Figure 1.
Tissue-specific pattern of MCMV-specific CD8 T cell responses.
(A) C57BL/6 mice were infected with MCMV-Δm157 and virus titers were determined in the spleen, lungs, liver and salivary glands (SG) at days 7, 14, 28 and 150. In a separate experiment, virus titers were determined in the axillary lymph nodes at days 4, 6 and 12 post infection (B). The horizontal lines represent the respective detection limits. (C) The percentage of M38- (black bars) and M45-specific CD8 T cells (white bars) were measured by tetramer staining in the spleen, lungs and inguinal lymph nodes at the indicated time points post MCMV infection. For spleen and lungs, bars show averages of three mice per group ± SEM, whereas for lymph nodes bars represent cells pooled from three mice. (D) Representative plots showing KLRG-1 and IL7Rα expression on M38- and M45-specific CD8 T cells in the spleen, lungs and inguinal lymph nodes on day 150 post infection. All data are representative of at least three independent experiments.
Figure 2.
Generation of MHC class I-restricted TCR transgenic mice with specificity for the M38 epitope of MCMV.
(A) Splenocytes from WT C57BL/6 mice and from the transgenic mouse lines Mini and Maxi were stained with M38-tetramer (top panels) and with anti-Vβ10 antibody (lower panels). Percentages of CD8+ M38+ and CD8+ Vβ10+ are depicted in the plots and are representative of at least 10 individual mice. (B) 6×105 Mini CD8 T cells and 6×104 Maxi CD8 T cells were CFSE labeled and incubated with DCs loaded with the indicated concentration of M38 peptide or with medium for three days. The percentages of CFSElow cells are depicted. Bars show averages of three mice per group ± SEM, and one of two independent experiments is shown as representative. (C) 105 Mini CD8 T cells or 104 Maxi CD8 T cells expressing the CD45.1 congenic marker were adoptively transferred into CD45.2 recipient C57BL/6 mice one day prior to infection with MCMV. The percentages of endogenous (black circles) and transferred (white circles) M38-specific CD8 T cells were measured by tetramer staining from blood samples collected at the indicated time points after infection. Each point shows the average of three to five mice per group ± SEM. One of at least five (for Mini CD8 T cells) and two (for Maxi CD8 T cells) independent experiments are shown as representative.
Figure 3.
Antigen presentation by non-hematopoietic cells is essential for M38-specific CD8 T cell inflation.
WT→WT and WT→H-2Kb−/− chimeric mice were generated by irradiating WT and H-2Kb−/− mice followed by reconstitution with WT bone-marrow. One day prior infection with MCMV-Δm157, 105 CD45.1+ Mini CD8 T cells were adoptively transferred. (A) The percentage of M38- (pregated on CD45.1+ cells) and M45-specific CD8 T cells among total CD8 T cells were measured in the blood on days 7, 12, 15, 28 and 65 post infection. Every point represents the average of four individual mice ± SEM. The mice shown in (A) were sacrificed on day 100 post infection and the percentage of M38- (pregated on CD45.1+ cells) and M45-specific CD8 T cells among total CD8 T cells (B) and their total numbers (C) were determined in the lungs. Bars indicate averages of four mice per group ± SEM. The relative expression of IL7Rα and CD62L on M38- and M45- specific CD8 T cells is indicated in (D) in the same groups of mice shown in (A) for lungs and spleen. (E) MCMV latent genomes were quantified in the spleen (upper panel) and in the lungs (lower panel) of WT→WT (black circles) and WT→H-2Kb−/− mice (white circles) on day 60 post infection or from naïve mice by qPCRs using primers specific for β-actin and the MCMV-encoded M38 gene. The relative amount of viral genome was calculated using the delta-delta Ct method, where 10−6 was the highest value detected for naïve mice and used as detection limit. Data pooled from two independent experiments are shown. (F) Virus titers were determined in spleen, lungs, liver and salivary glands on day 7 (top panel) and day 60 (bottom panel) in WT→WT (black circles) and WT→H-2Kb−/− (white circles) mice. Lines represent the geometric means of three individual mice. One of at least four independent experiments are shown for (A to D) and of two independent experiments for (E to F). Significances were analyzed by Student's t test. *, P<0.05; **, P<0.01; *** P<0.001; ****, P<0.0001.
