Granzyme B-producing B cells: a bidirectional relationship with breast cancer cells and implications for immunotherapy

Granzyme B (GrB)-producing B cells have dual roles in tumor immunity, either killing tumor cells or suppressing antitumor responses by eliminating effector T cells. In this study, we aimed to investigate how breast cancer cells influence GrB-producing B cells from tumor-draining lymph nodes and whether B cell activation enhances their cytotoxic potential. Mononuclear cells were isolated from 14 fresh axillary lymph node samples by density gradient centrifugation using Ficoll-Hypaque. Lymphocytes were co-cultured with breast tumor cell lines (MCF-7 and MDA-231) in the presence of recombinant interleukin-21 (rIL-21) and anti-B cell receptor (BCR). B cell granzyme B production was measured by flow cytometry, while tumor cell (MCF-7) apoptosis was assessed using calcein AM release assays. Direct co-culture of lymphocytes with MCF-7 or MDA-MB-231 significantly reduced the frequency of GrB-producing B cells (P=0.001 and P=0.031, respectively), while tumor supernatants alone had no effect. When B cells were pre-stimulated with IL-21 and anti-BCR for 24 hours before direct co-culture, GrB expression was maintained at baseline levels (no significant difference vs. control). Additionally, B cells activated with IL-21 and anti-BCR caused significant apoptosis in MCF-7 cells (38±8.9%, P=0.023). In conclusion, breast cancer cells suppress GrB⁺ B cell responses via direct contact, but this suppression is reversible through B cell activation. Importantly, pre-activated B cells exhibit direct cytotoxic activity against tumor cells, highlighting their potential as an effector population for breast cancer immunotherapy.