Fig. 3: Spontaneous production of IFNα by limiting the fatty acid biosynthesis pathway triggers a type I IFN response in CD4⁺ T cells.

a The amount of IFNα in the cell supernatant was measured by ELISA. Data are means ± SD. b qRT-PCR analyses of the relative expression of ISGs in WT and ACC1 KO Th1 cells. 10 μg/ml IFNAR neutralizing antibody was treated for 72 h after TCR stimulation. Relative expression (normalized to Hprt) with SD is shown. c Intracellular staining and flow cytometry analyzing of IRF7 in Th1 cells treated with IFNAR neutralizing antibody as in (b) are shown. Mean fluorescence intensity (MFI) of IRF7 are shown. Isotype means isotype-matched control antibody. d Summary data of four independent experiments of IRF7 expression are shown. Each dot represents one experiment. Data are means ± SD. (n = 4 per each group biologically independent sample). e Co-culture experiment was designed as Supplementary Fig. 3a. Co-culture was started after 48 TCR stimulation. Under the WT mix conditions, WT and littermate Th1 cells were cultured in a same well. Under the ACC1^−/− mix conditions, WT and ACC1^−/− Th1 cells were cultured in a same well. After 72 h, cells were harvested and sorted by flow cytometer based on the expression of Ly5.1(WT) and Ly5.2 (littermate or ACC1^−/−). qRT-PCR analyses of the relative expression of ISGs in sorted Th1 cells. Relative expression (normalized to Hprt) with SD is shown. f qRT-PCR analyses of the relative expression of ISGs in WT and ACC1^−/− Th1 cells. 1 nM JAK inhibitor ruxolitinib was treated for 72 h. Relative expression (normalized to 18S) with SD is shown. g Intracellular staining and flow cytometry analyzing of IRF7 in Th1 cells treated with JAK1 inhibitor as in (f) are shown. Mean fluorescence intensity (MFI) of IRF7 are shown. Isotype means isotype-matched control antibody. h Summary data of four independent experiments of IRF7 expression are shown here. Each dot represents one experiment. Data are means ± SD. (n = 4 per each group biologically independent sample). i MLE-15 cells were cultured with supernatant of Th1 cells for 24 h as in Supplementary Fig. 3d. qRT-PCR analysis of ISGs in MLE-15 cells was performed. Relative expression (normalized to Hprt) with SD is shown. Three independent experiments for each group were performed with similar results. j MLE-15 cells were cultured as in (i) and infected with ×31. Virus titers were determined 72 h p.i. (n = 3 per each group biologically independent sample). More than three independent experiments were performed and showed similar results (a–j). More than three technical replicates were performed with quantitative RT-PCR and relative expression (normalized to Hprt) with SD is shown (a, b, e, f and i).