomarker, there is significant variability in CAR T-cell detection methods as well as the frequency and intervals of testing. Furthermore, heterogeneity in the reporting of quantitative data adds layers of complexity that limit intertrial and interconstruct comparisons. We sought to assess the heterogeneity of CAR T-cell expansion and persistence data in a scoping review using the PRISMA-ScR checklist. Focusing on 21 clinical trials from the USA, featuring a Food and Drug Administration-approved CAR T-cell construct or one of its predecessors, 105 manuscripts were screened and 60 were selected for analysis, based on the inclusion of CAR T-cell expansion and persistence data. Across the array of CAR T-cell constructs, flow cytometry and quantitative PCR were identified as the two primary techniques for detecting CAR T-cells. However, despite apparent uniformity in detection techniques, the specific methods used were highly variable. Detection time points and the number of evaluated time points also ranged markedly and quantitative data were often not reported. To evaluate whether subsequent manuscripts from a trial resolved these issues, we analyzed all subsequent manuscripts reporting on the 21 clinical trials, recording all expansion and persistence data. While additional detection techniques–including droplet digital PCR, NanoString, and single-cell RNA sequencing–were reported in follow-up publications, inconsistencies with respect to detection time points and frequency remained, with a significant amount of quantitative data still not readily available. Our findings highlight the critical need to establish universal standards for reporting on CAR T-cell detection, especially in early phase studies. The current reporting of non-interconvertible metrics and limited provision of quantitative data make cross-trial and cross-CAR T-cell construct comparisons extremely challenging. Establishing a standardized approach for collecting and reporting data is urgently needed and would represent a substantial advancement in the ability to improve outcomes for patients receiving CAR T-cell therapies.

An important paradigm in allogeneic hematopoietic cell transplantations (allo-HCTs) is the prevention of graft-vs-host disease (GVHD) while preserving the graft-vs-leukemia (GVL) activity of donor T cells. From an observational clinical study of adult allo-HCT recipients, we identified a CD4+/CD8+ double positive T cell (DPT) population, not present in starting grafts, whose presence was predictive of ≥ grade 2 GVHD. Using an established xenogeneic transplant model, we reveal that the DPT population develop from antigen stimulated CD8 T cells which become transcriptionally, metabolically and phenotypically distinct from single-positive CD4 and CD8 T cells. Isolated DPTs were sufficient to mediate xeno-GVHD pathology when re-transplanted into naive mice but provided no survival benefit when mice were challenged with a human B-ALL cell line. Overall, this study reveals human DPTs as a T cell population directly involved with GVHD pathology.One Sentence SummaryHuman CD4+/CD8+ double positive T cells (DPTs) mediate xenogeneic GVHD but possess limited GVL activity.