B-cell activating factor plays a critical role in CAR-T cell-associated cytokine release syndrome

Introduction : Despite the growing success of chimeric antigen receptor (CAR) T cell therapy in treating previously incurable cancers, toxicities remain a major concern. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the most common and potentially life-threatening adverse events (AEs) associated with CAR-T cell therapy and are related to a heightened immune effector state. We explored a previously unidentified role of B-cell activating factor (BAFF), a cytokine that plays a prominent role in B-cell tumor microenvironments, in the pathophysiology of these AEs. Methods : A Luminex multi-analyte assay was used to measure serum levels of BAFF and known CRS cytokines in patients who experienced CAR-T CRS. To identify the cell types contributing to the release of BAFF in vitro, monocytes, CD19 CAR-T cells, and Jeko-1 cancer cells were incubated alone or at a 1:1:1 ratio, and a multiplex assay was performed on the supernatant. After IFN-gamma stimulation of monocytes, BAFF secretion and expression of the BAFF receptors (BAFF-R, TACI, BCMA) were measured by ELISA and flow cytometry, respectively. To determine the impact of BAFF on cytokine release, this tri-culture was co-incubated in the presence of a BAFF or BCMA neutralizing antibody, and cytokine release was measured by multiplex assay (IL-6, IL-1beta, GM-CSF, IL-10, CXCL8, CCL2). To determine if BAFF neutralization interferes with CAR-T cell function, cancer cells were cocultured with CD19 CAR-T cells in the presence of monocytes and a BAFF-neutralizing antibody. Cytotoxicity was measured by propidium iodide uptake in cancer cells, and activation and degranulation of CD3+ cells was measured by percent positivity of CD69 and CD107a by flow cytometry. Results : First, we observed that patients who experienced CAR-T cell-related CRS have elevated serum BAFF levels that coincide with increased IL-6 and other known CRS and ICANS-associated cytokines. In the tri-culture system of monocytes, cancer cells, and CAR-T cells, monocytes were the primary cellular producer of BAFF. Mechanistically, we show that IFN-gamma, produced by activated CAR-T cells, stimulates monocytes to release BAFF. Additionally, monocytes derived from CRS patients express BCMA, which is further induced by IFN-gamma stimulation. In the tri-culture system of cancer cells, monocytes, and CD19 CAR-T cells, neutralization of BAFF or BCMA significantly reduces production of various CRS and ICANS-related cytokines without impairing CAR-T cell function. Conclusion : We demonstrate that BAFF plays a role in CAR-T-cell-related AEs, and that its neutralization may be a novel strategy for treating both CRS and ICANS. In our model, we suggest that activated CAR-T cells secrete IFN-gamma, which can increase the expression of BCMA on monocytes as well as the secretion of BAFF. BAFF binds to BCMA and induces increased release of cytokines that contribute to CRS and ICANS.