Three experimental replicates were conducted per time interval

ontrol for the functional integrity of the cells to respond to other chemokines. These results cannot rule out the possibility that gp120 binds to other cell surface molecules but do clearly show that cell-bound X4-gp120 or X4-HIV-1 forms gp120-CD4-CXCR4 ternary surface complexes on qCD4s for prolonged periods. immunoblotted with anti-IgG Ab. As shown in Fig. 2C, IgG was only 2883-98-9 web detected in rCD4 lysates from HIV-1+ Pts but not from healthy donors. The level of IgG detected by immunoblotting correlated with the mean fluorescence intensities of surface IgG on rCD4s as detected by FACS. Furthermore, utilizing confocal microscopy, we found that Igs colocalized with surface CD4 on rCD4s purified from HIV-1+ Pts. These results collectively confirm that Igs are attached to CD4 on peripheral blood rCD4s in HIV-1+ Pts. cICs in the Serum of Viremic HIV-1+ Pts are Sufficient to form sICs on B Cells but not Resting CD4+ T Cells It has been reported that B cells and T cells from HIV-1+ Pts are covered with complement-opsonized cICs or auto-Abs. Therefore, we tested whether serum from HIV-1+ Pts contains sufficient levels of cICs or auto-Abs to form sICs on rCD4s. Before proceeding with the experiments, we first sought to determine whether complement receptors or the Fc receptor were expressed in B cells and rCD4s. As shown in Slow Turnover of Ig-gp120 sICs on Dense Resting CD4+ T Cells Several studies have shown that in the presence of serum from HIV-1+ Pts, sICs can form on HIV-1-infected cells or gp120exposed uninfected cells. We tested whether patient serum contains sufficient anti-env Abs to allow the formation of sICs on gp120-pre-exposed qCD4s. Although the amount of sICs on qCD4s was proportional to the concentration of exposed gp120, the levels of sICs varied among patients, reflecting different levels of anti-env Abs in the serum of HIV-1+ Pts. We then examined the turnover of cellbound gp120. The turnover of cell-bound gp120 was not significantly affected, even in the presence of patient serum or open diamonds vs. open squares ). Collectively, serum from HIV-1+ Pts always contained sufficient levels of anti-env Abs to form sICs, and the kinetics of surface gp120 were extremely slow in qCD4s regardless of whether cellbound gp120 formed sICs. Resting CD4+ T Cells from Acutely and Chronically HIV-1infected Subjects are Coated with IgG and IgM If gp120 turnover on qCD4s in vivo is similar to that observed in vitro, we should detect sICs on qCD4s from the peripheral blood of HIV-1+ Pts. For technical convenience, to easily detect qCD4s by FACS, we examined the presence of sICs in peripheral blood CD252 CD692 CD4+ CD3+ cells. We utilized biotinylated anti-IgG F2 and/or anti-IgM F2 Abs to prevent non-specific surface binding through the Fc portion. Sixteen individuals with asymptomatic chronic HIV-1 infection, four individuals with acute HIV-1 infection, and ten healthy individuals were examined. In agreement with previous PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19649022 studies, means of 78.18611.77% and 42.18619.73% of peripheral blood rCD4s from sixteen chronic HIV-1+ Pts stained positive with anti-IgG and anti-IgM, respectively, whereas no sIC+ rCD4s were detected in healthy donors. In contrast, means of 48.22622.69% and 72.1069.20% of peripheral blood rCD4s from four acute HIV-1+ Pts were positive for anti-IgG and anti-IgM, respectively. To more clearly demonstrate that peripheral blood rCD4s from HIV-1+ Pts were coated with Ig, rCD4s were purified from HIV-1+ Pts, lysed, and The Dynamics of