Figure 8.
Figure 8. bTRM-mediated rapid virus clearance depends on IFN-γ and perforin. (A) Mice were infected with rLCMV i.c to generate bTRM. At least 6 wk later, mice were treated with anti-CD8a depleting antibody i.p. before LCMVwt i.c. challenge or left unchallenged. Brains were analyzed 3 d later. (B, left) Representative flow cytometry plot of IFN-γ production and degranulation as measured by surface exposure of CD107a in bTRM (CD8+CD44+CD69+) of unchallenged (bTRM) or LCMVwt i.c.-challenged (bTRM + LCMVwt i.c.) WT mice with (+) or without (–) in vitro NP396-404 peptide stimulation. Numbers indicate frequencies (%) of cells in corresponding gates. (right) Frequencies (mean + SEM) of IFN-γ–positive (black bars), CD107a positive (gray bars), and double-positive (blue bars) bTRM with (+) or without (–) LCMVwt i.c. challenge and with (+) or without (–) in vitro NP396-404 peptide stimulation (n = 3–4 mice per group). (C–G) IFN-γ–competent (control)- or GKO-rLCMV memory mice were generated and treated as in A. (C) Frequencies of TM cells (CD127+KLRG1–) among bTRM (CD8+CD44+CD69+) as determined by flow cytometry in unchallenged IFN-γ–competent (control) and GKO mice. Each symbol represents an individual mouse and bars indicate mean (n = 4 mice per group). (D) Representative images of immunostained brain sections for CD8 (top, arrows) or LCMV (bottom). (E) Quantification of CD8+ T cells, (F) LCMV+ cells, or (G) LCMV RNA by qPCR (normalized to naive mice infected with LCMVwt i.c. = 1) in brains of GKO and control mice with (+) or without (–) LCMVwt i.c. challenge. Data represent mean + SEM (n = 3–10 mice per group). (H–M) Perforin-competent (control)- or PKO-rLCMV memory mice were generated and treated as in A. (H) Frequencies of TM cells among bTRM (as in C) in unchallenged perforin-competent (control) and PKO mice. Each symbol represents an individual mouse and bars indicate mean (n = 4 mice per group). (I) Frequencies of GzmB-expressing cells among bTRM as determined by flow cytometry 3 d after LCMVwt i.c. challenge of control or PKO mice. Each symbol represents an individual mouse, and bars indicate mean (n = 4–6 mice per group). (J) Representative images of immunostained brain sections for CD8 (top, arrows) or LCMV (bottom). (K) Quantification of CD8+ T cells, (L) LCMV+ cells, or (M) LCMV RNA by qPCR (normalized to naive mice infected with LCMVwt i.c. = 1) in brains of PKO and control mice with (+) or without (–) LCMVwt i.c. challenge. Data represent mean + SEM (n = 4–6 mice per group). *, P < 0.05; **, P < 0.01, asterisk colors represent multiple comparisons performed, two-way ANOVA with Tukey’s multiple comparisons (B), one way ANOVA with Sidak’s multiple comparisons test (I), unpaired two-tailed Student’s t test (C, F, G, H, L, and M). One representative (B–F and J–M) or pool (G) of two independent experiments is shown. (H and I) Experiments were performed once. Bars, 100 µm; (inset) 20 µm.

bT RM -mediated rapid virus clearance depends on IFN-γ and perforin. (A) Mice were infected with rLCMV i.c to generate bTRM. At least 6 wk later, mice were treated with anti-CD8a depleting antibody i.p. before LCMVwt i.c. challenge or left unchallenged. Brains were analyzed 3 d later. (B, left) Representative flow cytometry plot of IFN-γ production and degranulation as measured by surface exposure of CD107a in bTRM (CD8+CD44+CD69+) of unchallenged (bTRM) or LCMVwt i.c.-challenged (bTRM + LCMVwt i.c.) WT mice with (+) or without (–) in vitro NP396-404 peptide stimulation. Numbers indicate frequencies (%) of cells in corresponding gates. (right) Frequencies (mean + SEM) of IFN-γ–positive (black bars), CD107a positive (gray bars), and double-positive (blue bars) bTRM with (+) or without (–) LCMVwt i.c. challenge and with (+) or without (–) in vitro NP396-404 peptide stimulation (n = 3–4 mice per group). (C–G) IFN-γ–competent (control)- or GKO-rLCMV memory mice were generated and treated as in A. (C) Frequencies of TM cells (CD127+KLRG1–) among bTRM (CD8+CD44+CD69+) as determined by flow cytometry in unchallenged IFN-γ–competent (control) and GKO mice. Each symbol represents an individual mouse and bars indicate mean (n = 4 mice per group). (D) Representative images of immunostained brain sections for CD8 (top, arrows) or LCMV (bottom). (E) Quantification of CD8+ T cells, (F) LCMV+ cells, or (G) LCMV RNA by qPCR (normalized to naive mice infected with LCMVwt i.c. = 1) in brains of GKO and control mice with (+) or without (–) LCMVwt i.c. challenge. Data represent mean + SEM (n = 3–10 mice per group). (H–M) Perforin-competent (control)- or PKO-rLCMV memory mice were generated and treated as in A. (H) Frequencies of TM cells among bTRM (as in C) in unchallenged perforin-competent (control) and PKO mice. Each symbol represents an individual mouse and bars indicate mean (n = 4 mice per group). (I) Frequencies of GzmB-expressing cells among bTRM as determined by flow cytometry 3 d after LCMVwt i.c. challenge of control or PKO mice. Each symbol represents an individual mouse, and bars indicate mean (n = 4–6 mice per group). (J) Representative images of immunostained brain sections for CD8 (top, arrows) or LCMV (bottom). (K) Quantification of CD8+ T cells, (L) LCMV+ cells, or (M) LCMV RNA by qPCR (normalized to naive mice infected with LCMVwt i.c. = 1) in brains of PKO and control mice with (+) or without (–) LCMVwt i.c. challenge. Data represent mean + SEM (n = 4–6 mice per group). *, P < 0.05; **, P < 0.01, asterisk colors represent multiple comparisons performed, two-way ANOVA with Tukey’s multiple comparisons (B), one way ANOVA with Sidak’s multiple comparisons test (I), unpaired two-tailed Student’s t test (C, F, G, H, L, and M). One representative (B–F and J–M) or pool (G) of two independent experiments is shown. (H and I) Experiments were performed once. Bars, 100 µm; (inset) 20 µm.

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