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 non-relapse complication after allo-HSCT is graft-versus-host disease (GVHD). The use of T regulatory (Treg) 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 one year after leukemia treatment, we report single-cell mRNA 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 transplant and this difference persisted through six months after treatment. scRNA-seq analysis 3 weeks post-transplant identified increased expression of FOXP3 and Helios amongst CD4+CD25- T conventional (Tcon) cells in Orca-T treated patients. Using flow cytometry, we then confirmed the increased frequency of this novel population of CD4+CD25-FOXP3+Helios+ Tcons 3 weeks post-treatment in patients receiving Orca-T. Further, 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 post-treatment, regardless of which therapy patients received. Overall, this study identifies a novel T cell subset which 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.