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. 2012 Feb;86(4):2165-75.
doi: 10.1128/JVI.06042-11. Epub 2011 Dec 7.

The NK cell response to mouse cytomegalovirus infection affects the level and kinetics of the early CD8(+) T-cell response

Maja Mitrović  1 , Jurica Arapović, Stefan Jordan, Nassima Fodil-Cornu, Stefan Ebert, Silvia M Vidal, Astrid Krmpotić, Matthias J Reddehase, Stipan Jonjić
Affiliations

The NK cell response to mouse cytomegalovirus infection affects the level and kinetics of the early CD8(+) T-cell response

Maja Mitrović et al. J Virol. 2012 Feb.

Abstract

Natural killer (NK) cells and CD8(+) T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8(+) T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8(+) T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8(+) T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8(+) T cells has only a minor effect on the early control of wild-type MCMV, CD8(+) T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8(+) T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8(+) T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H-m157 interaction.

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Figures

Fig 1
Fig 1
NK cell-specific recognition of MCMV affects the CD8+ T-cell response. (A) Indicated strains of mice were i.v. injected with 2 × 105 PFU of either WT or Δm157 MCMV. On days 1 and 5 p.i., mice were i.p. injected with phosphate-buffered saline (PBS) or depleting anti-CD8 MAb. On day 7 p.i., viral titers in spleens and salivary glands were determined by plaque assay. (B) C57BL/6 mice were i.v. injected with 2 × 105 PFU of either WT or Δm157 MCMV. On day 1 p.i., mice were i.p. injected with PBS or depleting anti-CD8 MAb. On day 3.5 p.i., viral titers in spleens and lungs were determined by plaque assay. Titers of individual mice (circles) and median values (horizontal bars) are shown. Representative data of at least two independent experiments with three to five mice per group are shown. Differences are regarded as significant for P values of <0.05. Dotted line, detection limit.
Fig 2
Fig 2
Virus load affects the CD8+ T-cell-mediated control of MCMV infection in C57BL/6 mice. (A) C57BL/6 mice were i.v. injected with different doses of WT MCMV (2 × 105 PFU, 2 × 106 PFU, or 4 × 106 PFU) or with a single dose of 2 × 105 PFU of Δm157 MCMV. On day 3.5 p.i., viral titers in spleens and lungs were determined by plaque assay. (B) C57BL/6 mice were injected as described above, and on days 1 and 5 p.i. they were i.p. injected with PBS or depleting anti-CD8 MAb. On day 7 p.i., viral titers in spleens were determined by plaque assay. (C) C57BL/6 mice were i.v. injected with 2 × 105 PFU of either WT or Δm157 MCMV. Indicated groups were depleted of NK cells, CD8+ T cells, or both NK cells and CD8+ T cells. On day 7 p.i., viral titers in spleen and salivary glands were determined by plaque assay. Ø, untreated C57BL/6 mice. Data are compiled from two independent experiments with two to five mice per group. Titers of individual mice (circles) and median values (horizontal bars) are shown. Dotted line, detection limit.
Fig 3
Fig 3
Impact of Ly49H–m157 signaling on the NK cell response during early MCMV infection. (A) C57BL/6 mice, either NK cell depleted with anti-NK1.1 MAb or left undepleted, were i.v. injected with 2 × 105 PFU of either WT or Δm157 MCMV. On days 1.5 and 3.5 p.i., viral titers in spleens were determined by plaque assay. Titers of individual mice (circles) and median values (horizontal bars) are shown. (B) On days 1.5, 3.5, and 4.5 p.i., splenocytes were assessed for IFN-γ production by Ly49H+ and Ly49H− subsets of CD3ε− NK1.1+ NK cells. (C to F) The absolute numbers of IFN-γ+ CD3ε− NK1.1+ NK cells (C), the percentages of CD69+ CD3ε− NK1.1+ NK cells (D), the absolute numbers of proliferating bromodeoxyuridine-positive (BrdU+) CD3ε− NK1.1+ NK cells (E), and the absolute numbers of CD3ε− NK1.1+ NK