Enriched circulating and tumor-resident TGF-beta(+) regulatory B cells in patients with melanoma promote FOXP3(+) Tregs

B cells are emerging as key players of anti-tumor adaptive immune responses. We investigated regulatory and pro-inflammatory cytokine-expressing B cells in patients with melanoma by flow cytometric intracellular cytokine, CyTOF, transcriptomic, immunofluorescence, single-cell RNA-seq, and B:T cell co-culture analyses. We found enhanced circulating regulatory (TGF-beta(+) and PD-L1(+)) and reduced pro-inflammatory TNF-alpha(+) B cell populations in patients compared with healthy volunteers (HVs), including lower IFN-gamma(+):IL-4(+) and higher TGF-beta(+):TNF-alpha(+) B cell ratios in patients. TGF-beta-expressing B cells in the melanoma tumor microenvironment assembled in clusters and interacted with T cells via lymphoid recruitment (SELL, CXCL13, CCL4, CD74) signals and with Tregs via CD47:SIRP-gamma, and FOXP3-promoting Galectin-9:CD44. While reduced in tumors compared to blood, TNF-alpha-expressing B cells engaged in crosstalk with Tregs via TNF-alpha signaling and the ICOS/ICOSL axis. Patient-derived B cells promoted FOXP3(+) Treg differentiation in a TGF-beta-dependent manner, while sustaining expression of IFN-gamma and TNF-alpha by autologous T-helper cells and promoting T-helper cell proliferation ex vivo, an effect further enhanced with anti-PD-1 checkpoint blockade. Our findings reveal cytokine-expressing B cell compartments skewed toward regulatory phenotypes in patient circulation and melanoma lesions, intratumor spatial localization, and bidirectional crosstalk between B and T cell subsets with immunosuppressive attributes.