Fig. 4: CD28-mediated second signaling is required for cytokine production but not for enhancement of cytotoxicity. | Communications Biology

Fig. 4: CD28-mediated second signaling is required for cytokine production but not for enhancement of cytotoxicity.

From: CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters

Fig. 4: CD28-mediated second signaling is required for cytokine production but not for enhancement of cytotoxicity.

a, b Splenic T cells were introduced by ζ.- or CD28ζ.CAR-HaloTag and PKCθ-EGFP, conjugated with CD80-expressing or not expressing EL-4-hCD19 cells and real-time imaged by confocal microscopy 20 min after contact. Histograms show fold FI of CAR and PKCθ on the diagonal yellow lines (a). Conjugated pairs in (a) were categorized by location of PKCθ at T-EL-4 cell borderline (n = 30) (b). c–e The T cells in (a) were plated onto an SLB containing ICAM-1– and hCD19–GPI with or without CD80–GPI and real-time imaged by confocal microscopy 2 min or 20 min after contact. Histograms show fold FI of CAR and PKCθ on the diagonal yellow lines (c). The scatter plot summarizing the PCC values in (c) (2 min). PCC was calculated between CAR/PKCθ in the indicated pattern by 20 randomly plotted profiles on 10 cells. Horizontal bars, average (d). The graph shows the frequency of distribution patterns of CAR and PKCθ at T cell–SLB interface, as seen in (c) (20 min, merge) (n = 25) (e). f AND-TCR T cell hybridomas (2D12) expressing ζ. or CD28ζ.CAR were conjugated with EL-4-hCD19 cells not expressing or expressing CD80. The WCLs were blotted for phosphorylated (p) or total CD3ζ in CARs, pErk, or Erk. The number below each line represents the intensity ratio, pζ/ζ in CARs or pErk/Erk. g Splenic CD4+ or CD8+ were introduced by ζ. or CD28ζ.CAR and cocultured with EL-4-hCD19 cells not expressing or expressing CD80 at low (CD80Lo) or high (CD80Hi) level. At 6 h after coculture, the concentration of IL-2 in each supernatant was measured by ELISA. h The concentration of IFNγ after 18 h coculture in (g). i CD8+ CAR-T cells in (g) were cocultured with EL-4 cells as in (g). The graph shows percent specific lysis at the indicated E/T ratios 18 h after coculture. All data are representative of three independent experiments. Bars, 5 μm. Statistical analysis was performed using one-way ANOVA. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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