Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders.
ABSTRACT Infection with hepatitis C virus (HCV), a leading cause of chronic liver diseases, can associate with B lymphocyte proliferative disorders, such as mixed cryoglobulinemia and non-Hodgkin lymphoma. The major envelope protein of HCV (HCV-E2) binds, with high affinity CD81, a tetraspanin expressed on several cell types. Here, we show that engagement of CD81 on human B cells by a combination of HCV-E2 and an anti-CD81 mAb triggers the JNK pathway and leads to the preferential proliferation of the naïve (CD27-) B cell subset. In parallel, we have found that B lymphocytes from the great majority of chronic hepatitis C patients are activated and that naïve cells display a higher level of activation markers than memory (CD27+) B lymphocytes. Moreover, eradication of HCV infection by IFN therapy is associated with normalization of the activation-markers expression. We propose that CD81-mediated activation of B cells in vitro recapitulates the effects of HCV binding to B cell CD81 in vivo and that polyclonal proliferation of naïve B lymphocytes is a key initiating factor for the development of the HCV-associated B lymphocyte disorders.
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ABSTRACT: CD100, also known as Sema4D, is a member of the semaphorin family and has important regulatory functions that promote immune cell activation and responses. The role of CD100 expression on B cells in immune regulation during chronic hepatitis C virus (HCV) infection remains unclear. We longitudinally investigated the altered expression of CD100, its receptor CD72, and other activation markers CD69 and CD86 on B cells in 20 chronic HCV-infected patients before and after treatment with pegylated interferon-alpha (Peg-IFN-α) and ribavirin (RBV) by flow cytometry. The frequency of CD5+ B cells as well as the expression levels of CD100, CD69 and CD86 was significantly increased in chronic HCV patients and returned to normal in patients with sustained virological response after discontinuation of IFN-α/RBV therapy. Upon IFN-α treatment, CD100 expression on B cells and the two subsets was further up-regulated in patients who achieved early virological response, and this was confirmed by in vitro experiments. Moreover, the increased CD100 expression via IFN-α was inversely correlated with the decline of the HCV-RNA titer during early-phase treatment. Peripheral B cells show an activated phenotype during chronic HCV infection. Moreover, IFN-α therapy facilitates the reversion of disrupted B cell homeostasis, and up-regulated expression of CD100 may be indirectly related to HCV clearance.PLoS ONE 12/2014; 9(12):e113338. · 3.53 Impact Factor
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ABSTRACT: Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood worldwide and Mexico has reported one of the highest incidence rates. An infectious etiology has been suggested and supported by epidemiological evidences; however, the identity of the involved agent(s) is not known. We considered that early transmitted lymphotropic herpes viruses were good candidates, since transforming mechanisms have been described for them and some are already associated with human cancers. In this study we interrogated the direct role of EBV, HCMV, HHV6, and HHV7 human herpes viruses in childhood ALL. Viral genomes were screened in 70 bone marrow samples from ALL patients through standard and a more sensitive nested PCR. Positive samples were detected only by nested PCR indicating a low level of infection. Our result argues that viral genomes were not present in all leukemic cells, and, hence, infection most likely was not part of the initial genetic lesions leading to ALL. The high statistical power of the study suggested that these agents are not involved in the genesis of ALL in Mexican children. Additional analysis showed that detected infections or coinfections were not associated with prognosis.BioMed Research International 01/2014; 2014:548097. · 2.71 Impact Factor
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ABSTRACT: Despite the development of potent antiviral drugs, HCV remains a global health problem; global eradication is a long way off. In this review, we discuss the immune response to HCV infection and particularly, the interplay between viral strategies that delay the onset of antiviral responses and host strategies that limit or even eradicate infected cells but also contribute to pathogenesis. Although HCV can disable some cellular virus-sensing machinery, IFN-stimulated antiviral genes are induced in the infected liver. Whereas epitope evolution contributes to escape from T cell-mediated immunity, chronic high antigen load may also blunt the T cell response by activating exhaustion or tolerance mechanisms. The evasive maneuvers of HCV limit sterilizing humoral immunity through rapid evolution of decoy epitopes, epitope masking, stimulation of interfering antibodies, lipid shielding, and cell-to-cell spread. Whereas the majority of HCV infections progress to chronic hepatitis with persistent viremia, at least 20% of patients spontaneously clear the infection. Most of these are protected from reinfection, suggesting that protective immunity to HCV exists and that a prophylactic vaccine may be an achievable goal. It is therefore important that we understand the correlates of protective immunity and mechanisms of viral persistence.Journal of Leukocyte Biology 07/2014; · 4.30 Impact Factor
Activation of naı ¨ve B lymphocytes via CD81,
a pathogenetic mechanism for hepatitis C
virus-associated B lymphocyte disorders
Domenico Rosa*†, Giulietta Saletti*†, Ennio De Gregorio*†, Francesca Zorat‡, Consuelo Comar‡, Ugo D’Oro*,
Sandra Nuti*, Michael Houghton§, Vincenzo Barnaba¶, Gabriele Pozzato‡, and Sergio Abrignani*?**
*Chiron Vaccines, 53100 Siena, Italy;‡Institute of Internal Medicine, University of Trieste, 34127 Trieste, Italy;§Chiron Corporation,
Emeryville, CA 94608-2916; and¶Fondazione Andrea Cesalpino, Institute of Internal Medicine, University of Rome, 00158 Rome, Italy
Communicated by Rino Rappuoli, Chiron Corporation, Siena, Italy, October 28, 2005 (received for review September 30, 2005)
Infection with hepatitis C virus (HCV), a leading cause of chronic
liver diseases, can associate with B lymphocyte proliferative dis-
orders, such as mixed cryoglobulinemia and non-Hodgkin lym-
phoma. The major envelope protein of HCV (HCV-E2) binds, with
high affinity CD81, a tetraspanin expressed on several cell types.
