Both percentage and MFI (geometric mean) of GFP were determined for GFP+ cells. Statistical analysis Nonparametric statistical tests were used due to small sample sizes. through reduction in apoptosis and accumulation of virus-producing cells in follicular areas, is unknown. The present study was PAX3 designed to evaluate two hypotheses: (1) CXCR5+ CD4+ lymphoid tissue cells express more Bcl-2 than CXCR5? cells and (2) HIV-1-producing follicular cells express more Bcl-2 than HIV-1-producing CXCR5? cells. Materials and Methods Clinical specimens Tonsils were obtained from discarded pathologic specimens of children without known HIV-1 infection undergoing elective tonsillectomies at Children’s Hospital Denver in accordance with local IRB regulations. Tonsils were first visually inspected, necrotic material was removed, and specimens were mechanically disaggregated in sterile phosphate-buffered saline (PBS, Mediatech, GSK-269984A Manassas, VA). The cell suspension was filtered through a 70-m filter (Fisher Scientific, Denver, CO) and washed with PBS. infection with HIV-1 green fluorescent protein (GFP) reporter viruses The HIV-1 NL4-3-based CXCR4-tropic (X4) GFP GSK-269984A reporter virus NLENG1-IRES and CCR5-tropic (R5) GFP reporter virus NLYUV3-GFP have been described elsewhere.19,33 Virus stocks were generated by transfection of 293T cells using Effectene (Qiagen, Valencia, CA), and p24 concentrations were GSK-269984A determined by ELISA (PerkinElmer, Shelton, CT). Freshly isolated tonsil cells were cultured with 5?g/ml of phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO) at a concentration of 2 million cells/ml for 48 to 72?h in R10 medium consisting of RPMI (Mediatech), 1% l-glutamine, 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), 1% penicillin, GSK-269984A 1% streptomycin, and 10 units of IL-2/ml (Roche, Nutley, NJ). Cells were pelleted and resuspended in fresh medium at a concentration of 1107 cells/ml. Then 0.5?ml to 1 1.5?ml of either R5-tropic reporter virus stock (ranging from 400 to 1 1,050?ng of p24 antigen/ml) or X4-tropic reporter virus stock (ranging from 380 to 1 1,050?ng p24 antigen/ml) was added for 2?h at 37C. Samples were diluted to 2106 cells/ml in R10 medium and cultured for an additional 48?h. Flow cytometric analyses Cells were stained with antibodies including CD3-Pacific Orange (Invitrogen, Camarillo, CA), CD4-APC-Cy7, CD8-Pacific Blue, and CXCR5-AF647 [all from Becton Dickinson (BD) Biosciences, San Diego, CA] for 30?min, then washed and fixed with 2% paraformaldehyde (Sigma) solution. To characterize Bcl-2 expression, cells were stained with the above antibodies, fixed for 15?min in 100?l of solution A (Fix & Perm, Invitrogen), washed, and resuspended GSK-269984A in 100?l of solution B (Invitrogen). Following this, cells were incubated for 30?min with unconjugated Bcl-2 antibody (Epitomics, Burlingame, CA), washed, treated with goat anti-rabbit-PE (Invitrogen) for 30?min, then washed and fixed prior to flow cytometry. Data were acquired on a FACS Aria (BD, San Jose, CA) and analyzed using FlowJo (Tree Star, Ashland, OR). GFP+ cells were detected in the FITC channel. This antibody panel was optimized by methods described previously.34 Spectral overlap was determined to be no greater than 45%. A fluorescence minus one or FMO was used to identify gating for CXCR5 and Bcl-2 using uninfected cells. Percentages of antibody-staining cells were determined with the exception of Bcl-2, which was evaluated by using the geometric mean fluorescence intensity (MFI). In all populations analyzed, the MFI of Bcl-2 in the FMO was subtracted from the measured MFI of Bcl-2. Both the percentage and MFI (geometric mean) of GFP were determined for GFP+ cells. Statistical analysis Nonparametric statistical tests were used due to small sample sizes. Wilcoxon-signed rank two tailed test was used for unpaired observations. For determining correlations, Spearman’s correlation was used. A value <0.05 was considered statistically significant. Data were analyzed using Graphpad Prism (La Jolla, CA). Results Bcl-2 expression was elevated in CXCR5+CD4+ T cells in human tonsils Tonsils were obtained from 20 children with a median age of 10 years (range, 2 to 16 years). Figure 1 illustrates the gating strategy for identifying CXCR5+ cells and characterizing Bcl-2 expression. Flow cytometry analyses of disaggregated tonsil cells revealed that a median of 26% (range, 8% to 58%) of CD3+CD4+ cells expressed CXCR5. In freshly isolated tonsil cells, MFI of Bcl-2 was 50% higher in CXCR5+ (median, 292) compared to CXCR5? CD4+ T cells (median, 194) (Fig. 2). Open in a separate window FIG. 1. Representative flow cytometry plot demonstrating the gating strategy for CXCR5 and Bcl-2. Open in a separate window FIG. 2. Bcl-2 expression in CD3+CD4+, follicular (CXCR5+), and extrafollicular (CXCR5) CD4+ T cells isolated from tonsils of subjects at low risk for HIV-1 infection before and after stimulation with phytohemagglutinin (PHA) and interleukin-2 (IL-2). Bcl-2 expression increased in CD4+ T cells from tonsils following in vitro stimulation PHA+IL-2 stimulation for 48?h resulted.