cd30 apc  (Miltenyi Biotec)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Humanized CD30-Targeted Chimeric Antigen Receptor T Cells Exhibit Potent Preclinical Activity Against Hodgkin’s Lymphoma Cells

doi: 10.3389/fcell.2021.775599

Figure Lengend Snippet: CARs with humanized HRS3-specific scFv recognize CD30 + tumor cells. (A) B cell lymphoma and Hodgkin’s lymphoma (HL) cell lines were stained with a monoclonal antibody specific for the CD30 antigen and analyzed using flow cytometry. CD30 expression was evident in a majority of the cell lines after staining with an anti-CD30 monoclonal antibody (Pink) when compared to staining with control antibody (Blue). Raji was used as negative control. (B) Flow-cytometry analyses in a representative donor showing CAR expression. (C) T cells expressing hHRS3-CAR and HRS3-CAR exhibited cytotoxicity against the non-HL and HL cell lines in a 24-h assay. Graphs show mean ± SEM of duplicate wells. One of two representative experiments with different donor cells and similar results is shown. (D) IFN-γ concentrations in the supernatants of CD30-CAR-T-cells after stimulation with Raji, L428, and L540 cells for 24 h, analyzed by ELISA. (E,F) In vitro cytotoxic assay to evaluate cytolytic activity of NC T cells (Blue line), CD19-CAR-T cells (Purple line), HRS3-CAR-T cells (Red line) and hHRS3-CAR-T cells (Black line) on CD30 + lymphoma cell lines, namely L428 and L540 cells. The results are presented as the mean volume ±SD, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 vs CD19 (CD19 as negative control). (G) Diagram of the full-length overexpressing human CD30 vector. (H,I) Detection of Raji and Raji-CD30 cell killing and cytokines (CD19 as positive control). “NC” represents uninfected T cells (negative control). “MOCK” indicates the transgenic plant that contained only the vector construct (negative control). The data were derived from different donor cells and the in vitro experiments were repeated independently at least three times. The results are presented as the mean volume ±SD, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 vs NC.

Article Snippet: The following antibodies were used: PE-anti-CD3 (Clone UCHT1, BD Biosciences or Biolegend, 981002), APC-anti-CD4 (BioLegend, 357408), FITC-anti-CD8 (BioLegend, 344704), PE-anti-CD45RO (Biolegend, 304206), APC-anti-CCR7 (BioLegend, 353214), and CD30 APC (Clone BerH8, BD Biosciences).

Techniques: Staining, Flow Cytometry, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, In Vitro, Activity Assay, Plasmid Preparation, Positive Control, Transgenic Assay, Construct, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Humanized CD30-Targeted Chimeric Antigen Receptor T Cells Exhibit Potent Preclinical Activity Against Hodgkin’s Lymphoma Cells

doi: 10.3389/fcell.2021.775599

Figure Lengend Snippet: Central memory cells (Tcm) expressing hHRS3-CAR were enriched in mice and conferred long-lasting immunity to HL. (A) Survival of mice is shown as a Kaplan-Meier curve (data shown combined from two independent experiments, n = 12/group). Survival was not statistically different for NC mice versus CD19; p = 0.8037. p < 0.0001 for the comparison of HRS3 group versus CD19 mice. p < 0.0001 for the comparison of hHRS3 group versus CD19 mice. NC, non-transduced T cells. (B) Flow cytometry detection of tumor cell involvement in the liver of mice in each group. The control group was CD19 group ( n = 3), and the experimental group was HRS3 and hHRS3 groups ( n = 4). (C) Spleen, (D) blood, and (E) bone marrow from surviving CD30-CAR-treated mice ( n = 3) were analysed for the number of CAR-T cell subsets using flow cytometry (mean ± SD). T cells are functionally divided into four subsets: naïve (CCR7 + CD45RO − ); central memory (CCR7 + CD45RO + ); effector memory (CCR7 − CD45RO + ); and terminal effector (CCR7 − CD45RO − ), according to the cell surface expression of CCR7 and CD45RO. Each experiment included 6 mice per group and was repeated twice (total n = 12 mice per group). (B) data shown are representative or (C–E) combined from two independent experiments ( n = 12). * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 vs control (CD19).

Article Snippet: The following antibodies were used: PE-anti-CD3 (Clone UCHT1, BD Biosciences or Biolegend, 981002), APC-anti-CD4 (BioLegend, 357408), FITC-anti-CD8 (BioLegend, 344704), PE-anti-CD45RO (Biolegend, 304206), APC-anti-CCR7 (BioLegend, 353214), and CD30 APC (Clone BerH8, BD Biosciences).

Techniques: Expressing, Flow Cytometry