<i>L. monocytogenes</i>nfection modulates chemokine receptor expression <i>in vivo</i> and increases chemokine secretion by macrophages <i>in vitro.</i>

<p>A) Spleen and bone marrow cells were harvested from uninfected or infected animals, and stained for flow cytometry. NK cells were defined as NKR-P1A⁺CD3⁻, further gated as either Ly49s3⁺ or Ly49s3⁻ NK cells. The median fluorescence intensities of CCR5, CXCR3, or CXCR4 by Ly49s3⁺ or Ly49s3⁻ NK cells at 0, 24, and 48 hrs p.i. are presented. The data represents three individual experiments with two animals at each time point, ± SEM. B) The release of chemokines by uninfected or <i>L. monocytogenes</i> infected R2 macrophages was analyzed in supernatants harvested at 24–72 hrs of culture with a multiplex cytokine assay. C) The ability of supernatants from infected R2 macrophages to induce NK cell chemotaxis was analysed in a Transwell chemotaxis assay. Supernatants from uninfected macrophages were used as negative controls. The data are presented as the ratio of migrated cells in response to infected supernatants versus uninfected supernatants. Mean ± SEM from three independent experiments performed in duplicates. <i>p</i> = 0.0075 (one way ANOVA with Tukey's multiple comparisons test).</p

Production of IFN-γ in response to <i>L. monocytogenes</i> infection is Ly49-independent.

<p>Splenocytes from rats either uninfected or infected with <i>L. monocytogenes</i> for 24–48 hrs were harvested and the production of IFN-γ by (A) bulk NK cells or (B) Ly49s3⁺ or Ly49s3⁻ NK cells was determined by flow cytometry. The data in A) represents the mean ± SEM of two independent experiments, with two animals in each group (n = 4). Data were analyzed with the one-way ANOVA with Tukey's multiple comparisons test, ***: <i>p</i><0.001.</p

<i>L. monocytogenes</i> infection leads to up-regulation of MHC class I molecules and down-modulation of Ly49s3 surface expression on NK cells.

<p>Mononuclear leukocytes were isolated from the spleen, blood, bone marrow, and cervical lymph nodes of uninfected (filled histograms) and infected rats, Rats were infected with <i>L. monocytogenes</i> for 24 (grey line) or 48 hrs (black line). Cells were stained with antibodies and analysed for A) Ly49s3 expression on NK cells, B) NKp46⁺ expression by spleen cells, and MHC class I expression on C) OX41⁺ macrophages and D) NK cells. n = 4.</p

Production of IFN-γ by NK cells is cell-contact dependent.

<p>A) R2 macrophages (Mφ) were either left untreated or infected with <i>L. monocytogenes</i> (LM) for 24 hrs prior to co-incubation with freshly isolated splenic lymphocytes for another 18 hrs. Alternatively, splenocytes were incubated with supernatants from infected R2 macrophages alone. As negative and positive controls, splenocytes were stimulated with IL-2 alone or IL-2 + IL-12, respectively. IFN-γ production by NK cells was measured by intracellular FACS staining gating on NKR-P1A⁺CD3⁻ NK cells. B) Measurement of IL-1α, IL-6, IL-12p70, and TNF-α in the supernatants of uninfected or infected R2 macrophages cultured for 24–72 hrs was done with multiplex cytokine assay analysis.</p

The percentage of Ly49s3⁺ NK cells increases in the spleen and decreases in the bone marrow following <i>L. monocytogenes</i> infection.

<p>A) Mononuclear leukocytes were harvested from spleen, blood, and bone marrow from uninfected and infected animals 24 and 48 hrs p.i. Cells were stained with antibodies to NKR-P1A, CD3, and Ly49s3. NK cells were gated as NKR-P1A⁺CD3⁻ cells, and the percentage of Ly49s3⁺ NK cells was analysed by flow cytometry. The data represent the mean ± SEM from three independent experiments, with 2 animals at each time point. Data were analyzed with the one-way ANOVA with Tukey's multiple comparisons test. B) The activation status of NK cells from uninfected (filled histogram) or infected animals 24 (grey line) and 48 hrs p.i. (black line) was tested by flow cytometry. An antibody towards the activation marker CD25 was employed.</p

CD53 induces CD2-independent homotypic clustering of NK cells.

<p>A) Purity of 12 day-old LAK cells, as determined by staining with anti-CD3 and anti-NKR-P1A. LAK cells were cultured for 2 h in the presence of soluble antibodies towards CD53, CD2, NKR-P1A, or an isotype control IgG. B) Cluster formation was photographed with a light microscope, or C) quantified by counting the number of free, non-aggregated cells in treated samples relative to cells cultured in medium using a hemocytometer. D and E) LAK cells were co-cultured for up to 2 h with antibodies towards CD53 and CD2 or with CD53 and isotype control antibody. Cluster formation was assessed qualitatively by microscopy (D), or quantitatively by hemocytometer (E) as described above. Results are presented as the mean±SEM of 3 independent experiments with samples in duplicates (n = 6). Comparisons within an experimental group were performed with the One-way analysis of variance (ANOVA) and a <i>post hoc</i> Tukey's multiple comparisons test to compare treated samples to untreated sample. *, p<0.05; **, p<0.005; ***, p<0.001.</p

Increased proliferative activity of NK cells in response to CD53 ligation.