Here, we show that engagement of CD81 on human B cells by a
combination of HCV-E2 and an anti-CD81 mAb triggers the JNK
pathway and leads to the preferential proliferation of the naı ¨ve
and that naı ¨ve cells display a higher level of activation markers
than memory (CD27?) B lymphocytes. Moreover, eradication of
HCV infection by IFN therapy is associated with normalization of
the activation-markers expression. We propose that CD81-medi-
ated activation of B cells in vitro recapitulates the effects of HCV
binding to B cell CD81 in vivo and that polyclonal proliferation of
naı ¨ve B lymphocytes is a key initiating factor for the development
of the HCV-associated B lymphocyte disorders.
monoclonal antibody ? multimeric engagement ? B cell antigen receptor ?
with one large translational ORF encoding a single polyprotein,
which is processed by host and viral proteases into at least three
structural and seven nonstructural proteins with various enzymatic
activities (1). Two heavily N-glycosylated proteins E1 and E2 are
virion-envelope proteins and form heterodimers in vitro (2). An
(3). HCV infection is associated with the development of chronic
hepatitis, cirrhosis, and hepatocellular carcinoma (4). B cell abnor-
malities, including cryoglobulinemia (5) and an increased risk of B
cell non-Hodgkin lymphoma (6, 7), have been reported in a
minority of HCV infections.
Until very recently, it was not possible to grow HCV in cell
been surrogated by the assessment of binding and entry of HCV
recombinant glycoproteins (8) or virus pseudotypes (9).
We have previously reported that HCV-E2 protein binds with
high affinity to the large extracellular loop of human CD81 (CD81-
LEL) and that ‘‘bona fide’’ HCV particles bind human CD81 (10).
Recently, it has been demonstrated that CD81 is required for entry
and infection of human cells by in vitro-generated infectious HCV
(11). CD81 is a widely distributed cell-surface tetraspanin that
participates in different molecular complexes on various cell types,
CD81 is known to form a costimulatory complex with CD19 and
CD21 (13, 14) and that coligation of the B cell antigen receptor
(BCR) with any of the components of this costimulatory complex
lowers the threshold required for BCR-mediated B cell prolifera-
tion (15). We have proposed that HCV exploits CD81 not only to
invade hepatocytes but also to modulate the immune system.
Flaviviridae family (1). The HCV genome is 9.6 kb in length,
mAbs leads to costimulation of human T cells (16) or inhibition of
human NK cells (17). In this study, we investigated whether
engagement of CD81 by recombinant HCV-E2 protein and?or
anti-CD81 mAbs had any effect on human B cell function. We
found that multimeric engagement of CD81 activates B lympho-
polyclonal stimuli, CD81 engagement preferentially induced pro-
liferation of naı ¨ve B cells. We also found that, in the great majority
of HCV-infected patients, B cells display an activated phenotype
that disappears after therapeutic eradication of HCV.
Materials and Methods
Patients. We analyzed peripheral blood B lymphocytes from 64
patients (55% males and 45% females; median age of 62.5 years)
with chronic HCV infection, as demonstrated by the presence of
HCV-RNA and elevated serum levels (?40 units?liter) of alanine
aminotransferase. Most of the patients (61%) were infected with
HCV genotype 1; the remainder (39%) were infected by genotypes
2 and 3. A fraction of these patients (26 of 64) were diagnosed with
cryoglobulinemia, as indicated by the presence of dosable levels of
cryoglobulins (?1.0%) accompanied by weakness, arthralgias, and
purpura (see Table 3, which is published as supporting information
antiviral therapy for ?12 months at the time of the sampling. None
of them had clinical evidence of cirrhosis.
