CD39-associated myeloid-T-cell coordination within an immunoregulatory neighborhood linked to treatment resistance in triple-negative breast cancer

Immunotherapy efficacy in triple-negative breast cancer (TNBC) remains limited, partly due to mechanisms of immune suppression within the tumor microenvironment (TME). Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, encoding cluster of differentiation 39, CD39) is a key immunoregulatory ectonucleotidase, but its treatment-associated distribution and coordination across myeloid and T-cell states in TNBC remain incompletely characterized. We re-analyzed a single-cell RNA sequencing (scRNA-seq) dataset comprising 156120 immune cells from 27 tumor and metastatic samples across 14 patients with TNBC. Trajectory and cell-cell communication analyses were complemented by NicheNet, independent single-cell and bulk transcriptomic validation, CosMx spatial transcriptomics, and multiplex immunofluorescence. CD39 protein expression and its association with survival were evaluated in an independent cohort of 112 patients with TNBC using immunohistochemistry (IHC), Kaplan-Meier analysis, and multivariable Cox proportional hazards regression. Three signed-R 2 -based summary indices-treatment contribution (TC), ENTPD1 dynamic index (EDI), and tumor response index (TRI)-were applied to summarize treatment-associated immune dynamics. ENTPD1 expression was observed across macrophage and regulatory T-cell states, including CD4 + CD39 + Tregs. Higher macrophage ENTPD1 expression was associated with M2-related programs, attenuated stimulator of interferon genes (STING)-type I interferon signaling, and reduced antigen-presentation programs. Cell-cell communication analysis revealed preferential interactions between myeloid cells and CD4 + CD39 + T cells. Inferred lymphotoxin (LT) signaling involving CD4 + CD39 + T cells and myeloid populations was higher in non-responders. Cross-cohort and patient-level analyses supported coordinated CD39-associated immune programs, while spatial transcriptomics and representative multiplex immunofluorescence imaging visualized the implicated populations within shared tumor regions. In the independent IHC cohort, high tissue-level CD39 expression remained independently associated with shorter overall survival (OS) after adjustment for age group and American Joint Committee on Cancer (AJCC) stage. ENTPD1/CD39 was associated with coordinated myeloid and T-cell states, an adenosine-related immunoregulatory context, and reduced response to anti-programmed death-ligand 1 (PD-L1) therapy plus chemotherapy in TNBC. These findings support further evaluation of ENTPD1/CD39-associated immune states in the context of immune checkpoint blockade (ICB). High tissue-level CD39 expression was independently associated with shorter OS in an independent clinical cohort.