Regulatory-like FOXP3+Helios+CD4+ T conventional cells correlate with T-cell activation after Orca-T immunotherapy

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematologic malignancies. The primary nonrelapse complication after allo-HSCT is graft-versus-host disease (GVHD). The use of regulatory T cells to prevent GVHD has emerged as a promising allogeneic T-cell immunotherapy in the form of Orca-T. However, the precise differences in immune activation, which may influence infection, GVHD, and relapse after Orca-T compared with unmanipulated peripheral blood stem cell (PBSC) grafts, remain unexplored. Using peripheral blood specimens longitudinally collected between 3 weeks and 1 year after leukemia treatment, we report single-cell RNA sequencing (scRNA-seq) and flow cytometric analysis of 51 HLA-matched patients receiving either Orca-T or unmanipulated PBSC grafts. Orca-T recipients exhibited increased frequencies of effector memory CD4+ T cells 3 weeks after transplantation, and this difference persisted through 6 months after treatment. scRNA-seq analysis 3 weeks after transplantation identified increased expression of FOXP3 and Helios among CD4+CD25- T conventional cells (Tcon) in Orca-T-treated patients. Using flow cytometry, we then confirmed the increased frequency of this novel population of CD4+CD25-FOXP3+Helios+ Tcon 3 weeks after treatment in patients receiving Orca-T. Furthermore, we discovered that this T-cell subset possessed a regulatory-like phenotype and correlated significantly with the frequencies of activated CD4+ and CD8+ T-cell populations 3 months after treatment, regardless of which therapy patients received. Overall, this study identifies a novel T-cell subset that is enriched very early after cellular therapy for leukemia and may be predictive of long-term immune activation after Orca-T and PBSC-derived T-cell infusion.