IMMUNOBIOLOGY

Clonal B cells in patients with hepatitis C virus–associated mixedcryoglobulinemia contain an expanded anergic CD21low B-cell subset

Edgar D. Charles,1,2 Claudia Brunetti,1,3 Svetlana Marukian,1 Kimberly D. Ritola,1 Andrew H. Talal,2 Kristen Marks,2

Ira M. Jacobson,2 Charles M. Rice,1 and Lynn B. Dustin1

1Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, Rockefeller University, New York, NY; 2Department of Medicine, Division ofGastroenterology and Hepatology, Weill Medical College of Cornell University, New York, NY; and 3University of Bari, Bari, Italy

Hepatitis C virus (HCV) is associated withthe B-cell lymphoproliferative disordersmixed cryoglobulinemia (MC) and non-Hodgkin lymphoma. We have previouslyreported that HCV�MC� patients haveclonal expansions of hypermutated, rheu-matoid factor–bearing marginal zone-likeIgM�CD27� peripheral B cells using theVH1-69 gene. Here we coupled transcrip-tional profiling with immunophenotypicand functional studies to ascertain thesecells’ role in MC pathogenesis. Despitetheir fundamental role in MC disease,these B cells have overall transcriptional

features of anergy and apoptosis insteadof neoplastic transformation. Highly up-regulated genes include SOX5, CD11C,galectin-1, and FGR, similar to a previ-ously described FCRL4� memory B-cellsubset and to an “exhausted,” anergicCD21low memory B-cell subset in HIV�

patients. Moreover, HCV�MC� patients’clonal peripheral B cells are enrichedwith CD21low, CD11c�, FCRL4high, IL-4Rlow

memory B cells. In contrast to the func-tional, rheumatoid factor–secretingCD27�CD21high subset, the CD27�CD21low

subpopulation exhibits decreased cal-

cium mobilization and does not efficientlydifferentiate into rheumatoid factor–secreting plasmablasts, suggesting thata large proportion of HCV�MC� patients’clonally expanded peripheral B cells isprone to anergy and/or apoptosis. Down-regulation of multiple activation path-ways may represent a homeostaticmechanism attenuating otherwise uncon-trolled stimulation of circulating HCV-containing immune complexes. This studywas registered at www.clinicaltrials.govas #NCT00435201. (Blood. 2011;117(20):5425-5437)

Introduction

Hepatitis C virus (HCV) chronically infects approximately170 million people worldwide and is the leading indicator for livertransplantation in the United States. Although hepatocytes are theprimary target for HCV infection, the B-cell lymphoprolifera-tive disorder mixed cryoglobulinemia (MC) affects up to 50% ofHCV patients.1 MC is characterized by the aberrant productionof monoclonal rheumatoid factor (RF)–containing immunecomplexes that deposit on vascular endothelium of organs, suchas skin, kidneys, and peripheral nerves, eliciting a complementC1q-mediated vasculitis.2 HCV has also been associated withB-cell non-Hodgkin lymphoma (NHL),3 most frequently oflow-grade marginal zone or mucosa-associated lymphoid tissuesubtypes, although associations with higher-grade NHL havebeen reported.

HCV-induced B-cell dysregulation probably represents a con-tinuum from the relatively benign clonal B-cell expansion of MC toovert NHL. The continued presence of HCV is necessary forabnormal B-cell lymphoproliferation, as eradication of HCVtypically results in resolution of both HCV-related MC and NHL.4

Clonal B-cell populations are present in the liver and peripheralblood of HCV�MC� patients5; such B cells demonstrate biasedusage of the RF-encoding VH1-69 and V�3-20 gene segments,6 asdo B cells isolated from lymph nodes of HCV-NHL patients.7 Itremains unclear why B cells undergo clonal proliferation duringchronic HCV infection.

It is probable that HCV-induced B-cell lymphoproliferation isnot the result of direct B-cell infection or transformation, but rather,an indirect process arising from chronic antigenic stimulation of alimited pool of preexisting autoreactive B cells. We have proposedthat persistently high levels of HCV-containing immune complexesstimulate the proliferation of RF-bearing B cells,6 but the preciseantigen(s) and stimulatory mechanisms have remained elusive. Wehave previously shown that HCV�MC� patients’ clonal B cells arepredominantly IgM memory B cells expressing modestly hypermu-tated immunoglobulin genes; phylogenetic analysis supports aprocess of antigen-directed affinity maturation. However, many ofthese clonal cells have decreased expression of CD21, the CR2complement receptor.6 Because CD21 augments B-cell receptor(BCR)-mediated signaling as part of the B-cell coreceptor com-plex, its down-regulation may confer a state of relative anergy tothese cells, as has been demonstrated among CD21low naive B cellsfrom patients with chronic variable immunodeficiency and rheuma-toid arthritis.8

To better understand how HCV elicits the expansion of autore-active B-cell clones, we have performed transcriptional, immuno-phenotypic, and functional analyses on HCV�MC� patients’ clonalB cells. Contrary to expectations, these cells have a globaltranscriptional profile suggestive of anergy and apoptosis, and alarge proportion of them have immunophenotypic features ofanergy. Taken together, our data suggest that, although HCV�MC�

Submitted October 11, 2010; accepted March 6, 2011. Prepublished online asBlood First Edition paper, March 18, 2011; DOI 10.1182/blood-2010-10-312942.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2011 by The American Society of Hematology

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patients clearly have expanded peripheral B cells capable ofdifferentiating into RF-secreting plasmablasts, these cells do nothave transcriptional features of neoplastic transformation, and asignificant proportion of this clonal population may be refractory toongoing antigenic stimulation.

Methods

Patients

The studies were approved by the Institutional Review Boards at theRockefeller University and New York Presbyterian Hospitals. Donors gavewritten informed consent according to the Declaration of Helsinki beforeenrollment. We enrolled HCV Ab�, HCV Ab�/HCV RNA�, and HCVAb�/HCV RNA� volunteers. No subjects received interferon or immuno-suppressive therapy within 6 months of enrollment. Blood was obtained byperipheral blood draw and leukapheresis. Peripheral blood mononuclearcells (PBMCs) were prepared as previously described.6

Clinical tests

HCV RNA was quantified clinically by the Roche Amplicor assay (Version2.0; Roche Diagnostics); results are standardized to international units.Liver biopsies were evaluated by pathologists according to the Scheuersystem. These tests, in addition to serum alanine aminotransferase measure-ments, were performed as part routine medical care. Testing for MC wasperformed as previously described.6

IgM���CD27� B-cell isolation

IgM��� B cells were isolated from PBMCs by negative selection tominimize transcriptional changes effected by BCR signaling. All steps wereperformed at 4°C. B cells were immunomagnetically isolated using a B CellIsolation Kit (Miltenyi Biotec). These were incubated with phycoerythrin-conjugated anti-IgG, anti-IgA, and anti-�, then with anti–phycoerythrin-conjugated microbeads, and the negative fraction was magnetically puri-fied. The CD27� fraction was immunomagnetically isolated using anti–CD27-conjugated microbeads.

RNA extraction, cDNA synthesis, amplification, and labeling

RNA was extracted from 5000 to 10 000 cells using the RNeasy Plus MicroKit (QIAGEN) with on-column DNase digestion. RNA integrity andconcentration were determined using Lab on a Chip Pico. Samples withRNA integrity numbers � 9.0 were used for downstream processing. A totalof 2 ng RNA was reverse-transcribed with random hexamers as primers andamplified using the WT-Ovation Pico Kit (Nugen), and 5 �g cDNA labeledusing uracyl-N-glycosylase (Epicentre Biotechnologies) and biotinylatedaldehyde-reactive probe.

Microarray procedures

Human V3 BeadChips (Illumina) were hybridized with 1.5 �g cDNA.Chips were scanned on an Illumina Beadstation and analyzed with IlluminaBeadStudio software (Version 3.2). Datasets were analyzed using Gene-Spring GX Version 11.1 (Agilent Technologies). Raw signal values werelog-transformed, chips were normalized to the 50th percentile, and genesnormalized to the median signal. This dataset was filtered to include geneswith signals above background. Welch t test (P � .05, Benjamini-Hochbergfalse discovery rate � 0.05) was used to test for differences in genesbetween groups. The resulting set was filtered to include genes that were2-fold up- or down-regulated. Hierarchical clustering was performed usingthe weighted pairwise group method with centroid average, using thePearson correlation as the distance metric. Statistics were calculated usingGeneSpring GX and Prism (GraphPad Software).

