Fig 6. Control of chronic LCMV infection by innate CD8⁺ T cells.

(A) T-E and T-T P14 cells were isolated from the spleens of each BM chimera as shown in Fig 5 and transferred into Thy1.2⁺ congenic C57BL/6 mice. One day after transfer, the mice were infected with 5 x 10⁵ PFU of LCMV CL–13 per mouse. (B-D) Thy1.1⁺ CD8⁺ donor P14 cells were gated for following analysis. (B) CellTrace Violet (CTV)-labelled donor T-E and T-T P14 cells were analyzed at 2.5 DPI by flow cytometry. Frequency of Thy1.1⁺ CD8⁺ P14 cells among splenocytes are depicted. The gated donor Thy1.1⁺ CD8⁺ P14 cells are examined for CTV dilution along with CD44 expression. Division time and frequency of CD44^hi cell population are indicated in histogram plot. (C) Lymphocytes were isolated from the spleens of T-E and T-T P14 chimeric mice at 5 and 18 DPI and analyzed by flow cytometry. The number of Thy1.1⁺ P14 cells in the spleen and PD–1 expression among CD8⁺ T cells were analyzed at indicated DPI. Numbers in the plots indicate PD–1⁺ or PD–1^- Thy1.1⁺ transferred cells. PD–1 expression level (MFI) on Thy1.1⁺ transferred cells is summarized in the graph. (D) Splenocytes of T-E and T-T P14 chimeric mice were restimulated in vitro with GP33 for P14 cells, and frequency of IFN-γ- and TNF-α-producing Thy1.1⁺ transferred cells were analyzed. Numbers in the plots indicate the percentage of TNF-α⁺ and TNF-α^- CD8⁺ T cells producing IFN-γ, respectively. Frequency of Thy1.1⁺ transferred cells producing both IFN-γ and TNF-α was summarized in the graph. (E) Serum viral titers were checked in T-E and T-T P14 chimeras at the indicated DPI. Dashed line indicates the virus detection limit. Undetectable samples were given a half of detection limit. (F-G) CD8⁺ T cells were gated for following analysis. (F) PD–1 expression on GP276 tetramer-positive CD8⁺ T cells was analyzed at indicated DPI. Numbers in the plots indicate PD–1⁺ or PD–1^- GP276 tetramer-positive cells among CD8⁺ T cells. PD–1 expression level (MFI) on GP276 tetramer-positive CD8⁺ T cells is summarized in the graph. (G) Splenocytes of T-E and T-T P14 chimeric mice were restimulated in vitro with GP276 peptide for endogenous CD8⁺ T cells, and frequency of IFN-γ- and TNF-α-producing CD8⁺ T cells was analyzed. Numbers in the plots indicate the percentage of TNF-α⁺ and TNF-α^- CD8⁺ T cells producing IFN-γ, respectively. Frequency of CD8⁺ T cells producing both IFN-γ and TNF-α was summarized in the graph. Bar graphs show mean + SD. Data are representative of more than two independent experiments (n≥3 per group in each experiment). NS, not significant; *P<0.05; **P<0.01; ***P<0.001.