Twenty-two of 64 subjects were selected among therapy-naı ¨ve
patients and underwent antiviral therapy. Patients received 1.5 ?g
of peginterferon ?-2b (PEG-Intron, Shering–Plough) per kg of
body weight s.c. once weekly and oral ribavirin (Rebetol, Shering–
Plough) at 1,000–1,200 mg?day (those weighing ?75 kg received
the higher dose). Treatment lasted 6 months for patients with
genotypes 2 and 3 (13 of 22) and 12 months for patients with
genotype 1 (9 of 22). Analyses were performed before and at the
end of therapy. In responders to therapy, as indicated by HCV-
RNA negativity at PCR, an additional analysis after 12 months of
follow-up was performed to identify patients who achieved sus-
tained virological response. The main biochemical and virological
characteristics of HCV-infected patients at the moment of enroll-
Conflict of interest statement: D.R., G.S., E.D.G., U.D., S.N., and M.H. are employees of
Chiron Corporation; S.A. is a consultant of Chiron Vaccines.
Freely available online through the PNAS open access option.
Abbreviations: BCR, B cell antigen receptor; CFSE, carboxyfluorescein succinimidyl ester;
HCV, hepatitis C virus; NK, natural killer; PBMC, peripheral blood mononuclear cell; PE,
phycoerythrin; SAC, Staphylococcus aureus Cowan I.
†D.R., G.S., and E.D.G. contributed equally to this work.
?Present address: National Institute of Molecular Genetics, 20122 Milan, Italy.
**To whom correspondence should be addressed at: National Institute of Molecular
Genetics, Via Francesco Sforza 28, 20122 Milan, Italy. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
December 20, 2005 ?
vol. 102 ?
and 16 patients with chronic hepatitis B, matched for age and sex,
were used as control groups. Study protocols were approved by the
ethical committee of Trieste University School of Medicine.
All subjects provided written informed consent.
Clinical Tests. Alanine aminotransferase was measured by using
standard clinical tests. HCV RNA was determined by using RT-
PCR by Amplicor HCV assay (Roche Diagnostics) and quantified
with a REAL QUANT C kit (Genedia, Munich). HCV genotypes
were determined by using the line-probe assay (Inno-Lipa HCV,
Innogenetics, Ghent, Belgium). To evaluate the presence of cryo-
globulins, blood samples were allowed to clot at 37°C; serum was
centrifuged for 10 min at 350 ? g at 4°C in graduated Wintrobe
was measured by rate nephelometry.
Cell Preparation and Purification. Peripheral blood mononuclear
cells (PBMCs) were obtained from the blood of patients and
healthy volunteers by Ficoll-Hypaque (Amersham Pharmacia Bio-
tech) gradient centrifugation. B lymphocytes were negatively pu-
rified from PBMCs of healthy donors. Briefly, the cells were first
stained with purified mAbs specific for CD2 (clone RPA-2.10),
CD14 (clone M5E2), CD16 (clone 3G8), and CD56 (clone B159)
(BD Biosciences) at 4°C for 30 min; after washing, the cells were
incubated with magnetic beads coated with goat anti-mouse IgG
antibodies (Miltenyi Biotec, Auburn, CA) according to the manu-
facturer’s instructions. The purity of selected B lymphocytes was
?98%, as confirmed by FACS analysis using peridinin–
BD Biosciences). Purified B lymphocytes were stained with phy-
coerythrin (PE)-anti-CD27 mAb (clone L-128, BD Biosciences),
and naı ¨ve (CD27?) and memory (CD27?) subsets were sorted by
FACSVantage SE (Becton Dickinson) with ?96% purity. Human
tonsils were obtained from patients undergoing routine tonsillec-
beads coated with an anti-CD2 mAb (Dynabeads, Oxoid, Basing-
stoke, U.K.). More than 97% of the isolated cells expressed CD19.
Generation of mAbs Specific for Human CD81-LEL. Monoclonal anti-
bodies (MG81 and N81, IgG1 isotype) were obtained in our
laboratory from BALB?c mice immunized with a recombinant
human CD81-LEL fused with thireodoxin (10) by using standard
to CD81 on CD81-transfected NIH 3T3 cell line (American Type
Culture Collection). The F(ab?)2 fragments were prepared by
pepsin digestion by using the ImmunoPure F(ab?)2 kit (Pierce)
according to the manufacturer’s instructions. Retention of the
ability of the F(ab?)2fragment of N81 and MG81 mAbs to bind
CD81 on the cell surface was confirmed by flow cytometry.
Cell Cultures and Reagents. All cultures were performed in RPMI
medium 1640 (GIBCO?BRL) supplemented with vitamin, strep-
tomycin, and glutamine, all from GIBCO?BRL and 10% FCS
(HyClone) and incubated at 37°C in a humidified atmosphere
containing 5% CO2. For the immunoglobulin-secretion experi-
ment, complete medium containing 5% of ultralow IgG FCS
(GIBCO?BRL) was used.
