Single-Cell Profiling of CD8± T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion

Comprehensive investigation of CD8 & PLUSMN; T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we per-formed single-cell RNA profiling of CD8 & PLUSMN; T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) patients with AML. Cells coexpressing canonical exhaustion markers formed a cluster constituting <1% of all CD8 & PLUSMN; T cells. We identified two effector CD8 & PLUSMN; T-cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resis-tance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8 & PLUSMN; T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated patients with AML, suggesting that the bona fide state of CD8 & PLUSMN; T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Further-more, CD8 & PLUSMN; T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived anti-gen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8 & PLUSMN; T cells are less differentiated and have greater capacity for clonotype transitions.