Fig. 1

The functions of 2nd-generation CAR-T cells are significantly impaired in MPE/MA environment, and it is partially related to MPE/MA-induced PD-L1 expression. a The cytotoxicity of HER CAR-T cells (HER2.28ζ and HER2.BBζ) that incubated with SKOV3 or A549 cells for 24 h in cytokine-free medium (culture medium) was analyzed by CCK-8 assay. b Summary of IFN-γ, IL-2 and TNF-α release by HER2.28ζ and HER2.BBζ CAR-T cells after 24 h of co-incubation with SKOV3 or A549 (E: T = 2: 1) as measured by ELISA assay. c The cytotoxicity of HER2.28ζ CAR-T cells was analyzed by CCK-8 assay, after 48 h of co-culture with SKOV3 cells (E: T = 2: 1) in different components of patient-derived MPE or MA samples (Supplementary Table. S1). d The cytotoxicity of HER2.28ζ CAR-T cells was analyzed by CCK-8 assay, after 24 h of co-culture with SKOV3 cells in culture medium or MPE-supernatant (Pt1, as Supplementary Table. S1). e The levels of cytokines (IFN-γ, IL-2 and TNF-α) in the supernatants from (c) were determined by ELISA. f The fold expansion of HER2.28ζ CAR-T cells were analyzed through FCM-counting beads, after stimulated by irradiated SKOV3 cells (E: T = 2: 1) in culture medium or MPE-supernatant (Pt1, as Supplementary Table. S1), and the portion of live CAR-T cells (Annexin-V^-/PI^-) on the 3rd day after stimulation was shown in g. h The cytotoxicity of ROR1.28ζ and CD19.28ζ CAR-T cells were co-cultured with SKOV3 and Raji cells respectively in culture medium or MPE-supernatant (Pt1, as Supplementary Table. S1) for 24 h, and analyzed by CCK-8 assay. i The mRNA expression of PD-L1 in cancer cells, fibroblasts, macrophages and T cells of human ovarian cancer ascites analyzed by single-cell RNA-sequencing (scRNA-seq) data deposited in public Gene Expression Omnibus (GSE146026). j PD-L1 mRNA expression levels in MPE/MA-cells were shown as fold change to 293 T, HUVEC and MDA-MB-468 cells (as controls). k The statistical analysis of PD-L1 expression on clinically derived MPE/MA-cells (Supplementary Table. S1) was analyzed by flow cytometry. l Representative flow cytometry histogram of PD-L1 expression on CD45^- and CD45⁺ cells from MPE/MA samples (Supplementary Table. S1). m Representative flow cytometry histogram of PD-L1 expression on healthy donor-derived CD3⁺ T cells that cultured in MPE/MA-supernatant (Supplementary Table. S1) continuously for 7 days. (Pre: pre-culturing; Post: post-culturing) Pt, patient; MPE, malignant pleural effusion; MA, malignant ascites. P-values were determined by unpaired two-tailed t-test a, b, d, f, g, h or one-way ANOVA with Tukey’s multiple comparison test adjusted P value c, e. *P <0.05; **P <0.01; ns, not significant. Data show the mean ± SD from three independent experiments