Dissecting B-cell responses in high grade serous ovarian cancer.

Abstract

e14562 Background: T cell-based immunotherapies have demonstrated great success for the treatment of many solid tumors, but not all tumor types and patients respond well or durably. Reasons for these discrepancies are unclear. High grade serous ovarian cancer (HGSOC) is an example of a deadly disease that has exhibited poor responses to T cell targeted therapies, like checkpoint inhibitors. Thus, it would be an ideal model for studying other parts of the immune ecosystem that could contribute to anti-tumor immunity. Tumor infiltrating B cells (TIL-Bs) are being increasingly studied in HGSOC because of their association with improved prognosis, especially when differentiated into germinal center (GC) B cells. However, the true effector functions of B cells, including antigen presentation, cytokine production, and antibody production, in ovarian and other solid tumors remains understudied. Here, we study tumor antigen-specific B cell responses in a mouse model of HGSOC. Methods: We have engineered a murine HGSOC cell line ID8 p53 -/- brca2 -/- (ID8) to express a membrane bound mode antigen, hen egg lysozyme (mHEL), at two different affinities to primary HEL-specific B cells (MD4 B cells) from MD4 transgenic mice. We use flow cytometry to characterize HEL-specific B cell responses to ID8 mHEL-expressing cells in vitro . We use a syngeneic mouse model of HGSOC in which we intraperitoneally inject ID8 cells (with or without mHEL) and use flow cytometry to identify immune phenotypes of B and T cells in tumor tissue, spleen, lymph nodes, and ascites. We also use ELISA to measure HEL-specific antibody production in the ascites and serum of these mice. Results: We demonstrate that MD4 B cells can upregulate canonical B cell receptor signaling elements in response to stimulation with ID8 mHEL in vitro , and that these responses can be titrated based upon the affinity of the mHEL presented. We also show that the syngeneic ID8 p53 -/- brca2 -/- mouse model produces tumors in the ovaries and peritoneum that are enriched for B cells, and that the ascites is specifically enriched for antigen specific B2 B cells (with a much smaller innate like B1 compartment as compared to the peritoneal lavage of non-tumor controls). We plan to inject host mice with ID8 mHEL tumors and compare tumor progression in mice that subsequently receive intraperitoneal adoptive transfer of MD4 HEL-specific B cells versus not. We also plan to compare the phenotypes of B and T cells in the tumor, spleen, lymph nodes, and ascites of these mice, as well as the serum and ascites levels of HEL antibodies. Conclusions: We have generated a model antigen expressing ovarian cancer cell line that produces antigen-specific B cell responses in vitro , and have shown that there is an enrichment in antigen-specific B cells in these ovarian tumors and ascites; future in vivo studies will elucidate the important effector functions of TIL-Bs in ovarian cancer and other solid tumors.