Chronic HCV-infected subjects (n = 25 for bulk B cell functional and phenotypic assays, and an overlapping set of n = 25 for purified B cell subset, Ki67 expression, and Bcl2 expression assays) had detectable serum HCVAb for at least 6 mo, had detectable HCV RNA by PCR, and were not previously treated for HCV infection. Both HCV-infected and healthy control subjects (n = 25) provided written informed consent for venous blood sampling under approval of the institutional review boards for human studies at the Cleveland VA Medical Center and University Hospitals of Cleveland (Cleveland, OH). HCV-infected subjects were mainly infected with genotype 1 HCV (88.5%). HCV level ranged from 85,000–7,000,000 IU/ml. Alanine aminotransferase ranged from 16–302 U/l. Aspartate aminotransferase (AST)/platelet ratio index ranged from 0.07–1.31. One study subject had impaired renal function attributed to HCV-associated membranoproliferative glomerulonephtritis and immune complex deposition, whereas other subjects carried no clinical or laboratory diagnosis of exptrahepatic HCV manifestation (renal impairment, arthritis, monoclonal gammopathy). Rheumatoid factor (RF) had been previously tested on n = 23 HCV-infected subjects, and 13 of these were RF seropositive.
PBMCs were prepared from fresh peripheral blood specimens using Isoprep (Fisher Scientific, Hudson, NH). Bulk B cells were prepared from PBMCs using a negative bead selection B cell Isolation Kit II (Miltenyi Biotec, Auburn, CA) depleting CD2, CD14, CD16, CD36, CD43, and CD235a (glycophorin A). Flow cytometric-based cell sorting of CD3-depleted PBMCs (RosetteSep Human CD3 cell depletion; StemCell Technologies, Vancouver, British Columbia, Canada) was performed using an FACSAria (BD Biosciences, San Jose, CA) housed inside a BSL2 Bioprotect II Biosafety Cabinet (Baker, Sanford, ME) within our Cleveland VA and Center for AIDS Research BSL2 core facility (Cleveland, OH).
Serum IgG, IgA, and IgM by ELISA, B cell total Ab ASC, and Ag ASC frequency by ELISPOT
Serum samples were collected using Vacutainer SST tubes (BD Biosciences) and frozen at −70°C. Serum samples were analyzed using the Total Human IgG, IgA, and IgM Elisa kits (Cygnus Technologies, Southport, NC).
Negatively selected B cells were stimulated in a 24-well plate for 4 d at 37°C with 2.5 μg/ml 2006 CpG (Operon, Huntsville, AL) and 1:10,000 Staphylococcus aureus Cowan (SAC) (EMD Chemicals, Gibbstown, NJ) in complete RPMI 1640 (penicillin/streptomycin and L-glut) with 10% FCS. ELI-SPOT plates (Whatman, Piscataway, NJ) were precoated with 10 μg/ml HCV core (aa 2–120), 10 μg/ml NS3 (aa1192-1457), and 10 μg/ml NS4 (aa 1569–1931) protein (Chiron, Emeryville, CA), 1:400 dilution of tetanus Ag (Wyeth, New York, NY), or 5 μg/ml goat anti-human κ and Λ Ab (Rockland, Gilbertsville, PA) in sterile PBS. Plates were blocked with 5% FCS in RPMI 1640 for2 h at room temperature, then precultured B cells were added. ASC frequency was measured after overnight incubation at 37°C, washing with PBS-0.025%Tween, and additional overnight incubation at 4°C with secondary Ab (100 μl 1:20,000 donkey anti-human IgG biotinylated Ab in 1% FCS in PBS-Tween; Jackson ImmunoResearch Laboratories, West Grove, PA). Plates were washed with PBS-0.025% Tween and incubated for 1 h at room temperature with 100 μl 1:2000 streptavidin-HRP (DakoCytomation, Carpinteria, CA) in PBS plus 1% BSA, washed with PBS, and developed with 200 μl 3-amino-9-ethylcarboazole. Spots were analyzed using an ELISPOT image analyzer (Cellular Technology Limited, Cleveland, OH).
BCR-dependent and CD40L-dependent B cell CD80/CD86 expression
A total of 300,000 PBMCs were incubated in a 96-well round-bottom plate for 3 d in the presence of media, 104 CD40L-expressing NIH 3T3 fibroblasts (supplied by Dr. Peter Heeger, Mount Sinai, New York, NY), 80 μg/ml goat anti-human IgG+IgA+IgM (IgGAM) (Jackson ImmunoResearch Laboratories), or CD40L-expressing 3T3 cells and anti-IgGAM. Cells were removed and stained with anti-CD19 allophycocyanin-Cy7, anti-CD27 APC, anti-CD80 PE-Cy5, and anti-CD86 PE (BD Biosciences) for flow cytometric analysis on an LSRII flow cytometer (BD Biosciences) with FACSDiva Software (BD Biosciences). CD40L dependence for activity was verified using CD40L-blocking Ab (Ancell Corporation, Bayport, MN) in comparison with isotype control.
