Study on the effect of γδ T cells expanded in vitro to kill hepatocellular carcinoma cells.

Background: γδ T cells for tumor cell immunotherapy has recently become a hot topic. Objective: To investigate the stimulation of expanded γδ T cells in vitro to kill liver cancer cells and its mechanism, and in vivo validation. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and amplified. The proportion of γδ T cells in T cells was determined using flow cytometry. γδ T cells were selected as effector cells, and HepG2 cells as target cells in the cytotoxicity experiment. NKG2D blocker was used to block effector cells from identifying target cells, and PD98059 was used to block intracellular signaling pathways. The nude mice tumor model was established in two batches, the tumor growth curve was drawn, and the tumor formation effect was tested using small animal imager to verify the killing effect of γδ T cells. Results: The γδ T cells in the three experimental groups exhibited a large amount of amplification (P < 0.01). In the killing experiment, the killing rate of γδ T cells stimulated by zoledronate (ZOL) in the experimental group was significantly higher than that in the HDMAPP group and the Mycobacterium tuberculosis H37Ra strain (Mtb-Hag) group (P < 0.05). The blocking effect of PD98059 is stronger than that of the NKG2D blocker (P < 0.05). Among them, in the HDMAPP group, when the target ratio was 40:1, the NKG2D blocker exhibited a significant blocking effect (P < 0.05). Alternatively, in the ZOL group, when the effect ratio was 10:1, the effector cells were blocked significantly after treatment using PD98059 (P < 0.05). In vivo experiments verified the killing effect of γδ T cells. According to the tumor growth curve, there was a difference between the experimental and control groups after cell treatment (P < 0.05). Conclusion: ZOL has high amplification efficiency and a positive effect on killing tumor cells.

c-Met specific CAR-T cells as a targeted therapy for non-small cell lung cancer cell A549.

Non-small cell lung cancer (NSCLC) is considered to be one of the most prevalent and fatal malignancies, with a poor survival rate. Chimeric antigen receptor T cell (CAR-T) cell therapy is one of the most exciting directions in the field of Cellular immunotherapy. Therefore, CAR-T cells that target c-Met have been developed for use in NSCLC therapy and might be a potential therapeutic strategy. The anti c-Met scFv structure was fused with the transmembrane and intracellular domains. Using a lentiviral vector to load the c-Met CAR gene, then transfected the c-Met CAR lentiviral into human T cells to obtain the second generation c-Met CAR-T expressing CARs stably. In vitro co-culture, experiments revealed that CAR-T cells have high proliferative activity and the potential to secrete cytokines (IL-2, TNF-α, and IFN-γ). c-Met CAR-T cells showed special cellular cytotoxicity in LDH release assay. A subcutaneous tumor model in nude mice was used to test the anticancer effectiveness of c-met CAR-T cells in vivo. For c-Met positive NSCLC tissue, according to tumor volume, weight, fluorescence intensity, and immunohistochemical detection, c-Met CAR-T cells had stronger tumor growth suppression compared to untransduced T cells. HE staining revealed that c-Met CAR-T cells did not produced side effects in nude mice. Taken together, we provided useful method to generate c-Met CAR- T cells, which exhibit enhanced cytotoxicity against NSCLC cells in vitro and in vivo. Thus, providing a new therapeutic avenue for treating NSCLC clinically.Highlights (1) c-Met CAR-T capable of stably expressing c-Met CARs were constructed.(2) c-Met CAR-T have strong anti-tumor ability and proliferation ability in vitro.(3) c-Met CAR-T can effectively inhibit the growth of A549 cells subcutaneous xenografts.