Quantitative RT-PCR

RNA was prepared from isolated B cells, as described under “RNAextraction, cDNA synthesis, amplification, and labeling.” Random-primed

cDNA was synthesized using Superscript III (Invitrogen). Primers (supple-mental Table 1, available on the Blood Web site; see the SupplementalMaterials link at the top of the online article) were constructed using thePrimerBank Database (www.pga.mgh.harvard.edu/primerbank) and weredesigned to span exon-intron borders to reduce the possibility of genomicDNA amplification. SYBR Green PCR Master Mix (Applied Biosystems)was used for quantitative reverse-transcribed polymerase chain reaction(RT-PCR). We normalized all samples to RPS11 and to the target gene inUniversal Human Reference RNA (Stratagene). Fold change expressionwas calculated using the 2���Ct method. Groups were compared using theKruskal-Wallis test; when Kruskal-Wallis P .05, the HCV�MC� andHCV�MC� groups were compared using the Dunn post-test.

Flow cytometry

Cells were stained with monoclonal antibodies (mAbs) in phosphate-buffered saline supplemented with 2% (weight/volume) bovine serumalbumin (Fraction V; Fisher Biotech) and 0.02% NaN3. All antibodies andreagents were from BD Biosciences, except for G6 (provided by R. Jefferis),F(ab)2 anti-FCRL4 biotin (provided by G. Erhardt and M. Cooper), andKi-67 fluorescein isothiocyanate (Invitrogen). Conjugation of G6 to biotinand AlexaFluor-594 was performed using commercial kits (Pierce Chemi-cal, Invitrogen). Analysis was performed within 1 hour on a BD LSRII flowcytometer (BD Biosciences).

G6� B-cell subset isolation

PBMCs were stained with anti-CD20, anti-CD27, anti-CD21, G6 mAbs,and 4,6-diamidino-2-phenylindole (to exclude dead cells). LiveCD27�/�CD21high/low G6�, CD20� B cells were bulk-sorted on a BDFACSAria II (BD Biosciences). For assessment of postsort viability,sorted samples were restained with 4,6-diamidino-2-phenylindole andwere reanalyzed by flow cytometry. Postsort analysis confirmed morethan 85% viability and more than 99% purity of sorted populations.

Cell cycle analysis

B cells were negatively isolated from PBMCs using EasySep Human B CellEnrichment Kit (StemCell Technologies) and incubated with biotinylatedG6 mAb. G6� and G6� B-cell subsets were purified using streptavidin-conjugated immunomagnetic beads. Cells were fixed in 80% ethanol. Afterincubation with FITC-labeled Ki-67, cells were resuspended in PBScontaining 10 mg/mL RNase A and incubated at 37°C. Propidium iodide20 �g/mL was added before flow cytometry.

Electron microscopy

Cells were fixed in 2% glutaraldehyde, incubated in 1% osmium, dehy-drated in a graded alcohol series, embedded in spur resin, and then treatedwith 2% uranyl acetate and Reynold lead citrate. Transmission electronmicroscopy was performed at �2000 and �10 000 magnification.

Calcium mobilization assay

A total of 2 � 106 B cells were incubated with 1�M Indo-1 for 30 minutes.Cells were then labeled with anti-CD19, IgG, CD27, and CD21 mAbs andsuspended in HBSS with Ca2� and 1% bovine serum albumin. Emission at405 and 495 nm was measured to obtain a baseline, and then for 5 minutes.After addition of 10 �g/mL goat F(ab)2 anti-IgM, 405/495 nm emissionratios of IgG� B-cell subsets were analyzed with FlowJo software Version9.2 (TreeStar).

Annexin V apoptosis assay

A total of 2 � 106 PBMCs were incubated with 1 �g/mL anti-CD95 ormouse IgG1 and 2 �g/mL Protein G (Invitrogen) in RPMI/10% fetal calfserum at 37°C for 0 and 6 hours. Cells were washed with phosphate-buffered saline and resuspended in 0.01M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (pH 7.4), 0.14M NaCl, 2.5mM CaCl2, and 2% fetal

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calf serum. After incubation with annexin V–phycoerythrin and 7-amino-actinomycin at room temperature, cells were stained with anti-CD20 FITCanti-CD21–allophycocyanin, and G6-biotin at room temperature. Afterstaining cells with streptavidin-Cy7-allophycocyanin, flow cytometry wasperformed.

Immunoglobulin secretion assays

Cells (50 000/well in 96-well round-bottom plates) were cultured for 6 daysin RPMI supplemented with 10% fetal calf serum, 2mM L-glutamine,100 U/mL penicillin/streptomycin, and 0.25 �g/mL amphotericin B, withthe addition of 6 U/mL IL-2 (R&D Systems), 200 ng/mL IL-10 (R&DSystems), and 1 �g/mL flag-tagged CD40L with 2 �g/mL mouse IgG1

anti-flag Ab (Alexis Biochemicals).For ELISPOT, cells were washed with RPMI, placed on MultiScreen

filter plates (Millipore), coated with goat F(ab)2 anti–human IgM (JacksonImmunoResearch Laboratories), and incubated at 37°C for 6 hours. Plateswere then incubated with horseradish peroxidase-labeled anti–human IgM,and the assays were developed with 3-amino-9-ethylcarbazole (Sigma-Aldrich). Spots were counted using an ImmunoSpot Analysis Instrument(Cellular Technology).

For ELISA, cell culture supernatants were added to MaxiSorb plates(Nunc) coated with anti-IgM (Bethyl Laboratories), or for RF assay, humanIgG1� (Sigma-Aldrich), and incubated at room temperature for 1 hour.Plates were then incubated with HRP-labeled goat-anti–human IgM, andthe assays were developed with TMB (BioFX Laboratories). After stoppingreactions with 1N H2SO4, A450 was measured on a FLUOstar Omegamicroplate reader (BMG Laboratories).

Accession numbers

Microarray data are accessible through NCBI Gene Expression Omnibusaccession number GSE18084 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc � GSE18084).

Results

Characteristics of study subjects

Five HCV Ab� volunteers, 7 sustained virologic responders(SVRs), and 27 persons with chronic HCV infection were enrolledfor gene transcriptional analyses. Three additional HCV�MC� and2 additional HCV�MC� subjects were enrolled only for immuno-phenotypic and functional analyses (Table 1). Sixteen HCV�

subjects were MC�, and all 16 had evidence of clonal B-cellpopulations, as demonstrated by complementarity-determiningregion 3 PCR.6 Four of these subjects had lymphadenopathy;lymph node biopsies performed by their physicians confirmedlow-grade B-NHL (subjects 1116, 1308, 1716, and ECH 529).Fifteen of 16 patients had evidence of clonal IgM gammopathy byserum immunofixation electrophoresis and were classified as beingIgM MC�. Subject ECH 529 had evidence of clonal IgA gammopa-thy by immunofixation electrophoresis. In addition, cervical lymphnode biopsy in this patient revealed abnormal numbers of IgA���

B cells. Plasma from 10 of 12 HCV RNA� and 25 of 27 HCVRNA� patients had detectable anti-Epstein-Barr virus nuclearantigen 1 IgG, indicating previous exposure to Epstein-Barr virus.HCV� IgM MC� subjects had significantly expanded populationsof IgM��� peripheral B cells, although overall B-cell numberswere not increased, consistent with our earlier report6 (supplemen-tal Figure 1).

IgM���CD27� B cells from HCV�MC� patients have a distincttranscriptional profile

A total of 69 unique genes (33 up-regulated, 36 down-regulated)were found to be more than 2-fold differentially expressed in

IgM���CD27� B cells from IgM MC�, compared with IgM MC�,subjects (Figure 1). Notably, the transcriptional profile of 2 HCVRNA�MC� patients (ECH 516 and ECH 522) shared severalfeatures with that of the MC� population.

Several of the differentially expressed genes were grouped accord-ing to broad function (Table 2). The overall transcriptional pattern wassuggestive not of oncogenesis, but of dampened activation and aug-mented proapoptotic pathways. Unsurprisingly, several IFN-inducedgenes were up-regulated: growth interferon-inducible protein X (PY-HIN1), myeloid nuclear differentiation antigen (MNDA), and 2, 5-oligoadenylate synthetase 1 (OAS1). Several genes associated withB-cell anergy were up-regulated: galectin-1 (LGALS1), lymphocytetransmembrane adapter 1 (LAX1), and CD200 receptor 1 (CD200R1),an inhibitory receptor highly expressed on memory B cells andplasmablasts. Significantly up-regulated proapoptotic genes included:galectin-1, the interferon-response gene, PYHIN1, death-associatedprotein kinase 2 (DAPK2), and MNDA. The prosurvival gene, T-celllymphoma 1A (TCL1A), was markedly down-regulated.

In addition to having an overall transcriptional program sugges-tive of B-cell anergy and apoptosis, HCV�MC� patients’IgM���CD27� B cells demonstrated differential regulation ofseveral genes previously reported increased in patients with NHL.Up-regulated genes included: SRY-box 5 (SOX5), �-X integrin(ITGAX, CD11C), and MNDA. Down-regulated genes included:L-selectin (SELL), LIM only 2 (LMO2), forkhead box 1 (FOXP1),and TCL1A. Also down-regulated was IL-4 receptor (IL-4R),polymorphisms of which have been associated with diffuse largeB-cell lymphoma25 and which may be down-regulated in mantlecell lymphoma.9 In addition, BTB and CNC homology 1, basicleucine zipper transcription factor 2 (BACH2) was down-regulated;both BACH2 and IL-4R are reported to be down-regulated inB cells from patients with Waldenstrom macroglobulinemia.10

Because many of the up-regulated genes (eg, CD11C, CD84,CD200R1, and bone morphogenetic protein receptor 1A [BMPR1A])are known to be expressed in activated and/or memory B cells,11,26-28

we hypothesized that their up-regulation reflected a particular stageof differentiation of HCV�MC� patients’ clonally expandedB cells. Several of the most significantly up-regulated genes(SOX5, Gardner-Rasheed feline sarcoma viral oncogene ho-molog [FGR], and CD11C) have previously been found to behighly up-regulated in FCRL4-expressing tonsillar B cells, arecently described memory B-cell subset thought to play animportant role in mucosal defense.29 Despite this transcriptionalsimilarity, our microarray data did not reveal differences inFCRL4 transcript between MC� and MC� patients’IgM���CD27� peripheral B cells. However, quantitative RT-PCR of unamplified cDNA confirmed the up-regulated expres-sion of SOX5, FGR, and CD11C in HCV�MC� patients’expanded IgM���CD27� B cells (Figure 2; supplemental Table2). In addition, quantitative RT-PCR confirmed the up-regulation of galectin-1 and OAS1 and the down-regulation ofIL-4R and TCL1A, and it detected no significant difference inFCRL4 expression. We did not detect Epstein-Barr virus nuclearantigen 2 or latent membrane protein 1 transcripts by quantita-tive RT-PCR (data not shown).

A significant proportion of clonal cells from IgM MC� subjectsare CD21low, CD11c�, FCRL4high, IL-4Rlow memory B cells

We and others have previously shown that clonally expandedIgM���CD27� B cells from IgM MC� patients preferentially usethe VH1–69 gene segment.6,30 We used the G6 mAb, whichrecognizes the complementarity-determining region 2 of Ig VH1-

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69,31 to more precisely immunophenotype HCV�MC� patients’clonally expanded B cells. We have previously confirmed thespecificity of G6 for VH1-69 by RT-PCR.32 These G6� B cells fromHCV�MC� patients are frequently CD21low and IgM��� (supple-mental Figure 2). They are also morphologically normal, nonprolif-erating, and predominantly CD20high, CD10�, CD21low, CD27�,CD11c�, FCRL4high, and IL-4Rlow (Figure 3). We immunopheno-typed B cells from 11 MC� (SVR � 2, HCV Ab� � 3, HCVRNA� � 6) and 9 HCV RNA�MC� subjects (Figure 4). Asexpected, we found that MC� patients had a significant (P .005)

expansion of G6� B cells (median, 25.9% of B cells) comparedwith HCV RNA�MC� patients (median, 5.1%), SVR (median,3.0%), and HCV Ab� (median, 3.8%) patients. In 3 MC� patients(LDU 125, 110, and 1432), more than 50% of total peripheralB cells were G6�. When we examined G6� and G6� B cells fromeach person, we confirmed that G6�, compared with G6�, B cellsfrom HCV�MC� patients were predominantly CD21low, CD11c�,FCRL4high, and IL-4Rlow. Interestingly, G6�, compared with G6�,B cells from MC� persons were also disproportionately CD21low.However, they did not have significantly increased expression

Table 1. Characteristics of the study patients

Condition/subjectno. Age, y Sex Ethnicity HCV RNA GT Stage (0-4) Treatment history Clonal CDR3

HCV Ab-

LDU 099 35 Male White NA NA NA NA No

LDU 128 40 Male Black NA NA NA NA No

ECH 503 35 Male Asian American NA NA NA NA No

ECH 527 50 Female White/Hispanic NA NA NA NA No

ECH 528 51 Female White NA NA NA NA No

SVR

543 48 Female White 50 1 4 pIFN/RBV 2001 No

731 52 Female White 50 2b 2 pIFN/RBV 2003 No

856 61 Female White 50 1a 2 pIFN/RBV 2003 No

1154 56 Female Asian American 50 1a 2 pIFN/RBV 2003 No

1197 38 Female White 50 2a 1 pIFN/RBV 2003 No

ECH 521 43 Male White 50 1 ND pIFN/RBV 2005 No

ECH 542 38 Female Black 50 1 ND pIFN/RBV 2007 No

HCV�, IgM MC�

1235 44 Female White 3.85 � 106 2b ND Naive No

1330 54 Female White/Hispanic � 7 � 105 1a 2 Naive No

1419 48 Female White 0.39 � 106 1 3 Naive No

1864 58 Male White 0.02 � 106 4 3 Naive No

LDU 107 50 Male White/Hispanic � 7 � 105 2 2 Naive No

ECH 507 54 Male White/Hispanic � 7 � 105 1 ND Naive No

ECH 512* 62 Male White � 7 � 105 1 ND Naive No

ECH 516 57 Male White/Hispanic 2.4 � 106 1b 2 Naive No

ECH 519 52 Female White/Hispanic � 7 � 105 1 ND Naive No

ECH 520 57 Male Black � 5 � 106 1b 2 Naive No

ECH 522 50 Female White/Hispanic � 7 � 105 1 ND Naive No

ECH 529†‡ 52 Female White � 7 � 105 3 4 Naive Yes

ECH 530 49 Male White/Hispanic � 7 � 105 1 ND Naive No

ECH 536* 50 Male White/Hispanic 0.2 � 106 1 1 Naive No

ECH 537* 54 Male Black 0.9 � 106 1a ND Naive No

HCV�, IgM MC�

110 53 Male White/Hispanic 1.70 � 106 1 3 Naive Yes

880 54 Female White 3 � 106 1b 2 Naive Yes

1116‡ 37 Male White 3 � 104 2b 2 Naive Yes

1308‡ 55 Female White 0.72 � 106 1 1 Naive Yes

1403 44 Female White/Hispanic 0.50 � 106 1 2 Naive Yes

1432 69 Female White 0.03 � 106 1 4 Naive Yes

1540 37 Male White/Hispanic 0.28 � 106 1 ND Naive Yes

1716‡ 30 Female White 1.45 � 106 1a ND Naive Yes

1931 56 Female White 2.2 � 106 1b 2 Naive Yes

92200 57 Female White 2 � 106 2 2 Naive Yes

LDU 125 60 Male White 0.2 � 106 3 ND Naive Yes

ECH 531 56 Male White � 7 � 105 1 ND Naive Yes

ECH 532 51 Female Black � 7 � 105 1 ND Naive Yes

ECH 533 63 Male White 3.8 � 106 1 0 pIFN/RBV 2007 Yes

ECH 535 57 Male White 0.74 � 106 1 ND Naive Yes

ECH 546* 52 Male White � 7 � 105 1 3 pIFN/RBV 2008 ND

ECH 559* 58 Female White/Hispanic 1.27 � 106 1 4 pIFN/RBV 2001 ND

GT indicates genotype; CDR3, Ig complementarity determining region 3; pIFN/RBV, pegylated interferon/ribavirin; NA, not applicable; and ND, not done.*Subjects used for immunophenotypic and functional assays only.†Subject with �� IgA MC.‡Subjects with marginal zone B-cell NHL (documented on lymph node biopsy).

