OBJECTIVE The aim of this study was to assess the consequence of sequence variations in HLA-C*03:04-presented Rabbit polyclonal to PDXDC1. HIV-1 p24 Gag epitopes about binding of the inhibitory NK cell receptor KIR2DL2 to HLA-C*03:04. HIV-1 epitopes has on KIR2DL2 binding and KIR2DL2+ NK cell function was identified PI-103 using KIR2DL2-Fc constructs and NK cell degranulation assays. RESULTS Several novel HLA-C*03:04 binding epitopes were identified within the HIV-1 p24 Gag consensus sequence. Three of these consensus sequence peptides (Gag144-152 Gag163-171 and Gag295-304) enabled binding of KIR2DL2 to HLA-C*03:04 and resulted in inhibition of KIR2DL2+ main NK cells. Furthermore naturally occurring minor variants of epitope Gag295-304 enhanced KIR2DL2 binding to HLA-C*03:04. CONCLUSIONS Our data display that naturally happening sequence variations within HLA-C*03:04-restricted PI-103 HIV-1 p24 Gag epitopes can have a significant impact on the binding of inhibitory KIR receptors and main NK cell function. three were and one was Main NK cells were isolated by incubating whole blood with RosetteSep? NK cell enrichment cocktail (Stem Cell) and carrying out a Histopaque?-1077 (Sigma) density gradient centrifugation. NK cells were subsequently incubated over night in RPMI medium 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS Sigma-Aldrich) 2500 U/mL penicillin 2500 ug/mL streptomycin 100 L-Glutamine (Cellgro) and 1.0 ng/mL IL-15 (Cellgro). Next NK cells (1×105) were co-incubated with peptide-pulsed 221-ICP47-C*03:04 cells (5×105) at an effector-target-ratio of 1 1:5 in RPMI comprising anti-human CD107a-PE-Cy7 (12.5 μL/mL). Cells were incubated for 30 minutes at 26°C in 5% CO2 after which monensin (1.5 μL/mL BD GolgiStop?) was added followed by an additional 5 hours of incubation at 26°C in 5% CO2. Cells were stained with anti-CD3-PB anti-CD16-BV785 anti-CD56-BV605 anti-CD14/19-BV510 anti-KIR2DL2/3-PE and anti-KIR2DL3-APC washed fixed with 4% paraformaldehyde and analyzed by circulation cytometry. Data acquisition and analysis Circulation cytometry data was acquired using BD PI-103 3 Laser LSRII (BD Biosciences) and analyzed using FlowJo software version 9.4.4 (Tree Celebrity Inc.). For statistical analysis GraphPad PI-103 Prism 5.0d (GraphPad Software Inc.) was used. HLA stabilization ideals are offered as relative mean ± SEM. Relative mean was determined by dividing the mean fluorescence intensity (MFI) of a given OLP from the MFI of the bad control ‘no peptide’ in the respective assay. KIR2DL2 binding is definitely offered in percentages KIR2DL2-Fc+ cells and NK cell degranulation ideals are indicated as normalized degranulation. Normalized degranulation was determined by dividing the percentage CD107a+ cells of a given sample from the percentage CD107a+ cells of bad control sample ‘GKL’ after reduction of background CD107a+ manifestation. Statistical assessment between organizations was performed using repeated steps ANOVA. Results Several p24 Gag-derived peptides stabilize HLA-C manifestation on 220-C*03:04 cells In order to investigate whether HIV-1 peptides offered by HLA-C*03:04 can effect the binding of KIR2DL2 we in the beginning screened for HIV-1 p24 Gag peptides that stabilized HLA-C manifestation on HLA-C*03:04-expressing tapasin-deficient 220 cells. We used 222 10-mer peptides overlapping by 9 amino acids that spanned the entire HIV-1 p24 Gag consensus sequence (based on sequences published in the Los Alamos database http://www.hiv.lanl.gov/content/index see Supplemental table 1). Two previously explained HLA-C*03-offered self-peptides GAL (GAVDPLLAL) and GKL (GAVDPLLKL) were also included as positive settings (observe supplemental number 1) . Table 1 presents the seven OLPs that induced strong HLA-C stabilization (i.e. high DT9 fluorescence intensity) in 220-C*03:04 cells which were also confirmed in 221-ICP47-C*03:04 cells. Given that 9 amino acid-long peptides have been described to have the ideal size for HLA-C*03:04 binding  we investigated whether 9-mer peptides contained within these seven 10-mers could stabilize HLA-C*03:04 more efficiently. PI-103 In most cases one of the respective 9-mer peptides stabilized HLA-C*03:04 manifestation more so than its 10-mer counterpart (number 1A). Therefore in subsequent KIR2DL2-Fc-binding experiments and practical assays we used the most strongly stabilizing peptides. From your peptide testing in 220-C*03:04 cells an HLA-C stabilization (DT9) score was generated and determined as the median DT9 fluorescence intensity of a given OLP divided from the median DT9 fluorescence intensity of all.