Anti-CD20 CAR mRNA enhances exPBNK in vitro cytolytic activity against CD20+ B-NHL cells and rituximab-resistant cells. exPBNK were electroporated in the.

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Anti-CD20 CAR mRNA enhances exPBNK in vitro cytolytic activity against CD20+ B-NHL cells and rituximab-resistant cells. exPBNK were electroporated in the absence (mock, gray) or in the presence of anti-CD20 CAR mRNA (CAR, black). Anti-CD20 CAR mRNA enhances exPBNK in vitro cytolytic activity against CD20+ B-NHL cells and rituximab-resistant cells. exPBNK were electroporated in the absence (mock, gray) or in the presence of anti-CD20 CAR mRNA (CAR, black). A, MOCK and CAR exPBNK were incubated for 4 hours with the BATDA-labeled CD20+ NK-sensitive Ramos (top left), CD20+ NK-resistant Daudi (top middle), and U-698-M (top right) cells at the indicated E:T ratios. In vitro cytotoxicity was measured by standard europium release assay. At indicated by E:T ratios, cytotoxicity of exPBNK expressing anti-CD20 chimeric receptors was significantly higher than that of mock exPBNK. B, MOCK and CAR exPBNK were incubated for 4 hours with the BATDA-labeled CD20− NK-sensitive Jurkat (bottom left) and CD20− NK-resistant Rs4;11 (bottom right) cells at the indicated E:T ratios. In vitro cytotoxicity was measured by standard europium release assay. C, anti-CD20 CAR expressing NK cells inhibit Ramos-cell recovery. MOCK or CAR exPBNK were incubated overnight with Ramos cells at E:T = 3:1. Ramos cells incubated with medium only were used as controls. The total living cells were gated by 7-AAD negative. The living Ramos cells were gated by CD19+ 7-AAD− by flow cytometry analysis. Each data point represents the mean (± SEM; n = 3) percentage of living CD19+ cells versus total living cells. P values using the unpaired Student t test are noted in A, B, and C, respectively. D, MOCK and CAR exPBNK were incubated for 2 hours with the BATDA-labeled CD20+ rituximab-sensitive Raji and rituximab-resistant Raji-2R and Raji-4RH cells at the E:T ratio = 10:1. In vitro cytotoxicity was measured by standard europium release assay. As indicated by E:T ratios, cytotoxicity of exPBNK expressing anti-CD20 chimeric receptors was significantly higher than that of exPBNK without anti-CD20 CAR (P < 0.001) against Raji, Raji-2R, and Raji-4RH cells. Each data point represents the mean (± SEM; n = 4) percentage of specific europium release after culture. P values using the unpaired Student t test are noted. E, expression of HLA class I antigens in Raji, Raji-2R, and Raji-4RH cells was determined by flow cytometry with allophycocyanin-conjugated anti-human HLA-ABC antibody (BD Biosciences). Representative staining results are shown. Yaya Chu et al. Cancer Immunol Res 2015;3:333-344 ©2015 by American Association for Cancer Research