Cutting edge: Priming of NK cells by IL-18

Abstract

Recent evidence suggests that NK cells require priming to display full effector activity. In this study, we demonstrate that IL-18 contributed to this phenomenon. IL-18 signaling-deficient NK cells were found to be unable to secrete IFN-gamma in response to ex vivo stimulation with IL-12. This was not due to a costimulatory role of IL-18, because blocking IL-18 signaling during the ex vivo stimulation with IL-12 did not alter IFN-gamma production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN-gamma upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN-gamma transcription by NK cells was comparable in IL-18 signaling-deficient and -sufficient NK cells. This suggests that priming by IL-18 leads to an improved translation of IFN-gamma mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.

Figure 1. MyD88 is required for IFN-γ production by NK cells in response to IL-12

NK cells from wild-type and MyD88 KO mice were stimulated in the indicated conditions. (A–C) The expression of NK1.1, CD3, CD107a and IFN-γ were measured. (A) Representative FACS analysis of NK1.1 and IFN-γ expression by gated CD3⁻ cells. Cytokine stimulation did not induce CD107a surface exposure (data not shown). (B) Representative FACS analysis of IFN-γ expression and CD107a surface exposure by gated NK1.1⁺ CD3⁻ cells. (C) Mean production of IFN-γ (left) and CD107a surface exposure (right) by gated MyD88 NK cells, expressed as the percentage of the wild-type value in each condition. Mean +/− SD of n = 8 mice. (D) IFN-γ level was measured by ELISA in the culture supernatant over time. Mean +/− SD for n = 4 MyD88 KO mice and n = 3 WT mice. (E) Detection of IFN-γ transcripts as assessed by quantitative RT-PCR upon 4 hours of stimulation. The relative quantity of IFN-γ transcripts was determined by normalization to Hprt1 mRNA. Mean +/− SD of n = 3.

Figure 2. MyD88 and IRAK4 have an intrinsic role in NK cell response to IL-12

NK cells from mixed bone marrow chimera mice (wild-type CD45.1 with MyD88 KO or Pococurante or IRAK4 KO) were stimulated as indicated. The expression of NK1.1, CD45.1, CD3, CD107a and IFN-γ were measured. (A) Mean production of IFN-γ (left) and CD107a surface exposure (right) by gated MyD88 KO NK cells, expressed as the percentage of the wild-type value in each condition. Mean +/− SD of n = 8 mice. (B) Mean production of IFN-γ (left) and CD107a surface exposure (right) by gated Pococurante or IRAK4 KO NK cells, expressed as the percentage of the wild-type value in each condition. Mean +/− SD of n = 4 mice. ND: not determined.

Figure 3. IL-18 signalling is required for IL-12-induced IFN-γ production by NK cells

NK cells from wild-type, IL-18R KO or IL-18 KO mice were stimulated as indicated. The expression of NK1.1, CD3, and IFN-γ were measured. Results show the mean production of IFN-γ by gated NK cells from IL-18R KO mice (A) or from IL-18 KO mice (B). Results are expressed as the percentage of the wild-type value in each condition. Mean +/− SD of n = 10 mice for each genotype.

Figure 4. Endogenous IL-18 does not costimulate NK cells stimulated with IL-12

(A–B) NK cells isolated from wild-type mice were stimulated 4 hours in vitro as indicated in the presence or absence of anti-IL-18 antibody (A) or anti-IL-18Rα mAbs (B). Results show the mean production of IFN-γ by gated NK cells, expressed as the percentage of the control (no antibody) value. Mean ± SD of (A) n = 7; (B) n = 8.

Figure 5. In vitro or in vivo NK cell pre-activation restores IL-12 induced IFN-γ production by MyD88 KO NK cells

(A) NK cells from WT and IL-18 KO mice were cultured 4 hours in vitro with IL-18, extensively washed and then stimulated 4 hours as indicated. The percentage of IFN-γ⁺ NK cells was measured. n = 4–5. (B) NK cells from Poly(I:C) injected wild-type and MyD88 KO mice were stimulated 4 hours in vitro as indicated. The percentage of IFN-γ⁺ NK cells was measured. n = 4. (C) LAK cells from wild-type and MyD88 KO NK cells were stimulated 4 hours in vitro as indicated. The percentage of IFN-γ⁺ LAK cells was measured. n = 4.