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. 2015 Aug;89(15):7922-31.
doi: 10.1128/JVI.00199-15. Epub 2015 May 20.

Cytokine-Mediated Activation of NK Cells during Viral Infection

Bailey E Freeman  1 , Hans-Peter Raué  1 , Ann B Hill  2 , Mark K Slifka  3
Affiliations

Cytokine-Mediated Activation of NK Cells during Viral Infection

Bailey E Freeman et al. J Virol. 2015 Aug.

Abstract

Natural killer (NK) cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as interleukin 12 (IL-12) and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting gamma interferon (IFN-γ) production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced 8 days after acute LCMV infection but recovered to preinfection levels by 60 days postinfection. In contrast, during MCMV infection, NK cell responses to cytokines remained robust at all time points examined. Ly49H-positive (Ly49H+) NK cells recognizing viral ligand m157 showed preferential proliferation during early MCMV infection. A population of these cells was still detected beyond 60 days postinfection, but these divided cells did not demonstrate enhanced IFN-γ production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b or CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation and highlight variations in NK cell function during two unrelated viral infections.

Importance: Natural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN-γ production by NK cells. Ly49H+ NK cells, which can be directly activated by MCMV protein m157, preferentially proliferated during MCMV infection but did not show enhanced IFN-γ production following direct ex vivo cytokine stimulation. Instead, mature CD11b+ and/or CD27+ NK cells responded similarly to innate cytokine stimulation regardless of Ly49H expression. Collectively, our data provide a better foundation for understanding cytokine-mediated NK cell activation during viral infection.

