Additional file 1 of Chimeric antigen receptor T cells targeting PD-L1 suppress tumor growth
Creators: name: Qin, Le
name: Zhao, Ruocong
name: Chen, Dongmei
name: Wei, Xinru
name: Wu, Qiting
name: Long, Youguo
name: Jiang, Zhiwu
name: Li, Yangqiu
name: Wu, Haipeng
name: Zhang, Xuchao
name: Wu, Yilong
name: Cui, Shuzhong
name: Wei, Wei
name: Yao, Huihui
name: Liu, Zixia
name: Cao, Su
name: Yao, Yao
name: Zhang, Zhenfeng
name: Li, Peng
Publisher: name: figshare
Year: 2020
Subjects: subject: Biophysics
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Supplemental Figure 1. Transduction efficiency of CAR-T cells and GFP and PD-L1 expression of GL transduced cancer cell lines. (A) Transduction efficiency of CAR-T cells used in in vitro cytotoxicity assays. (B) Transduction efficiency of CAR-T cells used to evaluate the anti-tumor efficacy of the combination of CARMSLNz T and dPD1z T and the combination of CARMSLNz T and CARPD-L1z T cells in NSCLC (P4) PDX. (C) Schematic diagram of the GL vector. FACS analysis of (D) GFP and (E) PD-L1 expression levels in multiple cancer cell lines transduced with GL. Supplemental Figure 2. PD-L1 expression in primary tumor samples. (A) qRT-PCR and (B) FACS analysis PD-L1 expression in primary NSCLC cells (P1), gastric cancer cells (P2) and hepatoma carcinoma cells (P3). (C) The expression of PD-L1 and Mesothelin (MSLN) in primary NSCLC (P4) cells. Supplemental Figure 3. dPD1z T cells inhibit tumor growth in gastric cancer and hepatoma carcinoma PDXs. (A) IHC images of a normal spleen (left) and a spleen with metastatic tumors (right). (B) Images of spleens from gastric cancer PDXs after treatment with dPD1z T, CAR19z T or untreated controls (blank). (C) Tumor volumes and (D) tumor weights of hepatoma carcinoma PDXs (P3) after treatment with dPD1z T, CAR19z T cells or untreated controls (Blank). NSI mice were transplanted with hepatoma carcinoma cells at day 0, subsequently, dPD1z T or CAR19z T (5 × 106) cells were infused twice at day 15 and day 20. Tumor volumes were monitored at indicated days and tumor weights were measured after mice euthanasia. The result of tumor volume represent mean ± SEM, and was compared by two-way ANOVA with Tukey’s multiple comparisons test. * P &lt; 0.05. The result of tumor weight represent mean ± SD, and was compared by unpaired t-test. ** P &lt; 0.01. Supplemental Figure 4. The production of IL-2 and IFN-γ of CARMSLNz T, CARPD-L1z T, the combination of CARMSLNz T and CARPD-L1z T or CAR19z T cells post co-cultured with H460-MSLNGL cells. (A) FACS detection of Mesothelin (MSLN) expression of H460GL and H460-MSLNGL cells. The production of (B) IL-2 and (C) IFN-γ after CARMSLNz T, CARPD-L1z T, the combination of CARMSLNz T and CARPD-L1z T or CAR19z T cells co-cultured with H460-MSLNGL cell line for 24 h at a definitive E: T ratio (1: 1). Error bars denote SD, and the results were compared by unpaired t-test. * P &lt; 0.05, ** P &lt; 0.01, and *** P &lt; 0.001. Supplemental Figure 5. Percentages of CAR T cells in the spleen of NSCLC PDXs (P4) after treated with CARMSLNz T, CARPD-L1z T, the combination of CARMSLNz T and CARPD-L1z T or CAR19z T cells (gated on live cells). Supplemental Figure 6. The expression of PD-L1 in the activated T cells. Percentage of PD-L1+ T cells in (A) CD4+ T cells (gated on CD3+CD8− cells) and (B) CD8+ T cells (gated on CD3+CD8+ cells) post activated by CD3 and CD28 antibodies. FACS detection of PD-L1 expression at indicated time points. Supplemental Figure 7. The expression of PD-L1 in CARMSLNz T cells post co-cultured with H460-MSLNGL cells. Percentage of PD-L1+ T cells in (A) CD4+ CARMSLNz T cells (gated on CD3+GFP+CD4+ cells) and (B) CD8+ CARMSLNz T cells (gated on CD3+GFP+CD8+ cells) post co-cultured with H460-MSLNGL cells. CARMSLNz T cells were co-cultured with H460-MSLNGL for 0 h, 16 h, 24 h, 40 h and 48 h at a definitive E: T ratio (1: 1), then the expression of PD-L1 was detected by FACS. Supplemental Figure 8. Overexpression PD-L1 in T cells. (A) Percentage of CD25+CD69+ T cells in CARPD-L1z T and CAR19z T cells (gated on CD3+GFP+ cells) post activated by CD3 and CD28 antibodies for 16 h. (B) Percentage of CD25+CD69+ T cells in CAR19z T cells (gated on CD3+GFP+ cells) post co-cultured with NALM6 cells for 24 h at a definitive E: T ratio (2: 1), and percentage of CD25+CD69+ T cells in CARPD-L1z T cells (gated on CD3+GFP+ cells) post co-cultured with H460GL cells for 24 h at a definitive E: T ratio (2, 1). (C) Schematic diagram of uPD-L1 vector. FACS detection of the expression of (D) CD19 and (E) PD-L1 in T cells after transduced with uPD-L1.