CD19 CAR-macrophage suppressed the progression of DLBCL via modulating SYK/SHP2/TBK1/TFEB signaling.

To explore the specific molecular mechanisms of CD19 CAR-Macrophage (CD19 CAR-M) in treating Diffuse Large B-cell Lymphoma (DLBCL). Differential expression and pathway enrichment analyses of GEO dataset GSE66770 were performed to construct a SYK/SHP2/TBK1/TFEB interaction network. CD19 CAR-M were generated by lentiviral transduction of THP-1-derived macrophage-like cells and co-cultured with U2932 cells. CCK-8, colony formation and Transwell assays were used to evaluate U2932 proliferation, invasion and migration; IL-6 and IFN-γ levels were measured by ELISA; ROS was detected by DHE staining. RT-qPCR quantified macrophage markers CD14 (M0), CD163 and CD206 (M2), and M1 marker IL-12. Western blot analyzed p-SYK, p-SHP2, NOX2, p-TBK1, TFEB, LC3B and phosphorylated/total protein ratios; immunofluorescence assessed endosomal markers EEA1 and LAMP1. Phagocytosis was evaluated by fluorescent microsphere assay; U2932 apoptosis was determined by flow cytometry after FcγR blockade. Flow cytometry was additionally performed to validate the maintenance of macrophage phenotype, CAR transduction efficiency, and the purity of cell separation after co-culture. NOD-scid mouse DLBCL xenografts were subjected to in vivo imaging for therapeutic validation. Bioinformatic analysis identified TBK1 as a key immune-evasion node positively correlated with SYK and negatively with SHP2. Flow cytometric analysis confirmed successful construction of the CD19 CAR-M cell model, with stable macrophage phenotype and specific CAR expression. CD19 CAR-M significantly inhibited U2932 proliferation, invasion and migration, elevated IL-6, IFN-γ and ROS, and induced M1 polarization. Phosphorylation of SYK/SHP2/TBK1/TFEB axis proteins and LC3B expression were markedly increased; EEA1/LAMP1 fluorescence intensities were enhanced. Fluovrescent microsphere uptake was elevated and reversible by FcγR inhibitor elsubrutinib, accompanied by increased U2932 late-apoptosis rate. In vivo experiments confirmed that CD19 CAR-M reduced tumor burden via activation of the SYK/SHP2/TBK1/TFEB axis. CD19 CAR-M kills DLBCL through macrophage phagocytosis mediated by the SYK/SHP2/TBK1/TFEB axis.