Schematic a shows B16-gp33 or MC38-gp33 tumor bearing B6.Cas9 mice receiving P14 control or P14 IRF8 knockout cells at day 7 with tumor growth monitored. Line graphs b and c show tumor volume in cubic millimeters over time for B16-gp33 and MC38-gp33 models across PBS, P14 control, and P14 IRF8 knockout groups, where IRF8 knockout groups show higher tumor volumes, confirmed by mean tumor volume graphs on the right with significant differences marked. Schematic d shows B16-gp33 mice receiving both P14 control and P14 IRF8 knockout cells at day 7 with FACS at day 14. Paired line graphs f show percentage TPEX and TEX among PD-1 positive cells with significant differences, supported by SLAMF6 versus TIM3 contour plots. Paired line graphs g show percentage IFN gamma-positive and Granzyme B-positive cells rising from control to IRF8 knockout in both tumor and draining lymph node with significant differences and representative histograms. Paired line graphs h show IRF8 and TOX geometric mean fluorescence intensity times 10 to the power of 3 declining from control to IRF8 knockout with significant differences, while PD-1 shows no significant change, with scatter plot i confirming significant IRF8 decline in draining lymph node. Schematic j shows wildtype mice receiving P14 control and P14 IRF8 overexpression cells at day 7 with FACS at day 14. Paired line graphs l show percentage TPEX and TEX shifting significantly with IRF8 overexpression supported by contour plots showing TEX proportions of 75.3 and 84.8 percent. Paired line graphs m show IRF8 rising significantly while PD-1 and TOX geometric mean fluorescence intensity times 10 to the power of 3 show significant changes with IRF8 overexpression. Paired line graphs n show percentage IFN gamma positive, Granzyme B positive, IL-2 positive, and TNF alpha positive cells all declining significantly with IRF8 overexpression.
IRF8 expression in CD8 + T cells promotes exhaustion and reduces anti-tumor function. (a) Schematic representation of experimental procedure used to assess tumor control capacity of IRF8 KO CD8+ T cells. (b and c) Tumor growth curves in B6-Cas9 mice that received a mock PBS injection, control-transduced P14 CD8 T cells, or IRF8 KO P14 CD8 T cells 7 days after B16-gp33 (b) or MC38-gp33 (c) tumor engraftment and mean of control and IRF8 KO P14 CD8 T cells transferred mice (bars show SEM). Numbers of mice for each condition are indicated in the figure, and data are issued from two pooled independent experiments in b and representative of two independent experiments in c. (d) Schematic representation showing experimental procedure used to address IRF8 KO effect in P14-Cas9 CD8+ TILs, with FACS plots showing gating strategy for differentiating KO, control, and endogenous cells among CD8+ cells in the B16-gp33 tumor of B6-Cas9 mice. (e) Recovered (output) versus injected (input) CD90.1+ control or KO CD8+ T cells represented as ratio output/input in tumor and DLN from co-transfer experiment illustrated in d. (f) Percentages of TPEX and TEX populations in IRF8 KO and control-transduced cells. (g–i) Flow cytometric analysis of IRF8, TOX, and PD-1 (gMFI) and IFNγ+ and GrzB+ (percentage) in P14 IRF8 KO and P14 control CD8+ T cells in tumor and DLN. Representative data from three independent experiments (n = 7). (j) Schematic representation showing experimental procedure used to address IRF8 OE effect in P14 CD8+ TILs in B16-gp33 tumors in WT B6 mice. (k) Recovered (output) versus injected (input) CD90.1+ control or OE CD8+ T cells represented as ratio output/input in tumor and DLN from co-transfer experiment illustrated in j. (l) Percentages of TPEX and TEX populations in IRF8 OE and control-transduced cells. (m and n) Flow cytometric analysis of IRF8, TOX, and PD-1 (gMFI), and IFNγ+, GrzB+, IL-2+, and TNFα+ (percentage) in P14 IRF8 OE and P14 control CD8+ T cells in tumor. Data from two pooled independent experiments (n = 10). Statistical analysis for b and c was done by repeated measures two-way ANOVA. In f–i and l–n, statistical analysis was done with paired two-tailed Student’s t test. Statistical analysis in e and k was done with two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.