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1.
Fig. P1.

Fig. P1. From: Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming.

(A) T-cell response to viral infection normally involves two steps dependent on MHC class I expression. In the priming step (1), MHC class I molecules on virus-infected cells present a viral peptide recognized by a TCR (not shown for clarity) of a rare virus-specific T cell, which then undergoes clonal expansion. Virus-specific T cells then migrate to sites of infection and recognize cells displaying the specific MHC class I-viral peptide combination (2), activating T-cell effector responses to kill the infected cell and produce cytokines. (B) Viruses capable of inhibiting MHC class I expression on an infected cell do not affect virus-specific T-cell priming, most likely because of cross-priming, which occurs when uninfected CD8α+ (or CD103+) dendritic cells (DCs) pick up antigen from infected cells and present virus-derived peptides on their MHC class I molecules. However, virus-specific T cells cannot perform their effector functions, because their TCRs cannot recognize their specific MHC-viral peptide ligands on infected cells.

Maria D. Gainey, et al. Proc Natl Acad Sci U S A. 2012 November 20;109(47):E3260-E3267.
2.
Fig. 5.

Fig. 5. From: Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming.

WT CPXV-primed CD8+ T cells can control Δ12Δ203 but not WT CPXV lesions. Adoptive transfer experiments. (A) Nine-week-old female B6 mice were infected i.n. with 10,000 pfu WT CPXV or were infected i.p. with 100,000 pfu of MCMV. Spleens were harvested 7 d postinfection and were enriched by negative selection for CD8+ T cells, which then were transferred into naive Rag-1–deficient mice. At 24 h after transfer, mice were infected on two adjacent areas of skin with 10-uL droplets containing 20,000 pfu of WT or Δ12Δ203 CPXV. Lesions were monitored daily. Representative photographs of mice that received MCMV- or CPXV-immune CD8+ T cells are shown on days 5 and 9 PI. Data are representative of two independent experiments. n = 3 mice. (B) Negatively enriched CD8+ T cells from mice infected with CPXV as described in A were transferred into Rag-1–deficient mice, and 16 h later mice were treated with an anti-CD8β-depleting or control antibody. At 24 h after transfer, mice were infected as described in A. Photographs of two representative mice per treatment group on day 9 PI are shown. Data are representative of two independent experiments. n = 5 or 6 mice.

Maria D. Gainey, et al. Proc Natl Acad Sci U S A. 2012 November 20;109(47):E3260-E3267.
3.
Fig. 4.

Fig. 4. From: Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming.

CPXV-primed CD8+ T cells display less control of WT CPXV lesions. (A) Scab loss. Nine-week-old female B6 mice were infected on two adjacent areas of skin with 10-uL droplets containing 20,000 pfu of WT or Δ12Δ203 CPXV. Lesions were monitored daily; scab loss for each lesion is shown. n = 20 mice until day 9, then n = 10 mice. Data are representative of four independent experiments. (B) Viral titers. Skin lesions were collected separately on day 6, 9, and 13 PI, and viral titers were determined by plaque assay. The only Δ12Δ203 lesion with a scab still remaining on day 9 and the two WT lesions with scabs remaining on day 13 are denoted by arrows. Data were analyzed with an unpaired Student t test; P value between comparison groups is shown. (C) Photographs of skin lesions in two representative mice from the experiment described in A and B on days 6 and 9 PI (D and E) Mice were treated with an anti-CD8β-depleting antibody or a control antibody 2 d before infection as described in A. (D) Percentage of scab loss on day 9. (E) Lesion titers on day 9. Data in D and F are representative of two independent experiments; n = 10 mice. (F) Pictures of two representative mice from each treatment group.

Maria D. Gainey, et al. Proc Natl Acad Sci U S A. 2012 November 20;109(47):E3260-E3267.
4.
Fig. 3.

Fig. 3. From: Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming.

CD8+ T cells produce more IFN-γ in the lungs of Δ12Δ203 CPXV-infected mice than in the lungs of WT CPXV-infected mice. (A) IFN-γ levels in lung homogenates during infection. Nine-week-old female B6 mice were infected i.n. with 5,000 pfu of WT or Δ12Δ203 CPXV as indicated. Lung homogenates were generated and used in IFN-γ ELISA on the indicated day PI. Data are representative of two independent experiments and are shown as means ± SEM. n = 3 or 4 mice. (B) IFN-γ production is CD8+ T-cell dependent. Nine-week-old female B6 mice were infected i.n. with 10,000 pfu of WT or Δ12Δ203 CPXV as indicated. Mice were treated 1 d before infection and on day 4 PI with an anti-CD8β-depleting antibody or control antibody. Lungs were harvested on day 6 PI, and homogenates were used in IFN-γ ELISA. Data are shown as mean ± SEM. (C) Viral lung titers were determined in the mice shown in B. Bars represent the mean of each sample group. (D) Direct ex vivo IFN-γ intracellular staining. Nine-week-old female B6 mice were mock infected or infected with 10,000 pfu of WT or Δ12Δ203 CPXV. Lungs were harvested on day 6 PI, and single-cell suspensions were intracellularly stained directly ex vivo without additional stimulation. Dot plots from two single representative mice from each infection group are shown. Numbers represent the percentage of IFNγ+CD8+ T cells in the sample. (E) Total number of IFN-γ+CD8+ T cells per lung. Analysis was performed as in D. Data are the combined results from two independent experiments and are shown as mean ± SEM. n = 4 mock, n = 9 WT CPXV, and n = 10 Δ12Δ203-infected mice. CPXV. The data were analyzed with an unpaired Student t test; P values between comparison groups are shown.