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Figures

FIG 1
FIG 1
Activation of NK cells following exposure to innate cytokines. Splenocytes from naive mice were stimulated in vitro with the indicated cytokines (10 ng/ml). At 6 h, IFN-γ production was assessed by intracellular cytokine staining and flow cytometry. (A) NK cells from BALB/c mice produce IFN-γ following exposure to a variety of cytokine combinations. Dot plots were gated on live, CD3− cells, and the number in the top right quadrant of each dot plot represents the percentage of CD3− DX5+ cells producing IFN-γ in response to the indicated cytokine combination. The mean fluorescence intensities (MFI) of IFN-γ+ cells are shown in parentheses. (B) NK cells from BALB/c and C57BL/6 mice exhibit a similar hierarchy of IFN-γ responses to cytokine stimulation. Each data point represents the percentage of CD3− DX5+ NK cells from BALB/c or C57BL/6 mice that produced IFN-γ in response to the indicated cytokine combination and is the average of 4 to 6 mice. (C) Cytokine-induced IFN-γ production by CD3− DX5+ cells and CD3− NK1.1+ cells in C57BL/6 mice. Data points represent the frequency of IFN-γ+ cells within each population following exposure to the indicated cytokine pair and are the averages of 8 mice.
FIG 2
FIG 2
Cytokine-mediated activation of NK cells during LCMV infection. Splenocytes from uninfected (naive) or LCMV-infected (8 days or >60 days postinfection) BALB/c mice were stimulated directly ex vivo with the indicated cytokine combinations at 10 ng/ml for 6 h. (A) IFN-γ production by NK cells following exposure to innate cytokines. Light gray bars represent IFN-γ responses to the unpartnered individual cytokines indicated on the x axis, and the corresponding black bars represent IFN-γ responses to each cytokine in combination with the cytokine indicated at the top of each panel. Spontaneous production of IFN-γ in medium-only controls was typically <1%, and this background was subtracted prior to preparing the graphs. IFN-γ responses to each cytokine pair were compared to responses after stimulation with the individual cytokines using a two-tailed t test. Cytokine pairs that induced NK cell responses that were significantly different (P < 0.05) from both responses to the individual cytokines within the pair are indicated with an asterisk. (B) Differential induction of cell surface CD69 expression and IFN-γ expression in NK cells. (C) Cytokine-mediated regulation of CD25 expression on CD3− DX5+ NK cells. Each data point is the average of 4 to 6 mice per group.
FIG 3
FIG 3
BrdU incorporation and cytokine-induced IFN-γ production by Ly49H+ NK cells. Mice were infected with MCMV and given BrdU daily for 3 to 6 days postinfection. Uninfected mice received BrdU daily for 4 days prior to necropsy. (A) Representative flow cytometry dot plots showing BrdU incorporation by DX5+ CD3− NK cells from naive and MCMV-infected mice. Numbers represent the percentage of cells in each quadrant, and numbers in parentheses represent the percentages of Ly49H− or Ly49H+ cells that are BrdU+. (B) Comparison of BrdU incorporation by Ly49H− and Ly49H+ NK cells before and during MCMV infection. Significant increase in BrdU+ Ly49H+ cells at 7 days postinfection (P < 0.05) is indicated with an asterisk. (C) IFN-γ production by BrdU+ and BrdU− Ly49H+ NK cells following direct ex vivo exposure to innate cytokines. Splenocytes from naive or MCMV-infected mice were stimulated with the indicated cytokine combinations, and IFN-γ expression by NK cells was assessed by flow cytometry. Values are averages ± standard deviations (SD) (error bars) for the groups of mice (four mice per group).
FIG 4
FIG 4
Cytokine-mediated IFN-γ production and cell surface phenotype of Ly49H+ and Ly49H− NK cells during viral infection. (A) IFN-γ production by Ly49H+ and Ly49H− NK cells following exposure to innate cytokines. Splenocytes from naive, MCMV-infected, or LCMV-infected mice were stimulated directly ex vivo with the indicated cytokines (10 ng/ml) for 6 h. IFN-γ responses of Ly49H− and Ly49H+ NK cells to each cytokine pair were compared using a two-tailed t test. Cytokine pairs that induced IFN-γ responses that were significantly different (P < 0.05) in Ly49H− and Ly49H+ NK cells are indicated with an asterisk. (B) Cytokine-mediated induction of CD69 surface expression on NK cells. Each data point represents the percentage of total, Ly49H−, or Ly49H+ CD3− DX5+ cells expressing CD69 or IFN-γ following exposure to the indicated cytokine pair. (C) CD25 and IFN-γ expression by total CD3− DX5+ NK cells, Ly49H− CD3− DX5+, or Ly49H+ CD3− DX5+ NK cells following cytokine exposure. (D) Surface phenotype and IFN-γ production by Ly49H+ and Ly49H− NK cells from naive and MCMV-infected mice. Dot plots are pregated on live CD3− DX5+ cells, and IFN-γ+ and IFN-γ− cells are represented by red and green dots, respectively. (E) CD11b+ Ly49H+ and CD11b+ Ly49H− NK cell populations display matched responses to cytokine stimulation. Dot plots were gated on CD11b+ NK cells prior to analysis of IFN-γ production by Ly49H+ and Ly49H− subsets. Values are averages ± SD for the groups (four mice per group).
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References

    1. Lodoen MB, Lanier LL. 2006. Natural killer cells as an initial defense against pathogens. Curr Opin Immunol 18:391–398. doi: 10.1016/j.coi.2006.05.002. - DOI - PMC - PubMed
    1. French AR, Yokoyama WM. 2003. Natural killer cells and viral infections. Curr Opin Immunol 15:45–51. doi: 10.1016/S095279150200002X. - DOI - PubMed
    1. Wu J, Lanier LL. 2003. Natural killer cells and cancer. Adv Cancer Res 90:127–156. doi: 10.1016/S0065-230X(03)90004-2. - DOI - PubMed
    1. Biron CA. 2012. Yet another role for natural killer cells: cytotoxicity in immune regulation and viral persistence. Proc Natl Acad Sci U S A 109:1814–1815. doi: 10.1073/pnas.1120528109. - DOI - PMC - PubMed
    1. Loh J, Chu DT, O'Guin AK, Yokoyama WM, Virgin HW IV. 2005. Natural killer cells utilize both perforin and gamma interferon to regulate murine cytomegalovirus infection in the spleen and liver. J Virol 79:661–667. doi: 10.1128/JVI.79.1.661-667.2005. - DOI - PMC - PubMed

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