Exhausted CD4+T cells are associated with CCL4-driven immunosuppressive macrophage accumulation in enzootic bovine leukosis

In enzootic bovine leukosis (EBL), a B-cell lymphoma caused by bovine leukemia virus (BLV) infection, immune remodeling within tumor-affected lymph nodes is currently poorly understood. Here, we analyzed chemokine production and macrophage phenotypes in tumor-affected lymph nodes in EBL, focusing on CCL4. Intracellular cytokine staining revealed that, compared with healthy lymph nodes, EBL tumor-affected lymph nodes showed increased proportions of CCL4 expressing cells, particularly among CD4+ T cells and B cells. Functional migration assays showed that recombinant CCL4 induced robust monocyte migration, with a comparatively modest T-cell migration. To identify lymph node macrophages, we characterized CD11bhiCD172a+ cells and confirmed their macrophage identity based on high expression levels of CD11c, CD14, CD16, and CD68. Using this definition, we found that the CD11bhiCD172a+ population was markedly increased in tumor-affected lymph nodes. Phenotypic analysis revealed an increased proportion of CD163+ and CD163+PD-L1+ macrophages, consistent with the acquisition of an immunosuppressive phenotype. In parallel, macrophages in tumor-affected lymph nodes showed a shift from MHC class I/IIhi to MHC I/IIlo subsets and reduced co-expression of MHC molecules with CD80 and CD86, indicating impaired antigen-presenting features. Collectively, these findings suggest that elevated CCL4 production in tumor lesions induced by EBL is associated with monocyte recruitment and the accumulation of phenotypically altered macrophages, highlighting a chemokine-driven remodeling of the tumor immune microenvironment.