Clonal expansion of immunoglobulin M CD27 B cells in HCV

IMMUNOBIOLOGY

Clonal expansion of immunoglobulin M�CD27� B cells in HCV-associatedmixed cryoglobulinemia

Edgar D. Charles,1,2 Rashidah M. Green,1 Svetlana Marukian,1 Andrew H. Talal,2 Gerond V. Lake-Bakaar,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; and 2Department of Medicine, Divisionof Gastroenterology and Hepatology, Weill Medical College of Cornell University, New York, NY

Hepatitis C virus (HCV) is associatedwith B-cell lymphoproliferative disor-ders such as mixed cryoglobulinemia(MC) and B-cell non-Hodgkin lymphoma(B-NHL). The pathogenesis of these dis-orders remains unclear, and it has beenproposed that HCV drives the pro-liferation of B cells. Here we demon-strate that certain HCV�MC� subjectshave clonal expansions of immuno-globulin M (IgM)��IgDlow/�CD21low

CD27� B cells. Using RT-PCR to amplify

Ig from these singly sorted cells, weshow that these predominantly rheuma-toid factor-encoding VH1-69/JH4 andV�3-20 gene segment-restricted cellshave low to moderate levels of somatichypermutations. Ig sequence analysissuggests that antigen selection drivesthe generation of mutated clones. Thesefindings lend further support to the no-tion that specific antigenic stimulationleads to B-cell proliferation in HCV MCand that chronic B-cell stimulation may

set the stage for malignant transforma-tion and the development of B-NHL. Thefinding that these hypermutated, mar-ginal zone-like IgM�CD27� B cells areclonally expanded in certain subjectswith MC offers insight into mechanismsof HCV-associated MC and B-cell malig-nancy. This study was registered atwww.clinicaltrials.gov as NCT00219999.(Blood. 2008;111:1344-1356)

© 2008 by The American Society of Hematology

Introduction

Up to 170 million people worldwide are chronically infected withhepatitis C virus (HCV), which is the leading indication for livertransplantation in the United States and Europe.1,2 Althoughhepatocytes are the primary target for HCV infection, mixedcryoglobulinemia (MC), considered a benign B-cell proliferativedisorder, can affect up to 50% of HCV patients.3,4 HCV MC ispredominantly characterized by cold-precipitable complexes ofmonoclonal immunoglobulin (Ig) M kappa (�) rheumatoid factor(RF), polyreactive IgG, HCV RNA, and HCV structural pro-teins.3,5,6 MC symptoms range from weakness, arthralgias, andpalpable purpura to more severe manifestations, such as neuropa-thy and membranoproliferative glomerulonephritis.7 HCV infec-tion is also associated with the development of B-cell non-Hodgkinlymphoma (B-NHL).8-14

The mechanisms by which HCV causes B-cell proliferation(reviewed in Dustin and Rice15) remain puzzling. Severalstudies have demonstrated the presence of abnormal clonalB-cell populations in the liver and blood of HCV–infectedpersons.16-18 Patients with HCV MC and B-NHL frequently haveclonal B cells that use Ig heavy chain variable (VH) 1-69 and Ig�V�3 gene segments,19-22 which can encode RF of the WAidiotype.23 Chronic antigenic stimulation by HCV may select aclonally expanded B-cell receptor (BCR) repertoire, which laterprogresses to B-NHL,24 similar to Helicobacter pylori–inducedmucosa-associated lymphoid tissue lymphomas.25 Consistentwith this idea, Ig cloned from an HCV� B-NHL patient wasshown to bind to the HCV structural protein, E2. However, this

Ig does not cross-react with IgG.26 It has also been suggestedthat HCV E2 directly activates polyclonal B cells by binding toCD81, a tetraspanin that forms part of the B-cell coreceptor.27

Furthermore, it was reported that the memory B-cell compart-ment from HCV� persons is polyclonally activated and that itcontains decreased steady-state levels of CD27� memory cells,which rapidly differentiate independently of the BCR.28 How-ever, these latter 2 studies fail to account for the clonal IgM�restriction frequently seen in HCV MC and B-NHL. Others haveproposed that direct HCV infection of B cells may causeoncogenic transformation, particularly through t(14;18) translo-cation.29,30 Although t(14;18)-positive cells have been detectedin HCV-infected patients, they are also present in up to 50% ofhealthy persons.31 Moreover, it is controversial whether HCVcan infect B cells (Lanford et al32 and L.B.D., unpublished data,2007), and this hypothesis also fails to explain the clonal B-cellexpansions frequently seen in HCV infection.

In support of the polyclonally activated B-cell model, it wasrecently reported that HCV� patients have high levels ofactivated naive B cells in the blood and that levels of total B-cellactivation do not significantly differ between MC� and MC�

persons.27 These findings contrasted with our earlier finding thatmost HCV patients had no evidence of circulating activated Bcells.33 This discrepancy led us to analyze peripheral B cellsfrom HCV�MC� and HCV�MC� patients. We have found that,in most HCV patients, circulating B cells show no evidence ofcurrent or prior activation. However, certain HCV�MC� pa-

Submitted July 17, 2007; accepted October 12, 2007. Prepublished onlineas Blood First Edition paper, October 17, 2007; DOI 10.1182/blood-2007-07-101717.

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.