Figure 4.
Restriction of antigen presentation to DCs abrogates memory inflation.
WT→WT, DC-MHCI→DC-MHCI, WT→DC-MHCI, DC-MHCI→WT chimeric mice were generated by reconstituting WT and DC-MHCI irradiated mice with WT and DC-MHCI bone marrow. All groups (3–5 mice per group) were infected with MCMV-Δm157 and the M38-specific CD8 T cell response was measured by tetramer staining from blood samples obtained on days 7, 9, 13, 20 and 45 post infection (A). The experiment was terminated on day 120 post infection and M38-, IE3-, and M45-specific CD8 T cell responses were quantified in the lungs by tetramer staining (B). One of two independent experiments is shown. Significances were analyzed by Student's t test. *, P<0.05; **, P<0.01; *** P<0.001.
Figure 5.
Non-hematopoietic cells promote extensive and systemic cell proliferation in the early phase of MCMV infection, but only local during latency.
105 Mini-CD8 T cells were transferred in WT→WT and WT→H-2Kb−/− chimeric mice one day prior to infection with MCMV-Δm157. (A) The percentage of M38-specific CD8 T cell response was measured in the blood on days 7, 9, 11, 13, 17, 21, 27, 42 and 60 post infection. Colors indicate different phases of the M38-specific response: primary expansion in yellow; secondary expansion in pink; maintenance phase in blue. Bcl-2 (B) and Ki67 (C) expression levels were measured on M38-specific CD8 T cells depicted in (A). One of two independent experiments is shown. (D) Representative FACS plots showing Bcl-2 and Ki67 expression on M38- and M45-specific CD8 T cells in the lungs and inguinal lymph nodes of WT→WT chimeric mice on day 12 post infection (upper panels) and day 130 post infection (lower panels). (E) The percentage of ki67+ cells in WT→WT and WT→H-2Kb−/− chimeric mice is shown for M38- and M45-specific CD8 T cells in the lungs (left graph) and in the lymph nodes (right graph). One of three independent experiments is shown. (F) The percentage of M38-specific cells expressing Ki67 during latency is shown for the lungs and the lymph nodes. Data pooled from three independent experiments ± SEM are shown. (G) Preferential antigen-driven proliferation of TCM M38-specific CD8 T cells during latency. Experimental layout: CD45.2+ C57BL/6 mice were adoptively transferred with CD45.1+ Maxi CD8 T cells and during latency TCM Maxi CD8 T were isolated from the LNs and the spleen, whereas TEM Maxi CD8 T cells were isolated from the lungs. Isolated cells were labeled with CFSE and transferred into naïve or latently infected CD45.2+ C57BL/6 mice (Number of transferred cells: TCM = 20′000/mouse, TEM = 80′000/mouse). CFSE dilution was measured 30 days later in the lungs. (H) Representative plots showing the population of transferred cells for each of the four experimental groups (gated on total CD8 T cell) and their respective CFSE dilutions, and (I) Graphical summary showing percentage of CFSElow cells among total transferred Maxi CD8 T cells. Data in (I) are pooled from two independent experiments.
Figure 6.
MCMV genome localizes preferentially in non-hematopoietic cells.
C57BL/6 mice were infected with MCMV-Δm157 and on day 7, 14 and 28 post infection total cells from the spleen and the lungs were sorted into CD45+ (black circles) and CD45- (white circles) cell populations. MCMV latent genomes were quantified by qPCR using primers specific for β-actin and the MCMV-encoded M38 gene. One of two independent experiments is shown.