Figures S1–S4 for additional data. " width="100%" height="100%">
Journal: Cell Reports Medicine
Article Title: Solid tumor immunotherapy using NKG2D-based adaptor CAR T cells
doi: 10.1016/j.xcrm.2024.101827
Figure Lengend Snippet: In vitro characterization of NKG2D/Dap10-12 CAR T cells (A) Structure of NKG2D/Dap10-12 CAR, making comparison with the NKG2D/Dap10 complex. NKG2D encoded by the NKG2D/Dap10-12 vector is also expected to associate with endogenous Dap10 present in T cells, giving rise to additional complexes, two of which are shown. (B) Flow cytometric analysis of NKG2D expression in CD4 + T cells following transduction with the NKG2D/Dap10-12 CAR or NKG2D alone, making comparison with untransduced cells (median + interquartile range). Number of biological replicates is indicated on this and subsequent panels. ∗∗∗∗ p < 0.0001 (Kruskal-Wallis). (C) Normalized MFI of NKG2D expression in CD4 + T cells following transduction with the NKG2D/Dap10-12 CAR or NKG2D alone. Data (median + interquartile range) were normalized to expression in NKG2D/Dap10-12 CAR T cells (set to 100). ∗∗∗∗ p < 0.0001 (Mann-Whitney). (D) CD4/CD8 analysis on day 10 of culture (median + interquartile range). ∗∗∗∗ p < 0.0001 (Mann-Whitney). (E) NKG2D/Dap10-12 CAR T cells were labeled with CDTR and incubated with an equal number of CFSE-labeled T cells that expressed NKG2D/Dap10-12 , NKG2D or were untransduced. Proportions of these cells remaining in the co-culture were analyzed by flow cytometry after 48 h (representative of three independent replicates). (F) Expression of KLRG1 (median + interquartile range) on NKG2D/Dap10-12 CAR T cells, making comparison with control cells that were untransduced or in which NKG2D alone was over-expressed. ∗∗ p < 0.01 (Friedman test). (G) Expression of CD57 (median + interquartile range) on the same T cell populations. ∗ p < 0.05; ∗∗ p < 0.01 (Friedman test). (H) Expression of CD27 (mean ± SEM) on the same T cell populations. ∗ p < 0.05; ∗∗∗∗ p < 0.0001 (one-way ANOVA). (I) Cytotoxicity assays were carried out in which the indicated T cells were co-cultivated for 24 h at the specified target to effector ratio with listed tumor cell lines. Tumor cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (mean ± SEM). ∗∗∗∗ p < 0.0001 (two-way ANOVA). (J) Indicated T cells were co-cultivated for 24 h at a 1:1 target to effector ratio with BxPC3 tumor cells, supplemented with increasing concentrations of recombinant MICA (mean ± SEM, n = 2). Tumor cell viability was determined as in (I). Data are representative of three independent replicates. (K) IL-2 concentration was analyzed in supernatants collected after 24 h from co-cultures of indicated tumor cells and T cells (target to effector ratio 1:1; median + interquartile range). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001; NS - not significant (Kruskal-Wallis). (L) IFN-γ concentration was analyzed in supernatants collected after 72 h from co-cultures described in (K) (median + interquartile range). ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001 (Kruskal-Wallis). (M) Maximum (Max.) fold expansion of indicated T cells following twice weekly re-stimulation on specified tumor cell lines (median + interquartile range). ∗∗∗∗ p < 0.0001; NS, not significant (Kruskal-Wallis). (N) Number of effective re-stimulation (restim.) cycles achieved by indicated T cells when re-stimulated twice weekly on specified tumor cell lines. Re-stimulations were considered successful if <60% of tumor cells remained viable (median + interquartile range). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001 (Kruskal-Wallis). See Figures S1–S4 for additional data.
Article Snippet: Anti-human CD8a Alexa Fluor 700 (OKT8) , eBioscience , Cat# 56-0086-82 RRID: AB_657756.
Techniques: In Vitro, Comparison, Plasmid Preparation, Expressing, Transduction, MANN-WHITNEY, Labeling, Incubation, Co-Culture Assay, Flow Cytometry, Control, MTT Assay, Recombinant, Concentration Assay
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Figures S1–S4 for additional data. " width="250" height="auto" />