Granzyme B production by activated B cells derived from breast cancer-draining lymph nodes

B lymphocytes with regulatory or effector functions synthesize granzyme B (GZMB). We investigated the frequency and phenotype of GZMB-producing B cells in breast tumor-draining lymph nodes (TDLNs). Mononuclear cells were isolated from 48 axillary lymph nodes and were stimulated with anti-BCR (B cell receptor), recombinant interleukin (IL)-21 and CD40 L alone or in combination. Flow cytometry was used to evaluate the expression of GZMB in B cells, and in 4 samples the phenotype of GZMB⁺ B cells was determined. B cells produced GZMB only when stimulated with a combination of IL-21 and anti-BCR for at least 16 h. Adding CD40 L to IL-21 and anti-BCR stimuli resulted in lower GZMB production in B cells. A small fraction of B cells was able to produce perforin in all stimulation conditions, and the majority of GZMB⁺ B cells were perforin-negative. Both naïve (CD24^lowCD27^-) and active/memory (CD24^hiCD27⁺) B cells expressed GZMB. In patients with invasive ductal carcinoma, the frequency of GZMB⁺ B cells was significantly lower in metastatic compared to non-metastatic lymph nodes. The frequency of GZMB⁺ B cells did not significantly correlate with prognostic factors such as stage, tumor size or Her2 expression. In summary, a subpopulation of both naïve and memory B cells expressed GZMB in breast TDLNs. Our findings underscore the need to investigate the function of GZMB⁺ B cells in breast tumor immunity.