Clonal b cells in patients with hepatitis c virus–associated mixed

IMMUNOBIOLOGY
Clonal B cells in patients with hepatitis C virus–associated mixed
cryoglobulinemia 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 of
Gastroenterology and Hepatology, Weill Medical College of Cornell University, New York, NY; and 3University of Bari, Bari, Italy
Hepatitis C virus (HCV) is associated with
the B-cell lymphoproliferative disorders
mixed cryoglobulinemia (MC) and non-
Hodgkin lymphoma. We have previously
reported that HCV؉MC؉ patients have
clonal expansions of hypermutated, rheu-
matoid factor–bearing marginal zone-like
IgM؉CD27؉ peripheral B cells using the
VH1-69 gene. Here we coupled transcrip-
tional profiling with immunophenotypic
and functional studies to ascertain these
cells’ role in MC pathogenesis. Despite
their fundamental role in MC disease,
these B cells have overall transcriptional
features of anergy and apoptosis instead
of neoplastic transformation. Highly up-
regulated genes include SOX5, CD11C,
galectin-1, and FGR, similar to a previ-
ously described FCRL4؉ memory B-cell
subset and to an “exhausted,” anergic
CD21low memory B-cell subset in HIV؉
patients. Moreover, HCV؉MC؉ patients’
clonal peripheral B cells are enriched
with CD21low, CD11c؉, FCRL4high, IL-4Rlow
memory B cells. In contrast to the func-
tional, rheumatoid factor–secreting
CD27؉CD21high subset, the CD27؉CD21low
subpopulation exhibits decreased cal-
cium mobilization and does not efficiently
differentiate into rheumatoid factor–
secreting plasmablasts, suggesting that
a large proportion of HCV؉MC؉ patients’
clonally expanded peripheral B cells is
prone to anergy and/or apoptosis. Down-
regulation of multiple activation path-
ways may represent a homeostatic
mechanism attenuating otherwise uncon-
trolled stimulation of circulating HCV-
containing immune complexes. This study
was registered at www.clinicaltrials.gov
as #NCT00435201. (Blood. 2011;117(20):
5425-5437)
Introduction
Hepatitis C virus (HCV) chronically infects approximately
170 million people worldwide and is the leading indicator for liver
transplantation in the United States. Although hepatocytes are the
primary target for HCV infection, the B-cell lymphoprolifera-
tive disorder mixed cryoglobulinemia (MC) affects up to 50% of
HCV patients.1 MC is characterized by the aberrant production
of monoclonal rheumatoid factor (RF)–containing immune
complexes that deposit on vascular endothelium of organs, such
as skin, kidneys, and peripheral nerves, eliciting a complement
C1q-mediated vasculitis.2 HCV has also been associated with
B-cell non-Hodgkin lymphoma (NHL),3 most frequently of
low-grade marginal zone or mucosa-associated lymphoid tissue
subtypes, although associations with higher-grade NHL have
been reported.
HCV-induced B-cell dysregulation probably represents a con-
tinuum from the relatively benign clonal B-cell expansion of MC to
overt NHL. The continued presence of HCV is necessary for
abnormal B-cell lymphoproliferation, as eradication of HCV
typically results in resolution of both HCV-related MC and NHL.4
Clonal B-cell populations are present in the liver and peripheral
blood of HCVϩMCϩ patients5; such B cells demonstrate biased
usage of the RF-encoding VH1-69 and V␬3-20 gene segments,6 as
do B cells isolated from lymph nodes of HCV-NHL patients.7 It
remains unclear why B cells undergo clonal proliferation during
chronic HCV infection.
It is probable that HCV-induced B-cell lymphoproliferation is
not the result of direct B-cell infection or transformation, but rather,
an indirect process arising from chronic antigenic stimulation 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 precise
antigen(s) and stimulatory mechanisms have remained elusive. We
have previously shown that HCVϩMCϩ patients’ clonal B cells are
predominantly IgM memory B cells expressing modestly hypermu-
tated immunoglobulin genes; phylogenetic analysis supports a
process of antigen-directed affinity maturation. However, many of
these clonal cells have decreased expression of CD21, the CR2
complement 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 to
these cells, as has been demonstrated among CD21low naive B cells
from 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 HCVϩMCϩ patients’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 together, our data suggest that, although HCVϩMCϩ
Submitted October 11, 2010; accepted March 6, 2011. Prepublished online as
Blood 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 charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by The American Society of Hematology
5425BLOOD, 19 MAY 2011 ⅐ VOLUME 117, NUMBER 20
For personal use only.on March 7, 2015.by guestwww.bloodjournal.orgFrom

patients 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 population may be refractory to
ongoing antigenic stimulation.
Methods
Patients
The studies were approved by the Institutional Review Boards at the
Rockefeller University and New York Presbyterian Hospitals. Donors gave
written informed consent according to the Declaration of Helsinki before
enrollment. We enrolled HCV AbϪ, HCV Abϩ/HCV RNAϩ, and HCV
Abϩ/HCV RNAϪ volunteers. No subjects received interferon or immuno-
suppressive therapy within 6 months of enrollment. Blood was obtained by
peripheral blood draw and leukapheresis. Peripheral blood mononuclear
cells (PBMCs) were prepared as previously described.6
Clinical tests
HCV RNA was quantified clinically by 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 measure-
ments, were performed as part routine medical care. Testing for MC was
performed as previously described.6
IgM؉␬؉CD27؉ B-cell isolation
IgMϩ␬ϩ B cells were isolated from PBMCs by negative selection to
minimize transcriptional changes effected by BCR signaling. All steps were
performed at 4°C. B cells were immunomagnetically isolated using a B Cell
Isolation 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 CD27ϩ 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 Micro
Kit (QIAGEN) with on-column DNase digestion. RNA integrity and
concentration were determined using Lab on a Chip Pico. Samples with
RNA integrity numbers Ͼ 9.0 were used for downstream processing. 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
aldehyde-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 Illumina
BeadStudio software (Version 3.2). Datasets were analyzed using Gene-
Spring GX Version 11.1 (Agilent Technologies). Raw signal values were
log-transformed, chips were normalized to the 50th percentile, and genes
normalized to the median signal. This dataset was filtered to include genes
with signals above background. Welch t test (P ϭ .05, Benjamini-Hochberg
false discovery rate ϭ 0.05) was used to test for differences in genes
between groups. The resulting set 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 calculated using
GeneSpring GX and Prism (GraphPad Software).
Quantitative RT-PCR
RNA was prepared from isolated B cells, as described under “RNA
extraction, 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 Supplemental
Materials link at the top of the online article) were constructed using the
PrimerBank Database (www.pga.mgh.harvard.edu/primerbank) and were
designed to span exon-intron borders to reduce the possibility of genomic
DNA 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 in
Universal Human Reference RNA (Stratagene). Fold change expression
was calculated using the 2Ϫ⌬⌬Ct method. Groups were compared using the
Kruskal-Wallis test; when Kruskal-Wallis P Ͻ .05, the HCVϩMCϩ and
HCVϩMCϪ 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 serum
albumin (Fraction V; Fisher Biotech) and 0.02% NaN3. All antibodies and
reagents were from BD Biosciences, except for G6 (provided by R. Jefferis),
F(abЈ)2 anti-FCRL4 biotin (provided by G. Erhardt and M. Cooper), and
Ki-67 fluorescein isothiocyanate (Invitrogen). Conjugation of G6 to biotin
and AlexaFluor-594 was performed using commercial kits (Pierce Chemi-
cal, Invitrogen). Analysis was performed within 1 hour on a BD LSRII flow
cytometer (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). Live
CD27ϩ/ϪCD21high/low G6ϩ, CD20ϩ B cells were bulk-sorted on a BD
FACSAria II (BD Biosciences). For assessment of postsort viability,
sorted samples were restained with 4,6-diamidino-2-phenylindole and
were reanalyzed by flow cytometry. Postsort analysis confirmed more
than 85% viability and more than 99% purity of sorted populations.
Cell cycle analysis
B cells were negatively isolated from PBMCs using EasySep Human B Cell
Enrichment Kit (StemCell Technologies) and incubated with biotinylated
G6 mAb. G6ϩ and G6Ϫ B-cell subsets were purified using streptavidin-
conjugated immunomagnetic beads. Cells were fixed in 80% ethanol. After
incubation with FITC-labeled Ki-67, cells were resuspended in PBS
containing 10 mg/mL RNase A and incubated at 37°C. Propidium iodide
20 ␮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 treated
with 2% uranyl acetate and Reynold lead citrate. Transmission electron
microscopy was performed at ϫ2000 and ϫ10 000 magnification.
Calcium mobilization assay
A total of 2 ϫ 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 and
suspended in HBSS with Ca2ϩ and 1% bovine serum albumin. Emission at
405 and 495 nm was measured to obtain a baseline, and then for 5 minutes.
After addition of 10 ␮g/mL goat F(abЈ)2 anti-IgM, 405/495 nm emission
ratios of IgGϪ B-cell subsets were analyzed with FlowJo software Version
9.2 (TreeStar).
Annexin V apoptosis assay
A total of 2 ϫ 106 PBMCs were incubated with 1 ␮g/mL anti-CD95 or
mouse IgG1 and 2 ␮g/mL Protein G (Invitrogen) in RPMI/10% fetal calf
serum at 37°C for 0 and 6 hours. Cells were washed with phosphate-
buffered saline and resuspended in 0.01M N-2-hydroxyethylpiperazine-NЈ-
2-ethanesulfonic acid (pH 7.4), 0.14M NaCl, 2.5mM CaCl2, and 2% fetal
5426 CHARLES et al BLOOD, 19 MAY 2011 ⅐ VOLUME 117, NUMBER 20

calf serum. After incubation with annexin V–phycoerythrin and 7-amino-
actinomycin at room temperature, cells were stained with anti-CD20 FITC
anti-CD21–allophycocyanin, and G6-biotin at room temperature. After
staining cells with streptavidin-Cy7-allophycocyanin, flow cytometry was
performed.
Immunoglobulin secretion assays
Cells (50 000/well in 96-well round-bottom plates) were cultured for 6 days
in RPMI supplemented with 10% fetal calf serum, 2mM L-glutamine,
100 U/mL penicillin/streptomycin, and 0.25 ␮g/mL amphotericin B, with
the addition of 6 U/mL IL-2 (R&D Systems), 200 ng/mL IL-10 (R&D
Systems), 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(abЈ)2 anti–human IgM (Jackson
ImmunoResearch Laboratories), and incubated at 37°C for 6 hours. Plates
were 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, human
IgG1␭ (Sigma-Aldrich), and incubated at room temperature for 1 hour.
Plates were then incubated with HRP-labeled goat-anti–human IgM, and
the assays were developed with TMB (BioFX Laboratories). After stopping
reactions with 1N H2SO4, A450 was measured on a FLUOstar Omega
microplate reader (BMG Laboratories).
Accession numbers
Microarray data are accessible through NCBI Gene Expression Omnibus
accession number GSE18084 (www.ncbi.nlm.nih.gov/geo/query/
acc.cgi?acc ϭ GSE18084).
Results
Characteristics of study subjects
Five HCV AbϪ volunteers, 7 sustained virologic responders
(SVRs), and 27 persons with chronic HCV infection were enrolled
for gene transcriptional analyses. Three additional HCVϩMCϪ and
2 additional HCVϩMCϩ subjects were enrolled only for immuno-
phenotypic and functional analyses (Table 1). Sixteen HCVϩ
subjects were MCϩ, and all 16 had evidence of clonal B-cell
populations, as demonstrated by complementarity-determining
region 3 PCR.6 Four of these subjects had lymphadenopathy;
lymph node biopsies performed by their physicians confirmed
low-grade B-NHL (subjects 1116, 1308, 1716, and ECH 529).
Fifteen of 16 patients had evidence of clonal IgM gammopathy by
serum immunofixation electrophoresis and were classified as being
IgM MCϩ. Subject ECH 529 had evidence of clonal IgAgammopa-
thy by immunofixation electrophoresis. In addition, cervical lymph
node biopsy in this patient revealed abnormal numbers of IgAϩ␬ϩ
B cells. Plasma from 10 of 12 HCV RNAϪ and 25 of 27 HCV
RNAϩ patients had detectable anti-Epstein-Barr virus nuclear
antigen 1 IgG, indicating previous exposure to Epstein-Barr virus.
