human duoset elisa kits  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: Therapeutic T cells with 3-in-1 strategy for the treatment of biliary tract cancer

doi: 10.1016/j.xcrm.2025.102349

Figure Lengend Snippet: Structure of ScTILs with native TCR and enhance receptor improved the function of T cells (A) The schematic diagram illustrates the structure of ScTILs, which includes the native TCR, an enhance receptor (ER) made up of an anti-PD-L1 scFv fused with a transmembrane region and the cytoplasmic fragment of CD28 molecule, and a CAR for expansion (CFE) that consists of scFv to CD19 fused to a transmembrane region, cytoplasmic fragment of 4-1BB, and the epsilon subunit of the CD3 complex. (B) Expression of ER and TCR or ER and CFE on cell surface demonstrated by flow cytometry analysis. Flow cytometry confirmed the expression of ER and TCR on the surface of ER-TCR-T cells and the expression of ER and CFE on the ER-CAR-T cells. The top three figures depict the detection of both ER and TCR on ER-TCR-T cells while the lower three figures show the expression of ER and CAR on ER-CAR T cells. In both cases, the ratio of double-positive cells was found to be above 40%. (C) Comparative cytotoxicity analysis of MOCK-T, CAR-T, and ER-CAR-T cells against Raji-PD-L1-CBG cells at different T:E ratios. The cytotoxicity levels of MOCK-T, CAR-T, and ER-CAR-T cells against Raji-PD-L1-CBG cells were analyzed at T:E ratios of 0.5:1, 1:1, and 2:1, respectively. The results show a comparative analysis of the cytotoxicity levels achieved by the three different cells. The p values less than 0.05, less than 0.01, and less than 0.001 are represented by ∗, ∗∗, and ∗∗∗, respectively. (D) Analysis of cytokine release from ER-TCR-T cells co-cultured with J82- NY or J82-NY-PD-L1 target cells. The ELISA assay was used to analyze the levels of IFN-γ and IL-2 released by ER-TCR-T cells when co-cultured with J82-NY or J82-NY-PD-L1 target cells. The analysis was done based on the supplier’s kit instructions, and the results represent the average (mean ± SD) obtained from three independent experiments. The p values less than 0.05, less than 0.01, and less than 0.001 are represented by ∗, ∗∗, and ∗∗∗, respectively. (E) Antitumor effect of MOCK-T, CAR-T, and ER-CAR-T cells on Raji tumor models. Mice inoculated with Raji tumor cells subcutaneously were intravenously infused with MOCK-T, CAR-T, or ER-CAR-T cells prepared from peripheral blood PD-1+ T cells after 30 min. The tumor volume was measured at different time points, and the results are presented as mean tumor volume (±SEM) for five mice per group. The numbers beside the horizontal axis show the number of mice with a complete response (CR) out of five mice in each group. (F) Evaluation of the antitumor effect of ER-TCR-T cells on J82-NY xenograft model. NSG mice were subcutaneously inoculated with J82-NY- GFP tumor cells. When the average tumor volume reached 100 mm 3 , mice were randomly split into groups and infused with MOCK-T, TCR-T, or ER- TCR-T cells. Tumor volume was measured at different time points, and the results are presented as mean tumor volume (±SEM) for five mice per group. The numbers beside the horizontal axis indicate the number of mice demonstrating a complete response (CR) out of five mice in each group. (G) T cells survived in tumor tissues in different treatments of animals.

Article Snippet: For cytokine production assays, supernatants were collected 24 h after co-culture and were assessed for human IL-2 and IFN-γ using the DuoSet ELISA Development Kit (R&D Systems).

Techniques: Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay