Schematic a shows LCMV Cl.13 infected V-beta-5 mice receiving P14 control and P14 IRF8 knockout cells at day 0 with FACS at day 21. Scatter plot b shows knockout to control ratios around 1 across spleen, lymph node, and liver with no significant differences. Paired line graphs c show TPEX and TEX percentages among PD-1 positive cells with SLAMF6 versus TIM3 contour plots showing TPEX at 30.2 and 33.1 percent and TEX at 69.5 and 66.6 percent for control and IRF8 knockout. Paired line graphs d on geometric mean fluorescence intensity times 10 to the power of 4 y-axes show IRF8 declining significantly and TOX declining significantly while PD-1 shows no significant change between control and IRF8 knockout, with representative histograms. Paired line graphs e show percentage IFN gamma positive, Granzyme B positive, TNF alpha positive, and IL-2 positive cells with Granzyme B rising while others remain largely unchanged, with representative histograms. Schematic f shows the equivalent experiment with P14 IRF8 overexpression cells in V-beta 5 mice with FACS at day 21. Scatter plot g shows overexpression to control ratios around 1 across spleen, lymph node, and liver with no significant differences. Paired line graphs h show TPEX decreasing and TEX increasing with IRF8 overexpression, with contour plots showing TEX at 89.2 and 83.7 percent. Paired line graphs i show IRF8, PD-1, and TOX all rising significantly on geometric mean fluorescence intensity times 10 to the power of 4 y-axes with representative histograms. Paired line graphs j show Granzyme B and TNF alpha rising while IFN gamma and IL-2 show minimal change with representative histograms. Schematic k shows Rag1 knockout mice receiving P14 control and IRF8 overexpression cells at day 0 with FACS at day 7. Scatter plot l shows CD90.1 positive output to input ratios declining significantly in spleen and lymph node with IRF8 overexpression. Paired line graphs m show IRF8 rising significantly on geometric mean fluorescence intensity times 10 to the power of 4 while TOX on geometric mean fluorescence intensity times 10 to the power of 3 shows no significant change. Paired line graphs n show percentage PD-1 positive cells rising significantly from ex vivo to 5 hour gp33 stimulation with representative histograms. Contour plots o show PD-1 versus IFN gamma, Granzyme B, TNF alpha, and IL-2 with gated percentages of 66.0, 18.1, 55.7, and 33.2 in control dropping to 40.0, 18.8, 34.8, and 15.8 in IRF8 overexpression, confirmed by paired line graphs p showing significant reductions across all four markers.
IRF8 has limited effect on exhausted CD8 + T cells during chronic infection but limits effector function in absence of persistent TCR stimulation. (a) Schematic representation showing experimental procedure used to address IRF8 KO effect in P14-Cas9 CD8+ T cells during chronic LCMV Cl13 infection in Vβ5 mice. (b) Accumulation of IRF8 KO cells compared with control cells represented as ratio KO/control in spleen, LN, and liver at day 21 after infection. (c–e) Percentage of TPEX and TEX (c), percentage of IFNγ+, GrzB+, TNFα+, and IL-2+ (e), and gMFI of IRF8, PD-1, and TOX (d) in IRF8 KO compared with control P14 cells from experiments illustrated in a. Representative experiment from two independent experiments (n = 5). (f) Schematic representation showing experimental procedure used to address IRF8 OE effect in P14 CD8+ T cells during chronic LCMV Cl13 infection in Vβ5 mice. (g) Accumulation of IRF8 KO cells compared with control cell represented as ration KO/control in spleen, LN, and liver at day 21 after infection. (h–j) Percentage of TPEX and TEX (h), percentage of IFNγ+, GrzB+, TNFα+, and IL-2+ (j), and gMFI of IRF8, PD-1, and TOX (i) in IRF8 KO compared with control P14 cells from experiment illustrated in f. Representative experiment from two independent experiments (n = 4). (k) Schematic representation showing experimental procedure used to address IRF8 OE effect in P14 CD8+ T cells in Rag1KO mice. (l) Recovered (output) versus injected (input) CD90.1+ control or OE CD8+ T cells represented as ratio output/input in LN and spleen from co-transfer experiment illustrated in k. (m–p) IRF8 and TOX gMFI (m), percentage of PD-1+ before and after ex vivo peptide restimulation (n), and percentage of IFNγ+, GrzB+, TNFα+, and IL-2+ (p) with representative FACS plots (o) in IRF8 OE compared with control P14 cells from experiment illustrated in k. Data from two pooled independent experiments (n = 8). In b and g, statistical analysis was done with a t test comparing the means with a theoretical value of 1, representing a fold change of 1. In c–e and m–p, statistical analysis was done with paired two-tailed Student’s t test. Statistical analysis in l was done with two-tailed Student’s t test. *P < 0.05 and ***P < 0.001.