HCVϩ IgM MCϩ subjects had significantly expanded populations
of IgMϩ␬ϩ peripheral B cells, although overall B-cell numbers
were not increased, consistent with our earlier report6 (supplemen-
tal Figure 1).
IgM؉␬؉CD27؉ B cells from HCV؉MC؉ patients have a distinct
transcriptional profile
A total of 69 unique genes (33 up-regulated, 36 down-regulated)
were found to be more than 2-fold differentially expressed in
IgMϩ␬ϩCD27ϩ B cells from IgM MCϩ, compared with IgM MCϪ,
subjects (Figure 1). Notably, the transcriptional profile of 2 HCV
RNAϩMCϪ patients (ECH 516 and ECH 522) shared several
features with that of the MCϩ population.
Several of the differentially expressed genes were grouped accord-
ing to broad function (Table 2). The overall transcriptional pattern was
suggestive not of oncogenesis, but of dampened activation and aug-
mented proapoptotic pathways. Unsurprisingly, several IFN-induced
genes were up-regulated: growth interferon-inducible protein X (PY-
HIN1), myeloid nuclear differentiation antigen (MNDA), and 2Ј, 5Ј-
oligoadenylate synthetase 1 (OAS1). Several genes associated with
B-cell anergy were up-regulated: galectin-1 (LGALS1), lymphocyte
transmembrane adapter 1 (LAX1), and CD200 receptor 1 (CD200R1),
an inhibitory receptor highly expressed on memory B cells and
plasmablasts. Significantly up-regulated proapoptotic genes included:
galectin-1, the interferon-response gene, PYHIN1, death-associated
protein kinase 2 (DAPK2), and MNDA. The prosurvival gene, T-cell
lymphoma 1A(TCL1A), was markedly down-regulated.
In addition to having an overall transcriptional program sugges-
tive of B-cell anergy and apoptosis, HCVϩMCϩ patients’
IgMϩ␬ϩCD27ϩ B cells demonstrated differential regulation of
several 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 large
B-cell lymphoma25 and which may be down-regulated in mantle
cell lymphoma.9 In addition, BTB and CNC homology 1, basic
leucine zipper transcription factor 2 (BACH2) was down-regulated;
both BACH2 and IL-4R are reported to be down-regulated in
B cells from patients with Waldenstro¨m 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 stage
of differentiation of HCVϩMCϩ patients’ clonally expanded
B 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 be
highly up-regulated in FCRL4-expressing tonsillar B cells, a
recently described memory B-cell subset thought to play an
important role in mucosal defense.29 Despite this transcriptional
similarity, our microarray data did not reveal differences in
FCRL4 transcript between MCϩ and MCϪ patients’
IgMϩ␬ϩCD27ϩ peripheral B cells. However, quantitative RT-
PCR of unamplified cDNA confirmed the up-regulated expres-
sion of SOX5, FGR, and CD11C in HCVϩMCϩ patients’
expanded IgMϩ␬ϩCD27ϩ B cells (Figure 2; supplemental Table
2). In addition, quantitative RT-PCR confirmed the up-
regulation of galectin-1 and OAS1 and the down-regulation of
IL-4R and TCL1A, and it detected no significant difference in
FCRL4 expression. We did not detect Epstein-Barr virus nuclear
antigen 2 or latent membrane protein 1 transcripts by quantita-
tive RT-PCR (data not shown).
A significant proportion of clonal cells from IgM MC؉ subjects
are CD21low, CD11c؉, FCRL4high, IL-4Rlow memory B cells
We and others have previously shown that clonally expanded
IgMϩ␬ϩCD27ϩ B cells from IgM MCϩ patients preferentially use
the VH1–69 gene segment.6,30 We used the G6 mAb, which
recognizes the complementarity-determining region 2 of Ig VH1-
B-CELLANERGY IN MIXED CRYOGLOBULINEMIA 5427BLOOD, 19 MAY 2011 ⅐ VOLUME 117, NUMBER 20

69,31 to more precisely immunophenotype HCVϩMCϩ patients’
clonally expanded B cells. We have previously confirmed the
specificity of G6 for VH1-69 by RT-PCR.32 These G6ϩ B cells from
HCVϩMCϩ patients are frequently CD21low and IgMϩ␬ϩ (supple-
mental Figure 2). They are also morphologically normal, nonprolif-
erating, and predominantly CD20high, CD10Ϫ, CD21low, CD27ϩ,
CD11cϩ, FCRL4high, and IL-4Rlow (Figure 3). We immunopheno-
typed B cells from 11 MCϪ (SVR ϭ 2, HCV AbϪ ϭ 3, HCV
RNAϩ ϭ 6) and 9 HCV RNAϩMCϩ subjects (Figure 4). As
expected, we found that MCϩ patients had a significant (P Ͻ .005)
expansion of G6ϩ B cells (median, 25.9% of B cells) compared
with HCV RNAϩMCϪ patients (median, 5.1%), SVR (median,
3.0%), and HCV AbϪ (median, 3.8%) patients. In 3 MCϩ patients
(LDU 125, 110, and 1432), more than 50% of total peripheral
B cells were G6ϩ. When we examined G6ϩ and G6Ϫ B cells from
each person, we confirmed that G6ϩ, compared with G6Ϫ, B cells
from HCVϩMCϩ patients were predominantly CD21low, CD11cϩ,
FCRL4high, and IL-4Rlow. Interestingly, G6ϩ, compared with G6Ϫ,
B cells from MCϪ persons were also disproportionately CD21low.