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of CD11c or FCRL4, nor did they have decreased expressionof IL-4R.

Reflective of having G6� B-cell expansions, HCV�MC� pa-tients had increased percentages of total B cells that were CD21low,CD11c�, FCRL4high, and IL-4Rlow. These markers were signifi-cantly correlated with the percentage of peripheral B cells that wereG6�. We did not detect any significant differences in any of thesecell surface markers among the total B cells of SVR, HCV Ab�,

and HCV RNA� patients, probably reflective of the low proportionof G6� B cells in these MC� persons.

FCRL4� memory B cells from normal tonsils and HIV-viremicblood are reported to be CD20highCD11c�CD95�CD21low.29,33 Inhealthy tonsils, these FCRL4� B cells have been variously reportedto be CD27� or CD27� memory B cells.34,35 Although we detectedincreased FCRL4 expression on G6�, compared with G6�, B cells,we did not detect significant differences in FCRL4 expression

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Expression

6 01

SOX5AEBP1SLC7A7PYHIN1SLC11A1ITGAX (CD11c)PNMA2TESCLGALS1 (Galectin-1)PPAP2BFGRHOXB7FLJ40869OAS1FCGR2AMNDAMIDNSTS-1LOC649199RASGRF1N/AN/ACD200R1CD84DAPK2LAX1TFECLRRN1OAS1SRGNC13orf15TUBB2ATUBB2AENC1DUSP5CD68TMEM44N/AADARB1TSPAN13SELLP2RY14N/ATCL1AIL4RC3orf37LMO2SPRY1ZNF135LARGESMAD3N/AEPB41L2VPREB3N/APPAPDC1BLIX1SSBP2MGC39372GCNT1TAPT1CRIM1CR1CR1C1orf162LOC647450LOC652493LOC652694N/APELI2CD1ANT5EIL13RA1N/ALOC652113N/ASERPINE2FOXP1BACH2FLJ14213IL15

Figure 1. Transcriptome analysis of IgM���CD27�

B cells. The transcripts differentially expressed byIgM���CD27� B cells from HCV�MC� and MC�

patients are displayed. Up- and down-regulated tran-scripts are indicated in red and blue, respectively. Themagnitude of expression is indicated by the color bar.

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among CD27�, CD27�, CD21high, or CD21low B-cell subsets(supplemental Figure 3). In an attempt to reconcile the findings ofunchanged FCRL4 transcript levels and increased FCRL4 proteinexpression in MC� compared with MC� B cells, we performedquantitative RT-PCR on RNA isolated from the bulk-sorted FCRL4�

and FCRL4� G6� B cells from subject LDU 125. However, wefailed to detect any difference in FCRL4 mRNA between these2 groups (data not shown).

CD21low B cells from healthy, HCV�MC�, and HCV�MC�

persons are anergic to BCR-mediated stimulation

CD21low memory B cells have been described in HIV-viremicpatients as being relatively anergic, “exhausted” B cells.33 Wesought to determine whether IgG�, CD21low B cells had attenuatedsignaling on BCR ligation with anti–human IgM. Data for onehealthy subject with an elevated (22%) CD21low B-cell frequency(ECH 503), 2 HCV�MC� (ECH 530, ECH 537) volunteers and5 HCV�MC� (ECH 546, ECH 559, 110, LDU 125, and 1931)volunteers are shown in Figure 5. In all but one (110), theCD27�CD21low subset had decreased Ca2� mobilization; thereason for this outlier is unclear. Furthermore, in all subjects, theCD27�CD21low B-cell subset had diminished amplitude of Ca2�

mobilization; ECH 546, for unknown reasons, had an intermediate

response to BCR triggering. Finally, in ECH 559 and 1931,CD27�CD21high cells had low responses to BCR stimulation; IgAmay possibly be a prominent non-IgG BCR in their CD27�CD21high

subsets. Overall, these data suggest that CD21low B cells, whetherfrom healthy, HCV�MC�, or HCV�MC� persons, are anergic toBCR-mediated stimulation.

G6� B cells are preferentially prone to death and apoptosis

Our microarray data suggested that HCV�MC� patients’IgM���CD27� B cells up-regulate several genes associated withapoptosis. We tested whether these cells were prone to apoptosis invitro by incubating them with either media, anti-CD95 mAb, orisotype control IgG (Figure 6). After 6 hours of incubation, PBMCswere stained with annexin V (to measure apoptosis), 7-amino-actinomycin D (7-AAD; to measure cell death), anti-CD20,and-CD21, and G6 Abs. After incubation with media alone, isotypecontrol, or anti-CD95, G6�, compared with G6�, B cells hadsignificantly higher binding of annexin V, either alone or incombination with 7-AAD. The percentage of annexin V�/7-AAD�

cells was highest in the anti-CD95-stimulated G6� B-cell subset.However, whereas G6� cells demonstrated a 2.1-fold increase inannexin V binding on stimulation with anti-CD95, compared withisotype control, G6� cells had a 1.4-fold increase (Figure 6A). This

Table 2. Functional grouping of differentially expressed genes

Functional group/gene Fold difference P Significance

Interferon response

PYHIN1 (IFIX), (growth interferon-inducible protein X) 2.8 .000052 Interferon-inducible HIN-200 gene family member17

MNDA (myeloid nuclear differentiation antigen) 2.2 .0092 HIN-200 gene family member17,19

OAS1 (2, 5 oligoadenylate cyclase) 2.1 .000052 IFN-inducible gene which activates RNaseL

B-cell anergy

LGALS1 (galectin-1) 3.6 .00038 Negatively regulates B-cell proliferation upon BCR ligation14;

up-regulated in anergic murine B cells12

CD200R1 (CD200 receptor 1) 2.4 .0034 Inhibitory receptor highly expressed on memory B cells and

plasmablasts; definitive role in B cell activation not established11

LAX1 (lymphocyte transmembrane adapter 1) 2.1 .032 Dampens B-cell response to BCR engagement16

B-cell apoptosis

LGALS1 3.6 .00038 Expressed in IgM� memory cells, reported to enhance apoptosis via

inhibition of Akt phosphorylation and up-regulation of Bim13

PYHIN1 2.8 .000052 As an HIN-200 gene family member, contains an N-terminal

PAAD/DAPIN/Pyrin domain that mediates binding with proteins

involved in apoptotic NF-�B and caspase signaling17

DAPK2 (death-associated protein kinase 2) 2.4 .043 Calcium/calmodulin-dependent protein kinase, which induces apoptosis15

MNDA 2.2 .0092 As an HIN-200 gene family member, contains an N-terminal

PAAD/DAPIN/Pyrin domain that mediates binding with proteins

involved in apoptotic NF-�B and caspase signaling17

B-cell survival

TCL1A (T-cell lymphoma antigen 1A) 0.34 .000075 Reported to enhance B-cell survival by induction of Mcl-113

B-cell lymphomagenesis

SOX5 (SRY-box 5) 3.6 .00062 Reported to be up-regulated in splenic follicular lymphoma20

ITGAX, (CD11C), (integrin, � X) 2.9 .00066 High expression associated with splenic marginal zone lymphoma21

MNDA 2.2 .0092 Expressed in marginal zone B cells, up-regulated in marginal zone

lymphoma22

BACH2 0.46 .022 Down-regulated in Waldenström macroglobulinemia10

FOXP1 0.44 .00072 Up-regulation of FOXP1 in mucosa-associated lymphoid tissue

lymphoma may be associated with neoplastic transformation23

TCL1A (T-cell lymphoma 1A) 0.34 .00075 Up-regulated in splenic marginal zone lymphoma18

SELL (L-selectin) 0.34 .00031 Up-regulated in splenic marginal zone lymphoma18

IL-4R (IL-4 receptor) 0.31 .00017 Down-regulated in mantle cell lymphoma9 and Waldenström

macroglobulinemia10

LMO2 (LIM domain only 2) 0.31 .0010 Up-regulation corresponds to increased survival in diffuse large B-cell

lymphoma24

IFN indicates interferon; PAAD/DAPIN, Pyrin, AIM (Absent in Melanoma), ASC (Apoptosis-associated-speck-like protein containing a caspase recruitment domain[CARD]) and death-domain (DD)-like Domain in Apoptosis and Interferon response; and NF�B, nuclear factor-�B.

