Article Figures & Data
Figures
- FIG. 1.
Protective immunity mediated by pp89-specific CD8+ T cells is dependent on the dose of infection and the timing of challenge. BALB/c mice were infected i.v. with 106 (◊) and 103 (⧫) PFU of MCMV or were left naive (○) as a negative control. (A) Thereafter, the pp89-specific CD8+-T-cell response was measured longitudinally in the blood. The percentage of CD8+ T cells staining with pp89 tetramer is plotted over time. Each line represents the mean of four to eight mice per experimental group. Error bars indicate the standard deviation (SD) within experimental groups. (B) At the indicated time points after MCMV infection, mice were challenged with Vac89 i.p., and the VV titers were measured in the ovaries 4 days later. Titers are given as the log10(PFU of Vac89/ovaries). The detection limit and the SD are indicated. Each line represents the mean of four or five mice per experimental group. The results from one of two similar experiments are shown; the second experiment was finished by day 120 after priming. To measure the expansion of specific T cells after challenge, the frequency of pp89-specific CD8+ T cells was determined by tetramer staining before and 4 days after challenge with Vac89 in the spleen at indicated time points after MCMV infection (Fig. 1C, blood, liver, and ovaries [data not shown]). The percentage of CD8+ T cells staining with pp89 tetramer is plotted over time. Each line represents the mean of three to four mice per experimental group. Error bars indicate the SD within experimental groups.
- FIG. 2.
Efficient induction of specific CD8+ T cells after infection with recombinant MCMV. (A) Western blot analysis of expression of recombinant proteins from viruses in vitro. MEF were infected with wild-type (wt) MCMV, with MCMV-NP (NP), with MCMV-GP (GP) or, as a negative control, with a mutant MCMV lacking expression of IE2 (Δie2). To make IE2 expression detectable, protein transcription and translation were synchronized by the treatment of cells with cycloheximide for the first 3 h of infection and with actinomycin D 4 to 7 h postinfection. Protein expression was detected 7 h postinfection by using a mouse antiserum specific for IE2. The appropriate IE2 bands are marked on the figure. The NP-IE2 and GP-IE2 fusion proteins show a slightly higher molecular weight due to the additional NP and GP epitopes, respectively. (B) C57BL/6 mice were infected with 2 × 106 PFU of MCMV-GP (left panel) or MCMV-NP (right panel). At 10 days after infection, splenocytes were harvested, restimulated for 5 days in vitro, and then tested in a 5-h 51Cr-release assay on target cells pulsed with GP33 (▴), NP366 (▪), or an irrelevant peptide (○). Each line represents an individual mouse. The results for one of four experiments are shown. (C) NP tetramer staining of spleen cells from a mouse infected 10 days previously with MCMV-NP. The upper panel shows an ex vivo tetramer staining. For the lower panel, splenocytes from the same mouse were restimulated for 7 days in vitro with NP366-pulsed naive spleen cells. The numbers indicate the percentage of NP-specific cells of CD8+ T cells. Panels are gated on live lymphocytes without B cells. The results for one of seven mice are shown.
- FIG. 3.
Phenotypic characterization of NP-specific CD8+ T cells after infection with MCMV-NP or influenza. C57BL/6 mice were infected i.v. with 2 × 106 PFU of MCMV-NP or i.n. with 100 hemagglutination units of influenza virus. Spleen cells were harvested at the indicated time points and then stained with NP tetramer (NP-tet), anti-CD8, or anti-B220 and with anti-CD62L. Panels are gated on live lymphocytes without B cells (A) or on live CD8+ lymphocytes (B). The numbers indicate the percentages of NP-specific cells among CD8+ T cells (A) or the percentages of NP-specific CD8+ T cells expressing CD431B11 (B) or CD62L (C). The results of one of three similar experiments are shown.
- FIG. 4.
Increasing protective CD8+-T-cell-mediated immunity after infection with MCMV-NP. C57BL/6 mice were infected with 2 × 106 PFU of MCMV-NP i.v. (▪) or with 100 hemagglutination units of influenza virus i.n. (▾), or they were left naive (○) as a control. (A) Thereafter, the NP-specific CD8+-T-cell response was measured longitudinally in the blood. The percentage of CD8+ T cells staining with NP tetramer is plotted over time. Each line represents the mean of four to five mice per experimental group. Error bars indicate the SD within experimental groups. (B) At indicated time points after infection with influenza virus (upper panel) or MCMV-NP (lower panel), mice were challenged i.p. with VacNP, and the VV titers were measured in the ovaries 4 days later. Titers are given as the log10(PFU of VacNP/ovaries). The detection limit and the SD are indicated. Each line represents the mean of four or five mice per experimental group. The experiment was repeated with comparable results.
- FIG. 5.
Increasing protective CD8+-T-cell-mediated immunity after infection with MCMV-GP. C57BL/6 mice were infected i.v. with 2 × 106 PFU of MCMV-GP (▴) or i.v. with 5 × 106 of PFU VacGP (▵) or were immunized s.c. with the peptide GP33 and CFA (⧫), or they were left naive (○) as a control. (A) Thereafter, the GP-specific CD8+-T-cell response was measured longitudinally in the spleen by ICS. The percentage of CD8+ T cells staining for intracellular IFN-γ after stimulation with GP33-41 is plotted over time. Each line represents the mean of three to four mice per experimental group. Error bars indicate the SD within experimental groups. (B) At indicated time points after immunization with GP33+CFA (upper panel) or after infection with MCMV-GP (lower panel), mice were challenged i.p. with VacGP, and the VV titers were measured in the ovaries 4 days later. Titers are given as the log10(PFU of VacGP/ovaries). The detection limit was 1.6, and the SD is indicated. Each line represents the mean of four to five mice per experimental group.
Tables
- TABLE 1.
Total number of epitope-specific CD8+ T cells per spleen at different time points after infection with MCMV or influenza virus or after immunization with peptide GP33a
Time postinfection (days) Total no. of epitope-specific CD8+ T cells/spleen (104) ± SD with epitope: IE1/pp89 (PFU) NP366 GP33 MCMV (106) MCMV (103) MCMV-NP Influenza virus MCMV-GP GP33+CFA 8-11 56.8 ± 20 2.4 ± 0.3 2.5 ± 1.1 55.8 ± 28.5 1.1 ± 0.5 25.1 ± 9.7 18-22 31.3 ± 8.7 2.9 ± 0.8 2.9 ± 1.6 20.3 ± 10 2.0 ± 0.7 35-45 26.5 ± 14.4 7.9 ± 2.8 6.9 ± 3.7 4.8 ± 0.9 10.4 ± 3.7 18.7 ± 6.4 100-160 59 ± 13.1 24.5 ± 6.9 12.7 ± 4.8 5.2 ± 2.2 15.5 ± 6.3 10.9 ± 2.6 250-350 115 ± 28 45.8 ± 22 9.2 ± 5.3 3.9 ± 0.5 ↵a Mice were infected or immunized with the indicated virus or antigen. At different time points thereafter the total numbers of epitope-specific CD8+ T cells per spleen were measured by fluorescence-activated cell sorting by using tetramer staining (IE1/pp 89 and NP366) or ICS (GP33). Mean numbers ± the SD are given for 4 to 12 mice per group. Data from one to three experiments with the same antigen or virus were pooled for calculation. Values of 106 and 103 indicate the virus dose in PFU used for infection.
- TABLE 2.
Correlation between the frequency of epitope-specific CD8+ T cells, the level of protection measured by VV challenge, and the time after priming with MCMV-wt, MCMV-NP, or influenza virusa
Priming (PFU) n Spearman rank correlation of factor vs factor Frequency of specific cells vs time after priming (P) Frequency of specific cells vs level of pro- tection (P) Time after priming vs level of pro- tection (P) MCMV (103) 40 0.841 (<0.001) 0.521 (0.001) 0.483 (0.002) MCMV-NP 23 0.702 (<0.001) 0.507 (0.014) 0.513 (0.012) Influenza virus 23 −0.882 (<0.001) 0.760 (<0.001) −0.736 (<0.001) Overall 86 0.566 (<0.001) ↵a Mice were primed with the indicated virus, and the frequency of epitope-specific CD8+ T cells was monitored in the blood by tetramer staining. Frequencies used for calculation were measured 0 to 3 days before challenge. At 4 days after challenge, the VV titers were measured in the ovaries. The level of protection was calculated as described in Materials and Methods to correct for variation between different challenge experiments. Numbers indicate the correlation coefficients that were calculated with the Spearman's rank correlation using logarithmic scales for all variables. Two-tailed P values for each correlation are indicated in parentheses. Data from one to three experiments per virus and time point were included in the analysis.