However, they did not have significantly increased expression
Table 1. Characteristics of the study patients
Condition/subject
no. 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 Ͻ 50 1 4 pIFN/RBV 2001 No
731 52 Female White Ͻ 50 2b 2 pIFN/RBV 2003 No
856 61 Female White Ͻ 50 1a 2 pIFN/RBV 2003 No
1154 56 Female Asian American Ͻ 50 1a 2 pIFN/RBV 2003 No
1197 38 Female White Ͻ 50 2a 1 pIFN/RBV 2003 No
ECH 521 43 Male White Ͻ 50 1 ND pIFN/RBV 2005 No
ECH 542 38 Female Black Ͻ 50 1 ND pIFN/RBV 2007 No
HCV؉, IgM MC؊
1235 44 Female White 3.85 ϫ 106 2b ND Naive No
1330 54 Female White/Hispanic Ͼ 7 ϫ 105 1a 2 Naive No
1419 48 Female White 0.39 ϫ 106 1 3 Naive No
1864 58 Male White 0.02 ϫ 106 4 3 Naive No
LDU 107 50 Male White/Hispanic Ͼ 7 ϫ 105 2 2 Naive No
ECH 507 54 Male White/Hispanic Ͼ 7 ϫ 105 1 ND Naive No
ECH 512* 62 Male White Ͼ 7 ϫ 105 1 ND Naive No
ECH 516 57 Male White/Hispanic 2.4 ϫ 106 1b 2 Naive No
ECH 519 52 Female White/Hispanic Ͼ 7 ϫ 105 1 ND Naive No
ECH 520 57 Male Black Ͼ 5 ϫ 106 1b 2 Naive No
ECH 522 50 Female White/Hispanic Ͼ 7 ϫ 105 1 ND Naive No
ECH 529†‡ 52 Female White Ͼ 7 ϫ 105 3 4 Naive Yes
ECH 530 49 Male White/Hispanic Ͼ 7 ϫ 105 1 ND Naive No
ECH 536* 50 Male White/Hispanic 0.2 ϫ 106 1 1 Naive No
ECH 537* 54 Male Black 0.9 ϫ 106 1a ND Naive No
HCV؉, IgM MC؉
110 53 Male White/Hispanic 1.70 ϫ 106 1 3 Naive Yes
880 54 Female White 3 ϫ 106 1b 2 Naive Yes
1116‡ 37 Male White 3 ϫ 104 2b 2 Naive Yes
1308‡ 55 Female White 0.72 ϫ 106 1 1 Naive Yes
1403 44 Female White/Hispanic 0.50 ϫ 106 1 2 Naive Yes
1432 69 Female White 0.03 ϫ 106 1 4 Naive Yes
1540 37 Male White/Hispanic 0.28 ϫ 106 1 ND Naive Yes
1716‡ 30 Female White 1.45 ϫ 106 1a ND Naive Yes
1931 56 Female White 2.2 ϫ 106 1b 2 Naive Yes
92200 57 Female White 2 ϫ 106 2 2 Naive Yes
LDU 125 60 Male White 0.2 ϫ 106 3 ND Naive Yes
ECH 531 56 Male White Ͼ 7 ϫ 105 1 ND Naive Yes
ECH 532 51 Female Black Ͼ 7 ϫ 105 1 ND Naive Yes
ECH 533 63 Male White 3.8 ϫ 106 1 0 pIFN/RBV 2007 Yes
ECH 535 57 Male White 0.74 ϫ 106 1 ND Naive Yes
ECH 546* 52 Male White Ͼ 7 ϫ 105 1 3 pIFN/RBV 2008 ND
ECH 559* 58 Female White/Hispanic 1.27 ϫ 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 ␬ϩ IgA MC.
‡Subjects with marginal zone B-cell NHL (documented on lymph node biopsy).

of CD11c or FCRL4, nor did they have decreased expression
of IL-4R.
Reflective of having G6ϩ B-cell expansions, HCVϩMCϩ pa-
tients had increased percentages of total B cells that were CD21low,
CD11cϩ, FCRL4high, and IL-4Rlow. These markers were signifi-
cantly correlated with the percentage of peripheral B cells that were
G6ϩ. We did not detect any significant differences in any of these
cell surface markers among the total B cells of SVR, HCV AbϪ,
and HCV RNAϩ patients, probably reflective of the low proportion
of G6ϩ B cells in these MCϪ persons.
FCRL4ϩ memory B cells from normal tonsils and HIV-viremic
blood are reported to be CD20highCD11cϩCD95ϩCD21low.29,33 In
healthy tonsils, these FCRL4ϩ B cells have been variously reported
to be CD27Ϫ or CD27ϩ memory B cells.34,35 Although we detected
increased FCRL4 expression on G6ϩ, compared with G6Ϫ, B cells,
we did not detect significant differences in FCRL4 expression
110
1931
1308
1432
1716
1116
922000
LDU125
1540
ECH533
1403
880
ECH535
ECH532
ECH531
ECH516
ECH522
1154
LDU107
856
ECH528
ECH521
1864
ECH503
ECH527
ECH529
ECH507
ECH520
ECH530
ECH519
1197
543
731
1330
1419
1235
ECH542
LDU128
LDU099
IgM MC
+
IgM MC
-
Expression
6 01
SOX5
AEBP1
SLC7A7
PYHIN1
SLC11A1
ITGAX (CD11c)
PNMA2
TESC
LGALS1 (Galectin-1)
PPAP2B
FGR
HOXB7
FLJ40869
OAS1
FCGR2A
MNDA
MIDN
STS-1
LOC649199
RASGRF1
N/A
N/A
CD200R1
CD84
DAPK2
LAX1
TFEC
LRRN1
OAS1
SRGN
C13orf15
TUBB2A
TUBB2A
ENC1
DUSP5
CD68
TMEM44
N/A
ADARB1
TSPAN13
SELL
P2RY14
N/A
TCL1A
IL4R
C3orf37
LMO2
SPRY1
ZNF135
LARGE
SMAD3
N/A
EPB41L2
VPREB3
N/A
PPAPDC1B
LIX1
SSBP2
MGC39372
GCNT1
TAPT1
CRIM1
CR1
CR1
C1orf162
LOC647450
LOC652493
LOC652694
N/A
PELI2
CD1A
NT5E
IL13RA1
N/A
LOC652113
N/A
SERPINE2
FOXP1
BACH2
FLJ14213
IL15
Figure 1. Transcriptome analysis of IgM؉␬؉CD27؉
B cells. The transcripts differentially expressed by
IgMϩ␬ϩCD27ϩ B cells from HCVϩMCϩ and MCϪ
patients are displayed. Up- and down-regulated tran-
scripts are indicated in red and blue, respectively. The
magnitude of expression is indicated by the color bar.