© 2008 by The American Society of Hematology

1344 BLOOD, 1 FEBRUARY 2008 � VOLUME 111, NUMBER 3

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tients harbor a substantial population of clonally expandedIgM�Ig��IgDlow/�CD21lowCD27� B cells in the peripheralcirculation. Moreover, these B cells express RF-like Ig that is inmost cases hypermutated, indicating specific antigen-drivenmaturation. Furthermore, phylogenetic analysis suggests thatthis hypermutation is a result of antigen selection. An associa-tion of IgM� memory-like B cells with MC, which itself isassociated with B-NHL, is consistent with a model in whichchronic, specific antigenic stimulation of IgM� B cells predis-poses toward neoplastic transformation.

Methods

Patients

The studies were approved by the Institutional Review Boards at theRockefeller University and New York-Presbyterian Hospitals. Beforeenrollment, written informed consent was obtained from donors in accor-dance with the Declaration of Helsinki. We enrolled HCV Ab�, HCVRNA� subjects whose medical history or physical examination wasconsistent with arthralgias and/or palpable purpura. We additionallyscreened for sensorimotor neuropathy by neurologic examination. All HCVRNA� subjects were HIV Ab�. For controls, we enrolled HCV Ab�, HBV-immunized healthy volunteers at low risk for HIV. We also enrolled HCVRNA�, HCV Ab� patients who had undergone antiviral treatment withpegylated interferon and ribavirin and who subsequently had 2 separateconfirmations of HCV RNA less than 50 IU/mL at least 6 months aftercessation of treatment and were sustained virologic responders.

Blood collection and processing

A total of 50 mL of blood was collected into Vacutainer tubes containingacid-citrate-dextrose solution A (BD Biosciences, Franklin Lakes, NJ) and aserum separator tube (prewarmed to 37°C). We obtained serum by allowingblood to clot at 37°C followed by centrifugation at 800g. Plasma wasobtained by room-temperature centrifugation and was stored at �80°C. Toobtain peripheral blood mononuclear cells (PBMCs), cells were separatedon lymphocyte separation medium (ICN, Costa Mesa, CA) by centrifuga-tion at 800g. The mononuclear cell layer was washed 3 times inphosphate-buffered saline (PBS) supplemented with 1% N-2-hydroxyeth-ylpiperazine-N-2-ethanesulfonic acid (HEPES) and 2% fetal bovine serum.Cells were resuspended at a concentration of 107/mL in RPMI 1640supplemented with 0.5% HEPES, 20% fetal bovine serum, and 7.5%dimethylsulfoxide for cryopreservation. Cells were slowly frozen to �80°Cand maintained at �150°C. Viable cell recovery was typically more than90% as judged by incorporation of an amine-reactive violet dye (LIVE/DEAD Fixable Dead Cell Stain, Invitrogen, Carlsbad, CA).

Clinical tests

To test for MC, we incubated serum at 4°C for 1 week and examined it dailyfor the presence of a cryoprecipitate.34 To determine MC type, anestablished protocol was followed.35 Cryoprecipitate was spun in a 4°Cmicrocentrifuge at 650g for 15 minutes, the supernatant was removed, andthe pellet was vortexed 6 times with 5 mL of ice-cold PBS, each timespinning out the cryoprecipitate for 15 minutes at 650g. The sample wasthen resuspended in 100 �L of PBS and was redissolved by incubation at37°C for 1 hour. Immunofixation was performed for IgM, IgG, IgA, Ig�,and Ig� using an immunofixation kit (Binding Site, Birmingham, UnitedKingdom), following the manufacturer’s instructions. HCV RNA wasquantified by the Roche Amplicor assay (version 2.0, Roche Diagnostics,Branchberg, NJ). HCV genotypes were determined by sequencing of theHCV NS5B region. Liver biopsies were evaluated by pathologists accord-ing to the Scheuer system.36 These actions, in addition to serum alanineaminotransferase (ALT) measurements, were performed as part of HCVpatients’ medical care. We measured plasma IgM RF using ELISA (BethylLaboratories, Montgomery, TX).

Antibodies

The following mouse mAbs were used: anti-CD19, anti-CD20, anti-CD3,anti-IgM, anti-IgD, anti-CD27, anti-CD69, anti-CD5, anti-CD21, andanti-CD25. FITC, phycoerythrin (PE), PE-Cy7, peridinin chlorophyllprotein cyanin 5.5 (PerCP-Cy5.5), allophycocyanin, Alexa Fluor 700,Alexa Fluor 488, and allophycocyanin Cy7 were used as fluorophores.Conjugated mAbs were obtained from BD PharMingen (San Diego, CA).Goat polyclonal F(ab�)2 PE anti-� and biotin anti-� were from SouthernBiotech (Birmingham, AL), and LIVE/DEAD Fixable Dead Cell Stain wasfrom Invitrogen.

Flow cytometric analysis

Cells (7 � 106/sample) were stained with the indicated Abs at RT in PBSsupplemented with 2% (w/v) BSA (Fraction V; Fisher Biotech, Fair Lawn,NJ) and 0.02% NaN3. Cells were incubated with primary and secondaryAbs for 30 minutes each, and were washed thrice after each stain withincubation buffer. Cells were fixed in PBS/2% paraformaldehyde andanalyzed within 1 hour on an LSRII flow cytometer (BD ImmunocytometrySystems, San Jose, CA). Data were analyzed using FlowJo (Tree Star,Ashland, OR). Lymphocytes were identified by forward and side-anglelight scatter characteristics. Dead lymphocytes, identified by positiveLIVE/DEAD stain, were excluded from further analysis.

