The remission status of AML patients post alloSCT is associated with a distinct single-cell bone marrow T cell signature.

Acute myeloid leukemia (AML) is a hematologic malignancy for which allogeneic stem cell transplantation (alloSCT) often remains the only curative therapeutic approach. However, incapability of T cells to recognize and eliminate residual leukemia stem cells (LSCs) might lead to an insufficient graft-versus-leukemia (GvL) effect and relapse. Here, we performed single-cell RNA-sequencing on bone marrow (BM) T lymphocytes and CD34+ cells of six AML patients 100 days after alloSCT to identify T cell signatures associated with either imminent relapse (REL) or durable complete remission (CR). We observed a higher frequency of cytotoxic CD8+ effector and gamma delta (γδ) T cells in CR versus REL samples. Pseudotime and gene regulatory network analyses revealed that CR CD8+ T cells were more advanced in maturation and had a stronger cytotoxicity signature, while REL samples were characterized by inflammatory TNF/NF-κB signaling and an immunosuppressive milieu. We identified ADGRG1/GPR56 as a surface marker enriched in CR CD8+ T cells and confirmed in a CD33-directed chimeric antigen receptor (CAR)-T/AML co-culture model that GPR56 becomes upregulated on T cells upon antigen encounter and elimination of AML cells. We show that GPR56 continuously increases at the protein level on CD8+ T cells after alloSCT and confirm faster IFNγ secretion upon re-exposure to matched, but not unmatched recipient AML cells in the GPR56+ versus GPR56- CD8+ T cell fraction. Together, our data provide a single-cell reference map of BM-derived T cells post alloSCT and propose GPR56 expression dynamics as a surrogate for antigen encounter post alloSCT.