Cells were stimulated in vitro with the following reagents: anti-
recombinant purified HCV-E2384–715 protein (96% pure) (18);
anti-CD19 mAb (clone HIB19, BD Biosciences); anti-CD21 (clone
B ly4, BD Biosciences); recombinant human CD81-LEL (purity
?90%) (10); formalinized Staphylococcus aureus Cowan I (SAC)
(Pansorbin, Calbiochem); goat anti-human IgM F(ab?)2fragments
specific for ?-chains (Cappel ICN Biomedical).
B Cell Proliferation Assay.TriplicatesofpurifiedBlymphocyteswere
seeded at 2 ? 105cells per well in a 96-well flat-bottom plate
(Costar) and stimulated with the following reagents: anti-CD81
mAbs (MG81 and N81), HCV-E2 protein, anti-CD19, anti-CD21,
SAC, and anti-IgM. For inhibition experiments, MG81, HCV-E2,
SAC, and anti-IgM were preincubated with recombinant CD81-
LEL for 10 min at 37°C and added to the cells. After 4 days of
and cells were incubated for an additional 16 h and harvested onto
filter plates (Packard). [3H]thymidine incorporation was measured
by using a TopCount NXT ?-counter (Packard).
Flow Cytometry. PBMCs or purified B cells were stained in a
one-step procedure incubating cells for 20 min at 4°C with the
following FITC-, PE- and PerCP-conjugated mAbs: anti-CD19
(clone 4G7), anti-CD27, anti-CD69 (clone L78), anti-CD71 (clone
L01.1), anti-CD86 (clone 2331), and anti-CXCR3 (clone 49801).
Mouse isotype-matched FITC, PE, or PerCP were used as negative
controls. All mAbs were purchased from Becton Dickinson or
Pharmingen, except for the CXCR3 mAb, which was purchased
from R & D Systems. After washing, samples were acquired on a
FACSCalibur flow cytometer (Becton Dickinson), and data were
processed by using the program CELLQUEST (Becton Dickinson).
Analyses of B cells on total PBMCs were performed, gating on
and memory B lymphocytes were resuspended at 2 ? 107cells
concentration of 0.5 ?M (19). The CFSE-labeled cells were
incubated with complete medium, anti-CD81 mAbs (MG81 and
N81), or SAC for 96 h, stained with PE-anti-CD27, and analyzed
by flow cytometry by using FACSCalibur (Becton Dickinson).
Cell viability was measured by propidium iodide incorporation.
In Vitro Ig Production. For Ig measurements, 1 ? 106purified naı ¨ve
and memory B lymphocytes were cultured with CD81 mAbs
(MG81 and N81) or SAC. At day 10, IgM and IgG concentration
in culture supernatants were measured by standard ELISA tech-
niques, as described in ref. 20.
Western Blot Analysis. B lymphocytes purified from human tonsils
N81 (5 ?g?ml) antibodies or goat anti-human IgM F(ab?)2frag-
ments (IgM) (10 ?g?ml) at 37°C. At the indicated time, cells were
washed with cold PBS and lysed for 1 h on ice in 300 mM NaCl, 50
mM Tris?HCl (pH7.6), 1% Triton X-100, 1 mM NaF, 1 mM
Na3OV4, 10 mM sodium pyrophosphate, 1 mM PMSF, and 1?
EDTA-free complete protease-inhibitor mixture (Roche Diagnos-
tics). Equal amounts of protein extracts were resolved on 10%
SDS?PAGE gel and transferred on nitrocellulose membrane. Blot-
ting was performed by using horseradish-peroxidase-conjugated
4G10 anti-phosphotyrosine antibody (Upstate Biotechnology,
Lake Placid, NY), phospho-c-Jun (KM1) and total c-Jun (H-79)
antibodies (Santa Cruz Biotechnology), total CD19 and phospho-
CD19 (Tyr-531) antibodies (Cell Signaling Technology, Beverly,
MA), phospho JNK1 and -2 (pTpY183?185), and total JNK1
(BioSource International, Camarillo, CA). The JNK inhibition
for 10 min at 37°C with increasing concentrations of SP600125
(Calbiochem) or an equivalent amount of DMSO as control.
Statistical Analysis. Comparison of the percentages of activation
at the end of therapy, and at the end of follow-up was performed
by using two-tailed P values, Wilcoxson matched-pairs test
Rosa et al.
December 20, 2005 ?
vol. 102 ?
no. 51 ?
