Journal: Frontiers in Immunology

Article Title: CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation

doi: 10.3389/fimmu.2023.1110482

Figure Lengend Snippet: CARs with an intracellular CD3ζ chain are superior over γ−chain CARs in T cell activation despite lower cell surface expression and shorter half-life on the T cell surface. (A) Schematic representation of CARs with their respective transmembrane (TM) and intracellular (IC) signaling domains consisting of the respective CD3ζ (ζ) or the FcεRI γ (γ) chain. All CARs harbour the same extracellular domain (EC) and were expressed by the same promoter in the same retroviral vector backbone. Cells were engineered with the respective CARs. CAR expression was monitored by an anti−IgG Fc antibody that detects the common extracellular CAR IgG1-Fc spacer domain. Background staining was determined by an isotype control antibody. Data represent the mean values of mean fluorescence intensity (mfi) ± SD. (B) MD45 cells were engineered with a ζ or a γ CAR, respectively, and incubated with cycloheximide (10 µg/ml) to block protein synthesis. For comparison, the CARs were additionally cross-linked by an anti−IgG (anti−CAR) antibody, that binds to the extracellular CAR spacer domain, for 2 h to induce CAR internalization. At different time points, 10 7 cells were lysed and proteins separated by SDS-PAGE on 8% (w/v) polyacrylamide gels under non-reducing conditions. CARs were detected by the anti−IgG Fc-POD antibody (1:10,000), actin was detected by the anti−actin antibody (0.5 µg/ml). Relative density of CAR bands was quantified utilizing the ImageJ software 1.48 and presented as percent of the initial amount at t=0. Data from a representative experiment out of three are shown. (C) Pulse-chase CAR labeling. CAR transfected cells were surface-labeled with biotin as described in Materials and Methods, washed and stimulated at 37°C by an anti−IgG antibody (1 µg/ml) directed against the IgG extracellular CAR domain. Aliquots of cells (5 x 10 6 cells) were lysed at different time points and lysates were subjected to ELISA plates coated with an anti−IgG1 mAb (1 µg/ml) to capture the CAR. Bound labeled CARs were detected by streptavidin POD and visualized with ABTS. OD at time point 0 was set at 100% and relative ODs at indicated time points were calculated. Numbers represent the mean values of three independent experiments ± SD. (D) CAR redirected T cell activation. T cells with ζ- or γ−chain anti−CA19−9 CAR were expanded in the presence of IL−2 and co-cultivated (0.625–5 x 10 4 cells/well) for 48 h with CA19−9 + LS174T or CA19−9 - A375 tumor cells (5 x 10 4 cells/well). Supernatants were analyzed for IFN−γ by ELISA, target cell lysis was determined by the XTT assay. Data represent mean values ± SD of two independent experiments. w/o, without CAR. (E) Activation of CAR T cells in the presence of soluble CA19−9 antigen. Anti−CA19−9 CAR T cells (5 x 10 4 cells/well) were co-cultivated for 48 h with CA19−9 + LS174T cells (5 x 10 4 cells/well) in the presence of serial dilutions of supernatants of H498 tumor cells containing about 20,000 U/ml of soluble CA19−9 (sCA19−9). Target cell lysis was determined by the XTT assay. For control, cells were co-cultivated in the presence of supernatants of the CA19−9 - cell line H716 lacking soluble CA19−9 (w/o). Data represent mean values ± SD of technical triplicates. For comparison of two groups, significant differences were determined by Student´s T test. For comparisons of three or more groups, one-way ANOVA with Tukey’s post hoc test was used. p-values < 0.05 were considered statistically significant (**p<0.01; ***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: CAR engineered or non-modified Jurkat T cells (5 x 10 4 /well) were incubated in coated plates for 48 h and IL−2 in the supernatant was determined by ELISA with a solid phase anti−human IL−2 (2 µg/ml) capture and a biotinylated anti−human IL−2 detection antibody (0.5 µg/ml) (BD Bioscience).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Staining, Fluorescence, Incubation, Blocking Assay, SDS Page, Software, Pulse Chase, Labeling, Transfection, Enzyme-linked Immunosorbent Assay, Lysis, XTT Assay

