Sustained exposure to CAR-T cell secretome impairs human Hematopoietic Stem Cell function and is reversible by dual TNFα-IFNγ blockade

Abstract Prolonged cytopenias are a frequent complication of chimeric antigen receptor (CAR) T-cell therapies and are associated with increased infection risk and non-relapse mortality. Although inflammatory cytokines released during CAR-T cell activation have been implicated in immune effector cell-associated hematotoxicity (ICAHT), their direct effects on human hematopoietic stem and progenitor cells’ (HSPCs) function remains incompletely understood. Here, we established a reductionist model of CAR-T-associated hematotoxicity using conditioned media (CM) derived from activated CD19 CAR-T cells. Sustained exposure of human HSPCs to CAR-T-derived inflammatory secretome impaired HSPC expansion and reduced long-term repopulating capacity in xenotransplantation assays. In contrast, short-term exposure did not abrogate HSPC function, indicating that brief inflammatory signals can initiate durable reprogramming events, with functional consequences emerging during subsequent proliferative expansion. Mechanistically, CAR-T CM induced IFNγ- and TNFα-responsive transcriptional programs in HSPCs and promoted inflammatory myeloid skewing without evidence of apoptosis-dependent stem cell loss. Combined inhibition of IFNγ and TNFα restored HSPC expansion, normalized lineage output, reversed inflammatory transcriptional signatures, and rescued in vivo repopulating capacity without impairing CAR-T cytotoxic activity. These findings demonstrate that CAR-T-derived inflammatory signaling can directly impair human HSC function and identify dual TNFα/ IFNγ blockade as a potential strategy to mitigate CAR-T-associated hematotoxicity while preserving antitumor efficacy.