Schematic a shows P14 cell transfer at day negative 1 into LCMV Cl.13 or Armstrong infected mice with ex vivo gp33 restimulation and analysis at day 28. Grouped bar graph b on a geometric mean fluorescence intensity times 10 to the power of 4 y-axis up to 2 shows Armstrong with 6.7 and 8.9 times increases and Cl.13 with a 1.7 times increase from baseline to plus gp33 time points, with accompanying histograms showing IRF8, CD44, PD-1, and TOX distributions for both conditions. Genome browser tracks c show ATACseq and Cut and Tag H3K27ac signals at the Irf8 locus on chromosome 8 spanning 10 kilobases across Naive, Cl.13 day 30 post infection, and TIL conditions with variable peak heights across highlighted regions and TF binding sites annotated below. Bar graph d shows relative Irf8 versus Tox H3K27ac signal where B16-gp33 TILs day 21 values exceed 3 compared to LCMV Cl.13 day 21 values below 1. Grouped bar graph e on a geometric mean fluorescence intensity times 10 to the power of 4 y-axis up to 8 shows IRF8 levels dropping from approximately 5 at no IFN beta to 1 at IFN beta day 0 to 9 before recovering to around 3 at later time points, with significant differences marked and representative histograms shown. Schematic f shows B16-gp33 mice receiving P14 control and IFNAR1 knockout cells with intra-tumoral CpG at days 6, 9, and 12 and FACS at day 14, with contour plots confirming knockout efficiency. Paired line graphs g show IRF8 and IRF7 geometric mean fluorescence intensity times 10 to the power of 3 declining from control to IFNAR1 knockout with significant differences and representative histograms. Schematic h shows LCMV Cl.13 infected mice receiving anti-IFNAR1 treatments across early, mid, and late phases from day 3 to day 27. Paired line graph i on a geometric mean fluorescence intensity times 10 to the power of 3 y-axis up to 5 shows significant separation between isotype and anti-IFNAR1 at early and late time points with no significant difference at mid. Histograms j show IRF8 and IRF7 distributions at early, mid, and late time points with visible peak shifts between isotype and anti-IFNAR1 conditions.
IRF8 is epigenetically repressed in virus-specific CD8 + T cells during chronic infection. (a) Schematic representation for the experimental procedure used to assess the capacity of P14 CD8+ T cells recovered from acute LCMV Arm or chronic LCMV Cl13 infection to upregulate IRF8 after ex vivo gp33 stimulation. (b) IRF8 levels in P14+ CD8 T cells obtained as described in a at ex vivo baseline, 24, and 72 h after restimulation with gp33 peptide (left) with representative histograms showing IRF8, CD44, PD-1, and TOX expression (right). n = 8 mice per condition, data pooled from two independent experiments. Fold-change respective to baseline is indicated in brackets. (c) ATAC-seq and Cut&Tag (H3K27ac, GEO: GSE235007) tracks and TF-binding regions (mouse CD8+ T cells; https://chip-atlas.org) at the Irf8 locus. Purple highlights indicate ATAC peaks increased in TILs compared with LCMV cl13 P14+ T cells. (d) Signal quantification for H3K27ac tracks represented as relative Irf8/Tox ratio. (e) IRF8 levels of in vitro chronically TCR stimulated CD8 T cells cultured with or without IFNβ during the indicated period with representative histogram. n = 6 biological replicates, representative data from three independent experiments. (f) Schematic representation of experimental procedure used to assess the capacity of type I IFN to downregulate IRF8 levels in CD8+ TILs. P14-Cas9 IFNAR1 KO and control-transduced P14-Cas9 cells were co-transferred in B16-gp33–bearing B6-Cas9 mice that received intratumoral CpG injection at the indicated time points as described in the Materials and methods section. At the bottom, FACS plots showing expression of IFNAR1 and CD90.1, expressed only in transduced cells, in P14 control and P14 IFNAR1 KO cells. (g) Flow cytometric analysis of IRF8 and IRF7 levels in P14+CD90.1+ IFNAR1 KO or control transduced CD8 TILs with representative FACS histograms. n = 6, data pooled from two independent experiments. (h) Schematic representation showing experimental procedure used to assess IRF8 levels during chronic LCMV Cl13 infection upon αIFNAR1 antibody treatment at the indicated time point, as described in the Materials and methods. (i and j) IRF8 quantification (i) at the indicated time point as illustrated in h with representative FACS histograms for IRF8 and IRF7 expression (j). n = 4–5 mice per condition at each time point. Representative data from three independent experiments are shown. In b statistical analysis was done with two-way ANOVA with comparing means within each group. In e statistical analysis was done with one-way ANOVA comparing means with control mean (no IFNβ). In g, statistical analysis was done by paired two-tailed Student’s t test. In h, statistical analysis was done by unpaired two-tailed Student’s t test. *P < 0.05 and ***P < 0.001.