CDR3 PCR

DNA was recovered from 3 � 106 PBMCs using a QIAamp DNA BloodMini Kit (Qiagen, Valencia, CA). PCR amplification was performed onup to 0.5 �g of DNA with a seminested protocol of amplificationaccording to a well-established procedure.37 The upstream primer wascomplementary to the conserved third framework V region (FR3) of theIgH gene, and the downstream primer annealed to an outer conservedregion of the IgH joining (J) region in the first round of amplification andto an inner conserved sequence of the same J region in the secondround.38 All samples were tested in 2 separate PCR experiments, andclonal and polyclonal CDR3 DNA controls, as well as a wateronly control, were included. At the end of amplification, 15 �L of thereaction mixture was analyzed by 12% polyacrylamide gel electro-phoresis, stained with ethidium bromide, and optically analyzed byultraviolet transillumination. Gels were scanned using an Epson Expres-sion 1600 scanner (Epson America, Torrance, CA) and densitometricanalysis was performed with ImageJ software (National Institutes ofHealth [NIH], Bethesda, MD).39 Clonal expansion was determined bythe presence of one or more discrete, dominant bands within an 80- to120-bp size range, and a polyclonal population was indicated by thepresence of a smear with no specific dominant bands.

Single-cell sorting

For single-cell sorting, incubations and washes were performed as forimmunophenotyping, except incubations were performed on ice in theabsence of added 0.02% NaN3. Moreover, cells were not fixed inparaformaldehyde after staining. Either IgM��CD21lowCD27�,IgM��CD21lowCD27�, IgM��CD5�CD27�, IgM��CD5�CD27�, orIgM��CD27�, single cells were sorted using a MoFlo cell sorter (DakoCytomaton, Ft. Collins, CO), directly seeded into 96-well plates containing4 �L lysis solution (0.5� PBS containing 10 mM DTT, 2 U RNase Inhibitor(Invitrogen) and 3 U Prime RNase Inhibitor (Eppendorf)), and immediatelyfrozen on dry ice. Samples were stored at �80°C.

VH and V� RT-PCR

We followed an established protocol for cloning VH and V� from singleB cells.40 Briefly, RNA was reverse transcribed in 14 �L reactionscontaining random hexamer primers and 50 U Superscript II RT (Invitro-gen) for 55 minutes at 40°C. RT-PCR reactions, primer sequences, andcloning strategy were as previously described.40 For VH, we used a pool offamily-specific sense primers directed to the 5� conserved leader and Vregions, and a downstream antisense primer directed to the first constant

CLONAL EXPANSION OF IgM�CD27� B CELLS IN HCV MC 1345BLOOD, 1 FEBRUARY 2008 � VOLUME 111, NUMBER 3




region of IgM (C�). Amplification was performed using Taq polymerase(Invitrogen). A second round of PCR was performed on this reactionproduct with VH- and JH-specific primers. For V�, we used pooledfamily-specific primers directed to the 5� conserved leader and V� regions,and a downstream antisense primer to the conserved C� region. Weamplified this PCR product with a 5� pan V� primer and a downstream 3� C�

primer. IgH and Ig� variable regions were sent for sequencing (BioticSolutions, New York, NY).

Analysis of somatic hypermutation and phylogeny

The International ImMunoGeneTics information system41 and NIHJoinsolver42 were used to identify the V, D, and J segments and somaticmutations of Ig genes.43 The ratio of replacement (R) and silent (S)

mutations was determined and analyzed according to a binomialdistribution model,44 using a web-based JAVA applet.45 Nucleotidesequences were aligned using Clustal W.46 Phylogenetic trees wereconstructed with MacVector (Accelrys, San Diego, CA), usingdistance-based neighbor-joining analysis performed on Tamura-Neineighbor estimates. Bootstrap analyses were conducted using1000 replicates.

Statistics

Values of clinical tests are reported as means (� SD). To test the nullhypothesis that clonal Ig was not more prevalent among HCV�MC�

subjects, Fisher exact test was used to compute a P value. All othercomparisons used the Kruskal-Wallis test, followed by the Dunn multiplecomparison test, as appropriate. We computed all statistics with Prism(GraphPad, San Diego, CA).

Accession numbers

GenBank accession numbers for the cloned Ig sequences are EF624068-EF624206, EF624208-EF624238, and EU146311-EU146395.47

Results

Characteristics of study subjects

Twenty-five patients with signs or symptoms suggestive ofcryoglobulinemic vasculitis, such as arthralgia, palpable pur-pura, peripheral neuropathy, and glomerulonephritis, were en-rolled. Subsequent tests revealed that 12 of these patients had

Table 1. Characteristics of subjects

CharacteristicHCV Ab�,

N � 16HCV�, Ab�,

N � 10HCV�,N � 25

Age, y

Mean (95% CI) 39.4 (33.8-45.0) 54.6 (47.0-62.3) 53.3 (49.7-56.7)

Median 37 54 54

Range 24-55 39-71 29-69

Males, no. (%) 7 (43.4) 3 (30.0) 9 (36.0)

Race or ethnic group, no. (%)

White 14 (87.5) 7 (70.0) 17 (68.0)

Black 1 (6.2) 0 (0.0) 3 (12.0)

Hispanic 1 (6.2) 2 (20.0) 5 (20.0)

Asian 0 (0.0) 1 (10.0) 0 (0.0)

MC positive, no. (%) 0 (0) 0 (0) 12 (48)

MC indicates mixed cryoglobulinemia.

