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. 2014 Apr 15;192(8):3676-85.
doi: 10.4049/jimmunol.1300837. Epub 2014 Mar 14.

Distinct requirements for activation of NKT and NK cells during viral infection

Aaron J Tyznik  1 , Shilpi Verma, Qiao Wang, Mitchell Kronenberg, Chris A Benedict
Affiliations

Distinct requirements for activation of NKT and NK cells during viral infection

Aaron J Tyznik et al. J Immunol. .

Abstract

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells, NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However, whether similar mechanisms govern activation of these two cell types, as well as the significance of NKT cells for host resistance, remain unknown. In this article, we show that, although both NKT and NK cells are activated via cytokines, their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient, whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results, GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection, consistent with their virtual lack of IL-12 production, whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo, NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV, whereas NK cells were still activated. In turn, splenic NK cell activation was more IL-18 dependent VSports手机版. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally, mice lacking NKT cells showed reduced control of MCMV, and depleting NK cells further enhanced viral replication. Taken together, our results show that NKT and NK cells have differing requirements for cytokine-mediated activation, and both can contribute nonredundantly to MCMV defense, revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses. .

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Figures

Figure 1
Figure 1
IFNγ production by iNKT and NK cells in DC co-cultures. Flt3L (A) or GM-CSF (B) derived BMDC from the indicated mouse strains were treated with CpG-ODN or control treated and then cultured with purified iNKT cells or NK cells for 48h. Flt3L (C) or GM-CSF (D) derived BMDC from the indicated mouse strains were mock or MCMV infected and cultured with purified iNKT cells or NK cells for 48h. Supernatants were analyzed for IFNγ by ELISA, and shown is a representative experiment of 6 performed. ELISA results represent the mean of one experiment with three replicate cultures measured in triplicate. Error bars represent SEM (n = 9 for each set of BMDC).
Figure 2
Figure 2
Activation of iNKT and NK cells by MCMV-infected DC does not require cell-cell contact. (A), IFNγ production by iNKT cells or NK cells cultured for 48h with supernatants from Flt3L or GM-CSF BMDC infected with MCMV. (B), iNKT cells were cultured with individual supernatants, a 1:1 mixture of the two APC types (Flt3L/GM-CSF cells) or with a 10:1 ratio of supernatants from MCMV-infected DC (Flt3L/GM-CSF supes). IFNγ ELISA results represent the mean of one experiment with 2 replicate cultures measured in triplicate. Error bars represent SEM (n = 6 for each culture condition). Shown is a representative experiment of 3 performed.
Figure 3
Figure 3
Cytokine production by DCs in response to CpG or MCMV. (A), Flt3L or GM-CSF BMDC were treated with CpG or control ODN (Non) for 48h, and supernatants were analyzed for IL-12 p70, IL-18 or IFNα by ELISA. (B), Same as in (A), but DC subtypes were mock or MCMV infected prior to analysis of cytokine levels by ELISA. Data are representative of 4 independent experiments. Results show the mean of one experiment with 3 replicate cultures of DC measured in duplicate for ELISA. Error bars represent SEM (n = 6 for each culture condition).
Figure 4
Figure 4
Activation threshold of purified iNKT and NK cells to cytokines. (A-B), Purified iNKT cells or NK cells were cultured with 10 pg/ml IL-12 and serially diluted amounts of IL-18 (A) or IFNβ (B). C-D, Purified iNKT cells or NK cells were cultured with 10 pg/ml IL-18 and serially diluted IFNβ (C) or IL-12 (D). E, Purified iNKT cells or NK cells were cultured with varying doses of IL-12, IL-18 or IFNβ. 48h later, supernatants were analyzed for IFNγ by ELISA. Data are representative of 3 independent experiments and show the mean of one experiment with 3 replicate cultures measured in triplicate for ELISA. Error bars represent SEM (n = 9 for each culture condition).
Figure 5
Figure 5
iNKT and NK cell activation following MCMV infection in vivo. (A), IFNγ expression by NK and iNKT cells from the spleen of indicated mouse strains 36h post-infection with MCMV (open histograms) compared with isotype controls (shaded histograms). Histograms are representative plots of three independent experiments of 3–8 mice per group. B, IFNγ mean fluorescence intensity (MFI) of 3–8 mice per group 36h post-infection with MCMV from a minimum of three independent experiments. Statistically significant differences using the equal variance Student t test are indicated with an asterisk (*, p≤ 0.01) comparing the responses of either WT NK or iNKT cells, respectively, to the corresponding responses in either of the mutant strains. Error bars represent SEM (n = 3–8 for each group).
Figure 6
Figure 6
Cytokine receptor expression by iNKT and NK cells. (A-C), Purified splenic iNKT or NK cells were analyzed by quantitative real-time PCR for their expression of IL-12 (A), IL-18 (B) and IFN-I (C) receptor mRNAs. Bar graphs show each receptor chain or receptor associated protein mRNA levels relative to the L32 ribosomal protein gene expression. Differences between groups were evaluated for statistical significance by the two-tailed unpaired Student t test. Data are the mean of 2 independent experiments performed in duplicate. Error bars represent SEM (n=4); =p<0.05. (D), Flow cytometric analysis of CD19− splenocytes for IL-12Rβ2 (left panel) and IL-18Rα (right panel) expression on tetramer+ iNKT cells (solid line), tetramer− NK cells (dashed line) and tetramer− αβ and γδ CD8+ T cells (shaded histogram). Histograms are representative plots of 3 mice per group from 2 independent experiments.
Figure 7
Figure 7
iNKT and NK cells mediate non-redundant, innate defense against MCMV. Groups of BALB/c Jα18+/+ iNKT cell-sufficient (WT) or BALB/c Jα18−/− iNKT cell-deficient littermate control mice were infected with MCMV, and viral pfu levels were determined in the liver (A), and spleen (B) 4 days later. (C-D), BALB/c Jα18−/− iNKT cell-deficient mice were depleted of NK cells (NKdep), or not, and subsequently infected with MCMV. MCMV replication levels in the liver (C), and spleen (D) were determined 4 days later. For (A-D), results are representative of 12 mice per group from a minimum of 2 independent experiments. Error bars represent SEM (n = 12 for each group). Differences between groups were evaluated for statistical significance by the two-tailed unpaired Student t test. Note that the data shown in (C) and (D) are from experiments carried out at separate times from those in (A) and (B), and therefore the absolute values of pfu cannot be directly compared.
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