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A. Flow cytometry histograms of CFSE (Carboxyfluorescein succinimidyl ester) cell proliferation assays showing CAR-T cell proliferation upon interaction with SSEA-4 + OVCAR4 cells in contrast to SSEA-4 − Caov-3 after 48 h. NT-T cells showed no proliferation in OVCAR4 and Caov-3 co-cultures. B and C . CFSE quantification of cells in A. D and E . Levels of IFNγ ( D ) <t>and</t> <t>IL-2</t> ( E ) detected by ELISA from the supernatants of OVCAR4 +/− NT-T or SSEA-4 CAR-T cells at 72 h. F . Antibody-dependent cellular cytotoxicity (ADCC) assay. SSEA-4 CAR-T cells were co-cultured with or without Natural Killer (NK) cells and Cetuximab antibody at the indicated concentrations. Quantification of SSEA-4 CAR-T cells was performed by flow cytometry using Protein L staining. ( B and C ) N=2. ( D-F ) N=3. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison tests. ( B ) * p=0.019; ( D-F ) **** p=0.0001 and *** p=0.001 . Not significant differences, n.s., were attributed to p⩾ 0.05 .
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A. Flow cytometry histograms of CFSE (Carboxyfluorescein succinimidyl ester) cell proliferation assays showing CAR-T cell proliferation upon interaction with SSEA-4 + OVCAR4 cells in contrast to SSEA-4 − Caov-3 after 48 h. NT-T cells showed no proliferation in OVCAR4 and Caov-3 co-cultures. B and C . CFSE quantification of cells in A. D and E . Levels of IFNγ ( D ) and IL-2 ( E ) detected by ELISA from the supernatants of OVCAR4 +/− NT-T or SSEA-4 CAR-T cells at 72 h. F . Antibody-dependent cellular cytotoxicity (ADCC) assay. SSEA-4 CAR-T cells were co-cultured with or without Natural Killer (NK) cells and Cetuximab antibody at the indicated concentrations. Quantification of SSEA-4 CAR-T cells was performed by flow cytometry using Protein L staining. ( B and C ) N=2. ( D-F ) N=3. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison tests. ( B ) * p=0.019; ( D-F ) **** p=0.0001 and *** p=0.001 . Not significant differences, n.s., were attributed to p⩾ 0.05 .

Journal: bioRxiv

Article Title: Efficacy and safety of glycosphingolipid SSEA-4 targeting CAR-T cells in solid tumors

doi: 10.1101/2022.09.02.506335

Figure Lengend Snippet: A. Flow cytometry histograms of CFSE (Carboxyfluorescein succinimidyl ester) cell proliferation assays showing CAR-T cell proliferation upon interaction with SSEA-4 + OVCAR4 cells in contrast to SSEA-4 − Caov-3 after 48 h. NT-T cells showed no proliferation in OVCAR4 and Caov-3 co-cultures. B and C . CFSE quantification of cells in A. D and E . Levels of IFNγ ( D ) and IL-2 ( E ) detected by ELISA from the supernatants of OVCAR4 +/− NT-T or SSEA-4 CAR-T cells at 72 h. F . Antibody-dependent cellular cytotoxicity (ADCC) assay. SSEA-4 CAR-T cells were co-cultured with or without Natural Killer (NK) cells and Cetuximab antibody at the indicated concentrations. Quantification of SSEA-4 CAR-T cells was performed by flow cytometry using Protein L staining. ( B and C ) N=2. ( D-F ) N=3. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison tests. ( B ) * p=0.019; ( D-F ) **** p=0.0001 and *** p=0.001 . Not significant differences, n.s., were attributed to p⩾ 0.05 .

Article Snippet: Human NK cells were isolated from PBMCs using the NK Cell Isolation Kit, human (#130-092-657, Miltenyi), according to the manufacturer’s instructions, and subcultured for 72 h in NK MACS medium supplemented with 5% human serum male AB plasma (#H4522, Sigma-Aldrich) and human IL-2 IS (200 IU/ml, # 130-097-746).

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