B cells are increasingly recognized regulators of antitumor immunity in melanoma, yet their differentiation states, lineage dynamics, spatial organization and integration within the immune microenvironment, and how these are influenced with immunotherapy remain incompletely defined.

We conducted deep profiling of tumor-resident B cell differentiation and evolutionary features in the melanoma tumor microenvironment using bulk, single-cell and spatial transcriptomic analyses, long-read antibody sequencing and mass cytometry (Cytometry by Time-Of-Flight, CyTOF). We characterized B cell differentiation trajectories, class-switching patterns, antibody isotype usage, signaling pathways, spatial niches against clinicopathological features and immune cell communication, before and after checkpoint blockade and in clinically defined responders and non-responders.

Melanoma-infiltrating B cells were enriched across primary and metastatic lesions, were preferentially associated with immune-rich tumors and correlated with T and natural killer (NK) cell densities. Long-read immunoglobulin sequencing demonstrated recurrent class switch recombination (CSR) trajectories with predominant IgG and IgA isotype usage, confirmed by bulk transcriptomic analyses. Across independent single-cell and bulk transcriptomic datasets we identified conserved class-switching, B cell receptor (BCR) activation, and pseudotime analyses showed differentiation trajectories progressing from naïve and memory states toward interferon (IFN)-driven terminal differentiation trajectories that persisted before and after immunotherapy. CyTOF analyses independently confirmed proliferative, germinal center (GC)-like, memory and class-switched B cell subsets, suggesting conserved inflammatory differentiation endpoints. Spatial transcriptomic deconvolution identified IFN-rich B cells within tertiary lymphoid structures (TLS). Following immunotherapy, B cells retained class-switched trajectories, displayed enriched GC-associated profiles, enhanced crosstalk with T cells and Dendritic Cells (DCs) and preserved or induced IFN-rich IgM + /IgD + or IgG4 + phenotypes, suggesting persistence or emergence of non-class switched and atypical class-switched isotype profiles. Transcriptomic analyses of B cells pointed to upregulated antigen-driven stimulation and BCR signaling, whereas in non-responders displayed IFN-rich B cells, IFN-associated genes, and IFN-pathway enrichment.

Melanoma-infiltrating B cells follow class-switched differentiation trajectories culminating in IFN signaling, atypical antibody expressing states that persist or emerge after immunotherapy, aligning dynamic B cell evolution and atypical lineage traits with immunotherapy.