BackgroundOur previous study showed that transmembrane tumor necrosis factor alpha (tmTNF-alpha) is overexpressed in primary breast cancers including triple-negative breast cancers (TNBCs). Chimeric antigen receptor engineered-T (CAR-T) cells have been successfully used mainly in B-cell malignancies.MethodsWe generated CAR-T cells targeting tmTNF-alpha but not secreted tumor necrosis factor alpha and assessed the antitumor effect of the CAR-T cells on tmTNF-alpha-expressing breast cancer cells in vitro and in vivo.ResultsOur tmTNF-alpha CAR-T cells showed potent cytotoxicity against tmTNF-alpha-expressing breast cancer cells but not tmTNF-alpha-negative tumor cells with increased secretion of interferon gamma (IFN-gamma) and interleukin (IL)-2 in vitro. In tmTNF-alpha-overexpressing TNBC-bearing mice, the tmTNF-alpha CAR-T therapy induced evident tumor regression, prolonged survival and increased serum concentrations of IFN-gamma and IL-2. However, we found thattmTNF-alpha induced programmed death-ligand 1 (PD-L1) expression through the p38 pathway via TNF receptor (TNFR) and through the NF-kappa B and AKT pathways via outside-to-inside (reverse) signaling, which might limit the efficacy of the CAR-T cell therapy. Blockage of the PD-L1/programmed death-1 (PD-1) pathway by PD-1 monoclonal antibody significantly enhanced the antitumor effect of the tmTNF-alpha CAR-T cell therapy in vitro and in vivo, and the combination was effective for antiprimary tumors and had a tendency to increase the antimetastasis effect of the CAR-T cell therapy.ConclusionOur findings suggest a potent antitumor efficacy of the tmTNF-alpha CAR-T cells that can be enhanced by anti-PD-L1/PD-1 because high PD-L1 expression in TNBC was induced by the tmTNF-alpha signaling, indicating a promising individual therapy for tmTNF-alpha-positive breast cancers including TNBC.