Journal: Frontiers in Immunology

Article Title: CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation

doi: 10.3389/fimmu.2023.1110482

Figure Lengend Snippet: ζ CARs did not rescue the CD3/TCR complex in CD3ζ KO Jurkat cells. (A) The CD3ζ locus in Jurkat cells was deleted by CRISPR/Cas9 engineering as described in Materials and Methods. Non-modified Jurkat cells (wt), Jurkat76 cells lacking TCR (TCR - ) and Jurkat E4 CD3ζ knock-out (KO) cells were tested by flow cytometry for intracellular CD3ζ expression and for surface expression of CD3ε and TCR, respectively. Histograms of a representative analysis are shown. (B) Western blots of genome edited Jurkat cells. Lysates of non-modified (wt) and CD3ζ KO Jurkat cells (5 µg protein lysate/lane) were separated by SDS PAGE, blotted, probed with a mouse anti−human CD3ζ antibody (1:500) and detected by a HRP-conjugated anti−mouse antibody (1:5,000). Blots were re-probed with an anti−β-actin antibody (1:20,000). (C) TCR + Jurkat (wt), Jurkat76 (TCR - ) and Jurkat E4 (CD3ζ KO) cells were engineered with the CD28ζ, ζ or γ CAR, respectively. Expression of CARs and surface expression of CD3 was recorded by flow cytometry and mean fluorescence intensity (mfi) was determined. Dot plots of a typical experiment and mean values of 5 independent experiments ± SD (D) are shown. Significant differences were determined by Student´s T test. (E) Peripheral blood T cells engineered with ζ−chain and γ−chain CAR, respectively, were stained for CAR and CD3 expression and analyzed by flow cytometry. CAR + and CAR - T cells were gated and mean fluorescence intensity (mfi) of CD3 was determined. Data represent mean values of 4 healthy donors ± SD. Statistical differences were determined by Student´s T test. (F) Jurkat (wt) and Jurkat E4 (CD3ζ KO) cells with and without CAR, respectively, were stimulated through the CAR and CD3 by incubation on 96-well plates (4 x 10 4 cells/well) coated with the agonistic anti−CD3 antibody OKT3 or anti−IgG Fc antibody (5 µg/ml each) that binds to the CAR extracellular domain. After 48 h supernatants were tested for IL−2 by ELISA. Values represent the means of technical triplicates ± SD. Significant differences were determined by Student´s T test. A representative experiment out of two is shown. p-values <0.05 were considered statistically significant (*p<0.05; **p<0.01; ***p<0.001; ****p<0.001; ns, not significant).

Article Snippet: CAR engineered or non-modified Jurkat T cells (5 x 10 4 /well) were incubated in coated plates for 48 h and IL−2 in the supernatant was determined by ELISA with a solid phase anti−human IL−2 (2 µg/ml) capture and a biotinylated anti−human IL−2 detection antibody (0.5 µg/ml) (BD Bioscience).

Techniques: CRISPR, Modification, Knock-Out, Flow Cytometry, Expressing, Western Blot, SDS Page, Fluorescence, Staining, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation

doi: 10.3389/fimmu.2023.1110482

Figure Lengend Snippet: The TCR/CD3 complex and ζ−chain CARs can complement in signaling. (A) T cells of healthy donors (5 x 10 4 cells/well) were engineered with a ζ or CD28ζ CAR, respectively, and cultivated for 48 h in micro-titer plates that were coated with serial dilutions (starting from 10 µg/ml) of an anti−IgG1 Fc antibody for CAR activation or an agonistic anti−CD3 and anti−TCR antibody, respectively. Data from a representative T cell donor are shown; data from four donors were accumulated in mean values ± SD. (B) T cells (5 x 10 4 cells/well) expressing an anti-HER2-Fc-CD28, anti-CEA-Fc-CD28 or anti-CEA-Fc-CD28ζ CAR were cultivated for 48 h in micro-titer plates coated with an agonistic anti−CD3 (1 µg/ml) and anti−CD28 (5 µg/ml) antibody, respectively, or the anti−idiotypic antibody BW2064/36 (8 μg/ml), directed against the binding domain of the anti−CEA CAR, or recombinant HER2-Fc protein (8 μg/ml) recognized by the anti−HER2 CAR, respectively. Combinations of antibodies and/or antigen were used as indicated. Culture supernatants were analyzed for IFN−γ or IL−2 by ELISA as indicated. Numbers represent mean values of technical triplicates ± SD. A representative experiment out of at least three experiments is shown.

Article Snippet: CAR engineered or non-modified Jurkat T cells (5 x 10 4 /well) were incubated in coated plates for 48 h and IL−2 in the supernatant was determined by ELISA with a solid phase anti−human IL−2 (2 µg/ml) capture and a biotinylated anti−human IL−2 detection antibody (0.5 µg/ml) (BD Bioscience).

Techniques: Activation Assay, Expressing, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay