The Acute Myeloid Leukemia microenvironment is defined by ineffective immune surveillance despite the presence of activated, clonally-expanded CD8 T cells with preserved effector function

Abstract T cell-based immunotherapies have shown limited efficacy in acute myeloid leukemia (AML), likely due to an incomplete understanding of how leukemia reshapes the T cell compartment. To address this, we performed a multi-platform study integrating high-dimensional proteomics, single-cell multiomics, and in vivo modeling to dissect CD8 T cell dynamics in AML. Using high-dimensional mass cytometry, we profiled diagnostic (untreated) bone marrow samples from 46 AML patients and 5 healthy donors with two panels: one assessing the broader hematopoietic landscape and another focused on T cell phenotypes. AML samples exhibited a marked depletion of naïve CD8 T cells and enrichment of terminal effector (TEMRA) cells characterized by CD57, granzyme B, and perforin. While reduced naïve T cells correlated with expansion of HSPC-like progenitor cells, no specific myeloid or progenitor phenotype was associated with the presence of TEMRA cells. To examine transcriptional states and clonal architecture, we performed single-cell RNA and TCR sequencing on marrow-derived CD45+CD3+ T cells from 16 AML patients. Differential gene expression revealed that CD8 T cells from patients who achieved remission after induction chemotherapy were enriched for activation and cytokine-response pathways, suggesting that a pre-existing activated T cell program may support leukemic clearance in response to chemotherapy. TCR sequencing uncovered highly expanded CD8 T cell clones in several patients. Two individuals with TP53-mutant AML exhibited extreme clonal dominance, with single clones comprising ~50% of their marrow CD8 T cells. Overall, highly expanded clones (>10% of the patient's CD8s) showed enrichment in granzyme-mediated cell death and myeloid leukocyte activation pathways, yet lacked classical markers of exhaustion such as PDCD1 or LAG3. These findings suggest a non-canonical effector state that is potentially functional, diverging from the exhausted T cell phenotypes described in solid tumors or chronic viral infection. We mapped TCR sequences against public databases and identified CMV-specific clones in three AML patients. Two of these comprised >10% of the marrow CD8 T cell population, while the third represented ~1% of its respective sample. Given that the bone marrow is both the primary site of disease and a reservoir for resident memory T cells, these findings suggest that the leukemic niche may remodel both tumor-specific and bystander viral-specific T cells. CMV-specific clones expressed higher levels of SLAMF6 and GZMB. In contrast, non-viral expanded clones (potentially leukemia reactive) expressed higher levels of GNLY and NFKB1, suggesting distinct effector programs shaped by viral vs tumor-associated antigen exposure in AML. To interrogate CD8 dynamics in vivo, we employed three unconditioned (non-irradiated) immunocompetent mouse models of AML representing diverse genetic drivers. Transformed progenitor cells expressing Mll-Af9 or overexpressing Meis1 and Hoxa9 robustly engrafted in CD45.1+ hosts, as did bone marrow cells from an inducible Flt3ITD-Npm1c double-mutant model (Bowman 2024). Across all models, leukemia reduced CD8 T cell frequency in blood, spleen, and lymph nodes, but CD8 T cells were relatively preserved, and even enriched, within the bone marrow as a proportion of CD45.1+ host cells. This marrow-specific retention suggests selective expansion or maintenance of CD8 T cells in the leukemic niche. Ex vivo stimulation of AML-exposed marrow CD8 T cells with PMA and ionomycin demonstrated preserved, and in some cases enhanced, IFNγ production. Transcriptomic profiling of effector CD8 T cells from Mll-Af9 leukemic mice showed upregulation of proliferation and inflammation pathways, including E2F targets and G2M checkpoints, mirroring the activation patterns observed in patient samples. These findings identify a conserved terminal effector CD8 T cell phenotype in AML across diverse human and mouse systems. This population is clonally expanded, transcriptionally active, and functionally competent. Highly clonal CD8 T cell populations expressed cytotoxic gene programs without classical features of exhaustion. The presence of expanded CMV-specific clones further suggests that the AML marrow niche also shapes bystander T cells. These insights highlight preserved cytotoxic potential within the AML-associated CD8 T cell pool and suggest opportunities to refine immunotherapy strategies that leverage this effector compartment.