Journal: bioRxiv

Article Title: Strain-Specific Human Natural Killer Cell Recognition of Influenza A Virus

doi: 10.1101/148528

Figure Lengend Snippet: (A) Cytokine concentrations assessed by Luminex®, values displayed represent the mean fluorescence intensity in pH1N1-infected/H3N2 infected conditions (MOI=3). Cytokines elevated by 2.5-fold over the level in mock-infected monocytes are plotted. (B) Impact of cytokine receptor blocking on the NK cell IFN-γ response evaluated by pre-incubating NK cells for 1 hr with blocking antibodies specific to IFNAR2, IL-12R, IL-15R, IL-18R, and IFNGR1 followed by co-culture with infected monocytes. 24 HPI, intracellular cytokine staining was used to assess NK cell IFN-γ + frequency compared to treatment with an isotype control antibody (H3N2: n =2-6, pH1N1: n =9). (C) NK cells were incubated for 1 hr with antibodies specific to IFNAR2 and CD226 or CD54 followed by co-culture with autologous infected monocytes. At 24 HPI, intracellular cytokine staining was used to assess IFN-γ production compared to treatment with an isotype control antibody ( n =6). * P < 0.05, ** P < 0.005, Wilcoxon signed-rank test. (D) Model of strain-specific NK cell recognition of influenza A infection. pH1N1-infection of monocytes virus does not downregulate CD54 and CD112 to the same extent as H3N2-infected monocytes. CD54 expression is retained on Flu-NP + cells while CD112 expression is preferentially retained on exposed, uninfected monocytes. pH1N1 infection of monocytes elicits enhanced IFN-α secretion and blockade of IFNAR2 dampens NK cell anti-pH1N1 IFN-γ production.

Article Snippet: IL-12R β 2 Ab (R&D Systems AF1959-SP) was used at a final concentration of 83.33 μg/mL.

Techniques: Luminex, Fluorescence, Infection, Blocking Assay, Co-Culture Assay, Staining, Control, Incubation, Virus, Expressing

Journal: PLoS ONE

Article Title: Increased Membrane Cholesterol in Lymphocytes Diverts T-Cells toward an Inflammatory Response

doi: 10.1371/journal.pone.0038733

Figure Lengend Snippet: ( A ) SDS-PAGE silver stain of pooled, negatively-sorted CD4 + splenic T-cell lysates from F1 mice (n = 4/group) untreated ( left lane ) or squalene treated (single dose of 180 µg, right lane ) 3 days post-injection shows no detectable quantitative alterations in the protein bands between the two groups of mice. ( B ) Tyrosine phosphorylation patterns of the same samples in panel A were blotted with anti-phosphorylated tyrosine Ab-HRP conjugate. Of note, the amount of 55–100 kDa phosphorylated protein bands is increased in mice treated with squalene. ( C ) Immunoprecipitation of pooled splenic CD4 + T-cell lysates from F1 mice treated or not with squalene (180 µg/mouse, n = 4/group) 3 days post-injection was carried out for IL-12Rβ2, IL-2Rα, IL-4Rα, CD28, or CD3 receptors using specific Abs, and probed with specific anti-phospho Abs for STAT4, STAT5, STAT6, PI3K, and ZAP-70 kinases. Only the phosphorylated STAT-4, STAT-5, and ZAP-70 in squalene treated mice were significantly enhanced. Shown is one of two representative experiments.

Article Snippet: To analyze receptor co-localization with the lipid rafts, resting CD4 T-cells from the squalene treated or untreated mice were triple stained with DAPI, GM1-CTB, and either anti IL-2Rα-APC, anti-IL-4Rα-PE, or anti-IL-12Rβ2-PE conjugates (BD Biosciences).

Techniques: SDS Page, Silver Staining, Injection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Increased Membrane Cholesterol in Lymphocytes Diverts T-Cells toward an Inflammatory Response

doi: 10.1371/journal.pone.0038733

Figure Lengend Snippet: Resting CD4 T-cells from individual F1 mice (n = 5/group) before and 7 days after squalene treatment (180 µg/mouse) were analyzed for interleukin receptor expression and distribution at the single-cell level by CLSM. Cells were stained with IL-4Rα-, IL-12Rβ2-, or IL-2Rα-PE conjugates, and co-stained for GM1 ganglioside by CTB- FITC conjugate and for nuclei with DAPI. First column indicates single-channel color for DAPI staining (blue), second column indicates GM1 staining (green), third column indicates interleukin receptor (IL-Rs) staining (red), and last column indicates merged channels at X63 magnification. Top-two rows , indicate cells from untreated ( upper row ) and squalene treated mice ( lower row ) stained for IL-4Rα. Middle-two rows , indicate cells from untreated ( upper row ) and squalene treated mice ( lower row ) stained for IL-12Rβ2. Bottom-two rows , indicate cells from untreated ( upper row ) and squalene treated mice ( lower row ) stained for IL-2Rα. Arrows indicate presence of IL-Receptor co-expression with the GM1 resident of lipid rafts. Enlargements of the merged channels are depicted to the right along with two different angles of the membrane for each IL-Receptor at X220 magnification. Shown are representative images in one of three experiments.