among CD27ϩ, CD27Ϫ, CD21high, or CD21low B-cell subsets
(supplemental Figure 3). In an attempt to reconcile the findings of
unchanged FCRL4 transcript levels and increased FCRL4 protein
expression in MCϩ compared with MCϪ B cells, we performed
quantitative RT-PCR on RNAisolated from the bulk-sorted FCRL4ϩ
and FCRL4Ϫ G6ϩ B cells from subject LDU 125. However, we
failed to detect any difference in FCRL4 mRNA between these
2 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-viremic
patients as being relatively anergic, “exhausted” B cells.33 We
sought to determine whether IgGϪ, CD21low B cells had attenuated
signaling on BCR ligation with anti–human IgM. Data for one
healthy subject with an elevated (22%) CD21low B-cell frequency
(ECH 503), 2 HCVϩMCϪ (ECH 530, ECH 537) volunteers and
5 HCVϩMCϩ (ECH 546, ECH 559, 110, LDU 125, and 1931)
volunteers are shown in Figure 5. In all but one (110), the
CD27ϩCD21low subset had decreased Ca2ϩ mobilization; the
reason for this outlier is unclear. Furthermore, in all subjects, the
CD27ϪCD21low B-cell subset had diminished amplitude of Ca2ϩ
mobilization; ECH 546, for unknown reasons, had an intermediate
response to BCR triggering. Finally, in ECH 559 and 1931,
CD27ϩCD21high cells had low responses to BCR stimulation; IgA
may possibly be a prominent non-IgG BCR in their CD27ϩCD21high
subsets. Overall, these data suggest that CD21low B cells, whether
from healthy, HCVϩMCϪ, or HCVϩMCϩ persons, are anergic to
BCR-mediated stimulation.
G6؉ B cells are preferentially prone to death and apoptosis
Our microarray data suggested that HCVϩMCϩ patients’
IgMϩ␬ϩCD27ϩ B cells up-regulate several genes associated with
apoptosis. We tested whether these cells were prone to apoptosis in
vitro by incubating them with either media, anti-CD95 mAb, or
isotype control IgG (Figure 6). After 6 hours of incubation, PBMCs
were 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, isotype
control, or anti-CD95, G6ϩ, compared with G6Ϫ, B cells had
significantly higher binding of annexin V, either alone or in
combination with 7-AAD. The percentage of annexin Vϩ/7-AADϪ
cells was highest in the anti-CD95-stimulated G6ϩ B-cell subset.
However, whereas G6Ϫ cells demonstrated a 2.1-fold increase in
annexin V binding on stimulation with anti-CD95, compared with
isotype control, G6ϩ 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 (2Ј, 5Ј 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 IgMϩ 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.

indicates that G6ϩ B cells’ propensity for apoptosis is not fully
dependent on CD95, although CD95 was upregulated on several
HCVϩMCϩ patients’ G6ϩ cells. A larger percentage of apoptotic
annexin Vϩ7-AADϪ cells had lower CD21 expression compared
with annexin VϪ7-AADϪ cells (Figure 6B). This was true for both
G6ϩ and G6Ϫ populations. Surface CD20 expression was similar
among all subsets, except for G6Ϫ annexin Vϩ7-AADϩ cells.
Importantly, 6 hours of incubation with anti-CD95 did not signifi-
cantly alter the percentages of CD20ϩ B cells that were G6ϩ (37 vs
29% at 0 and 6 hours, respectively), nor did it cause down-
regulation of CD21 on G6ϩ or G6Ϫ B cells (Figure 6C), indicating
that cell surface CD20, CD21, and G6 expression was stable and
not significantly affected by apoptosis or cell death. Taken together,
these data suggest that, in the absence of survival signals, G6ϩ
B cells, and in particular, the CD21low G6ϩ subset, are prone to
spontaneous cell death and apoptosis.
CD27؉CD21high, but not CD27؉CD21low, G6؉ B cells from
HCV؉MC؉ patients efficiently differentiate into
antibody-secreting plasmablasts
To determine whether CD27ϩCD21low B cells could proliferate and
differentiate in response to stimulatory signals, we stimulated
sorted CD27ϩCD21high, CD27ϩCD21low, CD27ϪCD21high, and
CD27ϪCD21low G6ϩ B cells from 4 HCVϩMCϩ subjects. For a
fifth patient, CD27ϩCD21low, CD27ϩCD21high, CD27Ϫ G6ϩ B cells
and total G6Ϫ B cells were sorted. We used 2 independent samples
to test whether our cell sorting procedure caused early cell death; in
all subsets, more than 85% of cells were viable, as measured by
4,6-diamidino-2-phenylindole staining (supplemental Figure 4).
Cells were cultured for 6 days with CD40L, IL-2, and IL-10 and
analyzed for differentiation toward antibody-secreting cells.
Using IgM ELISPOT, we found that the CD27ϩCD21high, com-
pared with CD27ϩCD21low and CD27Ϫ G6ϩ B-cell subsets, were
more efficient at differentiating to IgM-secreting plasmablasts on
CD40L/IL-2/IL-10 stimulation (Figure 7A). To confirm these
findings, we quantitated IgM in the cell culture supernatants using
ELISA. The CD27ϩCD21high cell culture supernatants all had
higher levels of IgM compared with their CD27ϩCD21low and
CD27Ϫ counterparts (Figure 7B). Class switch did not account for
the decreased IgM in these subsets, as we consistently detected
Ͻ 150 ng/mL IgG in cell culture supernatants.
We confirmed that the IgM produced by the stimulated G6ϩ
B-cell subsets had RF activity by demonstrating that they could
bind human IgG1 (Figure 7C). As a control, we tested patient LDU
125’s G6Ϫ B-cell supernatant. Although IgM was present at a total
concentration 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 CD27ϩCD21low, compared
with the CD27ϩCD21high, G6ϩ B-cell subset.
Discussion
We previously reported that HCVϩMCϩ patients have clonal
expansions of IgMϩ␬ϩCD27ϩ peripheral B cells.6 Here we have
transcriptionally profiled IgMϩ␬ϩCD27ϩ B cells isolated from
HCVϩMCϩ patients, and we have identified a set of 69 genes that
are differentially expressed. This set is notable for interferon
response genes, B-cell activation markers, transcription factors,
and glycoprotein synthesis genes. Although several genes previ-
ously implicated in B-cell neoplasia (eg, SOX5, CD11C, and
MNDA) are up-regulated among these cells, others (LMO2, IL-4R,
SELL, TCL1A, FOXP1, and BACH2) are down-regulated. Thus,
although IgMϩ␬ϩCD27ϩ B cells are responsible for MC pathogen-
esis, their overall gene expression pattern suggests that there exists
a 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 known
pleiotropic roles in innate and adaptive immune responses. Galec-
tin-1 (and CD11C) transcripts have been identified as being
up-regulated in anergic murine B cells.12 Moreover, galectin-1 has
been shown to be overexpressed in IgMϩCD27ϩ B cells, is further
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
HCVAb-
HCV+
MC-
HCV+
MC+
SVR
p < 0.05 p < 0.001
p < 0.001
p = n.s.