*Bonferroni corrected.

5430 CHARLES et al BLOOD, 19 MAY 2011 � VOLUME 117, NUMBER 20

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indicates that G6� B cells’ propensity for apoptosis is not fullydependent on CD95, although CD95 was upregulated on severalHCV�MC� patients’ G6� cells. A larger percentage of apoptoticannexin V�7-AAD� cells had lower CD21 expression comparedwith annexin V�7-AAD� cells (Figure 6B). This was true for bothG6� and G6� populations. Surface CD20 expression was similaramong all subsets, except for G6� annexin V�7-AAD� cells.Importantly, 6 hours of incubation with anti-CD95 did not signifi-cantly alter the percentages of CD20� B cells that were G6� (37 vs29% at 0 and 6 hours, respectively), nor did it cause down-regulation of CD21 on G6� or G6� B cells (Figure 6C), indicatingthat cell surface CD20, CD21, and G6 expression was stable andnot significantly affected by apoptosis or cell death. Taken together,these data suggest that, in the absence of survival signals, G6�

B cells, and in particular, the CD21low G6� subset, are prone tospontaneous cell death and apoptosis.

CD27�CD21high, but not CD27�CD21low, G6� B cells fromHCV�MC� patients efficiently differentiate intoantibody-secreting plasmablasts

To determine whether CD27�CD21low B cells could proliferate anddifferentiate in response to stimulatory signals, we stimulatedsorted CD27�CD21high, CD27�CD21low, CD27�CD21high, andCD27�CD21low G6� B cells from 4 HCV�MC� subjects. For afifth patient, CD27�CD21low, CD27�CD21high, CD27� G6� B cellsand total G6� B cells were sorted. We used 2 independent samplesto test whether our cell sorting procedure caused early cell death; inall subsets, more than 85% of cells were viable, as measured by4,6-diamidino-2-phenylindole staining (supplemental Figure 4).Cells were cultured for 6 days with CD40L, IL-2, and IL-10 andanalyzed for differentiation toward antibody-secreting cells.Using IgM ELISPOT, we found that the CD27�CD21high, com-pared with CD27�CD21low and CD27� G6� B-cell subsets, weremore efficient at differentiating to IgM-secreting plasmablasts onCD40L/IL-2/IL-10 stimulation (Figure 7A). To confirm thesefindings, we quantitated IgM in the cell culture supernatants usingELISA. The CD27�CD21high cell culture supernatants all had

higher levels of IgM compared with their CD27�CD21low andCD27� counterparts (Figure 7B). Class switch did not account forthe decreased IgM in these subsets, as we consistently detected 150 ng/mL IgG in cell culture supernatants.

We confirmed that the IgM produced by the stimulated G6�

B-cell subsets had RF activity by demonstrating that they couldbind human IgG1 (Figure 7C). As a control, we tested patient LDU125’s G6� B-cell supernatant. Although IgM was present at a totalconcentration of approximately 0.5 �g/mL, we detected no signifi-cant RF activity. Taken together, these results suggest that differen-tiation to pathogenic RF-secreting plasmablasts on CD40L/IL-2/IL-10 stimulation is less efficient in the CD27�CD21low, comparedwith the CD27�CD21high, G6� B-cell subset.

Discussion

We previously reported that HCV�MC� patients have clonalexpansions of IgM���CD27� peripheral B cells.6 Here we havetranscriptionally profiled IgM���CD27� B cells isolated fromHCV�MC� patients, and we have identified a set of 69 genes thatare differentially expressed. This set is notable for interferonresponse genes, B-cell activation markers, transcription factors,and glycoprotein synthesis genes. Although several genes previ-ously implicated in B-cell neoplasia (eg, SOX5, CD11C, andMNDA) are up-regulated among these cells, others (LMO2, IL-4R,SELL, TCL1A, FOXP1, and BACH2) are down-regulated. Thus,although IgM���CD27� B cells are responsible for MC pathogen-esis, their overall gene expression pattern suggests that there existsa subpopulation that is biased toward anergy and/or apoptosis.

The most highly up-regulated gene was the -galactoside–binding lectin galectin-1, which is intriguing given its knownpleiotropic roles in innate and adaptive immune responses. Galec-tin-1 (and CD11C) transcripts have been identified as beingup-regulated in anergic murine B cells.12 Moreover, galectin-1 hasbeen shown to be overexpressed in IgM�CD27� B cells, is further

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Figure 2. Relative expression of selected genes in IgM���CD27� B cells determined by quantitative RT-PCR. Values are normalized to RPS11 for cDNA content and toa universal standard RNA. Comparisons between groups were made using the Kruskal-Wallis test, and P values for HCV�MC� compared with HCV�MC� subjects werecomputed using Dunn post test. N.S. indicates not significant (P � .05).

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induced on BCR ligation, has been associated with Bim-mediatedapoptosis,13 and may negatively regulate B-cell proliferation andBCR-mediated tyrosine phosphorylation.14 Also up-regulated wereCD200R1, a potentially inhibitory immune receptor that is up-regulated in human tonsillar memory B cells and plasmablasts,11

and DAPK2, a calcium/calmodulin-dependent serine/threoninekinase responsible for IFN-�, TNF-�, and Fas ligand–inducedapoptosis.15 Similarly up-regulated was LAX1, a negative regulatorof BCR-mediated calcium flux, Akt activation, and cell survival

and proliferation.16 PYHIN1 (IFIX) and MNDA, 2 members of theIFN-inducible HIN-200 gene family implicated in apoptosis,17

were also up-regulated. One of the most down-regulated genes wasTCL1A, a proto-oncogene up-regulated in splenic marginal zonelymphoma cells.18 TCL1A is induced in naive B cells on BCRligation, and this may protect B cells from apoptosis.13 Thedown-regulation of IL-4R in HCV�MC� patients’ clonally ex-panded B cells is additionally supportive of a proapoptotic state,given that IL-4 is an effective antiapoptotic cytokine for B cells and

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Figure 3. Phenotypic analysis of HCV�MC� patients’ clonal B cells. PBMCs from one HCV�MC� (ECH 542, red) and one HCV�MC� patient (LDU 125, blue)representative of 6 samples each are shown. (A) Staining of PBMCs with anti-CD20 and G6 mAbs. (B) Forward light scatter analysis of G6� and G6� B cells from theHCV�MC� person. Scanning electron micrographs of immunomagnetically sorted G6� and G6� B cells from the HCV�MC� person. (C-D) Scale bars on the low and highpower magnifications represent 5 �m and 1 �m, respectively. Propidium iodide and Ki-67 staining of immunomagnetically sorted G6� and G6� B cells from the HCV�MC�

person for Ki-67 and propidium iodide (E-F). Staining for cell surface CD10, CD21, CD27, CD11c, FCRL4, and IL-4R (G). Electron micrographs (original magnifica-tion �10 000) were taken with a FEI Tecnai G2 Spirit BioTWIN Transmission Electron Microscope equipped with a Gatan ES500W Erlangshen CCD camera andDigitalMicrograph Software.

