Allele sequences were obtained by performing sequence-specific PCR amplification for both genes using peripheral blood leukocyte (PBL)-Panitumumab (anti-EGFR) supplier derived cDNA, as described elsewhere (Finstad et al., unpublished). Cells were cocultured for 6 h before being stained with CD3-Pacific Blue, Anti-Mouse CD11a Antibody Purity & Documentation CD56-PE-Cy7, CD16-allophycocyanin (APC)-Cy7, and CD4-Amcyan (BD Biosciences) to define the effector and target cell subsets. Data were analyzed using FlowJo (Tree Star, Ashland, OR). Background levels of NK cell activation by HIV-infected autologous cells were subtracted from experimental values, and a response was considered positive if the frequency of CD107a-expressing cells was at least 3-fold greater than 3 standard deviations over the mean of unstimulated NK cells. The 50 effective concentration (EC50) for wildtype b12 was estimated by extrapolating the curve to the same level (75 activation) PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/25832652?dopt=Abstract as the maximum level observed for NFb12. Human NK degranulation was assessed by flow cytometry, modified from a previously described method (1). Autologous CD4 cells (target cells) and NK cells (effector cells) were used to eliminate the possibility of non-self-recognition. Activated CD4 cells were infected with vesicular stomatitis virus (VSV)-pseudotyped full-length HIV JRCSF for 2 days (50 to 80 infection on day 2). The cells were cocultured at an effector-to-target ratio of 1:1 in the presence of 20 l/ml CD107a-phycoerythrin (PE)-Cy5, 0.3 g/ml GolgiStop (BD Biosciences, La Jolla, CA), and 0.5 g/ml of brefeldin A (Sigma, St. Louis, MO) in complete RPMI medium. Cells were cocultured for 6 h before being stained with CD3-Pacific Blue, CD56-PE-Cy7, CD16-allophycocyanin (APC)-Cy7, and CD4-Amcyan (BD Biosciences) to define the effector and target cell subsets. The cells were washed and fixed before measurement on a BD LSRII instrument. Data were analyzed using FlowJo (Tree Star, Ashland, OR). Background levels of NK cell activation by HIV-infected autologous cells were subtracted from experimental values, and a response was considered positive if the frequency of CD107a-expressing cells was at least 3-fold greater than 3 standard deviations over the mean of unstimulated NK cells. The 50 effective concentration (EC50) for wildtype b12 was estimated by extrapolating the curve to the same level (75 activation) as the maximum level observed for NFb12. Plasma viral loads. The quantity of SHIV viral RNA (vRNA) genomic copy equivalents (vRNA copy Eq/ml) in EDTA-anticoagulated plasma was determined using a quantitative reverse-transcription PCR (QRTPCR) assay (10). Briefly, vRNA was isolated from plasma, and QRT-PCR was performed using a SuperScript III Platinum One-Step Quantitative RT-PR System (Invitrogen, Carlsbad, CA). Reactions were run on a Roche LightCycler, version 2.0, instrument, and vRNA copy number was determined using LightCycler, version 4.0, software (Roche Molecular Diagnostics, Indianapolis, IN) to interpolate sample crossing points onto an internal standard curve prepared from 10-fold serial dilutions of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/25762297?dopt=Abstract a synthetic RNA transcript representing a conserved region of SIV Gag. Fc R genotyping. The genotypes for Fc RIIa and Fc RIIIa were determined for all animals.