Immune responses to modified T cells. Two assays were performed to detect Ab against the transgene in patient serum samples collected before and at serial time points after T-cell infusions. (A) An ELISA was performed testing for the presence of Ab binding to Leu16 mouse anti–human CD20 Ab (from which the αCD20-28-BB-ζ CAR is derived). Baseline patient serum was used as a negative control. Data represent the mean ± SEM of duplicate values. (B) A flow cytometric assay was performed in which serial patient serum samples were incubated with HEK-293 cells genetically modified to express the αCD20-28-BB-ζ CAR, untransfected HEK-293 cells (negative control), or autologous EBV-LCL (positive control), followed by FITC-conjugated goat anti–human F(ab′)₂ Ab. The median fluorescence intensity (MFI) for each sample at various time points at the 1:2 dilution is shown for each cell line. The black bar indicates the 14-day period of low-dose IL-2 injections. (C) The presence of cellular immune responses to infused T cells was assessed by stimulating serially collected patient PBMCs with irradiated autologous EBV-LCL modified to express the αCD20-28-BB-ζ CAR and NeoR gene products (UPN-02 and UPN-03) or irradiated autologous CAR⁺ T cells (UPN-04) at a 2:1 responder/stimulator ratio. After two 1-week stimulations, these PBMCs were used as effectors in ⁵¹Cr-release assays in which target cells were pre-infusion modified T cells, untransfected autologous EBV-LCL, CAR⁺ autologous EBV-LCL, or untransfected autologous PBMCs at E:T ratios of 25:1 (UPN-03 and UPN-04) or 12.5:1 (UPN-02). The lysis of CAR⁺ LCL is expressed as the difference between CAR⁺ and CAR⁻ LCL for patients UPN-02 and UPN-03. The mean ± SEM of triplicate wells is shown.