Fig. 3: IFN-γ-is by CD8⁺ T-cells at priming and is paracrine.

a–c WT mice were infected with LM-OVA and mice were either left untreated (Ctrl, grey) or treated with anti-IFN-γ 16-24 h (red) or at day 5 and 6 (orange) post-infection. Graphs show relative abundance (n = 13 Ctrl and 24h-anti-IFN-γ-treated animals, 12 day 5/6-anti-IFN-γ-treated animals) (a), normalized N4-tetramer gMFIs (n = 13 Ctrl/24h-anti-IFN-γ-treated animals, 12 day5/6-anti-IFN-γ-treated animals) (b), and CD3 gMFI (n = 6 Ctrl/24h-anti-IFN-γ-treated animals, 7 day5/6-anti-IFN-γ-treated animals) (c) of N4-tetramer⁺ CD8⁺ T-cells analyzed by flow cytometry 9 days post-infection. d–g WT mice were treated with depleting NK1.1 (brown) or control antibodies (grey) and infected with LM-OVA. d, e Relative abundance (d) and the normalized N4-tetramer gMFIs (e) of N4-tetramer⁺ CD8⁺ T-cells 9 days post-infection (n = 10 animals). f–g Splenocytes were re-stimulated in vitro with the indicated concentrations of N4 for 16 h. Quantification of relative IFN-γ expression (n = 5 animals) (f) and EC₅₀ of IFN-γ production (n = 10 animals) (g) by flow cytometry. h–k Control (Ctrl, blue) and CD8-IFN-γ^KO (pink) chimera mice were infected with LM-OVA. h, i Splenocytes were isolated after 24 hrs. h IFN-γ production by CD8⁺ T-cells and NK cells was quantified after ex-vivo PMA and Ionomycin stimulation (n = 3 animals). i STAT1 phosphorylation (pSTAT1) was quantified in activated CD8⁺ T-cells (n = 12 animals). pSTAT1 of BM chimeras was normalized to IFN-γKO pSTAT1 MFI by subtracting the background gMFI. j, k Splenocytes were isolated after 9 days. Relative abundance (n = 11 Ctrl, 12 CD8-IFN-γ^KO animals) (j) and normalized N4-tetramer gMFIs (n = 11 animals) (k) of N4-tetramer⁺ CD8⁺ T-cells. N4-tetramer gMFIs of IFN-γ- or NK-depleted samples were normalized to the mean Ctrl N4-tetramer gMFI within each independent experiment. Data are from ≥3 (a–e, i–k), 2 (f, g) or representative of 3 (h) independent experiments. Two-tailed unpaired Student’s t-test (d, e, g, j, k), one-way ANOVA with Tukey’s multiple comparison test (a–c) and two-way ANOVA and Šidák’s multiple comparison test (h). Error bars indicate the mean ± s.e.m.