Multimeric Engagement of CD81 Induces B Cell Activation and Prolif-
eration. It is well documented that CD81 cross-linking by HCV-E2
protein or by anti-CD81 mAbs inhibits NK cells (17) and costimu-
cross-linking on human B lymphocytes. Engagement of CD81 on
freshly purified B cells from healthy individuals through HCV-E2
coated on plastic or anti-CD81 mAb cross-linked by anti-mouse
antibodies did not affect expression of activation markers, matu-
ration, or proliferation (data not shown). However, when B lym-
phocytes were cultured for 5 days with a combination of two
anti-CD81 mAbs, MG81 and N81, in soluble form, we detected a
robust B cell proliferation (Fig. 1A). Both MG81 and N81 are
directed against the recombinant CD81-LEL protein; however,
only N81 is capable of neutralizing HCV-E2 binding to CD81. No
stimulation of proliferation was detected when B cells were treated
directed against the other components of the B cell coreceptor,
CD19 and CD21 (Fig. 1A). The stimulation of B cell proliferation
was preserved, although to a lesser extent, when N81 was replaced
by a recombinant form of the HCV-E2 glycoprotein, whereas
HCV-E2 alone did not have any effect (Fig. 1A). We found that B
cells stimulated with F(ab?)2MG81 and N81 mAbs proliferate as
efficiently as cells stimulated with intact immunoglobulins, exclud-
ing the involvement of Fc-mediated activities (Fig. 1B). To further
validate that B cell proliferation was mediated by CD81, recombi-
nant CD81-LEL protein was combined with HCV-E2 plus MG81,
SAC, or anti-IgM. The proliferation induced by CD81 engagement
was strongly inhibited (50% inhibition at 0.25 ?g?ml of CD81-
LEL), whereas CD81-LEL did not affect SAC or anti-IgM stimu-
replaced by N81 or when CD81-LEL was substituted with an
anti-HCV-E2 mAb, which is known to interfere with the binding of
HCV-E2 to CD81 (data not shown).
CD81 engagement resulted in an increased percentage of B cells
expressing the early activation marker CD69, the transferrin recep-
tor CD71, and the costimulatory molecule CD86. Similar results
were obtained when well characterized polyclonal B cell stimuli
(anti-IgM or SAC) were used (Table 1). Table 1 also shows that
CXCR3, which is expressed in a small fraction of resting B cells, is
up-regulated by anti-CD81 mAbs but not by anti-IgM and SAC.
Taken together, these results demonstrate that multimeric engage-
ment of CD81, in the absence of exogenous cytokines and BCR
engagement, induces activation, proliferation, and increased ex-
pression of CXCR3 molecules on B lymphocytes and that the
epitopes involved in HCV binding to target cells are required for
CD81 Engagement Preferentially Activates Naı ¨ve B Cell Proliferation.
subsets on the basis of CD27 expression (21). To better assess the
effect of CD81-mediated activation on these B cell subsets, periph-
eral blood B lymphocytes were separated as CD27?and CD27?
cells, labeled with CFSE, and activated by CD81, IgM, or SAC.
We first assessed the proliferative response through CFSE la-
beling. Fig. 2 shows that naı ¨ve and memory B lymphocytes dis-
played a similar proliferation rate when activated by SAC. In
contrast, CD81 engagement resulted in the preferential division of
in the memory B cell subset (15% vs. 9% control) (Fig. 2 Top). In
a control experiment, we found no differences in the cell death rate
of memory and naı ¨ve B cells activated through CD81 engagement
(data not shown).
CD81 multimeric engagement and SAC stimulation induced the
differentiation of the majority of naı ¨ve cells into memory B
lymphocytes. In addition, both CD81 and SAC treatments resulted
in the up-regulation of activation molecule CD71 in the naı ¨ve and
memory B cell subsets (Fig. 2 Middle).
We then measured the concentration of immunoglobulins in
?g?ml) and 5 ?g?ml N81 F(ab?)2(B); CD81-LEL (0.03–5 ?g?ml) combined with MG81 plus HCV-E2 (?) (5 ?g?ml each), goat anti-human IgM F(ab?)2fragments (‚)
(10 ?g?ml), or SAC (E) (1:30,000, vol?vol) (C). Cell proliferation was assessed by [3H]thymidine incorporation. In A and B, data are shown as the mean of cpm ?
103? SD of triplicate wells. In C, data are expressed as the percentage of inhibition of the B cell proliferation rate calculated in the absence of CD81-LEL. Data
are representative of at least five different experiments performed with independent donors.
