Salivary Gland NK Cells Are Phenotypically and Functionally Unique

Natural killer (NK) cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV). However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV) infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells

NK Cell–Like Behavior of Vα14i NK T Cells during MCMV Infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Vα14 invariant natural killer T cell response to MCMV has not been examined. We found that Vα14i NK T cells become activated and produce significant levels of IFN-γ, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Vα14i NK T cells into MCMV-infected CD1d−/− mice demonstrate that CD1d is dispensable for Vα14i NK T cell activation. In contrast, both IFN-α/β and IL-12 are required for optimal activation. Vα14i NK T cell–derived IFN-γ is partially dependent on IFN-α/β but highly dependent on IL-12. Vα14i NK T cells contribute to the immune response to MCMV and amplify NK cell–derived IFN-γ. Importantly, mortality is increased in CD1d−/− mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Vα14i NK T cells that act as effector T cells during bacterial infection, but have NK cell–like behavior during the innate immune response to MCMV infection

Vα14i NK T cells are activated but do not expand in response to MCMV infection in vivo.

<p><i>A</i>, Splenic and hepatic leukocytes were isolated from MCMV infected or vehicle treated mice at 20 and 40 hrs post-infection and the Vα14i NK T cell compartment was analyzed by staining with TCR-β and α-GalCer-loaded CD1d tetramer. <i>B</i>, Hepatic Vα14i NK T cells were analyzed for the surface expression level of CD25 at 20 and 40 hrs p.i. compared to vehicle treated mice. <i>C,</i> The average expression level of CD25 on the surface of Vα14i NK T cells, average MFI±SD is shown. <i>D,</i> Hepatic leukocytes were isolated from MCMV infected or vehicle treated mice at the indicated days post-infection. The Vα14i NK T cell compartment was analyzed by staining with TCR-β and α-GalCer-loaded CD1d tetramer and the NK cell compartment was analyzed by gating on the NK1.1⁺TCRβ⁻ cells. The percentage and absolute number of Vα14i NK T and NK cells is shown. Results are representative of 3 to 5 independent experiments.</p

The innate immune cytokine response is impaired in the absence of Vα14i NK T cells.

<p><i>A</i>, Splenic leukocytes were isolated from uninfected or MCMV infected CD1d^−/− and CD1d^+/− littermates or Jα18^−/− and Jα18^+/− littermates at 1.5 days post-infection and analyzed for intracellular IFN-γ. The percentage of IFN-γ⁺ NK cells is shown. The results are representative of 5 independent experiments (*, P<0.05). <i>B</i>, Serum levels of cytokines were measured by ELISA or using the cytometric bead array inflammation kit at 1.5 days post-infection (*, P<0.05). The results are representative of 3 independent experiments. <i>C</i>, Percent survival following high dose MCMV infection.</p

Optimal Vα14i NK T cell IFN-γ response requires IFN-α/β and IL-12.

<p><i>A,</i> Hepatic leukocytes isolated from B6, IL-12p40^−/−, and IFNα/βR1^−/− mice at 40 hrs p.i. were stained with α-GalCer CD1d tetramer, TCRβ, and NK1.1 followed by permeabilization and stained for intracellular IFN-γ and compared to vehicle treated mice. IFN-γ⁺ Vα14i NK T cells and NK cells are shown. <i>B</i>, The percent of IFN-γ⁺ Vα14i NK T cells and NK cells, average±SD, is shown for infected B6, IL-12p40^−/−, and IFNα/βR1^−/− mice. IFN-γ was not detectable in vehicle treated animals (data not shown). Results shown are representative of 2 to 5 independent experiments.</p

Vα14i NK T cells produce IFN-γ in response to MCMV infection.

<p><i>A</i>, Following surface staining, hepatic Vα14i NK T cells and NK cells were fixed, permeablized and stained for intracellular IFN-γ. <i>B,</i> The frequency and <i>C,</i> number of IFN-γ⁺ Vα14i NK T and IFN-γ⁺ NK cells, average±SD, is shown for infected B6 at day 1.5 post-infection. Results shown are representative of 2 to 5 independent experiments.</p

CD1d is dispensable for Vα14i NK T cell activation in response to MCMV.

<p><i>A</i>, B6 mice were injected with 300 µg of anti-CD1d mAb or isotype control and infected with 5×10⁴ pfu/mouse MCMV or vehicle control. Hepatic and splenic lymphocytes were isolated from the host animals at day 1.5 post-infection and examined for the percentage of IFN-γ⁺ Vα14i NK T cells and IFN-γ⁺ NK cells. IFN-γ was not detectable in vehicle treated animals (data not shown). The results are representative of 2 separate experiments. <i>B</i>, Hepatic leukocytes were isolated from wild type, congenic B6 mice and depleted of CD8⁺, CD11b⁺, CD19⁺, and CD11c⁺ cells to enrich for Vα14i NK T cells prior to injection via tail vein into CD1d^−/− or Jα18^−/− mice infected with 5×10⁴ pfu/mouse MCMV or vehicle control. Hepatic and splenic lymphocytes were isolated from the host animals at day 1.5 post-infection and examined for the percentage of IFN-γ⁺ Vα14i NK T cells. The results are representative of 4 separate experiments. <i>C</i>, Hepatic leukocytes were isolated from wild type, congenic B6 mice and CD5⁺NK1.1⁺ cells were sorted prior to injection via tail vein into CD1d^−/− or Jα18^−/− mice infected with 5×10⁴ pfu/mouse MCMV or vehicle control. Hepatic and splenic lymphocytes were isolated from the host animals at day 1.5 post-infection and examined for the percentage of IFN-γ⁺ Vα14i NK T cells. The results are representative of 3 independent experiments.</p