(E) The scatter plot illustrates the relationship between the levels of sera anti-HIV gp140 IgG and the percent of total NK cells, from a common donor, activated by the sera in the whole blood ADCC assay in the presence of gp140. to respond to Glimepiride HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate educated KIR3DL1+ NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate uneducated KIR3DL1+ NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies supplementation with the following cytokines and growth factors, for the five hour duration of the anti-HIV ADCC assay, was studied to assess their influence on Glimepiride NK cell activation profiles: IL-10 (50 ng/ml) (BD Biosciences), IL-15 (5 ng/ml) (R&D Systems), IL-4 (50 Units/ml) (BD Biosciences), GM-CSF (1 g/ml) (BD Biosciences), IL-12 (100 ng/ml) (R&D Systems), IL-7 (50 ng/ml) (BD Biosciences), TNF (200 ng/ml) (eBioscience) and LPS (1 g/ml) (Sigma). Statistical Analyses Data analyses were performed using GraphPad Prism Version 4.0 software. Data sets were tested for normal distribution using the Kolmogorov-Smirnov test. Parametric data was analyzed using T-tests or Glimepiride paired T-tests. Non-parametric data was compared using Mann-Whitney tests, Wilcoxon Matched Pairs tests, or Spearman correlations. Results Skewed ADCC-induced NK Cell Activation Profiles Mediated by Sera from HIV-infected Individuals NK cells exhibit a number of functions when activated by the Fc portion of ADCC antibodies, including the expression of cytokines and the degranulation and lysis of target cells. We hypothesized that ADCC-induced NK cell effector functions may be differentially regulated depending on the cytokine milieu of the plasma. To evaluate this hypothesis we simultaneously evaluated sera samples obtained from a cohort of 32 antiretroviral therapy naive HIV-infected subjects for their ability to activate fresh NK cells in blood obtained from a single healthy donor in the presence of Env peptide or protein antigens. We utilized a previously described flow cytometric assay of antibody-mediated NK cell activation [12], [15], [29], [30], [32]C[34]. This simple assay studies the ability of donor NK cells within whole blood to be activated by antibodies within HIV+ sera samples recognizing HIV peptides or Env proteins. The NK cell activation in this assay occurs only when both peptides/proteins and antibodies are present (Fig. 1a). Furthermore, this assay is not dependent on NK cell recognition of immune Glimepiride complexes. Open in a separate window Figure 1 Differential NK cell activation patterns by HIV-specific ADCC.The ability of NK cells to respond to anti-HIV ADCC antibodies was assessed using a flow-based assay. (A) Stimulated cells were stained with fluorochrome conjugated antibodies against CD3, CD2, CD56, CD107a and IFN. After collection on a FACS II Canto, lymphocytes were gated upon and Glimepiride NK cells were identified as CD3?CD2+CD56+. Cells within the NK Robo3 cell population were assessed for IFN production and CD107a expression prior to and following activation. (B) Zebra plots depict examples of the diverse anti-HIV ADCC responses obtained when NK cells from a common donor are stimulated with sera from different HIV-infected individuals in the presence of Env peptides (top) or gp140 protein (bottom). The numbers in the quadrants represent the percentages of responding NK cells that are mediating IFN+CD107a?, IFN?CD107a+ and IFN+CD107a+ responses. (C) The pie chart on the left illustrates the frequency with which IFN dominant, CD107a dominant and even response profiles were observed, when sera samples from 32 HIV-infected individuals were used to stimulate NK cells from.