CD81 engagement induces human B cell proliferation. B lymphocytes purified from healthy donor PBMCs were cultured for 5 days in the presence of
Table 1. In vitro analysis of B lymphocytes from healthy donors
stimulated by multimeric CD81 engagement
6 ? 1
17 ? 7
7 ? 2
8 ? 4
81 ? 5
77 ? 7
71 ? 7
27 ? 4
57 ? 2
56 ? 3
49 ? 4
4 ? 2
76 ? 10
79 ? 6
75 ? 2
9 ? 6
Data are shown as percentage of CD19 cells expressing the markers indi-
cated on the left (mean ? SD). B lymphocytes from healthy donors (n ? 10)
stimulated in vitro for 24 h by using:*, complete medium; †, MG81 mAb (5
?g?ml) ? N81 mAb (5 ?g?ml); ‡, SAC (1:30,000 vol?vol); §, Goat anti-human
IgM F(ab?)2(10 ?g?ml).
www.pnas.org?cgi?doi?10.1073?pnas.0509402102Rosa et al.
culture supernatants from naı ¨ve and memory B cells stimulated
through CD81 or SAC. As expected, activated naı ¨ve B cells did not
produce IgG but a low (in the case of SAC) or very low (in the case
amount of IgM and IgG in response to SAC activation and a low
amount of IgM and IgG in response to the modest CD81 activation
(Fig. 2 Bottom).
In summary, these results demonstrate that, although CD81
engagement up-regulates activation markers in both the naı ¨ve and
the memory B cell subsets, it promotes a preferential polyclonal
expansion of naı ¨ve B lymphocytes.
CD81-Mediated Activation of B Cells Triggers JNK Activity. We next
assessed the intracellular signaling events induced by multimeric
CD81 engagement on B cells. We found that, in contrast to BCR
stimulation by anti-IgM, multimeric CD81 engagement did not
result in a significant increase in total tyrosine phosphorylation
levels in purified tonsil-derived B cells (Fig. 3A). Despite the
extensive difference observed in the tyrosine phosphorylation
pattern, both CD81 and BCR engagement activated the JNK
pathway, as shown by the increased phosphorylation of JNK1 and
-2 and by the accumulation of the activated form of c-Jun (pJun)
2–4 h after treatment (Fig. 3A). When B cells from the same
experiment were monitored for activation markers and prolifera-
tion, we obtained the same results shown in Table 1 and Fig. 1 for
SP600125 (22) abolished both c-jun activation and proliferation of
tonsil B cells induced by CD81 engagement (see Fig. 6, which is
published as supporting information on the PNAS web site).
Because CD81 is part of the coreceptor complex with CD19, we
assessed whether multimeric CD81 engagement could signal
through the activation of CD19, which occurs through phosphor-
ylation of its cytoplasmic domain. BCR engagement by anti-IgM
stimulated CD19 phosphorylation, whereas CD81 engagement
resulted in a transient inhibition of CD19 phosphorylation (Fig.
3B). The absence of a significant change in tyrosine phosphoryla-
tion levels, together with the inhibition of CD19 activity, suggests
that CD81-mediated activation of B cells is independent from the
B cell receptor and coreceptor complexes. The activation of the
JNK pathway, monitored by phospho c-Jun accumulation, was
specific for multimeric CD81 engagement (MG81 plus N81) and
3C). In contrast, the transient dephosphorylation of CD19 was
observed in response to both multimeric engagement and N81
alone (Fig. 3C).
Chronic HCV Infection Associates with B Lymphocyte Activation. We
reasoned that polyclonal B cell activation obtained by multimeric
CD81 engagement in vitro could mimic what occurs in HCV-
infected patients when whole HCV particles bind CD81 on the B
cell surface in vivo. To test this hypothesis, we compared the
phenotype of peripheral blood B cells from 64 chronic hepatitis C
patients with (n ? 26) or without (n ? 38) cryoglobulinemia with
21 healthy controls and 16 patients with chronic hepatitis B. We
found that B cells from the great majority of HCV patients (54 of
64) expressed elevated levels of the activation markers CD69,
CD71, and CD86 and of the chemokine receptor CXCR3, whereas
B cells from all 16 patients with chronic hepatitis B had the same
levels of activation markers and CXCR3 as healthy controls (Table
associate with a particular HCV genotype, RNA levels (Table 3),
or the presence of cryoglobulinemia (Table 2). In contrast with
previous observations showing that the frequency of B cells is
higher in HCV patients (23), we found the same percentage of B
lymphocytes (CD19?cells) in healthy controls and HCV- and
hepatitis B virus (HBV)-infected patients (data not shown). We
then analyzed the relative expression of activation markers and
CXCR3 on naı ¨ve (CD27?) and memory (CD27?) subsets from
peripheral B cells of healthy controls and HCV patients. We found
more activated naı ¨ve and memory B cells in HCV-infected patients
than in healthy controls; however, the difference in the frequency
of B cells expressing activation markers and CXCR3 between
healthy controls and HCV patients was more pronounced in the
naı ¨ve B cell subset (Fig. 4). Because CD27-negative cells up-
vivo suggests that a continuous activation of naı ¨ve B cells occurs
lymphocytes. Purified naive (CD27?) or memory (CD27?) B cells were incu-
bated with complete medium (Control), MG81 and N81 mAbs (5 ?g?ml each)
(CD81), or SAC (1:30,000, vol?vol). (Top) Proliferation was measured as CFSE
5 ? 105events were acquired. Numbers indicate the percentage of dividing
stimulation was measured by flow cytometry. For each sample, 1 ? 105events
CD71-positive cells. (Bottom) IgM and IgG secretion in cell-culture superna-
tants was measured 10 days after stimulation by ELISA. Data are shown as
mean ? SD of duplicate wells and are representative of at least five experi-
ments performed with independent donors.
