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. 2016 Jan 11;213(1):75–92. doi: 10.1084/jem.20142350

Figure 8.

Figure 8.

The early IFN-γ production participates in the clustering of mCTLs after secondary infection. IFN-γ–blocking antibodies (anti–IFN-γ) or isotype control (Isot) were injected to memory mice 18 h before secondary infection. Spleens were harvested at 6 h after, cut in equal halves, and processed either for flow cytometry or confocal imaging. (A) Frequency of IL-12p40/70 and CXCL9-producing XCR1+ DCs in spleens. (B) Spleen sections were stained for GFP (green) and B220 (dark blue). Bar, 200 µm. Arrows show clusters. Graph represents percentage of OT-I mCTLs that form clusters in anti–IFN-γ-treated memory mice as mathematically determined. Data (mean ± SEM) are shown for one experiment representative of two independent ones, each with at least three mice per group. *, P < 0.05; ns, nonsignificant. (C) Spleens of memory mice were harvested 6 h after secondary infection. Circle diagram encompasses all cells that stained positive for IFN-γ. One representative experiment out of four with three mice per group is shown. (D and E) Spleens from memory mice were fixed 6 h after the infection, and sections were stained with anti-GFP (green), anti-dsRed (red), and anti-NKp46 (cyan; bar, 200 µm; D), or with anti-GFP (green), anti–IFN-γ (red) and anti-NKp46 (cyan; bar, 50 µm; E). One representative experiment out of two, each with three mice per group, is shown. (F-I) Impact of NK cell depletion. (F) Frequency of IL-12p40/70 and CXCL9-producing XCR1+ DCs in spleens 6 h after Lm-OVA secondary infection. Data (mean ± SEM) are pooled from two independent experiments, each with at least three mice per group. *, P < 0.05; **, P < 0.01. (G and H) Frequency of OT-I mCTLs (G) and of polyclonal CD44hi mCTLs (H) that produce IFN-γ 6 h post-secondary infection was measured as in Fig. 3. One experiment representative of three independent ones, each with five mice per group is shown. Data are represented as mean ± SEM. *, P < 0.05. (I) Statistical analysis of clustered OT-I mCTLs. Data (mean ± SEM) are pooled from two independent experiments, each with at least three mice per group.