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. 2020 Sep 5;29(1):75–85. doi: 10.1016/j.ymthe.2020.08.016

Figure 3.

Figure 3

In Vitro Characterization of Three Different 2nd-Gen NKG2D CAR T Cells

(A) Cytolytic activity. Human HCT116 colorectal cancer cells were labeled with DELFIA BATDA Reagent (DELFIA EuTDA Cytotoxicity Reagents, PerkinElmer) followed by co-culture with CAR-T cells at indicated E:T ratios. Cytotoxicity assay was carried out over 4 h (left) and 16 h (right), and percent cytotoxicity was then calculated by measuring Europium release signal from the target tumor cells using a plate reader. The results shown are from one representative experiment out of two independent experiments with 2 different donors. (B) Cytokine release. HCT116 tumor cells were co-cultured with CAR-T cells at a 1:1 ratio for 16 h. Supernatants were collected to determine cytokine production by using the CBA (Cytometric Bead Array) Human Th1/Th2 Cytokine Kit (BD Biosciences). Data in bar graph represent mean ± SD for 3 cytokines in supernatants tested with CAR-T cells from 3 different PBMC donors. (C) CAR-T cell expansion. Antigen-dependent expansion of NKG2D CAR-T cells with γ-irradiated K562C6 feeder cells in the absence or presence of exogenous IL-2 (300 IU/mL) was examined. Data indicate total cell numbers obtained at the end of 1-week expansion from day 14 to day 21 (top) and day 21 to day 28 (bottom) from cell counting by trypan blue exclusion assay. Data represent mean ± SD of three independent experiments with CAR-T cells from 3 different PBMC donors. (D) CFSE T cell proliferation assay. At day 21, CAR-T cells were labeled with CFSE and co-cultured with K562C6 at a 1:1 ratio with or without IL-2 for 7 days, and proliferation was assessed at day 28 by flow cytometry. Flow cytometry plots for CFSE peak dilution are indicated, with the CFSE staining of stimulated CAR-T cells indicated as histograms on the left and the CFSE staining of non-stimulated CAR-T cells indicated as histograms on the right. The results shown are for one representative experiment out of three independent experiments with 3 different donors. (E) Total number of CFSE peaks. Data in bar chart graphs represent mean ± SD of the total number of CFSE peaks (left) in CAR-T samples from 3 different PBMC donors. Statistical significance was evaluated by one-way ANOVA followed by multiple correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.