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 CAR engagement of antigen induced clustering without TCR integration. Anti−HER2 CAR T cells were labeled with either AF647−conjugated F(ab´) 2 goat anti−human IgG antibody that recognizes the CAR, anti−TCRα/β AF647-conjugated antibody, or AF647-conjugated anti−transferrin receptor (TfR) antibody. (A) Schematic representation of the used anti−HER2 CD28ζ CAR linked to GFP at the intracellular site (IC). (B) Cells were seeded onto surfaces coated with HER2 protein or anti−CD3 antibody OKT3 and contacts were formed for 20 minutes. Cells were fixed and TIRF microscopy was used to image the localization of CAR-GFP (green) and either CAR-AF647, TCR-AF647, or TfR-AF647 (red). Scale bar represents 5 µm. For each cell, the pixel-wise correlation of the brightness values recorded in the red and in the green channel were plotted along the x- and the y-axis, respectively. Pearson’s correlation coefficient (PCC) for each cell was calculated. Contact size (C) and Mean intensity (D) of anti−HER2 CAR-GFP plated on HER2 (blue) and OKT3 coated slides (grey) are displayed for n≥16 cells per group in a Whisker box plot. Statistical differences were determined by Wilcoxon-Mann-Whitney test with Python code (**p<0.01). (E) PCC for CAR-GFP and TCR-AF647 was calculated for anti−HER2 CAR T cells plated on HER2 (blue) and OKT3 (grey) coated slides and displayed for n≥24 cells per group in a Whisker box plot. Statistical differences were determined by Kruskal-Wallis test with Dunn’s post-hoc test performed with Python code (***p<0.001). (F) Labelled anti-HER2 CAR T cells were seeded on HER2 expressing N87 target cells plated on chambered coverslips. 3D fluorescence images of live anti-HER2 CAR T cells forming contacts with the tumor target were recorded in AiryScan Fast mode. Confocal images of each analyzed cell were recorded as controls. (G) Differential 3D distribution of CAR-GFP and either CAR-AF647, TCR-AF647, or TfR-AF647 in the synaptic contact region and the extrasynaptic membrane was normalized to total intensity (n CAR-GFP =11, n TCR-AF647 = 11, n CAR-AF647 = 10, n TfR-AF647 = 6; 3D images contained approximately 50-80 slices). (H) PCC for CAR-GFP and either CAR-AF647, TCR-AF647, or TfR-AF647 in the synaptic contact region and the extrasynaptic membrane was quantified separately for each slice and averaged for each individual cell and presented as the mean of multiple cells across 3 independent experiments. 2D AiryScan Fast images of unstimulated cells were used as control (n CAR_CAR =15, n TCR_CAR =7, n TfR_CAR =14). Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ns, not significant).
Article Snippet: AF647-conjugated transferrin receptor monoclonal antibody (MEM-75) was purchased from Invitrogen, Regensburg, Germany.
Techniques: Labeling, Microscopy, Whisker Assay, MANN-WHITNEY, Expressing, Fluorescence
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