Figure 7.

Effect of a PD-1 antibody combined with CAR-T therapy on primary tumors and metastasis. (A) CAR-T or NT cells were incubated with MDA-MB-231 cells at different E:T ratios for indicated time points. The lysis rate was detected by the LDH (lactate dehydrogenase)-release assay and represent means±SEM of three independent experiments. (B–D) NOD/SCID mice were inoculated subcutaneously with 2×10⁶ MDA-MB-231 cells on the right mamma pat (day 0). Intravenous infusion of 5×10⁶ tmTNF-α CAR-T cells or NT cells was on days 7 and 14. PD-1 mAb (200 µg) was intraperitoneally injected every 3 days, starting on day 7 prior to CAR-T cell infusion (n=6 each group). (B) Tumor growth curve. (C) Human CD3⁺ T cells in the blood were analyzed by flow cytometry. (D) Representative images of CD3⁺ T cells in tumor tissues (magnification ×200) detected by indirect immunofluorescence and quantitative data. The quantitative data represent means±SEM. *P<0.05, **P<0.01, ***P<0.001 versus NT. (E, F) NOD/SCID mice were intravenously injected with 5×10⁵ luciferase-labeled MDA-MB-231 cells (day 0). 5×10⁶ tmTNF-α CAR-T cells or NT cells were infused on days 1 and 8. PD-1 mAb was intraperitoneally injected every 3 days, starting on day 1 prior to infusion of T cells (n=5–8, each group). (E) A representative mouse for each group for firefly luciferase imaging of pulmonary metastases and quantitative data. (F) Representative H&E staining pictures of lung sections and the percentage of tumor area was calculated using Image J software (National Institutes of Health, Bethesda, Maryland, USA). The quantitative data represent median±SD. **P<0.01, ***P<0.001 versus NT. CAR-T, chimeric antigen receptor engineered-T; DAPI, 4′,6-diamidino-2-phenylindole; E:T, effector:target; mAb, monoclonal antibody; NT, non-transduced T; tmTNF-α, transmembrane tumor necrosis factor alpha.