Table 2. Characteristics of HCV� subjects

Subjectno. Sex Age, y

HCV riskfactor

HCVtreatment

statusALT,IU/mL HCV GT

HCV RNA,IU/mL � 106 Stage Extrahepatic conditions

Plasma [IgM RF],IU/mL

MCtype

HCV� MC�

108 M 57 IVDU Naive 38 1 0.3 ND Arthralgia 137.6 NA

159* M 59 Unknown NR 85 1a 10.1 3 Arthralgia 22.7 NA

162 M 51 IVDU NR 46 1b 0.29 0 Arthralgia 8.5 NA

1087 M 58 IVDU Naive 39 1 0.7 0 Arthralgia 13.6 NA

1148 F 48 Unknown Naive 41 1a 10 2 NHL (in remission), neuropathy 6.4 NA

1235 F 41 Unknown Naive 376 2b 3.9 ND Arthralgia 4.0 NA

1330 F 54 IVDU Naive 35 1a 10 2 Arthralgia, palpable purpura 8.3 NA

1339 M 58 IVDU Naive 60 1 4.25 4 Arthralgia 6.6 NA

1345 M 57 IVDU Naive 29 1a 0.3 ND Arthralgia 6.7 NA

1349 F 53 Transfusion Naive 27 2b 3.1 ND Arthralgia 7.5 NA

1373 F 49 INDU Relapser 29 1a 0.47 3 Palpable purpura 15.0 NA

1419 F 47 IVDU Naive 23 1a 0.83 3 Arthralgia, palpable purpura 6.5 NA

1446 F 67 Transfusion Naive 26 2b 0.6 0 Palpable purpura 60.9 NA

HCV� MC�

192 M 55 Unknown Naive 38 2b 0.66 1 Arthralgia, palpable purpura 10.0 III, IgM�

389* M 55 IVDU NR 52 1a 2 3 Arthralgia, palpable purpura, neuropathy 7.5 II, IgM�

734* M 53 Unknown Naive 117 1a 2.6 1 Arthralgia 32.6 ND

773* F 29 Unknown Naive 148 1b 0.38 4 Arthralgia, palpable purpura 68.8 II, IgM�

880* F 54 Unknown Naive 40 1b 3.08 ND Arthralgia, palpable purpura 46.2 II, IgM�

960* F 46 IVDU Relapser 33 1a 4.2 2 Arthralgia 93.3 II, IgM�

1073* F 46 Unknown NR 38 1a 1.34 2 Arthralgia, palpable purpura, neuropathy 37.0 II, IgM�

1308* F 54 IVDU Naive 95 1a 0.7 1 NHL 237.5 II, IgM�

1403* F 47 IVDU NR 13 1 1.34 4 Palpable purpura, GN, pulmonary hemorrhage 323.9 II, IgM�

1432* F 69 Transfusion Relapser 56 1a 0.03 4 Palpable purpura, hemolytic anemia 35.2 II, IgM�

11502 F 67 Transfusion Naive 41 1a 0.11 2 Arthralgia 21.4 ND

92200* F 57 Transfusion Naive ND 2b 3.1 2 Arthralgia, palpable purpura 5.3 III, IgM�

GT indicates genotype; IVDU, intravenous drug use; NA, not applicable; ND, not determined; NR, nonresponder; NHL, non-Hodgkin lymphoma; INDU, intranasal drug use;and GN, glomerulonephritis.

* Subjects with clonal Ig (as determined by PBMC DNA CDR3 PCR).

1346 CHARLES et al BLOOD, 1 FEBRUARY 2008 � VOLUME 111, NUMBER 3




demonstrable cryoprecipitates. Characteristics of the groups arepresented in Table 1. The HCV Ab� group was completely freeof arthralgia, palpable purpura, and neuropathy. Two HCV�,Ab� subjects had arthralgias. Only HCV� subjects had labora-tory evidence of MC. HCV�, Ab� subjects had previously beeninfected with HCV genotype 1 (n 7), 2b (n 1), 3b (n 1),or HCV of unknown genotype (n 1).

Table 2 describes the characteristics of the HCV-infected group.One HCV�MC� subject (1308) had a triclonal paraproteinemia onserum immunofixation electrophoresis and underwent bone mar-row biopsy, which revealed monotypic B cells with a normalkaryotype, consistent with low-grade NHL. Another HCV�MC�

patient (1148) had NHL in remission after treatment with cyclophos-phamide, doxorubicin, vincristine, and prednisone 1 year beforeenrollment. Compared with MC� subjects, MC� subjects tended tohave more than one MC related symptom or disease. Consistentwith an earlier report,48 monosymptomatic arthralgia was nonspe-cific for MC. IgM RF was not significantly elevated in HCV�MC�

subjects compared with HCV�MC� subjects. In the 10 subjectsin whom MC was typed, the cryoprecipitate was IgM�, and 8 of10 subjects had type II MC.

HCV�MC� patients have clonal B-cell expansions

We performed Ig CDR3 PCR on DNA isolated from PBMCs toassess for clonality (Figure 1). HCV�, Ab� patient samplesyielded DNA smears indicating polyclonality. Similarly, allHCV Ab� volunteers had evidence of polyclonal CDR3 (data

not shown). Consistent with previous reports,16,17 clonalCDR3 was highly correlated with the presence of MC. Monoclo-nal or oligoclonal bands with sizes between 80 to 120 basepairs were found in 10 of 12 HCV�MC� subjects, and in 1 of13 HCV�MC� subjects (P .001, Fisher exact test). Theseresults suggest that the elevated IgM and RF frequently seen inHCV�MC� patients is the result of clonal B-cell proliferation.Because, in the absence of overt malignancy, B-cell clonalexpansion requires activation through a B cell’s unique BCR,these data are consistent with the hypothesis that there isan antigenic stimulus that causes MC disease in differentHCV-infected patients.

