Graphs a and b show violin plots of regulon activity scores ranging from approximately 0.15 to 0.5 across TEX, TPEX, Effector Memory, Early Activated, and Naive-like subsets, with IRF8 displaying comparatively lower and narrower activity distributions in TEX cells relative to other regulons. Graph c shows Irf8 with expression levels reaching up to 3 on the y-axis, visibly broader and taller violin shapes compared to Irf1 through Irf7 and Irf9, particularly in Tex and Tpex populations. Flow cytometry plots d show dot plot distributions of TOX versus PD-1 and TOX versus IRF8 axes, where endogenous and P14 populations show distinct clustering patterns across the two axes. Graph e shows IRF8 gMFI values ranging from roughly 1000 to 6000 on the y-axis, with paired lines consistently rising from TOX negative to TOX positive cells in both endogenous and P14 populations. Graph f shows IRF8 gMFI values across five tumor models with paired lines trending upward from TOX negative to TOX positive across all five panels, with MIBC showing the widest range reaching above 3000. Flow cytometry plots g and h show dot plot distributions along TOX versus IRF8 and PD-1 axes in human melanoma samples, with graph h showing gMFI values rising from approximately 2000 to above 8000 between TOX negative and TOX positive cells. Flow cytometry plots i and k show gating along CD44 versus PD-1 and TOX versus IRF8 axes with distinct IRF8 high and IRF8 low gates visible. Graphs j and l show gMFI values for LAG3, PD-1, TIM3, and TOX on y-axes reaching up to 8000, with paired lines consistently higher in IRF8 high compared to IRF8 low cells across all four markers. Flow cytometry plots m show gating of IRF8 high and IRF8 low populations along TOX versus IRF8 axes with approximately 30 percent of cells falling within each gate, and graph n shows corresponding gMFI values up to 40000 for PD-1, with paired lines trending higher in IRF8 high cells across LAG3, PD-1, TIM3, and TOX.
IRF8 is highly expressed in exhausted CD8 + TILs in murine and human cancer. (a and b) Top eight discriminant regulon signature in TEX (a) and TPEX (b) CD8+ TILs based on publicly available datasets as described in the Materials and methods section. (c) IRF TF family members gene expression levels in CD8 TILs based on publicly available scRNA-seq datasets for different cancer studies, as described in the Materials and methods section. (d) Representative FACS plots showing TOX, PD-1, and IRF8 expression in endogenous and tumor-specific (P14) CD8+ TILs in indicated populations 14 days after B16-gp33 melanoma engraftment in C57/BL6 mice. (e) Quantification of IRF8 protein levels in TOX− and TOX+ CD8+ TILs among endogenous (left) and transferred tumor-specific P14 (right) CD8+CD44highPD-1+ with representative FACS histogram (TOX+ in red, TOX− in black). n = 7, representative data from three independent experiments. (f) Flow cytometric analysis of IRF8 expression levels comparing TOX+ and TOX− CD8+CD44highPD-1+ endogenous TILs population in B16F10 melanoma (n = 5), EL-4 thymoma (n = 5), LLC1 (n = 5), murine colon adenocarcinoma (MC38, n = 5), and orthotopic inducible MIBC (n = 7) with representative histograms. (g) Representative FACS plots showing TOX, PD-1, and IRF8 expression in human melanoma metastasis samples in indicated populations. IRF8 protein levels in TOX− and TOX+ CD8 TILs (among CD8+CCR7−CD45RA−PD-1high) from nine human melanoma metastasis samples with representative FACS histogram (TOX+ in red, TOX− in black). n = 9, data pooled from two independent experiments. (h, i, k, and m) Representative FACS plots showing gating strategy for IRF8high and IRF8low mouse CD8+ TILs in endogenous (i) and P14 (k) cells from experiment in d and e, and human CD8+ TILs cells from experiment in g, h, and m. (j, l, and n) Quantification of LAG3, PD-1, TIM3, and TOX protein levels comparing IRF8high versus IRF8low cells in the respective populations indicated above. (e, f, h, j, l, and n) Statistical analysis was done by paired two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.