CD81 engagement induces the preferential expansion of naı ¨ve B
B cells were stimulated with 5 ?g?ml MG81 plus 5 ?g?ml N81 (CD81) or with
10 ?g?ml anti-IgM (IgM). After the indicated time, equal amounts of protein
pJNK2), and anti-total JNK1 (JNK1) antibodies. Protein standards are ex-
pressed on the left in kDa. The asterisk indicates an unidentified protein
cross-reacting with the anti-phospho JNK1 and -2 antibody. (B) Tonsil-derived
extracts were subjected to Western blot analysis using anti-phospho-CD19
(pCD19) and anti-total CD19 (CD19) antibodies. (C) Tonsil-derived B cells were
stimulated with 5 ?g?ml MG81, 5 ?g?ml N81, or 5 ?g?ml both MG81 and N81
pJun, anti total c-Jun (Jun), pCD19, and CD19 antibodies. ctr, untreated cells.
Rosa et al.
December 20, 2005 ?
vol. 102 ?
no. 51 ?
during chronic HCV infection. Accordingly, we also found that
HCV patients display a higher percentage of memory (CD27?) B
cells compared with healthy controls and HBV patients (Table 2).
In a previous study performed on cryopreserved peripheral
blood lymphocytes from patients with chronic HCV infection,
activated B cells were not found (23). We therefore assessed B cell
separated cells or the same samples that had been cryopreserved.
Freshly isolated B cells displayed an increased expression of CD69,
CD71, CD86, and CXCR3, as described above. In contrast, cryo-
preserved B cells from the same samples, although viable and
responding to polyclonal stimuli, did not display the activated
on the PNAS web site). This finding indicates that assessment of
isolated rather than cryopreserved peripheral blood lymphocytes.
Eradication of HCV Associates with a Decreased Number of B Cells
Expressing CXCR3 and Activation Markers. To assess whether B cell
activation correlates with HCV replication, we investigated the B
cell phenotype in 22 patients with chronic HCV infection before
sustained virologic response (n ? 15), eradication of the virus
associates with the reduction of the number of B cells expressing
expressing CD69, CD71, CD86, and CXCR3 at the end of the
(P ? 0.005). Importantly, in these patients, the same expression
pattern persisted for the 12-month follow-up period (Fig. 5 and
In contrast, in nonresponder patients (n ? 7), the percentage of
peripheral blood B lymphocytes expressing CD69, CD71, CD86,
and CXCR3 did not change significantly before and after the end
of the therapy (Fig. 5 and Table 3). Altogether, these data establish
a correlation between the presence of HCV virus and the up-
regulation of activation markers and CXCR3 on circulating B cells.
We found that engagement of the HCV receptor CD81 activates
human B cells in the absence of BCR coligation. CD81-mediated
activation differs from other polyclonal B cell stimuli in that it
and SAC activate naı ¨ve and memory B cell proliferation equally
well, and CpGs selectively activate memory B cells (24).
CD81-mediated B cell activation occurred through a combina-
one anti-CD81 mAb. Because a single antibody should be able to
induce capping, our finding indicates that membrane reorganiza-
multimeric CD81 engagement required for activation of B cells is
in apparent contrast with the requirement of a single CD81 ligand
and a cross-linking agent in CD81-mediated inhibition of NK cells
(17) and costimulation of T lymphocytes (16). However, because
CD81 associates with specific complexes on different cell lineages
(12), it is likely that CD81 promotes cell-type-restricted signaling
events that might or might not require multiple ligands or receptor
lymphocytes from HCV patients (HCV) and healthy subjects (Control) were
acquired. Data is shown as the percentage of naı ¨ve B cells (CD19??CD27?) or
memory B cells (CD19??CD27?) expressing the indicated molecules (mean ?
SD). Numbers on bars represent the ratio between the level of expression of
each marker in HCV patients over control.
