Line graph a shows IRF8 gMFI times 10 to the power of 4 on the y-axis measured at 0, 24, 48, and 72 hours after anti-CD3 and anti-CD28 stimulation, where values rise from near 0 at 0 hours to approximately 11 at 48 hours before declining to around 9 at 72 hours, with accompanying histograms showing progressive broadening of IRF8 distribution from 0 to 72 hours. Contour plots b show PD-1 versus TIM3, SLAMF6 versus Granzyme B, LAG3 versus TOX, and IRF8 versus a fourth marker, displaying shifting population distributions across two overlaid conditions. Line graph c shows IRF8 gMFI on a logarithmic y-axis from 10 to the power of 3 to 10 to the power of 6 over 9 days after activation, where chronic TCR stimulation values start around 10 to the power of 5 at day 1 and decline gradually to around 10 to the power of 4 by day 9, while resting values begin lower and decline more steeply to near 10 to the power of 3 by day 9. Schematic d shows B16-OVA and B16-gp33 tumor bearing mice receiving preactivated P14 and OT-1 cells at day 7 with FACS analysis at day 14. Histograms e show pre-infusion phenotype of naive, P14, and OT-1 cells across CD44, CD25, PD-1, and IRF8 markers, where P14 and OT-1 show rightward shifted distributions compared to naive cells across all four markers. Paired line graphs f show PD-1 on a gMFI times 10 to the power of 3 y-axis up to 6, TOX on a gMFI times 10 to the power of 2 y-axis up to 20, and IRF8 on a gMFI times 10 to the power of 3 y-axis up to 5 in the B16-gp33 model, where paired lines consistently fall from P14 to OT-1 values across all 3 markers with significant differences marked, supported by representative histograms below. Paired line graphs g show the same 3 markers in the B16-OVA model with PD-1 and IRF8 lines rising from P14 to OT-1 values and TOX lines also rising, with significant differences marked for all 3 comparisons and representative histograms confirming the shifts.
TCR stimulation drives IRF8 expression in the TME. (a) Kinetics of IRF8 protein levels of enriched CD8 T cells activated with plate-bound αCD3 and soluble αCD28 antibodies with representative histogram (n = 3 biological replicates, representative data from two independent experiments). (b) Representative FACS plots showing PD-1, TIM3, SLAMF6, GZMB, LAG3, TOX, and IRF8 expression in resting (green) and in vitro chronically TCR-stimulated CD8+ T cells (black) as described in the Materials and methods. (c) IRF8 quantification in CD8+ T cells from experiment in b. n = 3 biological replicates, data representative of two independent experiments. (d) Schematic representation of experimental procedure used to address TCR dependency of IRF8 expression in vivo in the TME. B6 mice were engrafted with B16-gp33 and B16-OVA on opposite flanks. 106 preactivated P14 and 106 preactivated OT-I T cells were co-transferred at day 7 after tumor initiation. Readout was performed on day 14 after tumor engraftment. (e) FACS histograms showing expression of CD44, CD25, PD-1, and IRF8 on the day of ACT in naive (black dotted line), P14 (orange), and OT-I (purple) CD8+ T cells after 48 h of in vitro activation with gp33 or OVA peptide. (f and g) Flow cytometric analysis of PD-1, TOX, and IRF8 protein levels in P14+ and OT-I+ CD8 T cells recovered from B16-gp33 (f) or B16-OVA (g) tumors 7 days after co-transfer as described in d. n = 6, representative data from two independent experiments. In f and g, statistical analysis was done by paired two-tailed Student’s t test. **P < 0.01 and ***P < 0.001.