5432 CHARLES et al BLOOD, 19 MAY 2011 � VOLUME 117, NUMBER 20

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that IL-4R engagement can abrogate Fas-mediatedB cell apoptosis.36

We found that HCV�MC� patients’ IgM���CD27� B cells hadup-regulation of CD11C, SOX5, and FGR, similar to what wasrecently reported in an FCRL4� CD27� memory tonsillar B-cellsubset. FCRL4 is an orphan receptor whose intracellular domaincontains 3 immunoreceptor tyrosine-based inhibitory motifs thatcan inhibit BCR-mediated signaling.37 The primary location ofFCRL4� cells in the marginal zone equivalents of tonsillar andPeyer patch epithelia and in mucosa-associated lymphoid tissuelymphomas34 suggests that these cells may play a role in mucosaldefense against pathogens. FCRL4� B cells are reported to be large

CD20high, CD11c�, CXCR3�, CD95�, CD21low proliferatingB cells.29,33 Although we did not detect increased FCRL4 transcriptin clonally expanded G6� peripheral B cells, they shared severalfeatures with FCRL4� cells, as they expressed FCRL4 protein andwere predominantly CD20high, CD21low, CD11c� B cells. However,in contrast to the concordance between FCRL4 mRNA transcriptand FCRL4 protein expression that has been reported in healthypersons’ tonsillar B cells,35 FCRL4 protein, but not mRNA, waselevated in HCV�MC� patients’ peripheral B cells. We speculatethat an increased level of FCRL4 mRNA is not necessary tomaintain the relatively increased surface FCRL4 protein expressionon these cells. Further research is necessary to investigate whether

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Figure 4. Immunophenotypic profiles of MC� and MC� patients’ G6�, G6�, and total B cells. Data are flow cytometric analyses of PBMCs from HCV�MC� (n � 9) andMC� (n � 11; SVR � 2, HCV Ab� � 2, HCV RNA� � 6) subjects. Cell surface marker expression in each subject’s G6� and G6� B cells is shown in the column graphs. Thescatterplots represent expression of cell surface markers versus the proportion of total B cells that are G6�. In the scatterplots: Œ represents MC� patients; ‚, HCV RNA�

patients; and �, HCV RNA� patients. MFI indicates geometric mean fluorescence intensity. The P values for the column graphs were determined using the Kruskal-Wallis andDunn multiple comparison tests. *P .05. **P .01. n.s. indicates not significant (P � .05). Black bars represent medians. For the scatterplots, R was calculated using theSpearman correlation coefficient.

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HCV�MC� patients’ mucosal-associated and peripheral B cellsshare common transcriptional, immunophenotypic, and functionalcharacteristics.

Decreased CD21 expression is observed on apoptotic38 B cells.Increased CD21low B cells are seen in systemic lupus erythemato-sis,39 chronic variable immunodeficiency,8,40 and rheumatoid arthri-tis,8 and increases of CD21low, CD27�, FCRL4� B cells have beendescribed in HIV-viremic33 and Plasmodium falciparum–exposedpersons.41 Interestingly, the expanded B cells in chronic variableimmunodeficiency patients are activated, have up-regulated SOX5expression, and diminished calcium signaling and proliferativeresponses to BCR or CD40L stimulation.8,40 In HIV-viremicpatients, they have up-regulated CD95 expression and are prone toapoptosis.42 Moreover, these cells may be functionally “ex-hausted,” as they demonstrate less ability to proliferate on BCRligation with or without T-cell help and they have decreased abilityto differentiate into antibody-secreting cells on stimulation withStaphylococcus aureus Cowan and CpG.33

Given these facts, and because CD21, as part of the B-cellcoreceptor complex, augments BCR-mediated signaling, we sus-pected that HCV�MC� patients’ expanded CD21low G6� B-cellsubset represented a relatively anergic B-cell population. Inaddition, because transcriptional profiling revealed the up-regulation of several proapoptotic genes, we hypothesized thatthese B cells were prone to apoptosis. Indeed, we found that G6�,compared with G6�, B cells were more prone to apoptosis and celldeath and that these cells were skewed toward the CD21low subset.Our data are consistent with earlier reports suggesting that HCV�

patients’ memory B cells are prone to apoptosis and that this mayserve as a feedback inhibition mechanism to prevent exaggeratedautoreactive B-cell responses.43,44 In addition, CD21low B-cellsubpopulations from healthy, HCV�MC�, or HCV�MC� personswere relatively hyporesponsive to BCR stimulation, as measuredby Ca2� mobilization. Thus, the frequent down-regulation of CD21among G6� B cells suggests that these cells are relatively anergic.Moreover, the G6�CD21low subpopulation was only weakly in-duced to differentiate to IgM-secreting plasmablasts on stimulationwith CD40L, IL-2, and IL-10, suggesting that these cells in vivomay be refractory to stimulation by T cell–mediated signals in thecontext of BCR engagement. Whether other stimuli (eg, TLRagonists or IFN-�) can induce these cells to differentiate remainsan open question. We surmise that the down-regulation of CD21 isa homeostatic control mechanism that attenuates autoreactive RF�

B-cell responses to chronic HCV-containing immune complexstimulation.

To summarize, we have found that HCV�MC� patients’clonally expanded peripheral B cells have global transcriptionalfeatures not of proto-oncogenesis, but rather of stimulatory hypore-sponsiveness and anergy. Immunophenotypically, they resembleactivated memory B cells and share several features with previ-ously described CD21low and FCRL4� B-cell populations. Al-though we have shown that the overall clonal population is capableof differentiating into RF-secreting plasmablasts, the expandedCD21low fraction is prone to anergy. Together, our results suggestoverall attenuation mechanisms present in these autoreactive-proneB cells that limit their pathogenic expansion and differentiation in

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Figure 5. CD27�CD21low, compared with CD27�CD21high, B cells have attenuated Ca2� responses after BCR cross-linking. Analyses of B cells from one healthyvolunteer (ECH 503), 2 HCV�MC� patients (ECH 530 and ECH 537), and 5 HCV�MC� patients (ECH 546, ECH 559, 110, LDU 125, and 1931) are shown. PBMCs from1931 and LDU 125 were collected 4 and 10 months, respectively, after the cells collected for the microarray and primary immunophenotyping experiments. (Left panels) CD27 and CD21staining of IgG� B cells. Indo-1-AM-loaded cells were stained with anti-CD19, anti-IgG, anti-CD27, and anti-CD21, warmed to 37°C, and, after establishing a baseline for 30 seconds,stimulated with 10 �g/mL goat F(ab)2 anti–human IgM. Kinetic graphs represent ratios of bound/unbound Indo-1 over time for CD27�CD21high, CD27�CD21low, CD27�CD21high, andCD27�CD21low B-cell populations. Single arrows indicate injection of F(ab)2 anti–human IgM; and double arrow, injection of 10 �g/mL ionomycin.

5434 CHARLES et al BLOOD, 19 MAY 2011 � VOLUME 117, NUMBER 20

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vivo. Several clinical lines of evidence suggest that these B-cellattenuation mechanisms may serve to limit disease activity invivo. First, the incidence of clinically apparent MC duringchronic HCV infection is rather low (1 per 1000 person-years inUS veterans3), even in the setting of decades of infection.Second, clinical signs and symptoms of MC frequently wax andwane, with no apparent correlation with fluctuations in HCVRNA or liver inflammation.45 Third, even among patients withMC, progression to overt B-NHL is a relatively rare phenom-enon (6.6 per 1000 person-years in an Italian cohort46).

Most importantly, these attenuation mechanisms may, in part,explain the relatively ineffective anti-HCV humoral response. Ithas been shown that VH169 partially encoded Abs are enriched inanti-influenza47,48 and anti-HIV49 responses. One of the most potentcross-neutralizing anti-HCV mAbs, CBH5, is partially encoded by

VH169.50 VH169 encodes a distinct hydrophobic complementarity-determining region-H2 loop, and it has been speculated that thisconfers antiviral activity by binding to hydrophobic viral targets.47

As autoreactive RF is partially encoded by VH169, it is tempting tospeculate that homeostatic mechanisms that are up-regulated toattenuate self-reactivity have the unintended consequence of abro-gating effective VH169-mediated anti-HCV responses.

