Figure 1.
T cells can be engineered to modulate cytokine signaling. (A and B) Schematic of CRS regulation by CAR-T cells engineered to secrete cytokine modulators. Generated with BioRender. (A) Uncontrolled CAR-T cell activation may lead to immune overstimulation and result in CRS. (B) Self-regulating CAR-T cells secrete scFvs or antagonist proteins that block cytokine signaling, thus preventing immune overstimulation. (C–G) Cytokine- and cytokine-receptor–binding scFvs bind their intended targets. (C) Toci blocks IL-6–induced STAT3 signaling in primary human CD4+ T cells. T cells were coincubated with Toci for 3 h and then treated with or without 4 ng/ml human IL-6 protein for 30 min. Cell lysates were analyzed by western blot and probed for phosphorylated STAT3 (pSTAT3) and GAPDH. (D) Toci inhibits IL-6 signaling in IL-6 reporter HEK-Blue cells. IL-6 reporter HEK-Blue cells transfected with plasmids encoding the transduction marker EGFRt orToci were stimulated with the indicated concentration of IL-6 for 24 h. The amount of STAT3-induced SEAP secretion by the HEK-Blue cells was quantified by spectrophotometer. Triplicate wells were separately seeded, transfected, stimulated with IL-6, and quantified. Statistical significance was determined by two-way ANOVA corrected for multiple comparisons using Tukey’s method. (E) Adalimumab- and certolizumab-based scFvs block TNF-α–induced NF-κB signaling. NFκB-EGFP reporter Jurkat cells were incubated with indicated concentrations of TNF-α scFvs overnight before stimulation with 10 ng/ml of TNF-α. EGFP expression induced by NFκB signaling was quantified by flow cytometry, with percent EGFP+ and EGFP MFI shown. Triplicate wells were seeded, treated with the indicated scFvs, and quantified. Statistical significance was determined by two-tailed Student’s t tests comparing each condition against the “TNF-α only” control with correction for multiple comparisons using the two-stage step-up procedure by Benjamini, Krieger, and Yekutieli for false discovery rate < 1.00%. (F and G) Adalimumab- and certolizumab-based scFvs block TNF-α binding (F) and emapalumab-based scFv blocks IFN-γ binding (G). CD19 CAR-T cells were cocultured with Raji cells and donor-matched immune cells (monocytes, macrophages, and dendritic cells) to stimulate cytokine secretion. Addition of scFvs concentrated from HEK293T supernatant binds specific cytokines and blocks their detection by Cytometric Bead Array assay. Triplicate wells were separately seeded, treated with scFvs, and quantified. Pairwise comparisons against the no-scFv control were performed using Welch’s t test, with multiple-comparison correction by the Holm–Sidak method using a significance threshold of 0.05. In panels D–G, means of triplicates are shown with error bars indicating ± 1 standard deviation (SD). Statistical significance levels for all panels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The experiments in panels C–E were each performed once. Experiments shown in F and G were performed twice using two different healthy donors’ cells; data from one representative donor are shown. Source data are available for this figure: SourceData F1.

T cells can be engineered to modulate cytokine signaling. (A and B) Schematic of CRS regulation by CAR-T cells engineered to secrete cytokine modulators. Generated with BioRender. (A) Uncontrolled CAR-T cell activation may lead to immune overstimulation and result in CRS. (B) Self-regulating CAR-T cells secrete scFvs or antagonist proteins that block cytokine signaling, thus preventing immune overstimulation. (C–G) Cytokine- and cytokine-receptor–binding scFvs bind their intended targets. (C) Toci blocks IL-6–induced STAT3 signaling in primary human CD4+ T cells. T cells were coincubated with Toci for 3 h and then treated with or without 4 ng/ml human IL-6 protein for 30 min. Cell lysates were analyzed by western blot and probed for phosphorylated STAT3 (pSTAT3) and GAPDH. (D) Toci inhibits IL-6 signaling in IL-6 reporter HEK-Blue cells. IL-6 reporter HEK-Blue cells transfected with plasmids encoding the transduction marker EGFRt orToci were stimulated with the indicated concentration of IL-6 for 24 h. The amount of STAT3-induced SEAP secretion by the HEK-Blue cells was quantified by spectrophotometer. Triplicate wells were separately seeded, transfected, stimulated with IL-6, and quantified. Statistical significance was determined by two-way ANOVA corrected for multiple comparisons using Tukey’s method. (E) Adalimumab- and certolizumab-based scFvs block TNF-α–induced NF-κB signaling. NFκB-EGFP reporter Jurkat cells were incubated with indicated concentrations of TNF-α scFvs overnight before stimulation with 10 ng/ml of TNF-α. EGFP expression induced by NFκB signaling was quantified by flow cytometry, with percent EGFP+ and EGFP MFI shown. Triplicate wells were seeded, treated with the indicated scFvs, and quantified. Statistical significance was determined by two-tailed Student’s t tests comparing each condition against the “TNF-α only” control with correction for multiple comparisons using the two-stage step-up procedure by Benjamini, Krieger, and Yekutieli for false discovery rate < 1.00%. (F and G) Adalimumab- and certolizumab-based scFvs block TNF-α binding (F) and emapalumab-based scFv blocks IFN-γ binding (G). CD19 CAR-T cells were cocultured with Raji cells and donor-matched immune cells (monocytes, macrophages, and dendritic cells) to stimulate cytokine secretion. Addition of scFvs concentrated from HEK293T supernatant binds specific cytokines and blocks their detection by Cytometric Bead Array assay. Triplicate wells were separately seeded, treated with scFvs, and quantified. Pairwise comparisons against the no-scFv control were performed using Welch’s t test, with multiple-comparison correction by the Holm–Sidak method using a significance threshold of 0.05. In panels D–G, means of triplicates are shown with error bars indicating ± 1 standard deviation (SD). Statistical significance levels for all panels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The experiments in panels C–E were each performed once. Experiments shown in F and G were performed twice using two different healthy donors’ cells; data from one representative donor are shown. Source data are available for this figure: SourceData F1.

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