B cells from certain HCV�MC� subjects are biased toward arestricted IgM��IgDlow/�CD21lowCD27� phenotype

We used flow cytometry to identify the expanded B-cell clonalsubpopulation in this subset of HCV�MC� subjects. We testedwhether our cell freezing protocol altered the detectable B-cellimmunophenotype. We saw no difference in B-cell activationmarkers between fresh and frozen B-cell samples (Figure S1,available on the Blood website; see the Supplemental Materialslink at the top of the online article). We then determined whetherHCV� subjects with clonal Ig had increased percentages ofperipheral B cells (Table 3). After gating on lymphocytes, weexcluded dead cells, and we selected CD19�, CD20� B cells(Figure S2A). B-cell percentages (measured as the percentage oflymphocytes expressing CD19 and CD20) were slightly increased

Figure 1. Ig CDR3 PCR. DNA was isolated from PBMCs,amplified following an established protocol (see “CDR3PCR”) and run on a 12% polyacrylamide gel. PolyclonalCDR3s are indicated by the presence of DNA smearsbetween 80 and 120 bp, the expected size range ofCDR3. Monoclonal and oliglonal CDR3s are reflected bythe presence of solitary, or 2 to 3 bands, respectively, inthe expected size range (A). Densitometric analysis ofgel bands (B). *Patients with MC.

Table 3. B-cell subsets among subject groups

GroupCD19�,CD20�

cells,%

CD19�, CD20� cells, %

��, ��, % ��, CD27�,% IgM�,CD27�, % IgM�, IgDlow/�, % CD21low,CD27�, % % CD5�

HCV Ab�, n 16 14.74 � 5.82 47.42 � 11.30 17.23 � 9.78 13.43 � 7.59 8.21 � 6.20 3.41 � 2.29 22.18 � 5.82

HCV�, Ab�, n 10 13.75 � 6.02 52.07 � 4.98 17.33 � 6.47 10.12 � 4.66 5.61 � 4.41 3.47 � 3.41 13.75 � 6.02

HCV� clonal, n 11 18.55 � 6.73 65.23 � 20.84* 42.40 � 22.16† 35.90 � 25.53* 23.85 � 19.85 20.94 � 17.64* 18.55 � 6.73

HCV� polyclonal, n 14 20.14 � 8.17 51.72 � 6.53 19.22 � 15.33 15.61 � 13.03 9.73 � 9.92 4.86 � 2.80 20.14 � 8.17

Values are means (� SD).*P .05, Dunn multiple comparison test.†P .01, Dunn multiple comparison test.





in some HCV� patients compared with HCV Ab� volunteers andHCV�, Ab� patients. There were no differences in B-cell percent-ages between HCV�MC� and HCV�MC� groups. When wecalculated the absolute numbers of B cells by multiplying theB-cell percentages by the corresponding total lymphocyte counts,we found no differences among the groups (data not shown). Thesefindings confirmed our earlier study reporting normal B-cellfrequencies in most HCV� subjects.33 We did not detect signifi-cantly different percentages of the activation markers CD69 andCD25 among B cells from our patient groups (data not shown),suggesting that B cells are not polyclonally activated during HCVinfection.

Consistent with the fact that MC RF is IgM�, we found thatcertain HCV�MC� patients had B cells with a tremendous �light chain bias, exhibiting �/� ratios up to 90:10 (Figure S3A).This ratio was significantly increased among HCV� subjectswith clonal, as opposed to polyclonal, CDR3 (Table 3). Most ofthe �� and IgM� B cells in these patients expressed CD27(Figure S3B,C), which is a marker for memory B cells.49 HCV�

subjects with clonal, as opposed to polyclonal, CDR3 hadsignificantly increased percentages of ��CD27� and IgM�CD27�

B cells. Most of the expanded IgM� B cells wereIgDlow/� (Figure S3D). Interestingly, CD27� B cells from 5 of 11

HCV�MC� subjects, but from none of the HCV�MC� subjectsor controls, exhibited markedly diminished CD21 (Figure S3E).HCV� subjects with clonal, as opposed to polyclonal,CDR3 had significantly increased percentages of CD21lowCD27�

B cells. We thus identified a subset of HCV� patients with clonalIg that had an expansion of IgM��IgDlow/�CD21lowCD27�

B cells. We gated on ��CD27� and IgM�CD27� B-cell sub-sets in selected patients (Table 4; Figure S4). In selected HCV�

MC� patients, the expanded IgM��CD27� subset was pre-dominantly IgDlow/�.

Several groups have reported increased intrahepatic andperipheral CD5� B cells among HCV� subjects, and they havesuggested that this reflects increased “natural” Ab production inHCV infection.50,51 Although CD5� B-1a cells seem to beresponsible for the production of natural Abs in mice,52 theassociation between CD5� B cells and natural Abs in humans isnot as clear, and evidence suggests that CD5 expression isinducible.53 Despite these drawbacks, we measured CD5�

B cells by flow cytometry (Figure S2C). Consistent with anothergroup’s finding54 and our earlier report,33 the frequency of CD5�

B cells was not significantly increased among HCV� subjects,nor did frequencies significantly differ between HCV�MC� andHCV�MC� patients (Table 3). However, 2 HCV� patients(1446 and 960) had very high levels of CD5� B cells. Takentogether, these data confirm that the majority of HCV� patientsdo not have expansions of CD5� B cells.