OAS1 FCRL4IL4R TCL1A
LGALS1 SOX5 FGR CD11C
p < 0.001p < 0.001
0
1
2
3
4
5
6
7
0.0
2.5
5.0
7.5
10.0
12.5
0
10000
20000
30000
40000
0
10
20
30
p < 0.01
0
1
2
3
4
5
6
0
5
10
15
0
1000
2000
3000
4000
0.0
0.5
1.0
1.5
2.0
p < 0.0001
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 to
a universal standard RNA. Comparisons between groups were made using the Kruskal-Wallis test, and P values for HCVϩMCϪ compared with HCVϩMCϩ subjects were
computed using Dunn post test. N.S. indicates not significant (P Ͼ .05).

induced on BCR ligation, has been associated with Bim-mediated
apoptosis,13 and may negatively regulate B-cell proliferation and
BCR-mediated tyrosine phosphorylation.14 Also up-regulated were
CD200R1, 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/threonine
kinase responsible for IFN-␥, TNF-␣, and Fas ligand–induced
apoptosis.15 Similarly up-regulated was LAX1, a negative regulator
of BCR-mediated calcium flux, Akt activation, and cell survival
and proliferation.16 PYHIN1 (IFIX) and MNDA, 2 members of the
IFN-inducible HIN-200 gene family implicated in apoptosis,17
were also up-regulated. One of the most down-regulated genes was
TCL1A, a proto-oncogene up-regulated in splenic marginal zone
lymphoma cells.18 TCL1A is induced in naive B cells on BCR
ligation, and this may protect B cells from apoptosis.13 The
down-regulation of IL-4R in HCVϩMCϩ 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
CD10
G6
[log10]
[log10]
CD21
CD27
FCRL4
IL4R
CD20+
Propidium Iodide
Ki-67
CD20+
G6+
CD20+
G6-
0 50K 100K 150K 200K 250K
0
20
40
60
80
100
FSC-A
%ofMax
G6
CD20 Lymphocytes
HCV
+
MC
+
HCV
+
MC
-
G6+
G6-
A
B
G
[log10]
[log10]
Propidium Iodide
Ki-67
C
D
E
F
CD20+
0 50K 100K 150K 200K 250K
0
102
10
3
104
10
5
0.79
00.45
98.8
0 50K 100K 150K 200K 250K
0
102
10
3
104
105
0.88
0.0150.7
98.4
G6
CD11c
Figure 3. Phenotypic analysis of HCV؉MC؉ patients’ clonal B cells. PBMCs from one HCVϪMCϪ (ECH 542, red) and one HCVϩMCϩ 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 G6ϩ and G6Ϫ B cells from the
HCVϩMCϩ person. Scanning electron micrographs of immunomagnetically sorted G6ϩ and G6Ϫ B cells from the HCVϩMCϩ person. (C-D) Scale bars on the low and high
power magnifications represent 5 ␮m and 1 ␮m, respectively. Propidium iodide and Ki-67 staining of immunomagnetically sorted G6ϩ and G6Ϫ B cells from the HCVϩMCϩ
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 ϫ10 000) were taken with a FEI Tecnai G2 Spirit BioTWIN Transmission Electron Microscope equipped with a Gatan ES500W Erlangshen CCD camera and
DigitalMicrograph Software.

that IL-4R engagement can abrogate Fas-mediated
B cell apoptosis.36
We found that HCVϩMCϩ patients’IgMϩ␬ϩCD27ϩ B cells had
up-regulation of CD11C, SOX5, and FGR, similar to what was
recently reported in an FCRL4ϩ CD27Ϫ memory tonsillar B-cell
subset. FCRL4 is an orphan receptor whose intracellular domain
contains 3 immunoreceptor tyrosine-based inhibitory motifs that
can inhibit BCR-mediated signaling.37 The primary location of
FCRL4ϩ cells in the marginal zone equivalents of tonsillar and
Peyer patch epithelia and in mucosa-associated lymphoid tissue
lymphomas34 suggests that these cells may play a role in mucosal
defense against pathogens. FCRL4ϩ B cells are reported to be large
CD20high, CD11cϩ, CXCR3ϩ, CD95ϩ, CD21low proliferating
B cells.29,33 Although we did not detect increased FCRL4 transcript
in clonally expanded G6ϩ peripheral B cells, they shared several
features with FCRL4ϩ cells, as they expressed FCRL4 protein and
were predominantly CD20high, CD21low, CD11cϩ B cells. However,
in contrast to the concordance between FCRL4 mRNA transcript
and FCRL4 protein expression that has been reported in healthy
persons’ tonsillar B cells,35 FCRL4 protein, but not mRNA, was
elevated in HCVϩMCϩ patients’ peripheral B cells. We speculate
that an increased level of FCRL4 mRNA is not necessary to
maintain the relatively increased surface FCRL4 protein expression
on these cells. Further research is necessary to investigate whether
**
**
G6+
G6-
G6+
G6-
MC-
MC+
G6+
G6-
G6+
G6-
MC-
MC+
G6+
G6-
G6+
G6-
MC-
MC+
G6+
G6-
G6+
G6-
MC-
MC+
G6+
G6-
G6+ G6-
MC-
MC+
G6+
G6-
G6+
G6-
MC-
MC+
% G6+
(of CD20+
)
FCRL4MFI
r = 0.7386
p < 0.0005
%CD21high/int
% G6+
(of CD20+
)
r = -0.9167
p < 0.0001
*
% G6+
(of CD20+
)
%CD11c+
r = 0.5240
p < 0.05
**
r = 0.2422
p = n.s.
CXCR3MFI
% G6+
(of CD20+
)
r = 0.5233
p < 0.05
% G6+
(of CD20+
)
CD95MFI
% G6+
(of CD20+
)
%IL4R+
r = -0.4602
p < 0.05
n.s.
