Cutting edge: stage-specific requirement of IL-18 for antiviral NK cell expansion

Abstract

Although NK cells are considered part of the innate immune system, recent studies have demonstrated the ability of Ag-experienced NK cells to become long-lived and contribute to potent recall responses similar to T and B cells. The precise signals that promote the generation of a long-lived NK cell response are largely undefined. In this article, we demonstrate that NK cells require IL-18 signaling to generate a robust primary response during mouse CMV (MCMV) infection but do not require this signal for memory cell maintenance or recall responses. IL-12 signaling and STAT4 in activated NK cells increased the expression of the adaptor protein MyD88, which mediates signaling downstream of the IL-18 and IL-1 receptors. During MCMV infection, NK cells required MyD88, but not IL-1R, for optimal expansion. Thus, an IL-18-MyD88 signaling axis facilitates the prolific expansion of NK cells in response to primary viral infection, but not recall responses.

Figure 1. IL-18R-deficient NK cells mount a defective response to viral infection

A. WT and Il18r1^−/− NK cells were co-transferred into Ly49h^−/− mice and infected with MCMV. The relative ratio of populations is shown for each time point compared to day 0. B. Mixed WT:Il18r1^−/− chimeric mice were infected with MCMV and the relative ratio of Ly49H⁺ NK cells are shown for day 7 PI compared to uninfected. C. Percentages of co-transferred WT and Il18^−/− Ly49H⁺ NK cells are shown during MCMV infection. D. Percentages of co-transferred WT and Il18r1^−/− Ly49H⁺ NK cells are shown following infection with MCMV, MCMV-Δm157, VSV-m157, or VSV-Ova. E. WT and Il18r1^−/− NK cells were co-transferred into Rag×Il2rg^−/− mice and percentages of transferred Ly49H⁺ NK cells within the total cell population are shown. Data are mean ± s.e.m. representative of at least four independent experiments with at least n=3 biological replicates per condition. * p < 0.05 and ns, not significant, paired Student t-test.

Figure 2. IL-18 is necessary for optimal NK cell maturation and IFN-γ production following MCMV infection

A. Mixed WT:Il18r1^−/− chimeric mice were infected with MCMV and amount of IFN-γ and granzyme B in NK cells at day 1.5 PI are shown. B. WT and Il18r1^−/− NK cells were co-transferred into Ly49h^−/− mice and infected with MCMV. CD27 and CD11b staining is shown for WT and Il18r1^−/− Ly49H⁺ NK cells at day 7 PI. C. Equal numbers of purified effector WT and Il18r1^−/− NK cells (at day 7 PI) were co-transferred into a naïve Ly49h^−/−host. Percentages of Ly49H⁺ NK cells are shown. Data are mean ± s.e.m. representative of at least 3 independent experiments with at least n=3 biological replicates per condition. * p < 0.05 and ns, not significant, paired Student t-test.

Figure 3. MyD88-deficient NK cells exhibit defective proliferation during MCMV infection

A. WT: Myd88^−/− chimeric mice were infected with MCMV and percentages of splenic NK cells are shown for uninfected and day 7 PI. B. CD69 and IFN-γ are shown for wildtype and Myd88^−/− NK cells (compared to uninfected mice) at day 1.5 PI. C. CD27 and CD11b staining is shown for WT and Il18r1^−/− Ly49H⁺ NK cells at day 7 PI. D. Percentages of co-transferred WT (CD45.1) and MyD88^−/− (CD45.2) Ly49H⁺ NK cells are shown following MCMV infection. E. Percentages of co-transferred WT and Il1r^−/− Ly49H⁺ NK cells are shown during MCMV infection. Data are mean ± s.e.m. representative of three independent experiments with at least n=3 biological replicates per condition. * p < 0.05 and ns, not significant, paired Student t-test.

Figure 4. IL-12 signaling induces expression of Myd88 in NK cells

A. qRT-PCR analysis of Myd88 mRNA abundance in NK cells sorted from the spleen of WT mice following infection with MCMV (n=4 biological replicates per condition). Fold expression is shown relative to naïve mice. B. qRT-PCR analysis of Myd88 mRNA abundance in WT NK cells stimulated with IL-12 and IL-18 for 18 h (n=3 biological replicates per condition). Fold expression is shown relative to medium only. C. Vista browser image of mouse Myd88 promoter showing predicted STAT4 binding site. D. STAT4 binding at Myd88, Ifng, and control promoters as assessed through ChIP followed by qPCR in sorted WT NK cells stimulated with IL-12 and IL-18 for 18 h. STAT4 occupancy as percent of input is shown for target (Myd88) and control DNA (negative control: average of gene desert 50 kb upstream of Foxp3, Zfp42, and Utf1 promoters; positive control: Ifng promoter). Data were confirmed in two independent experiments. E. qRT-PCR analysis of Myd88 mRNA abundance in Stat4^−/− NK cells relative to WT NK cells from mice infected with MCMV (n=4 biological replicates per condition). Data are mean ± s.e.m. representative of three independent experiments. * p < 0.05, paired Student t-test.

Figure 5. IL-18 signaling is dispensable for recall response of NK cells

Memory WT and Il18r1^−/− NK cells were stimulated with Ly49D, IL-12 and IL18, or PMA and Ionomycin, and percent IFN-γ (A) or CD107a (B) expression shown. C. Percentage of recalled WT and Il18r1^−/− NK cells (at day 28 PI) are shown following secondary challenge with MCMV. Data are mean ± s.e.m. representative of five independent experiments with at least n=4 biological replicates per condition. * p < 0.05 or ns, not significant, paired Student t-test.