B cell activation of allogeneic memory CD4 cells
Negatively selected B cells (1.5 × 105
) were incubated on precoated IFN-γ ELISPOT plates for 3 d in the presence or absence of CD40L-expressing NIH 3T3 cells (104
). Negatively selected memory CD4 T cells (Miltenyi Biotec) obtained from a single allogenic healthy control donor were added at 150,000 cells/well for the final 16 h of culture, and IFN-γ–secreting cell frequency was measured as previously described (21
B and T lymphocyte frequency by flow cytometric analysis
Whole blood samples (200 μl) were stained with isotype control, anti-CD19 PE-Cy5 (or PerCP), anti-CD10 allophycocyanin, anti-CD20 allophycocyanin-Cy7, anti-CD27 PE (or PE-Cy7), anti-CD38 allophycocyanin (or PE-Cy7) (BD Biosciences), and anti-CD21 FITC (Beckman Coulter, Brea, CA) or PE (BD Biosciences) for 10 min at room temperature. Samples were incubated for 10 min with 2 ml 1:10 FACSLyse solution (BD Biosciences), then washed with 2 ml PBS with 0.01% BSA, then resuspended in 200 μl 1.0% paraformaldehyde solution (Electron Microscopy Science, Hatfield, PA) and 200 μl PBS with 0.01% BSA. Flow cytometric analysis was performed on an LSRII flow cytometer (BD Biosciences) with FACSDiva Software (BD Biosciences) for whole blood-based analysis of cell subset frequency. B lymphocytes were identified by forward and side scatter, and B cell subset frequencies were measured [proportion of CD19-gated cells that are CD10+ CD27− (immature transitional B cells), CD10− CD21+CD27− (naive B cells), CD10−CD21+CD27+ (resting memory B cells), CD20+CD21− CD27+ (mature activated B cells), CD10− CD21− CD27− (tissue like memory B cells), and CD20− CD38+ (plasma cells)].
For T lymphocyte phenotyping, cells were stained with anti-CD4 PerCP, anti-CD8 allophycocyanin, anti-CD45RA FITC, and anti-CCR7 PE-Cy7 (BD Biosciences). T lymphocytes were identified by forward and side scatter, and T cell subset frequencies were measured (proportion of CD4- or CD8-gated cells that are CD45RA−CCR7− [effector memory T cells], CD45RA−CCR7+ [central memory T cells], CD45RA+CCR7− [terminal effector T cells], and CD45RA+CCR7+ [naive T cells]).
Ki67 and Bcl2 intracellular flow cytometric analysis
Whole blood samples (200 μl) were stained with surface markers of B cell subsets as above for 10 min, then 2 ml 1:10 FACSLyse solution (BD Biosciences) was added for 10 min, followed by 2 ml 1:10 FACS Permeabilizing Solution 2 (BD Biosciences) for 10 min. Samples were washed in 2 ml PBS with 0.01% BSA and stained with isotype control, anti-Ki67 FITC, or anti-Bcl2 FITC (BD Biosciences) for 30 min. All of the above steps were completed at room temperature. B cell subset expression of Bcl2 and Ki67 was measured as proportion of cells staining positive, as well as by mean fluorescence intensity (MFI) of expression.
Serum IL-7 and BAFF by ELISA
Serum samples were stored at −70°C until analysis. IL-7 was measured using Quantikine HS Human IL-7 ELISA kit (R&D Systems, Minneapolis, MN). BAFF was measured using Human BAFF/BLyS/TNFSF13B Quantikine ELISA Kit (R&D Systems).
Flow-sorted B cell subset analysis for intrinsic cell death by Annexin V staining and flow cytometric analysis
Flow-sorted live B cell subsets were analyzed for cell death in direct ex vivo assays and in short-term culture (20 h at 37°C) by Annexin V staining. Purified B cell subsets were washed twice with 400 ml cold PBS, followed by addition of 100 μl cold 1× Annexin V binding buffer (BD Biosciences) and 1 μl Annexin V-FITC Ab (BD Pharmingen, San Diego, CA), then incubated 15 min at room temperature. Postincubation, 400 μl cold 1× Annexin V binding buffer was added, and samples were analyzed by flow cytometry within 1 h of staining.
We analyzed the data with SPSS for Windows, version 15.0 (SPSS, Chicago, IL) and Stata SE, version 9.2 (Stata, College Station, TX). We used conventional measures of central tendency and variability to describe the data. To compare continuous and categorical variables across groups, we used the Mann-Whitney U test or Kruskal-Wallis H test and Pearson’s χ2, respectively. We compared responses pre- and poststimulation with Wilcoxon’s signed-rank test and assessed the associations between continuous variables by Spearman’s rank correlation coefficient. All tests are two-sided, and a p value ≤0.05 was considered significant.