In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

Figure 7

NK- stimulation after coculture in the presence of NSG splenocytes. (a) Single-cell suspensions from NSG mice spleens were stained with an antibody cocktail and then analyzed by flow cytometry for expression of CD11b (Mac-1) versus Gr-1 (Ly-6G) and CD11b versus CD16/32 (FcRγRIII/FcRγRII). (b) NK-92 (NK-, NK-, or NK-) and splenocytes from NSG mice or the HER2 positive cell line BT474 were cocultured at an effector ratio of 5 : 1 and 2 : 1, respectively, in 96-well round bottom culture plates in RPMI 1640 supplemented with 10% FCS and IL2 (100 IU/ML). After 18 h, supernatants were collected and human INF-γ content was estimated using an ELISA kit (Affymetrix, e-biosciences). Data represent the mean with SD from three independent experiments.

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