Figure 6.

Tonic cGAS activity is required for IFN expression in iCD4+ T cells. (A) Western blot of cGAS, IRF3, IRF7, RELA, and actin in CD4⁺ T cells nucleofected with either control or cGAS targeting guides and subsequently transduced with control (GFP), RELA K5R or IRF3 lentivectors (n = 4 independent donors in two experiments). (B) IFN-I/III concentration following cGAMP (6 µg/ml) stimulation of CD4⁺ T cells nucleofected with control or cGAS gRNA and transduced with control, RELA K5R, or IRF3 lentivectors. Cells were pretreated with 5AZA (2 µM) for 48 h prior to cGAMP stimulation (n = 4 donors combined from two independent experiments). Each symbol represents one donor, bars represent geometric mean, paired one-way ANOVA with Tukey’s multiple comparison test. (C) cGAMP quantification by ELISA in CD4⁺ T cells transduced with either control (GFP), RELA K5R, or IRF3 lentivectors (n = 4 donors combined from two independent experiments). (D) DAPI, cGAS staining, and GFP localization by confocal microscopy in CD4⁺ T cells transduced with control (GFP), RELA K5R, or IRF3 lentivectors. Scale bar at 10 µm. Right: Magnification of cGAS channel showing contours used to delineate nuclear and cellular contents, scale bar at 5 µm (n = 4 donors combined from two independent experiments). (E) Quantification of the ratio of average intensity of nuclear to cytoplasmic cGAS signal in CD4⁺ T cells transduced with either control (GFP), RELA K5R, or IRF3 lentivectors (n = 4 donors combined from two independent experiments). Each symbol represents one cell, line represents median, mixed-effect analysis, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns, not significant. Source data are available for this figure: SourceData F6.