Cells were then stained with annexin V, 7-AAD, anti-CD20, anti-CD21, and G6 mAbs and were analyzed by circulation cytometry. chronic antigenic activation of a limited pool of preexisting autoreactive B cells. We have proposed that persistently high levels of HCV-containing immune complexes stimulate the proliferation of RF-bearing B cells,6 but the exact antigen(s) and stimulatory mechanisms have remained elusive. We have previously demonstrated that HCV+MC+ individuals’ clonal B cells are mainly IgM memory space B cells expressing modestly hypermutated immunoglobulin genes; phylogenetic analysis supports a process of antigen-directed affinity maturation. However, many of these clonal cells have decreased manifestation of CD21, the CR2 match receptor.6 Because CD21 augments B-cell receptor (BCR)-mediated signaling as part of the B-cell coreceptor complex, its down-regulation may confer a state of family member anergy to these cells, as has been demonstrated among CD21low naive B cells from individuals with chronic variable immunodeficiency and rheumatoid arthritis.8 To better understand how HCV elicits the expansion of autoreactive B-cell clones, we have performed transcriptional, immunophenotypic, and Pidotimod functional Pidotimod analyses on HCV+MC+ individuals’ clonal B cells. Contrary to expectations, these cells have a global transcriptional profile suggestive of anergy and apoptosis, and a large proportion of them have immunophenotypic features of anergy. Taken collectively, our data suggest that, although HCV+MC+ individuals clearly have expanded peripheral B cells capable of differentiating into RF-secreting plasmablasts, these cells do not have transcriptional features of neoplastic transformation, and a significant proportion of this clonal human population may be refractory to ongoing antigenic activation. Methods Individuals The studies were authorized by the Institutional Review Boards in the Rockefeller University or college and New York Presbyterian Private hospitals. Donors Pidotimod gave written informed consent according to the Declaration of Helsinki before enrollment. We enrolled HCV Ab?, HCV Ab+/HCV RNA+, and HCV Ab+/HCV RNA? volunteers. No subjects received interferon or immunosuppressive therapy Tagln within 6 months of enrollment. Blood was acquired by peripheral blood draw and leukapheresis. Peripheral blood mononuclear cells (PBMCs) were prepared as previously explained.6 Clinical tests HCV RNA was quantified clinically from the Roche Amplicor assay (Version 2.0; Roche Diagnostics); results are standardized to international units. Liver biopsies were evaluated by pathologists according to the Scheuer system. These tests, in addition to serum alanine aminotransferase measurements, were performed as part routine medical care. Screening for MC was performed as previously explained.6 IgM++CD27+ B-cell isolation IgM++ B cells were isolated from PBMCs by negative selection to minimize transcriptional changes effected by BCR signaling. All methods were performed at 4C. B cells were immunomagnetically isolated using a B Cell Isolation Kit (Miltenyi Biotec). They were incubated with phycoerythrin-conjugated anti-IgG, anti-IgA, and anti-, then with antiCphycoerythrin-conjugated microbeads, and the bad portion was magnetically purified. The CD27+ portion was immunomagnetically isolated using antiCCD27-conjugated microbeads. RNA extraction, cDNA synthesis, amplification, and labeling RNA was extracted from 5000 to 10 000 cells using the RNeasy Plus Micro Kit (QIAGEN) with on-column DNase digestion. RNA integrity and concentration were identified using Lab on a Chip Pico. Samples with RNA integrity figures 9.0 were utilized for downstream control. A total of 2 ng RNA was reverse-transcribed with random hexamers as primers and amplified using the WT-Ovation Pico Kit (Nugen), and 5 g cDNA labeled using uracyl-N-glycosylase (Epicentre Biotechnologies) and biotinylated Pidotimod aldehyde-reactive probe. Microarray methods Human being V3 BeadChips (Illumina) were hybridized with 1.5 g cDNA. Chips were scanned on an Illumina Beadstation and analyzed with Illumina BeadStudio software (Version 3.2). Datasets were analyzed using GeneSpring GX Version 11.1 (Agilent Systems). Raw transmission values were log-transformed, chips were normalized to the 50th percentile, and genes normalized to the median transmission. This dataset was filtered to include genes with signals above background. Welch test (= .05, Benjamini-Hochberg false discovery rate = 0.05) was used to test for variations in genes between organizations. The resulting arranged was filtered to include genes that were 2-fold up- or down-regulated. Hierarchical clustering was performed using the weighted pairwise group method with centroid average, using the Pearson correlation as the distance metric. Statistics were determined using GeneSpring GX and Prism (GraphPad Software). Quantitative RT-PCR RNA was prepared from isolated B cells, as explained under RNA extraction, cDNA synthesis, amplification, and labeling. Random-primed cDNA was synthesized using Superscript III (Invitrogen). Primers (supplemental Table 1, available on the web page; see the Supplemental Materials link at the top of the.