It remains unclear why only some HCV-infected personsdevelop MC and why these attenuation mechanisms fail to preventthe development of NHL in some HCV�MC� patients. It must beemphasized that we have studied patients’ peripheral B cells; itremains to be seen whether B cells at other anatomic sites, such asthe liver or perihepatic lymph nodes, display similar features ofattenuation. Further patient-oriented research will be necessary toanswer these central questions, as well as to determine whether the

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Figure 6. G6� B cells are prone to apoptosis and cell death, and the apoptotic cells are disproportionately CD21low. PBMCs were incubated for 6 hours in the presenceof media alone, mouse IgG1 (isotype control), or 1 �g/mL anti-CD95 with 2 �g/mL protein G. Cells were then stained with annexin V, 7-AAD, anti-CD20, anti-CD21, and G6mAbs and were analyzed by flow cytometry. Analyses of G6� and G6� CD20� B cells are shown. (A) Annexin V and 7-AAD staining of cells at baseline and after 6 hours ofstimulation. (B) Analysis of surface CD21 and CD20 expression on cells incubated for 6 hours with �-CD95/protein G; annexin V�7-AAD�, annexin V�7-AAD�, and annexinV�7-AAD� subsets. (C) Cell surface anti-CD20 (of total lymphocytes), G6 (of total B cells), and anti-CD21 (of G6� and G6� B cells) staining at baseline, compared with 6 hours,of stimulation. PBMCs from LDU 125 were collected 10 months after the cells collected for the microarray and primary immunophenotyping experiments. Data arerepresentative of 3 independent experiments.

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autoregulatory mechanisms described here depend on anatomiccontext, are specific for HCV�MC� patients or are common toother autoimmune diseases.

Acknowledgments

The authors thank the patient volunteers for their generosity; RoyJefferis for G6 monoclonal antibody; Gotz Ehrhardt for F(ab)2

anti-FCRL4 antibody; Donna Brassil, Veronica Whalen, and RhondaKost of the Rockefeller University Center for Clinical and Transla-tional Science for assistance with subject enrollment and studymanagement; Natasha Levenkova for statistical advice; and SantaMaria Di Vittorio for administrative assistance.

This study was supported in part by the National Institutes ofHealth/National Institute of Allergy and Infectious Diseases(R01AI60561, L.B.D.; and K08AI075031, E.D.C.), the Irma T.Hirschl/Monique Weill-Caulier Trust (L.B.D.), Center for Transla-tional Science Award (Pilot Grant CCL3001018) (E.D.C.), andCenter for Translational Science Award (grant UL1 RR024143, toRockefeller University), from the National Center for Research

Resources, a component of National Institutes of Health. Sortingon the FACSAria II was made possible by support from the EmpireState Stem Cell Fund (New York State Department of Healthcontract C023046).

Opinions expressed here are solely those of the authors and donot necessarily reflect those of the Empire State Stem Cell Fund,the New York State Department of Health, or the state of New York.

Authorship

Contribution: E.D.C., C.B., and L.B.D. devised and conducted theexperiments; E.D.C., K.M., A.H.T., and I.M.J. provided patientreferrals; E.D.C., L.B.D., C.M.R., S.M., K.D.R., K.M., A.H.T., andI.M.J. interpreted results; and E.D.C. and L.B.D. wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Lynn B. Dustin, Center for the Study ofHepatitis C, Laboratory of Virology and Infectious Disease,Rockefeller University, Box 64, 1230 York Ave, New York, NY10065; e-mail: dustinl@rockefeller.edu.

References

1. Agnello V, Chung RT, Kaplan LM. A role for hepa-titis C virus infection in type II cryoglobulinemia.N Engl J Med. 1992;327(21):1490-1495.

2. Sansonno D, Dammacco F. Hepatitis C virus, cryo-globulinaemia, and vasculitis: immune complex rela-tions. Lancet Infect Dis. 2005;5(4):227-236.

3. Giordano TP, Henderson L, Landgren O, et al.

Risk of non-Hodgkin lymphoma and lymphoprolif-erative precursor diseases in US veterans withhepatitis C virus. JAMA. 2007;297(18):2010-2017.

4. Hermine O, Lefrere F, Bronowicki JP, et al. Re-gression of splenic lymphoma with villouslymphocytes after treatment of hepatitis C virusinfection. N Engl J Med. 2002;347(2):89-94.

5. Charles ED, Dustin LB. Hepatitis C virus-inducedcryoglobulinemia. Kidney Int. 2009;76(8):818-824.

6. Charles ED, Green RM, Marukian S, et al. Clonalexpansion of immunoglobulin M�CD27� B cellsin HCV-associated mixed cryoglobulinemia.Blood. 2008;111(3):1344-1356.

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Figure 7. CD27�CD21low, compared with CD27� and CD27�CD21high, G6� B cells from HCV�MC� patients demonstrate decreased differentiation to IgMRF-secreting plasmablasts on CD40L/IL-2/IL-10 stimulation. Analyses of 5 HCV�MC� patients’ B cells are shown. B cells from LDU 125 were collected 10 months after thecells collected for the microarray and primary immunophenotyping experiments. CD27�CD21low, CD27�CD21high, CD27�CD21low, and CD27�CD21high G6� B cells werebulk-sorted and plated in a 96-well dish. For patient LDU 125, CD27�CD21low, CD27�CD21high, CD27� G6� B cells and total G6� B cells were sorted. After 6 days of incubationin media supplemented with CD40L, IL-2, and IL-10, cells and supernatants were collected. (A) IgM enzyme-linked immunospot of stimulated B cells. (B) IgM ELISA of cellculture supernatants. (C) IgM RF ELISA of cell culture supernatants.

5436 CHARLES et al BLOOD, 19 MAY 2011 � VOLUME 117, NUMBER 20

For personal use only.on March 7, 2015. by guest www.bloodjournal.orgFrom

7. Ivanovski M, Silvestri F, Pozzato G, et al. Somatichypermutation, clonal diversity, and preferentialexpression of the VH 51p1/VL kv325 immuno-globulin gene combination in hepatitis C virus-associated immunocytomas. Blood. 1998;91(7):2433-2442.

8. Isnardi I, Ng YS, Menard L, et al. Complementreceptor 2/CD21� human naive B cells containmostly autoreactive unresponsive clones. Blood.2010;115(24):5026-5036.

9. Ek S, Hogerkorp CM, Dictor M, Ehinger M,Borrebaeck CA. Mantle cell lymphomas expressa distinct genetic signature affecting lymphocytetrafficking and growth regulation as comparedwith subpopulations of normal human B cells.Cancer Res. 2002;62(15):4398-4405.

10. Gutierrez NC, Ocio EM, de Las Rivas J, et al.Gene expression profiling of B lymphocytes andplasma cells from Waldenstrom’s macroglobu-linemia: comparison with expression patterns ofthe same cell counterparts from chronic lympho-cytic leukemia, multiple myeloma and normal in-dividuals. Leukemia. 2007;21(3):541-549.

11. Rijkers ES, de Ruiter T, Baridi A, Veninga H,Hoek RM, Meyaard L. The inhibitory CD200R isdifferentially expressed on human and mouseT and B lymphocytes. Mol Immunol. 2008;45(4):1126-1135.

12. Clark AG, Chen S, Zhang H, et al. Multifunctionalregulators of cell growth are differentially ex-pressed in anergic murine B cells. Mol Immunol.2007;44(6):1274-1285.

13. Tabrizi SJ, Niiro H, Masui M, et al. T cell leukemia/lymphoma 1 and galectin-1 regulate survival/celldeath pathways in human naive and IgM� memoryB cells through altering balances in Bcl-2 family pro-teins. J Immunol. 2009;182(3):1490-1499.

14. Yu X, Siegel R, Roeder RG. Interaction of theB cell-specific transcriptional coactivator OCA-Band galectin-1 and a possible role in regulatingBCR-mediated B cell proliferation. J Biol Chem.2006;281(22):15505-15516.

15. Cohen O, Feinstein E, Kimchi A. DAP-kinase is aCa2�/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalyticactivity. EMBO J. 1997;16(5):998-1008.

16. Zhu M, Granillo O, Wen R, et al. Negative regula-tion of lymphocyte activation by the adaptor pro-tein LAX. J Immunol. 2005;174(9):5612-5619.

17. Ludlow LE, Johnstone RW, Clarke CJ. The HIN-200 family: more than interferon-inducible genes?Exp Cell Res. 2005;308(1):1-17.

18. Ruiz-Ballesteros E, Mollejo M, Rodriguez A, et al.Splenic marginal zone lymphoma: proposal ofnew diagnostic and prognostic markers identifiedafter tissue and cDNA microarray analysis. Blood.2005;106(5):1831-1838.

19. Briggs RC, Briggs JA, Ozer J, et al. The humanmyeloid cell nuclear differentiation antigen geneis one of at least two related interferon-induciblegenes located on chromosome 1q that are ex-pressed specifically in hematopoietic cells. Blood.1994;83(8):2153-2162.