<p>A) CFSE-labeled lymphocytes were cultured for 7 days with a sub-optimal dose of IL-2 in the presence of an anti-CD53 antibody or isotype control antibody at 10 µg/ml. The CFSE dilution profiles of NKR-P1A⁺CD3⁻ NK cells (upper panels) or CD3⁺NKR-P1A⁻ T cells (lower panels) were analyzed by flow cytometry. Data are representative of 3 independent experiments (n = 3). B) Flow cytometric analysis of CD53 expression by primary NK cells (upper panels) or T cells (lower panels) before and after culture in IL-2 for 24 h. NK cells were gated as NKR-P1A⁺CD3⁻ cells. Shown are stainings with anti-CD53 (solid line) and an isotype control antibody (grey line). n = 3.</p

CD53 ligation reduces NK cell degranulation and receptor-induced IFN-γ production.

<p>A) Degranulation of primary NKR-P1A⁺CD3⁻ NK cells in response to YAC-1 target cells was assessed by flow cytometry. Lymphocytes and YAC-1 cells mixed 1∶1 were co-cultured for 4 h with anti-CD107a antibodies in combination with anti-CD53 or isotype control antibodies. B) Cytotoxicity of RNK-16 cells (open symbols) or LAK cells (filled symbols) against Fc⁻ YAC-1 target cells (left panel) or Fc⁺ P388D1 target cells (right panel) in the presence of anti-CD53 or isotype control antibodies were determined with a standard 4 h ⁵¹Cr release assay. Data represent the mean values of triplicates ± SEM from one representative experiment out of three. C) Conjugate formation between CFSE-stained RNK-16 cells and SNARF-1 stained YAC-1 target cells was assessed by flow cytometry. Percent NK cells in conjugates with target cells was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097844#s2" target="_blank">Materials and methods</a>. The data are representative of 3 independent experiments, and presented as the mean±SEM. D) Ig-depleted spleen cells were stimulated for 6 h <i>in vitro</i> by the indicated plate-bound antibodies or cytokines, and the proportion of IFN-γ positive cells was assessed by flow cytometry gating on NKR-P1A⁺CD3⁻ NK cells. The data represents 3 independent experiments (n = 3), and are presented as the mean ± SEM. n = 3. Data were analysed using the two-tailed unpaired t-test. *, p<0.05; **, p<0.005.</p

CD53 enhances protein tyrosine phosphorylation induced by activating NK receptors, but not of the most proximal signalling molecules.

<p>A) LAK cells were stimulated with Dynabeads co-coated with combinations of anti-Ly49s3, anti-CD53 or control antibodies for 1 min as indicated. Cell lysates were subjected to immunoblotting with an anti-phosphotyrosine antibody, and show enhanced tyrosine phosphorylation of bands at ∼40 and 160 kDa ∼in sampled co-ligated with CD53 and Ly49s3 antibodies. B) NKR-P1A⁺ spleen cells were positively selected with NKR-P1A-coated Dynabeads on ice, and stimulation was induced at 37°C in the presence of either anti-CD53 or isotype control antibodies for 1 or 5 min. Cell lysates were subjected to immunoblotting with anti-phosphotyrosine. The western blots are representative for 3 independent experiments, and show enhanced tyrosine phosphorylation of bands at ∼25, 40, and 75 kDa in samples co-ligated with CD53 and NKR-P1A antibodies. C) LAK cells were stimulated with Dynabeads co-coated with combinations of anti-Ly49s3, anti-CD53 or control antibodies for 1 min. Cell lysates were immunoprecipitated with antibodies to Syk, PI3K, Vav, or PKC-θ, then immunoblotted with either an anti-phosphotyrosine antibody or the immunoprecipitating antibody as control. The data are representative for 3 independent experiments.</p

Elevated LFA-1 activation in response to CD53 ligation, but normal target conjugation and reduced migratory response.

<p>A) Enriched, primary NK cells, or B) LAK cells were pre-incubated with the indicated antibodies for 30 min on ice. Stimulation was induced for 5 min at 37°C in PBS supplemented with Ca²⁺ and Mg²⁺ with a crosslinking secondary F(ab)₂ anti-mouse antibody in the presence of a soluble rat ICAM-1/Fc-fusion protein. PBS with Mg²⁺/EGTA was used as positive control. Binding of ICAM-1 to NKR-P1A⁺CD3⁻ NK cells was assessed by flow cytometry. The data represents 3–5 independent experiments with samples in duplicates (n = 6–10), and are presented as the mean±SEM. Data were analysed using the two-tailed unpaired t-test. *, p<0.05; **, p<0.005.</p