Characterization of CAR-T cell factors that contribute to myeloid cell activation

Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in the treatment of hematologic cancers; however, their use is often accompanied by inflammatory toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). These toxicities, ranging from mild to life-threatening, are partly driven by bystander myeloid cell activation (BMCA) and the subsequent release of pro-inflammatory cytokines such as IL-6 and IL-1β. Although previous studies have described the individual contributions of GM-CSF, IFN-γ, and TNFα secreted by CAR-T cells, a comprehensive characterization of CAR-T-derived inflammatory factors has been lacking. In this study, we characterized the soluble factors secreted by activated CAR-T cells derived from human peripheral blood and assessed their role in BMCA. Comparative cytokine analyses across human T cell subsets, including CAR-T cells, identified multiple candidates involved in BMCA. Antibody-mediated neutralization confirmed that four factors, GM-CSF, IFN-γ, TNFα, and GPIbα, play dominant roles in driving BMCA. Furthermore, siRNA-mediated knockdown of these factors in CAR-T cells significantly reduced BMCA without impairing their anti-tumor activity. These findings are consistent with prior reports on the inflammatory roles of GM-CSF, IFN-γ, and TNFα during CAR-T cell therapy and, importantly, identify GPIbα as a previously unrecognized contributor to CAR-T-associated inflammatory toxicities. Targeting these factors through antibody blockade or genetic modification may represent a promising strategy to mitigate inflammatory toxicities and improve the safety of CAR-T cell therapies.