Schematic a shows a cell activation timeline from day 0 to day 9, with initial activation using anti-CD3, anti-CD28, and rhIL-2 at day 0, followed by restimulations with anti-CD3 and rhIL-2 at days 3, 6, 8, and 9. Grouped bar graph b shows geometric mean fluorescence intensity of IRF8 in P14 positive liver cells under baseline ex vivo, plus gp33 24 hours, and plus gp33 72 hours across Armstrong and Cl.13 conditions on a y-axis up to 60000, where Armstrong shows a 14.3 times increase and Cl.13 shows a 2 times increase from baseline to plus gp33 72 hours, with a 6 times difference between the two conditions. Genome browser tracks c show ATACseq chromatin accessibility at the Irf8 locus on chromosome 8 spanning 10 kilobases across Naive, Cl.13 day 8 and day 30 post infection, and TIL conditions alongside H3K27ac and TF binding region tracks, with highlighted regions showing variable peak heights and Cl.13 day 8 post infection displaying the tallest peaks. Genome browser tracks d show ATACseq and Cut and Tag H3K27ac signals at the Tox locus on chromosome 4 spanning 50 kilobases across Naive, Cl.13, and TIL conditions with TF binding regions annotated below, showing variable peak distributions throughout the locus. Line graphs e and f show geometric mean fluorescence intensity of IRF8 in CD8 positive cells at 0, 24, 48, and 72 hours after stimulation, where IFN gamma in graph e reaches approximately 30000 at 48 hours versus control at 10000, and in graph f IL-12 and IFN beta both reach approximately 100000 to 120000 at 48 hours substantially above control. Scatter plots g show IRF2, IRF4, and IRF7 geometric mean fluorescence intensity among CD8 positive cells across five IFN beta treatment timepoints, where IRF2 and IRF7 show significant differences across multiple timepoints on y-axes reaching 60000 and 40000 respectively, while IRF4 values up to 250000 show no significant differences.
Regulation of IRF8 expression in CD8 T cells is dependent on TCR stimulation and is repressed during chronic LCMV Cl13 infection. (a) Experimental setup of the chronic TCR stimulation in vitro used to assess IRF8 expression kinetics during artificially induced exhaustion. (b) IRF8 geometric mean fluorescence intensity (gMFI) in P14+ CD8 T cells recovered from livers of experiment presented in Fig. 4 a with fold change compared with baseline indicated in brackets. n = 8 mice per condition, data from two pooled independent experiments. (c) ATAC-seq tracks at the Irf8 locus of LCMV cl13 days 8 and 30 after infection P14+ T cells and TILs. Purple highlights indicate ATAC peaks decreasing in LCMV cl13 day 30 compared with LCMV cl13 day 8 P14+ T cells. TF-binding regions in CD8+ T cells (https://chip-atlas.org) are shown in green. (d) ATAC-seq and Cut&Tag (H3K27ac, GEO: GSE235007), tracks at the Tox locus of TILs and LCMV cl13 P14+ T cells. TF-binding regions in CD8+ T cells (https://chip-atlas.org) are shown in green. (e and f) IRF8 kinetics of enriched CD8 T cells activated with anti-CD3 and anti-CD28 antibodies with addition of IFNγ (e), IFNβ, or IL-12 (f) during 72 h compared with control (only IL-2). N = 3 biological replicates, e and f are two independent experiments. (g) IRF2, IRF4, and IRF7 gMFI of chronically TCR stimulated CD8 T cells (using protocol in a) with or without addition of IFNβ at different time points indicated in the figure. N = 6 biological replicates, representative data from three independent experiments. Two-way ANOVA comparing means within each condition was used to analyze data in b. One-way ANOVA was used in g. *P < 0.05, **P < 0.01, and ***P < 0.001.