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. 2016 Dec 7;3:16026. doi: 10.1038/mto.2016.26

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Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy

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Cassettes coexpressing GFP and VLR-CARs show a correlation between expression and activation. (a) The eGFP P2A CAR construct with either BCL or CD5-VLR. (b) Jurkat cells were transduced at various MOIs and a direct correlation between GFP expression and activation is observed in the eGFP P2A CD5-VLR-CAR transduced Jurkat cells, demonstrating dual expression of both proteins and activation of only VLR-expressing cells. (c) NK-92 cells were transduced with the GFP-p2a-CD5-VLR-CAR and sorted for GFP expressing cells. The isolated and expanded cells were followed over time for GFP expression. (d) Western blot using anti-CD3æ antibody on whole cell lysates of NK-92 cells shows the presence of CD5-VLR-CAR protein in the sorted cells. (e) Activation of eGFP P2A BCL-VLR-CAR transduced Jurkat cells when cocultured with target BCL cells. Naive Jurkat cells showed no activation when cocultured with BCL cells (left panel), cells modified but without coculture showed minimal activation (middle panel), and transduced cells cocultured with BCL cells (right panel) showed robust levels of activation. CAR, chimeric antigen receptor; eGFP, enhanced green fluorescence protein; MOI, multiplicity of infection; VLR, variable lymphocyte receptor.