Article Snippet: To analyze receptor co-localization with the lipid rafts, resting CD4 T-cells from the squalene treated or untreated mice were triple stained with DAPI, GM1-CTB, and either anti IL-2Rα-APC, anti-IL-4Rα-PE, or anti-IL-12Rβ2-PE conjugates (BD Biosciences).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Increased Membrane Cholesterol in Lymphocytes Diverts T-Cells toward an Inflammatory Response

doi: 10.1371/journal.pone.0038733

Figure Lengend Snippet: ( A ) Quantitative real-time RT-PCR of IL-4Rα, IL-12Rβ2, and IL-2Rα mRNA extracted from peripheral blood lymphocytes of individual F1 mice (n = 5/group) analyzed before squalene treatment (dark bars) and 7 days after squalene injection (180 µg/mouse) (light bars). Y axis indicates the mean fold increase in mRNA expression level relative to the endogenous 18S rRNA expression level (control ± SD). Shown are two combined separate experiments (* p values<0.05). ( B ) Aliquots samples in panel A were stained with CD4-FITC conjugate, co-stained either with IL-4Rα-PE or IL-12Rβ2-PE or IL-2Rα-PE conjugates, and analyzed by FACS at the single-cell level for the surface IL-Rs expression level based on MFI measurements. Shown are the IL-Rs MFI values ± SD measured in individual mice before and after squalene treatment. Of note, no significant changes occurred in the IL-Rs expression on cell surface after squalene treatment (* p values>0.05).

Article Snippet: To analyze receptor co-localization with the lipid rafts, resting CD4 T-cells from the squalene treated or untreated mice were triple stained with DAPI, GM1-CTB, and either anti IL-2Rα-APC, anti-IL-4Rα-PE, or anti-IL-12Rβ2-PE conjugates (BD Biosciences).

Techniques: Quantitative RT-PCR, Injection, Expressing, Staining

Journal: Immunology

Article Title: Differential activation of CD57-defined natural killer cell subsets during recall responses to vaccine antigens

doi: 10.1111/imm.12239

Figure Lengend Snippet: Interleukin-12 receptor β 2 (IL-12R β 2) and IL-18R α expression decrease with CD57 expression. Peripheral blood mononuclear cells were analysed ex vivo for IL-12R β 2 and IL-18R α expression. (a) Representative flow cytometry plots for IL-12R β 2 and IL-18R α . Frequency (b) and mean fluorescence intensity (MFI) (c) of IL-12R β 2 expression, and frequency (d) and MFI (e) of IL-18R α expression, were assessed by subset. Each data point represents one donor, n = 19. Lines indicate median values. CD56 dim subsets were analysed for linear trend with a repeated measures analysis of variance. **** P ≤ 0·0001.

Article Snippet: Anti-IL-12R β 2 monoclonal antibody was obtained from R&D Systems (Oxford, UK) and conjugated to PE-Cy5 using an Easylink PE/Cy5® Conjugation Kit (Abcam, Cambridge, UK).

Techniques: Expressing, Ex Vivo, Flow Cytometry, Fluorescence

Journal: Immunology

Article Title: Differential activation of CD57-defined natural killer cell subsets during recall responses to vaccine antigens

doi: 10.1111/imm.12239

Figure Lengend Snippet: The relationship between natural killer (NK) cell phenotype and functional responses to exogenous cytokines. NK cell subsets defined by CD56 and CD57 expression also differ in their expression of interleukin-18 receptor α (IL-18R α ), IL-12R β 2 and CD16 and in their ability to up-regulate CD25. Declining expression of cytokine receptors with increasing expression of CD57 results in gradual loss of the ability of the cells to secrete interferon- γ (IFN- γ ) after cytokine stimulation. The diameter of each cell reflects the approximate proportion of the entire NK population belonging to that subset. The shading of each cell reflects the capacity of that subset to produce IFN- γ in response to high-dose IL-12 + IL-18 (darkest shading denotes the highest IFN- γ production). NB: Our data indicate that CD107a expression does not differ significantly between subsets, whether induced by exogenous cytokines or receptor cross-linking.

Article Snippet: Anti-IL-12R β 2 monoclonal antibody was obtained from R&D Systems (Oxford, UK) and conjugated to PE-Cy5 using an Easylink PE/Cy5® Conjugation Kit (Abcam, Cambridge, UK).

Techniques: Functional Assay, Expressing