HCV�MC� subjects’ expanded IgM� ��CD21lowCD27� B cellsbear restricted, clonal Ig

We sorted single IgM��CD21lowCD27� B cells from subjectsLDU 077, LDU 095, 389, 773, 1403, 1308, and 1432. We wereunable to singly sort IgM��CD21lowCD27� B cells from otherHCV�MC� patients either because of a lack of cryopreserved

Figure 2. Phylogenetic analysis of VH (left) and V� (right) from subject 1432’s IgM�� CD27�CD21low B cells. Trees were constructed using neighbor-joining analysis ofTamura-Nei distances calculated from pairwise comparisons of Ig DNA sequences. Percentages of bootstrap support ( � 50%) from 1000 replicate samples (analyzed by theneighbor joining method) are indicated to the left of the support node. VH and V� from B cell A5 of patient 1403 serve as reference sequences.

Table 4. IgD� and CD5� subsets among selected subjects’ IgM�,CD27� B cells

SubjectIgM�, IgDlow/�

cells, %*� �, IgDlow/�

cells, %†CD5�, IgDlow/�

cells, %‡

HCV Ab� (LDU 095) 23.2 45.5 15.9

HCV� polyclonal (1419) 4.4 27.8 12.3

HCV� clonal (1308) 58.2 67.5 39.1

HCV� clonal (1403) 46.9 66.5 32.7

*Percentage of CD19�, CD20�, � �, CD27� cells.†Percentage of CD19�, CD20�, IgM�, CD27� cells.‡Percentage of CD19�, CD20�, IgM�, CD27� cells.





Table 5. IgH gene segment usages, nucleotide insertions, and CDR3 sequences in IgM��CD27� B cells

Subject no./status*/subset VH DH JH VD (P,N) DJ (P,N) IgH CDR3 sequence

LDU 077/HCV Ab� (CD21low)

Clones, no

1/8 3-74*01 3-16*02 4*02 0,13 1,2 CVRASWYYDRWGQG

1/8 1-46*01 3-16*02 4*02 1,6 1,3 CAREHGGGYDYWGQG

1/8 3-15*07 IR1*01R 2*01 0,30 1,0 CATGGRHSDYLLRGYFDLWGRG

1/8 3-30*03 6-06*01 6*02 0,0 0,11 CAKYSSTSNYYYGMDVWGQG

1/8 3-07*01 3-16*01 4*02 0,3 0,2 CASGLRYFDYWGQG

1/8 3-74*01 7-27*01 3*02 0,24 0,18 CARARGWAVETGSSEMIWAFDIWGQG

1/8 3-74*01 1-20*01 4*02 0,14 1,4 CVKDYNWNAGDYWGQG

1/8 4-04*02 4-17*01 4*02 0,3 0,0 CARGNGDYYFDYWGQG

LDU 095/HCV Ab� (CD21low)

Clones, no.

1/2 5-a 3-10*01 4*02 1,4 0,10 CARHGYGSGSYYDYWGQG

1/2 4-34*01 2-08*01R 6*02 0,34 0,0 CARVITRASPGTDGRTNMDVWGQG

389/HCV�MC� (CD21low)

Clones, no.

1/13 1-46*01 3-22*01 4*02 1,4 0,2 CARTSYYYDSSGYYPFDYWGQG

1/13 5-51*01 6-13*01 3*01 0,9 3,4 CARGQRAAAFDVWGQG

1/13 4-59*01 6-13*01 6*02 3,1 0,8 CARAPGIAAGNLPYYYYGMDVWGQG

1/13 3-21*01 3-22*01 4*02 1,5 0,1 CARYYYDSSGSYYFDYWGQG

1/13 4-34*01 3-22*01 2*01 1,10 1,8 CARGCGMHYDSSAGRYFDLWGRG

1/13 3-15*01 1-26*01 4*02 1,1 1,6 CTTAIVGAIHGDYWGQG

1/13 3-48*02 6-25*01 4*02 0,4 0,3 CARELGYSRPDYWGQG

1/13 3-49*03 4-23*01 4*02 0,6 0,2 CTSGRRWYLDYWGQG

1/13 3-21*01 4-17*01 6*02 1,8 0,5 CARDNQGTTGGYYYYGMDVWGQG

1/13 1-69*01 1-26*01 3*02 1,26 1,1 CAMAATREGSRKWELRAFDIWGQG

1/13 3-53*02 3-22*01 6*02 0,3 0,6 CARGITMNLHYYYGMDVWGQG

1/13 1-03*01 2-02*01 4*02 0,4 0,11 CARGYCSSTSCQYYSDYWGQG

1/13 3-23*01 1-07*01 6*02 1,24 0,15 CAKEGVPAAPRAYNWNSFFWSCMDVWGQG


Clones, no.

7/12† 1-69*01 6-13*01 4*02 0,0 0,8 CAREGIAAAVNPFDYWGQG

4/12† 1-69*06 2-15*01 4*02 1,4 1,14 CAREGRSGNVNPYDYWGQG

1/12 1-69*06 3-16*01 2*02 2,8 0,13 CAREGEAGGVGRNWAFDIWGQG

960/HCV�MC� (CD5�)

Clones, no.