*
** **
0
25
50
75
100
0 25 50 75 100
0
25
50
75
100
0
25
50
75
100
0 25 50 75 100
0
25
50
75
0
1000
2000
3000
0 25 50 75 100
0
1000
2000
0 25 50 75 100
0
1000
2000
3000
0
25
50
75
100
0 25 50 75 100
0
25
50
75
0 25 50 75 100
0
1000
2000
0
1000
2000
3000
0
1000
2000
3000
4000
0
1000
2000
3000
4000
**
Figure 4. Immunophenotypic profiles of MC؉ and MC؊ patients’ G6؉, G6؊, and total B cells. Data are flow cytometric analyses of PBMCs from HCVϩMCϩ (n ϭ 9) and
MCϪ (n ϭ 11; SVR ϭ 2, HCV AbϪ ϭ 2, HCV RNAϩ ϭ 6) subjects. Cell surface marker expression in each subject’s G6ϩ and G6Ϫ B cells is shown in the column graphs. The
scatterplots represent expression of cell surface markers versus the proportion of total B cells that are G6ϩ. In the scatterplots: Œ represents MCϩ patients; ‚, HCV RNAϩ
patients; and छ, HCV RNAϪ patients. MFI indicates geometric mean fluorescence intensity. The P values for the column graphs were determined using the Kruskal-Wallis and
Dunn multiple comparison tests. *P Ͻ .05. **P Ͻ .01. n.s. indicates not significant (P Ͼ .05). Black bars represent medians. For the scatterplots, R was calculated using the
Spearman correlation coefficient.

HCVϩMCϩ patients’ mucosal-associated and peripheral B cells
share common transcriptional, immunophenotypic, and functional
characteristics.
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, CD27Ϫ, FCRL4ϩ B cells have been
described in HIV-viremic33 and Plasmodium falciparum–exposed
persons.41 Interestingly, the expanded B cells in chronic variable
immunodeficiency patients are activated, have up-regulated SOX5
expression, and diminished calcium signaling and proliferative
responses to BCR or CD40L stimulation.8,40 In HIV-viremic
patients, they have up-regulated CD95 expression and are prone to
apoptosis.42 Moreover, these cells may be functionally “ex-
hausted,” as they demonstrate less ability to proliferate on BCR
ligation with or without T-cell help and they have decreased ability
to differentiate into antibody-secreting cells on stimulation with
Staphylococcus aureus Cowan and CpG.33
Given these facts, and because CD21, as part of the B-cell
coreceptor complex, augments BCR-mediated signaling, we sus-
pected that HCVϩMCϩ patients’ expanded CD21low G6ϩ B-cell
subset represented a relatively anergic B-cell population. In
addition, because transcriptional profiling revealed the up-
regulation of several proapoptotic genes, we hypothesized that
these B cells were prone to apoptosis. Indeed, we found that G6ϩ,
compared with G6Ϫ, B cells were more prone to apoptosis and cell
death and that these cells were skewed toward the CD21low subset.
Our data are consistent with earlier reports suggesting that HCVϩ
patients’ memory B cells are prone to apoptosis and that this may
serve as a feedback inhibition mechanism to prevent exaggerated
autoreactive B-cell responses.43,44 In addition, CD21low B-cell
subpopulations from healthy, HCVϩMCϪ, or HCVϩMCϩ persons
were relatively hyporesponsive to BCR stimulation, as measured
by Ca2ϩ mobilization. Thus, the frequent down-regulation of CD21
among G6ϩ B cells suggests that these cells are relatively anergic.
Moreover, the G6ϩCD21low subpopulation was only weakly in-
duced to differentiate to IgM-secreting plasmablasts on stimulation
with CD40L, IL-2, and IL-10, suggesting that these cells in vivo
may be refractory to stimulation by T cell–mediated signals in the
context of BCR engagement. Whether other stimuli (eg, TLR
agonists or IFN-␣) can induce these cells to differentiate remains
an open question. We surmise that the down-regulation of CD21 is
a homeostatic control mechanism that attenuates autoreactive RFϩ
B-cell responses to chronic HCV-containing immune complex
stimulation.
To summarize, we have found that HCVϩMCϩ patients’
clonally expanded peripheral B cells have global transcriptional
features not of proto-oncogenesis, but rather of stimulatory hypore-
sponsiveness and anergy. Immunophenotypically, they resemble
activated memory B cells and share several features with previ-
ously described CD21low and FCRL4ϩ B-cell populations. Al-
though we have shown that the overall clonal population is capable
of differentiating into RF-secreting plasmablasts, the expanded
CD21low fraction is prone to anergy. Together, our results suggest
overall attenuation mechanisms present in these autoreactive-prone
B cells that limit their pathogenic expansion and differentiation in
IgG
-
B Cells
0 100 200 300
0 100 200 300
0 100 200 300
ECH 530
HCV
+
MC
-
0 100 200 300
14.8 18.4
58.48.4
100 200 300
35.3
448.39
12.4
62
18.76.69
12.6 20.7
21.233.7
24.4
25.2
16.714.3
43.9
ECH 503
healthy
8.01 28.2
49.414.4
100 200 300
15.8 54.9
20.88.46
100 200 300100 200 300
ECH 503
11.3 14.5
66.37.88
50 100 150 200 250
14.5 10.7
62.911.8
CD21
CD27
TIme (seconds)
Ratio:
bound/unbound
4 1
3 2
Ionomycin
ECH 537
HCV
+
MC
-
ECH 559
HCV
+
MC
+
ECH 546
HCV
+
MC
+
110
HCV
+
MC
+
1931
HCV
+
MC
+
LDU 125
HCV
+
MC
+
Figure 5. CD27؉CD21low, compared with CD27؉CD21high, B cells have attenuated Ca2؉ responses after BCR cross-linking. Analyses of B cells from one healthy
volunteer (ECH 503), 2 HCVϩMCϪ patients (ECH 530 and ECH 537), and 5 HCVϩMCϩ patients (ECH 546, ECH 559, 110, LDU 125, and 1931) are shown. PBMCs from
1931 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 CD21
staining of IgGϪ 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(abЈ)2 anti–human IgM. Kinetic graphs represent ratios of bound/unbound Indo-1 over time for CD27ϩCD21high, CD27ϩCD21low, CD27ϪCD21high, and
CD27ϪCD21low B-cell populations. Single arrows indicate injection of F(abЈ)2 anti–human IgM; and double arrow, injection of 10 ␮g/mLionomycin.

