Dual requirement of cytokine and activation receptor triggering for cytotoxic control of murine cytomegalovirus by NK cells

Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells

IFN-I or IL-12 are required for GzmB and Prf1 induction in NK cells in a cell intrinsic manner during MCMV infection.

<p>(A and B) Mice deficient in IFNAR1 and/or IL-12p40 were infected with WT MCMV at 5000 PFU/mouse. After 3 days, splenocytes were harvested and analyzed for GzmB and Prf1 expression as indicated. Representative contour plots of individual mice are shown in (A). The median fluorescence intensity (MFI) is indicated for each plot and the cumulative data for one experiment is shown in (B). (C) GzmB levels of WT mice infected with 1x10⁵ PFU WT or Δm157 MCMV 2 days after infection. (D) 40x10⁶ WT or IFNAR1^-/-xIL-12Rβ2^-/- splenocytes were adoptively transferred to congenic host that was simultaneously infected with 1x10⁵ PFU MCMV/mouse. GzmB levels 2 days post-infection in host and transferred NK cells is depicted. Data are representative of three to four independent experiments with individual points representing a single mouse.</p

<i>In vitro</i> stimulation of NK cells with m157-Tg splenocytes alone induces DAP12-dependent degranulation.

<p>(A-C) Splenocytes from CD45.2 WT or indicated strain were mixed with CD45.1/2 congenic m157-Tg or WT splenocytes at a 1:1 ratio in the presence of the indicated cytokines. IFNβ was used at 100–200 U/ml, IL-12 was used at 10–20 ng/ml. NK cell degranulation was measured after a 6–9 hr incubation. (A) Representative FACS plots are shown on the left and percentage of CD107a⁺ cells within the Ly49H⁺ NK cell population of biological duplicates is shown on the right. (B) Jinx or B6.BxD8 splenocytes were mixed with m157-Tg or control splenocytes. For experiments with B6.BxD8 percentage of CD107a⁺ NK cells is calculated over the whole NK cell population. (C) DAP10^-/- or DAP12 KI splenocytes were mixed with adapter-expressing m157-Tg or control splenocytes. (D) Purified NK cells were cultured with m157-Tg or WT control murine embryonic fibroblasts (MEF) and analyzed as in (A). Data are representative of at least two independent experiments.</p

m157-specific cytotoxicity <i>in vivo</i> is greatly enhanced by poly(I:C) and dependent on IFN-I and degranulation.

<p>(A-D) Mice were i.p. injected with 100 μg poly(I:C), PBS, 200 μg anti-NK1.1, 200 μg anti-Ly49H and/or isotype control. After 16 hours 2x10⁶ differentially CFSE-labeled splenocytes were injected intravenously. Three hours after adoptive transfer splenocytes were analyzed by flow cytometry. (A) Representative histograms showing splenic CD19⁺CFSE⁺ cells. Numbers indicate percentage of m157-Tg CFSE^low and WT CFSE^high cells. (B-D) Specific lysis of m157-Tg targets. (E) Mice were injected IP with 100 ug poly(I:C) or PBS. After 16 hours the spleens were harvested and NK cells were analyzed for GzmB. Data are representative of two to three independent experiments with individual points representing a single mouse.</p

<i>In vitro</i> stimulation of NK cells with IFN-I and IL-12 induces GzmB expression in a cell-intrinsic manner.

<p>(A-D) Splenocytes from CD45.2 WT or indicated strain were mixed with CD45.1/2 congenic m157-Tg or WT splenocytes in the presence of the indicated cytokines. IFNβ was used at 100–200 U/ml, IL-12 was used at 10–20 ng/ml, unless otherwise indicated. NK cell GzmB was measured after 16–20 hr incubation. (A) Representative FACS plots are shown on the left and MFI of GzmB in NK cell population of biological duplicates is shown on the right. (B and C) Splenocytes were incubated with the indicated cytokine without stimulator splenocytes. (C) The concentration of the cytokines used were IFNα4 (200 U/ml), IFNβ (200 U/ml), IL-12 (20 ng/ml), IL-2 (200 U/ml), IL-6 (20 ng/ml), IL-15 (20 ng/ml), IL-18 (20 ng/ml). (D) IL-12Rβ2^-/- or IFNAR1^-/- splenocytes were mixed with cytokine receptor sufficient m157-Tg or control splenocytes. MFI = median fluorescence intensity. (E) Purified NK cells were cultured with m157-Tg or WT control MEF and analyzed as in (A). Data are representative of at least two independent experiments.</p

NK cell cytotoxic control of MCMV requires both IL-12 and IFN-I signaling.

<p>(A) Splenic viral titers and (B-F) escape (Δm157) frequency determined five days after a mixed MCMV infection (20000 PFU/mouse), as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005323#ppat.1005323.g001" target="_blank">Fig 1</a>. (D) Anti-IFNAR1 antibody (or isotype control) was administered where indicated 24hrs prior to infection. Data are combined from two to three independent experiments with individual points representing a single mouse. L = Limit of quantification. I = Input frequency (10%).</p

IL-12 or IFN-I are required for m157-specfic cytotoxicity <i>in vivo</i> during MCMV infection.

<p>Mice were i.p. injected with MCMV (10000 PFU/mouse) and at the indicated timepoint after infection an <i>in vivo</i> cytotoxicity assay was performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005323#ppat.1005323.g006" target="_blank">Fig 6</a>. <b>(A)</b> m157-specific rejection was analyzed at days 1–4 after MCMV infection. <b>(B)</b> Mice were infected with WT or Δm157 MCMV for 3 days, after which m157-specific rejection was analyzed. <b>(C)</b> m157-specific rejection was measured in mice deficient in IFNAR1, IL-12Rβ2, or both was analyzed at 3 days post-infection. Data are representative of three to four independent experiments with individual points representing a single mouse.</p