Figure 4.
KO of CD58 in tumor cells forms ineffective IS with CAR T cells and attenuates CAR signaling. (A) CD19 CAR T cells were stained with CellTrace Violet dye and incubated with WT Nalm6 (mCherry+) and CD58KO (GFP+) Nalm6 cells for 10 minutes or 30 minutes. Representative FACS and bar plots (gating from Violet+ cells) representing the quantification of conjugates formed by WT or CD58KO with CAR T cells (n = 3). (B) CD19 CAR T cells prestained with CellTrace Violet dye were cocultured with WT (mCherry+) or CD58KO (GFP+) Nalm6 cells for 10 minutes or 30 minutes. After incubation, the cells were analyzed for the expression of CellTrace Violet, GFP, mCherry, and phalloidin (AF647) using ImageStream. Phalloidin was used to stain F-actin. The gating strategy used for the identification of IS and a representative image of an IS are shown. The white arrow points to an IS. (C) Median fluorescence intensity (MFI) of phalloidin at IS (n = 3). (D) F-actin enrichment at IS with WT or CD58KO is reported as percent protein (n = 3). (E) Proximal signaling events of CAR-expressing Jurkat cells upon stimulation with WT or CD58KO Nalm6 cells. Similar results were obtained from 3 independent biological experiments. (F) Selected pathways of gene ontology enrichment analysis in the biological process category of differentially expressed genes in CD19 CAR T cells sorted by flow cytometry 3 days after stimulation with WT or CD58KO Nalm6 cells (n = 2 different peripheral blood mononuclear cell donors). (G) Heatmap showing select differentially expressed genes related to T-cell activation, T-cell differentiation, and cytokines in CD19 CAR T cells after 3 days of stimulation with WT or CD58KO Nalm6 cells (n = 2 different PBMC donors). Statistical comparisons were performed using a 2-way ANOVA test with multiple comparisons. The values are shown as the means plus or minus SD. ∗P < .05; ∗∗P < .01; ∗∗∗ P < .001. FACS, fluorescence-activated cell sorter; KO-FITC, CD58KO cells (Fluorescein Isothiocyanate); ns, not significant (P > .05); WT-PE, WT cells (phycoerythrin).

KO of CD58 in tumor cells forms ineffective IS with CAR T cells and attenuates CAR signaling. (A) CD19 CAR T cells were stained with CellTrace Violet dye and incubated with WT Nalm6 (mCherry+) and CD58KO (GFP+) Nalm6 cells for 10 minutes or 30 minutes. Representative FACS and bar plots (gating from Violet+ cells) representing the quantification of conjugates formed by WT or CD58KO with CAR T cells (n = 3). (B) CD19 CAR T cells prestained with CellTrace Violet dye were cocultured with WT (mCherry+) or CD58KO (GFP+) Nalm6 cells for 10 minutes or 30 minutes. After incubation, the cells were analyzed for the expression of CellTrace Violet, GFP, mCherry, and phalloidin (AF647) using ImageStream. Phalloidin was used to stain F-actin. The gating strategy used for the identification of IS and a representative image of an IS are shown. The white arrow points to an IS. (C) Median fluorescence intensity (MFI) of phalloidin at IS (n = 3). (D) F-actin enrichment at IS with WT or CD58KO is reported as percent protein (n = 3). (E) Proximal signaling events of CAR-expressing Jurkat cells upon stimulation with WT or CD58KO Nalm6 cells. Similar results were obtained from 3 independent biological experiments. (F) Selected pathways of gene ontology enrichment analysis in the biological process category of differentially expressed genes in CD19 CAR T cells sorted by flow cytometry 3 days after stimulation with WT or CD58KO Nalm6 cells (n = 2 different peripheral blood mononuclear cell donors). (G) Heatmap showing select differentially expressed genes related to T-cell activation, T-cell differentiation, and cytokines in CD19 CAR T cells after 3 days of stimulation with WT or CD58KO Nalm6 cells (n = 2 different PBMC donors). Statistical comparisons were performed using a 2-way ANOVA test with multiple comparisons. The values are shown as the means plus or minus SD. ∗P < .05; ∗∗P < .01; ∗∗∗ P < .001. FACS, fluorescence-activated cell sorter; KO-FITC, CD58KO cells (Fluorescein Isothiocyanate); ns, not significant (P > .05); WT-PE, WT cells (phycoerythrin).

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