vivo. Several clinical lines of evidence suggest that these B-cell
attenuation mechanisms may serve to limit disease activity in
vivo. First, the incidence of clinically apparent MC during
chronic HCV infection is rather low (1 per 1000 person-years in
US veterans3), even in the setting of decades of infection.
Second, clinical signs and symptoms of MC frequently wax and
wane, with no apparent correlation with fluctuations in HCV
RNA or liver inflammation.45 Third, even among patients with
MC, 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. It
has been shown that VH169 partially encoded Abs are enriched in
anti-influenza47,48 and anti-HIV49 responses. One of the most potent
cross-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 this
confers antiviral activity by binding to hydrophobic viral targets.47
As autoreactive RF is partially encoded by VH169, it is tempting to
speculate that homeostatic mechanisms that are up-regulated to
attenuate self-reactivity have the unintended consequence of abro-
gating effective VH169-mediated anti-HCV responses.
It remains unclear why only some HCV-infected persons
develop MC and why these attenuation mechanisms fail to prevent
the development of NHL in some HCVϩMCϩ patients. It must be
emphasized that we have studied patients’ peripheral B cells; it
remains to be seen whether B cells at other anatomic sites, such as
the liver or perihepatic lymph nodes, display similar features of
attenuation. Further patient-oriented research will be necessary to
answer these central questions, as well as to determine whether the
0h 6h
7AAD
Annexin V
G6+
G6-
A
CD21 CD20
Annexin V+
, 7AAD+
Annexin V-
, 7AAD-
Annexin V+
, 7AAD-
B
α-CD95Media ms IgG1
α-CD95: 6h
G6+
%ofMax
CD21
6 h
0 h
C
13.2 0.86
0.3285.6
4.06 0.087
0.3295.5
1.22 24.4
34.340.2
1.54 6.4
10.581.6
0.82 28.1
47.623.5
0.99 8.16
1773.9
0.55 20.2
64.314.9
0.12 4.98
36.158.8
G6-
Lymphocytes CD20+
B Cells
CD20 G6
α-CD95: 0h, 6h
G6+
B Cells G6-
B Cells
%ofMax%ofMax
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 presence
of 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 G6
mAbs and were analyzed by flow cytometry. Analyses of G6ϩ and G6Ϫ CD20ϩ B cells are shown. (A) Annexin V and 7-AAD staining of cells at baseline and after 6 hours of
stimulation. (B) Analysis of surface CD21 and CD20 expression on cells incubated for 6 hours with ␣-CD95/protein G; annexin Vϩ7-AADϩ, annexin Vϩ7-AADϪ, and annexin
VϪ7-AADϪ subsets. (C) Cell surface anti-CD20 (of total lymphocytes), G6 (of total B cells), and anti-CD21 (of G6ϩ and G6Ϫ 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 are
representative of 3 independent experiments.

autoregulatory mechanisms described here depend on anatomic
context, are specific for HCVϩMCϩ patients or are common to
other autoimmune diseases.
Acknowledgments
The authors thank the patient volunteers for their generosity; Roy
Jefferis for G6 monoclonal antibody; Go¨tz Ehrhardt for F(abЈ)2
anti-FCRL4 antibody; Donna Brassil, Veronica Whalen, and Rhonda
Kost of the Rockefeller University Center for Clinical and Transla-
tional Science for assistance with subject enrollment and study
management; Natasha Levenkova for statistical advice; and Santa
Maria Di Vittorio for administrative assistance.
This study was supported in part by the National Institutes of
Health/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.), and
Center for Translational Science Award (grant UL1 RR024143, to
Rockefeller University), from the National Center for Research
Resources, a component of National Institutes of Health. Sorting
on the FACSAria II was made possible by support from the Empire
State Stem Cell Fund (New York State Department of Health
contract C023046).
Opinions expressed here are solely those of the authors and do
not 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 the
experiments; E.D.C., K.M., A.H.T., and I.M.J. provided patient
referrals; E.D.C., L.B.D., C.M.R., S.M., K.D.R., K.M., A.H.T., and
I.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 of
Hepatitis C, Laboratory of Virology and Infectious Disease,
Rockefeller University, Box 64, 1230 York Ave, New York, NY
10065; e-mail: dustinl@rockefeller.edu.
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A
B
ECH 546 ECH 559 1931 110 LDU 125
21lo
21hi
27+
21lo
21hi
27
-
21lo
21hi
27+
21lo
21hi
27
-
21lo
21hi
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21lo
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27
-
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IgM(μg/ml)
IgM(μg/ml)
IgM(μg/ml)
IgM(ng/ml)
0
50
100
150
200
250
300
350
0
5
10
15
20
0
5
10
15
20
0
5
10
15
20
25
0
1000
2000
3000
4000
5000
6000
0
2500
5000
7500
10000
12500
0
500
1000
1500
2000
0
25
50
75
100
0
1
2
3
0
1
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0
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Dilution
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1:104
1:105
Dilution
1:101
1:102
1:103
1:104
1:105
Dilution
1:101
1:102
1:103
1:104
1:105
Dilution
1:101
1:102
1:103
1:104
1:105
Dilution
CD27+
CD21lo
CD27+
CD21hi
CD27
-
CD21lo
CD27
-
CD21hi
0.0
0.5
1.0
1.5
2.0
2.5
21lo
21hi
27+
G6
-
21lo
21hi
27+
27
-
0
250
500
750
1000
1250
0
1
2
3
CD27+
CD21lo
CD27+
CD21hi
CD27
-
G6
-
X
Figure 7. CD27؉CD21low, compared with CD27؊ and CD27؉CD21high, G6؉ B cells from HCV؉MC؉ patients demonstrate decreased differentiation to IgM
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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. Dustin
Edgar D. Charles, Claudia Brunetti, Svetlana Marukian, Kimberly D. Ritola, Andrew H. Talal, Kristen
B-cell subsetlowcryoglobulinemia contain an expanded anergic CD21
associated mixed−Clonal B cells in patients with hepatitis C virus
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