Plasmid maps a and b show PGQ-U6-IRF8sg2-Thy1.1 at 6346 base pairs and p5xNF-MSGV-Thy1.1-IRF8 at 7122 base pairs, containing key elements including U6 promoter, IRF8 sg2, gRNA scaffold, and Thy1.1 in map a, and IRF8 insert, 5xNFkB, and Thy1.1 in map b. Histograms c and d show CD44, CD25, PD-1, and IRF8 distributions where IRF8 knockout cells show a leftward shift and IRF8 overexpression cells show a rightward shift in IRF8 levels compared to their respective controls. Paired line graphs and histograms e show IRF8 geometric mean fluorescence intensity up to 50000 and TOX geometric mean fluorescence intensity up to 4000 with p values below 0.0001, and percentage PD-1 positive cells rising from approximately 5 to 30 percent between ex vivo and 5 hour gp33 conditions. Contour plots in the bottom panel show PD-1 versus IFN gamma, Granzyme B, TNF alpha, and IL-2 with gated percentages of 46.7, 1.51, 43.0, and 31.8 in control dropping to 22.9, 1.42, 26.6, and 12.8 in IRF8 overexpression, with paired line graphs confirming significant reductions in IFN gamma, TNF alpha, and IL-2 but no significant change in Granzyme B.
IRF8 level modulation did not change activation quality in CD8 + T cells. (a) Representation of the retroviral vector PQG-Thy1.1 used to induce KO in P14+CD8 T cells expressing the Cas9 endonuclease (see Materials and methods). The specific sgRNA sequence targeting the gene of interest (in this case IRF8) was inserted in front of the gRNA scaffold sequence under the control of a U6 promoter and followed by Thy1.1 (CD90.1) sequence, used as transduction marker, under the control of hPGK promoter. (b) Representation of the retroviral vector pMSGV-Thy1.1 used to induce IRF8 OE. The full IRF8 coding-sequence was inserted after the Thy1.1 sequence, separated by a P2A cassette. The control used was the same vector without the Irf8 sequence (empty). (c and d) Representative FACS histograms showing CD44, CD25, PD-1, and IRF8 expression in naïve, untransduced, control transduced, P14-Cas9 IRF8 KO transduced (c), or P14 IRF8 OE transduced (d) CD8+ T cells 48 h after activation with retroviral transduction being done at 24 h after activation, as described in the Materials and methods. (e) These data complement experiments presented in Fig. 5 e, IRF8 and TOX gMFI, percentage of PD-1+ before and after ex vivo peptide restimulation, and percentage of IFNγ+, GrzB+, TNFα+, and IL-2+ with representative FACS plots in IRF8 OE compared with control P14 cells recovered from LNs from experiment illustrated in Fig. 6, k–p. Representative data from two independent experiments (n = 8). Statistical analysis was done with paired two-tailed Student’s t test. **P < 0.01 and ***P < 0.001.