cells (F) in the total spleen cell population at the indicated time points are shown. (G) The maturation pattern of NK cells on days 1.5, 4.5, and 7 p.i. was determined by expression of cell surface markers CD27 and CD11b. Representative dot plots of splenic NK cells (CD3ε− NK1.1+) from two independent experiments with three or four mice per group are shown. (C to F) Data are presented as means ± standard error of the mean (SEM) of at least three mice per group from one of two independent experiments. Asterisks denote significant values as calculated by unpaired, two-tailed Student's t test: ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Dotted line, detection limit.
Fig 4
Fig 4
Absence of Ly49H–m157 signaling enhances the CD8+ T-cell response in MCMV-infected mice. C57BL/6 mice were infected intravenously (A to C) or at the left hind footpad (D and E) with 2 × 105 PFU of either WT or Δm157 MCMV. (A to D) On the indicated days p.i., splenic leukocytes (A to C) or immunomagnetically purified CD8+ T cells from the draining popliteal lymph node (PLN) (D) were stimulated in culture with the indicated peptides followed by intracellular IFN-γ staining in cytofluorometric analysis (A to C) or IFN-γ secretion in the ELISpot assay (D). (A) The percentages of IFN-γ+ CD8+ T cells specific for M45, m139, and M57 MCMV peptides among gated CD3ε+ CD8+ T cells are shown. (B and C) The percentages of proliferating BrdU+ CD8+ T cells (B) and CD69+ CD8+ T cells (C) among gated CD3ε+ CD8+ T cells are shown. (A to C) Data are presented as means ± SEM of at least three mice per group from one of at least three independent experiments. (D) Immunomagnetically purified CD8+ T cells from pools of PLN of five mice per group were stimulated for an IFN-γ-based ELISpot assay with EL-4 (H-2b) thymoma cells and a saturating concentration (10−6 M) of synthetic peptides corresponding to the epitopes indicated. Bars represent frequencies of responding CD8+ T cells; error bars indicate the 95% confidence intervals. (E) IE1 transcripts quantitated by RT-qPCR in draining PLN on day 3 after intraplantar infection. Symbols represent PLN of individual mice with the median value indicated. Throughout, asterisks denote significance values as calculated by unpaired, two-tailed Student's t test: ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 5
Fig 5
Impact of Ly49H–m157 signaling on cytokine production during early MCMV infection. C57BL/6 mice were i.v. injected with 2 × 105 PFU of either WT or Δm157 MCMV or left uninfected. On days 1.5 and 4 p.i., serum levels of the indicated cytokines were determined. Data are presented as means ± SEM of at least three mice per group from one of two independent experiments. Asterisks denote significant values as calculated by unpaired, two-tailed Student's t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Φ, not detected.
Fig 6
Fig 6
Ly49H–m157 signaling preserves splenic cDCs during MCMV infection. C57BL/6 mice were i.v. injected with 2 × 105 PFU of either WT or Δm157 MCMV or left uninfected. On days 1.5 and 4 p.i., splenic leukocytes were stained to assess DC subsets cytofluorometrically. (A) Representative dot plots of splenic cDCs (CD3− CD19− MHC-II+ CD11c+) and CD8α+ and CD11b+ subsets gated on cDCs are shown. (B and C) The absolute numbers of CD8α+ cDCs (B) and CD11b+ cDCs (C) in total splenic leukocytes are shown. (D) C57BL/6 mice, either NK cell depleted or left undepleted, were i.v. injected with 106 PFU of WT MCMV-GFP. The frequency of infected splenic cDCs (CD3− CD19− MHC-II+ CD11c+ GFP+) and infected CD8α+ and CD11b+ subsets gated on cDCs on days 1 and 4 p.i. are shown. (B to D) Data are presented as means ± SEM of at least four mice per group from one of three independent experiments. Asterisks denote significant values as calculated by unpaired, two-tailed Student's t test: **, P < 0.01; ***, P < 0.001.
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