HCV patients display high percentage of activated naı ¨ve B cells. B
Table 2. Ex vivo analysis of B lymphocytes from HCV-infected
MarkerControl* HCV cryo†
20 ? 4
5 ? 1
19 ? 6
12 ? 3
9 ? 2
43 ? 20
33 ? 11
52 ? 16
34 ? 14
44 ? 16
42 ? 20
35 ? 12
38 ? 13
31 ? 7
38 ? 12
23 ? 5
6 ? 2
17 ? 6
8 ? 2
9 ? 3
Data are shown as percentage of CD19 cells expressing the markers indi-
cated on the left (mean ? SD).*, Healthy donors (n ? 21); †, HCV-infected
patients with cryoglobulinemia (n ? 26); ‡, HCV-infected patients (n ? 38); §,
in Table 3. HBV, hepatitis B virus.
of activation markers and CXCR3. Ex vivo expression of activation molecules
ribavirin therapy (for details, see Materials and Methods) and 21 healthy
subjects (Control). Fifteen patients responded to therapy (Responders),
whereas seven did not eradicate HCV infection (Non Responders), as demon-
strated by HCV-RNA PCR. B lymphocytes from HCV-infected patients were
monitored before and soon after the end of therapy. In responders, an
up). All responders achieved sustained virological response. Data is shown as
the percentage of CD19-positive cells expressing the indicated markers
(mean ? SD). (*, P ? 0.005 as compared with patients before therapy)
Eradication of HCV infection associates with a decreased expression
www.pnas.org?cgi?doi?10.1073?pnas.0509402102 Rosa et al.
CD81 has very short cytoplasmic regions without any recogniz-
able signaling motif and is believed to signal through interaction
with associated partners. CD19 directly binds to CD81, represent-
ing an obvious candidate to exert such a function (25). Our data,
showing that CD19 is not activated during CD81 engagement,
identified (26, 27), but more work is required to assess whether
have found that CD81 engagement activates the JNK pathway
through a mechanism that, at variance with the BCR-dependent
JNK activation, is not associated with general tyrosine phosphor-
ylation, as detected by Western blot. The finding that the CD81
signaling pathway diverges from the BCR pathway upstream of
JNK activation might reveal new targets for the treatment of
lymphoproliferative diseases resulting from uncontrolled B cells
We have found that chronic HCV infection associates in 85% of
cases with a high percentage of activated B cells and that thera-
peutic eradication of HCV coincides with the reduction of this
activated B cell phenotype. Our data demonstrate that polyclonal
B cell activation is a general feature of chronic HCV infections,
because B cell activation is observed regardless of the presence of
mixed cryoglobulinemia. It is tempting to speculate that the poly-
clonal B cell activation obtained by multimeric CD81 engagement
binds CD81 on the B cell surface. Notably, our model does not
require the assumption that B cells are infected by HCV. Indeed,
the surface contact between HCV and CD81 could activate B cells
in the absence of infection. Our finding that CD81 engagement by
HCV envelope protein preferentially activates naı ¨ve B lymphocyte
proliferation may be relevant to explain the high rate of autoanti-
bodies found in HCV infections (28) and the association of HCV
independent activation of naı ¨ve B cells induced by HCV engage-
ment of CD81 is the first step toward the polyclonal expansion of
can be more readily activated in a bystander mode to produce
autoantibodies (24). In a few cases, this B cell autoreactivity, most
likely in combination with still unidentified genetic?environmental
factors, could evolve into frank lymphoproliferative disorders, such
as type II cryoglobulinemia (5) or non-Hodgkin lymphoma (6, 7).
We found that the chemokine receptor CXCR3 is up-regulated
at low level on B cells activated by SAC or BCR engagement. This
finding correlates with an increased expression of CXCR3 on B
cells derived from HCV-infected patients compared with those of
healthy controls. The ligands of CXCR3 (CXCL9, CXCL10, and
fashion together with locally produced cytokines could explain the
B lymphocyte follicles frequently observed in the livers of patients
with chronic HCV infection (32). Recently, in agreement with our
observations, an independent study has shown that CXCR3 is
those of healthy controls (33).
It is possible that the HCV–CD81 interaction plays a central role
for virus adaptation to the human host. We speculate that the
evolutionary pressure on the compact genome of HCV virus has
functions during infection. These activities include not only binding
and entry into target cells but also the modulation of the immune
system required to establish a chronic infection. The interaction
with the widely distributed tetraspanin CD81, a promiscuous
molecule forming different receptor complexes on different cell
types, may be a multifunctional strategy for HCV to achieve
multiple goals through the same envelope protein. Indeed, CD81
engagement by the HCV envelope protein inhibits NK cells (17),
costimulates T lymphocytes (16), and activates B cells (this article).
Interestingly, it has been recently reported that the engagement of
B cells by purified E2 induced double-strand DNA breaks specif-
ically in the variable region of Ig (VH) gene locus, leading to
hypermutation in the VHgenes of B cells (34). Altogether, one can
depict a scenario where interaction between HCV and CD81
mechanism for the virus through the inhibition of the innate
resulting from polyclonal expansion.
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