1/7 1-69*01 3-22*01 4*02 1,5 1,16 CARDMGSSAYFFFYWGQG

1/7 3-66*01 IR*01R 4*02 0,0 1,25 CARGGPSKEPFDCWGQG

1/7 3-11*01 6-19*01 4*02 2,4 2,0 CARDQLWLAYWGQG

1/7 3-23*01 6-13*01 6*02 0,12 2,2 CAAPRLAAAGPYYYYYYGMDVWGQG

1/7 3-23*01 6-06*01 4*02 0,12 0,13 CAKEGGAGAARLGSFSYWGQG

1/7 3-21*01 1-01*01R 3*02 0,0 2,10 CARSFSGAGAFDIWGQG

1/7 3-23*01 3-22*01 4*02 3,6 0,0 CAKDRGRYDSFDYWGQG

1073/HCV�MC� (All)

Clones, no.

1/10 3-74*01 IR1*01R 4*02 3,1 2,7 CTRGLWDHDYWGQG

1/10 3-72*01 6-19*01 6*03 1,15 0,23 CAIGPGFNSGRYYYYYMAVWGKG

1/10 3-74*01 3-09*01 4*02 0,29 0,0 CARDRYTFGTTPLDWGPG

1/10 3-30*04 5-12*01 3*02 1,8 1,6 CARDRAGVATIIGADAFDIWGQG

1/10 4-39*02 6-19*01 4*02 2,14 1,0 CTRRARGSGWYYWGQG

1/10 3-23*01 4-17*01 6*02 0,9 0,10 CAKGPSSVTRIKSYYYYGMDVWGQG

1/10 4-39*06 5-12*01 5*02 2,4 0,30 CARSEVATVVFVVLASQLFDWGQG

1/10 5-51*03 6-13*01 5*02 0,10 0,6 CARLGRSAAGTDLFDPWGQG

1/10 7-04-1*02 3-16*01 6*03 0,3 0,9 CARRDYEGHLDVWGKG

1/10 5-51*01 5-05*01 1*01 0,23 0,8 CARGFGRGLGASYWGSEYFQHWGQG


Clones, no.

15/15† 3-7*01 1-26*02 3*02 0,0 0,12 CARDGDLGSGSRSMGAFDIWGQG


Clones, no.

9/18† 1-69*01 3-22*01R 4*02 1,5 0,19 CARAYGNNPTEVYWVYWGQG

5/18† 1-18*01 2-15*01 6*02 0,9 0,34 CARDGAVCSGSSCRGNYYYYGLDVWGQG

VD (P,N) and DJ (P,N) indicate the number of palindromic (P) and nontemplated (N) nucleotides inserted at the VD and DJ joins, respectively. The bold type in the IgHCDR3 sequence indicates aa residues between position 106 and the conserved tryptophan in all JH segments.

*IgM�� CD27� B-cell subset.†in column 1 indicates clonal populations.





PBMCs (subjects 734, 880, and 92200), or because of the lowfrequency of these cell subsets. We were able to singly sortIgM��CD27� B cells from subject 1073. We cloned the entireexpressed Ig VH region (and for selected subjects, the entire IgV� region) from individual cells using a 2-stage, unbiased IgRT-PCR strategy.40 Results for VH and V� are summarized inTables 5, 6. In contrast to HCVAb� controls, IgM��CD21lowCD27�

B cells from the HCV�MC� patients exhibited marked VH, JH, and V�

gene segment usage restriction. Eleven of 12 IgM��CD21lowCD27�

B cells isolated from patient 773, 22 of 22 from subject 1432, and 10 of18 from patient 1403 used the RF-encoding gene segments VH1-69/JH4.There was a similar pattern of V�3-20 restriction in these subjects. AllIgM��CD21lowCD27� B cells isolated from patient 1308 used VH3-7/JH3 and V�3-15/J�1.

Earlier studies have shown that hepatic lymphoid aggregatesfrom HCV�MC� subjects consist predominantly of CD5�

B cells.55 As mentioned in “B cells from certain HCV�MC�

subjects are biased toward a restricted IgM��IgDlow/�CD27�

phenotype,” increased intrahepatic and peripheral CD5� B cellshave been reported among HCV� subjects,50,51 suggesting that

the B cells arising in the liver and stimulated by HCV antigensmay be CD5�. We therefore decided to clone Ig fromIgM��CD27�CD5� and IgM��CD27�CD5� B cells isolatedfrom subjects 1446 and 960 (who both had increased percent-ages of CD5� B cells) and from subject 1403 (to determinewhether unmutated clonal Ig was produced by CD5� cells). Wecould not recover any IgM��CD27�CD5� B cells from subject960. Of IgM��CD27�CD5� B cells, 1 of 7 clones from subject960, 2 of 6 from subject 1403, and 3 of 13 from the HCV�MC�

subject 1446 demonstrated VH1-69/JH4 gene segment usage,whereas no VH1-69/JH4 usage was seen among 10 clones fromsubject 1446’s IgM��CD27�CD5� B cells, suggesting thatthis gene segment restriction is not seen in CD5� cells. Of the2 VH1-69�/JH4� clones from 1403, one was unmutated and theother had only 2 mutations, suggesting that the unmutated clonalpopulation may arise from CD5� B cells.

