FcεRγI promotes canine CD8 chimeric antigen receptor T cell cytotoxicity through a Syk-NF-κB axis

Comparative oncology has advanced cancer immunotherapy, although cellular mechanisms governing chimeric antigen receptor T cell (CART) therapy in canines are poorly understood. In a first-in-canine trial, anti-CD20 CART with canine 4-1BB-CD3ζ (cBBζ) domains induced CD20-negative lymphoma outgrowth but did not persist or deplete B cells. Here we show that canine CARTs incorporating human BBζ (hBBζ) demonstrate superior therapeutic function, mediated by FcεRγI. hBBζ-CART showed greater cytolysis and CD8 T cell outgrowth than cBBζ-CART in repetitive killing assays and a canine B cell leukemia xenograft model. Transcriptional profiling revealed upregulation of FCER1G and innate-like genes in CD8 hBBζ versus cBBζ-CARTs. CRISPR-mediated FCER1G deletion and pharmacologic Syk/NF-κB inhibition indicated that Syk-NF-κB signaling regulates FcεRγI-mediated enhancement of hBBζ-CART cytotoxicity, associated with increased granzyme B and IFN-γ/TNF-α production. Syk-NF-κB signaling promotes FcεRγI expression in hBBζ CARTs, and CAR-TCR interactions potentiate NF-κB signaling to upregulate FcεRγI and enhance CART function. These studies identify a potent therapeutic subset of innate-like canine CARTs induced by hBBζ signaling, which holds potential to improve both canine and human CART therapy.