Cd39 overexpression in AML blasts elicits an immune suppressive bone barrow microenvironment via G3/G4 neutrophils and Th1 cells

Abstract Introduction:Cd39 expression in TIB-49 cells enhances the stemness and aggressiveness of AML blasts in vivo (PMID: 39157874, EHA2024-1113) via the Cd39-P2rx7-Wnt signaling axis. However, the impact of Cd39 overexpression in leukemic blasts within the bone marrow (BM) microenvironment still needs to be elucidated. Methods: Wild-type (WT) C57BL/6 mice were bred and maintained at Shandong Cancer Hospital and Institute. The syngeneic murine AML cell line TIB-49 from American Type Culture Collection was transduced with a lentiviral vector to overexpress Cd39 and labeled with TdTomato (hereafter referred to as TIB-Cd39high). WT mice were injected intravenously with 1×10⁶ TIB-49 or TIB-Cd39high cells, respectively. BM cells were harvested from leukemic mice for single-cell RNA sequencing (scRNA-seq, CapitalBio Technology, Beijing, China) and flow cytometry (FACS) analysis when showing leukemic symptoms. Results: Transcriptomic profiling analysis showed a significant increase in neutrophil proportions in the BM of TIB-Cd39high-bearing mice (BMC2) when compared to TIB-49-bearing mice (BMC1), which was also confirmed by FACS analysis. Neutrophils were then classified into five major subpopulations based on the expression of known marker genes: G1 (Prtn3, Elane), G2 (Ltf, Camp, Ngp, Chil3, Fcnb), G3 (Ltf, Camp, Ngp), G4 (Mmp8, Retnlg), and G5c (Fgl2, Gm2a, Gngt2), corresponding to progressive stages of neutrophil development and maturation (PMID: 32719519). G3 and G4 were found to the predominant populations in BM. Gm26917, G6pdx, Clec2d, and Ifi27l2a were identified as upregulated differentially expressed genes (DEGs) in G3/G4 subsets from the BMC2 compared to the cells from BMC1. Notably, Clec2d has been found to be associated with immunosuppression, directly inhibiting CD4⁺ T cell activity and proliferation via interaction with the Cd161 receptor (PMID: 38153903). We next investigated interactions between AML cells and neutrophils. Cytokine-receptor pair analysis revealed reduced Spp1-Cd44 signaling in the BMC2. Given the critical role of Spp1-Cd44 in neutrophil recruitment and differentiation, its downregulation in the BMC2 may impair neutrophil maturation or promote a pro-tumorigenic, immunosuppressive phenotype. Additionally, a higher proportion of Cd4⁺ T cells (Th1 subset) was observed in the BMC2 than that in the BMC1, which was also validated by FACS analysis. GO and KEGG enrichment analyses of these cells showed enhanced leukocyte degranulation but attenuated responses to external stimuli and chemical stress. KEGG pathways associated with neutrophil extracellular trap formation were upregulated, while immune-related pathways (e.g., IL-17 and TNF signaling) were downregulated, suggesting suppressed T cell proinflammatory functions. Our prior work (EHA2025-2202) highlighted the functions of Spp1-Cd44 axis in the interactions between TIB-Cd39high cells and Th1 cells in the BMC2, suggesting its role in AML-mediated immune modulation. Beyond AML-immune cell interactions, we analyzed neutrophil-Cd4⁺ T cell crosstalk. The Lgals9-Cd44 signaling from neutrophils to T cells was significantly upregulated in the BMC2. Lgals9 (Galectin-9) was found to regulate T cell functions, including Th1 expansion and apoptosis, further implicating this axis in BM immunosuppression. Conclusion: Our findings demonstrated that Cd39 overexpression in AML blasts elicited an immunosuppressive BM microenvironment through the expansion of G3/G4 neutrophils and Th1 cells, which was mediated by three key mechanisms: (1) the Spp1-Cd44 axis, which drive AML-induced effects on neutrophils and Th1 cells; (2) Clec2d-Cd161 signaling, facilitating neutrophil-mediated suppression of CD4⁺ T cells; and (3) Lgals9-Cd44 crosstalk, connecting neutrophil dysfunction to impaired T cell activity. These results suggested that targeting Cd39 could disrupt the immunosuppressive BM microenvironment mediated by leukemic blasts, neutrophils and CD4⁺ T cells, thereby enhancing anti-leukemic efficacy.