Maria D. Gainey, et al. Proc Natl Acad Sci U S A. 2012 November 20;109(47):E3260-E3267.
5.
Fig. 1.

Fig. 1. From: Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming.

CD8+ T-cell priming is not impaired during WT CPXV infection. (A) Tetramer analysis during infection. Eight-week-old female B6 mice were infected i.n. with 5,000 pfu of the indicated viruses. On days 4, 5, 6, and 8 PI, the number of CPXV-specific CD8+ T cells in the spleen, lung, and mediastinal lymph node was determined by H2Kb-TSYKFESV tetramer staining, as indicated. Data are representative of multiple individual time point experiments and are shown as mean ± SEM. n = 4 or 5 mice. (B) Tetramer analysis in Batf3-deficient mice. Female and male 8- to 11-wk-old Batf3-KO or B6 mice were infected with 5,000 pfu of the indicated viruses. On days 6 and 8 PI the number of CPXV-specific CD8+ T cells in the spleen was determined by H2Kb-TSYKFESV tetramer staining as indicated. Day 6 data are the combined results from two independent experiments. Day 8 data are combined results from three independent experiments. (C) CD8+ T-cell IFN-γ production. On day 8 splenocytes from the mice infected in B were stimulated with Δ12Δ203 CPXV-infected DC2.4 cells. The total number of IFN-γ–producing CD8+ T cells is shown. Data are the combined results of three independent experiments. The data were analyzed with an unpaired Student t test, P values between comparison groups are shown. Bars represent the mean for each sample group. Numbers followed by an “x” in B and C are the average fold reduction in the CD8+ T-cell response to the indicated virus in Batf3-KO mice.

Maria D. Gainey, et al. Proc Natl Acad Sci U S A. 2012 November 20;109(47):E3260-E3267.
6.
Fig. 2.

Fig. 2. From: Viral MHC class I inhibition evades CD8+ T-cell effector responses in vivo but not CD8+ T-cell priming.

CD8+ T cells generated during WT CPXV infection are functional. (A) Stimulation with Δ12Δ203 CPXV-infected DC2.4 cells. Female and male 9- to 11-wk-old B6 mice were infected i.n. with 5,000 pfu of WT or Δ12Δ203 CPXV. Mice were killed on day 8 PI, and single-cell lung suspensions were stimulated with DC2.4 cells infected with Δ12Δ203 CPXV. Then CD8+ T cells were stained for intracellular IFN-γ and TNF-α. (Upper) The total numbers of IFN-γ–producing CD8+ T cells are shown, representing the combined results from five independent experiments. (Lower) Percentages of cells producing both IFN-γ and TNF-α are shown, representing the combined results from three independent experiments. The bars represent the mean of each group. (B) Flow cytometry after stimulation with peptide-pulsed or infected DC2.4 cells. Single-cell lung suspensions were prepared as in A and were stimulated with DC2.4 cells that were pulsed with TSYKFESV peptide (Upper) or with DC2.4 cells infected with Δ12Δ203 CPXV (Lower) and were stained for intracellular IFN-γ. Representative dot plots are shown; the numbers represent the percentage of IFN-γ–producing CD8+ T cells. (C) Quantitation of stimulated cells and amount of IFN–γ produced by MFI. Cells were prepared and stimulated as in B. (Upper) Total number of IFN-γ+ CD8+ T cells. (Lower) MFI of IFN-γ staining. Data are the combined results of two independent experiments and are shown as mean ± SEM. n = 7 or 9 mice. (D) In vivo cytotoxicity assay. On day 8 PI mice mock infected or infected with indicated viruses were injected i.v. with CD45.1+ splenocytes that were labeled with 5 nM or 500 nM CFDA. The 500-nM–labeled cells were pulsed with TSYKFESV peptide before injection. Spleens were harvested 4 h after injection, and the number of CFDA-labeled cells was determined by flow cytometry. Histograms show the percentage of CD45.1+-, CFDA-high–, and CFDA-low–labeled cell populations at time of harvest from a representative experiment. (E) Target killing in combined experiments. The percentage of target cell (TSYKFESV peptide-pulsed cells) killing is shown. See Materials and Methods for the equation. Bars represent the mean of each sample group. Data shown are combined results from two independent experiments.

Maria D. Gainey, et al. Proc Natl Acad Sci U S A. 2012 November 20;109(47):E3260-E3267.

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