20. Storlazzi CT, Albano F, Lo Cunsolo C, et al. Up-regulation of the SOX5 by promoter swappingwith the P2RY8 gene in primary splenic follicularlymphoma. Leukemia. 2007;21(10):2221-2225.

21. Kost CB, Holden JT, Mann KP. Marginal zoneB-cell lymphoma: a retrospective immunopheno-

typic analysis. Cytometry B Clin Cytom. 2008;74(5):282-286.

22. Kanellis G, Roncador G, Arribas A, et al. Identifi-cation of MNDA as a new marker for nodal mar-ginal zone lymphoma. Leukemia. 2009;23(10):1847-1857.

23. Sagaert X, de Paepe P, Libbrecht L, et al. Fork-head box protein P1 expression in mucosa-associated lymphoid tissue lymphomas predictspoor prognosis and transformation to diffuselarge B-cell lymphoma. J Clin Oncol. 2006;24(16):2490-2497.

24. Lossos IS, Czerwinski DK, Alizadeh AA, et al.Prediction of survival in diffuse large-B-cell lym-phoma based on the expression of six genes.N Engl J Med. 2004;350(18):1828-1837.

25. Habermann TM, Wang SS, Maurer MJ, et al. Hostimmune gene polymorphisms in combination withclinical and demographic factors predict late sur-vival in diffuse large B-cell lymphoma patients inthe pre-rituximab era. Blood. 2008;112(7):2694-2702.

26. Postigo AA, Corbi AL, Sanchez-Madrid F,de Landazuri MO. Regulated expression andfunction of CD11c/CD18 integrin on human Blymphocytes: relation between attachment to fi-brinogen and triggering of proliferation throughCD11c/CD18. J Exp Med. 1991;174(6):1313-1322.

27. Tangye SG, van de Weerdt BC, Avery DT,Hodgkin PD. CD84 is up-regulated on a majorpopulation of human memory B cells and recruitsthe SH2 domain containing proteins SAP andEAT-2. Eur J Immunol. 2002;32(6):1640-1649.

28. Tomayko MM, Anderson SM, Brayton CE, et al.Systematic comparison of gene expression be-tween murine memory and naive B cells demon-strates that memory B cells have unique signalingcapabilities. J Immunol. 2008;181(1):27-38.

29. Ehrhardt GR, Hijikata A, Kitamura H, Ohara O,Wang JY, Cooper MD. Discriminating gene ex-pression profiles of memory B cell subpopula-tions. J Exp Med. 2008;205(8):1807-1817.

30. Carbonari M, Caprini E, Tedesco T, et al. HepatitisC virus drives the unconstrained monoclonal ex-pansion of VH1–69-expressing memory B cells intype II cryoglobulinemia: a model of infection-driven lymphomagenesis. J Immunol. 2005;174(10):6532-6539.

31. Potter KN, Li Y, Mageed RA, Jefferis R, CapraJD. Molecular characterization of the VH1-specific variable region determinants recognizedby anti-idiotypic monoclonal antibodies G6 andG8. Scand J Immunol. 1999;50(1):14-20.

32. Charles ED, Orloff MI, Dustin LB. A flow cytometry-based strategy to identify and express IgM fromVH1–69� clonal peripheral B cells. J Immunol Meth-ods. 2011;363(2):210-220.

33. Moir S, Ho J, Malaspina A, et al. Evidence forHIV-associated B cell exhaustion in a dysfunc-tional memory B cell compartment in HIV-infectedviremic individuals. J Exp Med. 2008;205(8):1797-1805.

34. Falini B, Tiacci E, Pucciarini A, et al. Expressionof the IRTA1 receptor identifies intraepithelial andsubepithelial marginal zone B cells of the mu-cosa-associated lymphoid tissue (MALT). Blood.2003;102(10):3684-3692.

35. Ehrhardt GR, Hsu JT, Gartland L, et al. Expres-sion of the immunoregulatory molecule FcRH4defines a distinctive tissue-based population of

memory B cells. J Exp Med. 2005;202(6):783-791.

36. Rothstein TL. Inducible resistance to Fas-mediated apoptosis in B cells. Cell Res. 2000;10(4):245-266.

37. Ehrhardt GR, Davis RS, Hsu JT, Leu CM,Ehrhardt A, Cooper MD. The inhibitory potentialof Fc receptor homolog 4 on memory B cells.Proc Natl Acad Sci U S A. 2003;100(23):13489-13494.

38. Segundo C, Medina F, Rodriguez C, Martinez-Palencia R, Leyva-Cobian F, Brieva JA. Surfacemolecule loss and bleb formation by human ger-minal center B cells undergoing apoptosis: role ofapoptotic blebs in monocyte chemotaxis. Blood.1999;94(3):1012-1020.

39. Wehr C, Eibel H, Masilamani M, et al. A newCD21low B-cell population in the peripheral bloodof patients with SLE. Clin Immunol. 2004;113(2):161-171.

40. Rakhmanov M, Keller B, Gutenberger S, et al.Circulating CD21low B cells in common variableimmunodeficiency resemble tissue homing, in-nate-like B cells. Proc Natl Acad Sci U S A. 2009;106(32):13451-13456.

41. Weiss GE, Crompton PD, Li S, et al. Atypicalmemory B cells are greatly expanded in individu-als living in a malaria-endemic area. J Immunol.2009;183(3):2176-2182.

42. Moir S, Malaspina A, Pickeral OK, et al. De-creased survival of B cells of HIV-viremic patientsmediated by altered expression of receptors ofthe TNF superfamily. J Exp Med. 2004;200(7):587-599.

43. Racanelli V, Frassanito MA, Leone P, et al. Anti-body production and in vitro behavior of CD27-defined B-cell subsets: persistent hepatitis C vi-rus infection changes the rules. J Virol. 2006;80(8):3923-3934.

44. De Re V, Pavan A, Sansonno S, Sansonno D,Racanelli V. Clonal CD27� CD19� B cell expan-sion through inhibition of FC gammaIIR inHCV(�) cryoglobulinemic patients. Ann N Y AcadSci. 2009;1173:326-333.

45. Cacoub P, Hausfater P, Musset L, Piette JC.Mixed cryoglobulinemia in hepatitis C patients:GERMIVIC. Ann Med Interne (Paris). 2000;151(1):20-29.

46. Monti G, Pioltelli P, Saccardo F, et al. Incidenceand characteristics of non-Hodgkin lymphomas ina multicenter case file of patients with hepatitis Cvirus-related symptomatic mixed cryoglobuline-mias. Arch Intern Med. 2005;165(1):101-105.

47. Sui J, Hwang WC, Perez S, et al. Structural andfunctional bases for broad-spectrum neutraliza-tion of avian and human influenza A viruses. NatStruct Mol Biol. 2009;16(3):265-273.

48. Corti D, Suguitan AL Jr, Pinna D, et al. Hetero-subtypic neutralizing antibodies are produced byindividuals immunized with a seasonal influenzavaccine. J Clin Invest. 2010;120(5):1663-1673.

49. Huang CC, Venturi M, Majeed S, et al. Structuralbasis of tyrosine sulfation and VH-gene usage inantibodies that recognize the HIV type 1 corecep-tor-binding site on gp120. Proc Natl Acad SciU S A. 2004;101(9):2706-2711.

50. Chan CH, Hadlock KG, Foung SK, Levy S.V(H)1–69 gene is preferentially used by hepatitisC virus-associated B cell lymphomas and by nor-mal B cells responding to the E2 viral antigen.Blood. 2001;97(4):1023-1026.

B-CELL ANERGY IN MIXED CRYOGLOBULINEMIA 5437BLOOD, 19 MAY 2011 � VOLUME 117, NUMBER 20

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online March 18, 2011 originally publisheddoi:10.1182/blood-2010-10-312942

2011 117: 5425-5437  

Marks, Ira M. Jacobson, Charles M. Rice and Lynn B. DustinEdgar D. Charles, Claudia Brunetti, Svetlana Marukian, Kimberly D. Ritola, Andrew H. Talal, Kristen 

B-cell subsetlowcryoglobulinemia contain an expanded anergic CD21associated mixed−Clonal B cells in patients with hepatitis C virus

 

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