Patients 1308 and 1432 had monoclonal IgM��CD21low

CD27� B cells, as indicated by the presence of identical CDR3 andpalindromic (P) and nontemplated (N) nucleotides at the VHDH,DHJH, and V�J� joins. We identified 2 clonal populations in subject

Table 5. IgH gene segment usages, nucleotide insertions, and CDR3 sequences in IgM��CD27� B cells (Continued)

Subject no./status*/subset VH DH JH VD (P,N) DJ (P,N) IgH CDR3 sequence

1/18 1-69*01 4-17*01 4*02 1,2 2,9 CAREGYGDYGRPYDYWGQG

1/18 3-20*01 1-26*01 6*02 2,1 0,0 CARAGGSYYYYYGMDVWGQG

1/18 3-21*01 3-22*01 4*02 3,11 1,6 CTRDGPYYDSSGFEYWGQG

1/18 3-23*01 2-15*01 6*02 1,13 1,8 CAKTLRSNVVVVATTHYYGMDVWGQG

1403/HCV�MC� (CD5�)

Clones, no.

2/6† 1-69*01 3-22*01R 4*02 1,5 0,19 CARAYGNNPTEVYWVYWGQG

1/6 3-23*01 3-10*01 4*02 0,12 0,4 CAKGGPRFTMVRGVKIYFDYWGQG

1/6 3-07*01 6-13*01 6*03 3,7 0,1 CARAQSFSWSMDVWGQG

1/6 1-18*01 2-15*01 6*02 0,9 0,34 CARDGAVCSGSSCRGNYYYYGLDVWGQG

1/6 3-23*01 3-10*01 2*01 0,3 1,23 CAGGSGSYSLLGPHWHIDLWGQG


Clones, no.

22/22† 1-69*01 3-03*01 4*02 0,16 0,2 CARDYGERESGYYFYWGQG

1446/HCV�MC� (CD5�)

Clones, no.

1/10 1-03*01 2-21*02 4*02 2,7 2,1 CARGVIYCGGDCYSGDYWGQG

1/10 3-23*01 2-15*01 6*02 0,2 0,11 CAKGGYAEGAYYYYGMDVWGQG

1/10 3-15*01 1-07*01 4*02 4,5 2,2 CTTDLFLLELRCDYWGQG

1/10 3-30*03 3-22*01 4*02 0,30 0,1 CAKGGDTAMVLSNYYDSSGVDYWGQG

1/10 5-51*01 3-10*01 4*02 0,10 0,21 CARRKSDYYGSGRAGFSDYWGQG

1/10 4-34*01 3-10*01 4*02 1,5 0,5 CARAPSITMVRGVINEFDYWGQG

1/10 3-21*01 4-17*01 4*02 0,5 0,3 CARDRPTVTTVFDYWGQG

1/10 3-15*01 3-10*01 6*03 2,2 0,0 CTTDRYYGSGSYYYGMDVWGQG

1/10 1-03*01 6-25*01 6*03 3,1 0,2 CARASGHYYYYMDVWGKG

1/10 3-15*01 6-19*01 4*02 0,7 0,13 CTTVGVIAVAGGFRGDYWGQG

1446/HCV�MC� (CD5�)

Clones, no.

3/13† 1-69*01 4-17*01 4*02 1,7 2,5 CAREGRATVTNPFDYWGQG

3/13† 3-11*03 IR1*01 4*02 0,14 2,0 CARGARLEGPVDYWGQG

2/13 3-15*01 IR1*01 5*02 0,2 0,14 CATGRDFYRFDPWGQG

1/13 1-18*01 4-17*01 4*02 0,14 1,6 CATRGRDTGDYEGDWGQG

1/13 3-23*01 4-17*01 4*02 1,11 0,13 CAKGRTSITVQYDSWGQG

1/13 3-15*01 3-22*01 4*02 2,5 1,7 CTTDPGITMIVVVPQGFDYWGQG

1/13 3-48*02 6-19*01 4*02 0,10 1,16 CAREARRSSGWYFHEIDYWGQG

1/13 4-34*01 2-15*01 4*02 1,7 1,29 CARTPRYCSGATCNKIMGLDHWGQG

VD (P,N) and DJ (P,N) indicate the number of palindromic (P) and nontemplated (N) nucleotides inserted at the VD and DJ joins, respectively. The bold type in the IgHCDR3 sequence indicates aa residues between position 106 and the conserved tryptophan in all JH segments.

*IgM�� CD27� B-cell subset.†in column 1 indicates clonal populations.





773. The largest, represented by 7 of 12 B cells, contained a CDR3exactly identical to that previously reported from a patient withsalivary gland mucosa–associated lymphoid tissue lymphoma.56

That identical CDR3 regions containing N nucleotides have beenidentified in 2 different patients strongly suggests a commonantigenic stimulus in the 2 patients, as it is extremely unlikely thatthese identical regions could arise by chance. The VH1-69/JH4clonal population in subject 1446’s IgM��CD27�CD5� B cellshad a CDR3 that differed by one amino acid from a CDR3previously recovered by another group from an unrelatedHCV�MC� subject’s expanded B cells.57 We identified 2 clonalpopulations among subject 1403’s IgM��CD21lowCD27� B cells.As mentioned, the predominant clonal VH1-69/JH4 population wasalso seen in 2 of 6 of 1403’s IgM��CD27�CD5� B cells. ClonalIg was not seen in subject 389’s IgM��CD21lowCD27� B cellsnor in subject 1073’s IgM��CD27� B cells. Because clonal Igwas seen in these subjects’ unfractionated PBMCs, this suggeststhat the clonally expanded B cells in these subjects were notCD21lowCD27�.

The expanded IgM��CD21lowCD27� B cells in certainHCV�MC� subjects contain somatically hypermutated Igs

The majority of expanded IgM��CD21lowCD27� B cellsexhibited low to moderate levels of somatic hypermutation(SHM; Table 7). An exception was subject 1403 whose clonalVH1-69/JH4 cells were unmutated. Within each clonalIgM��CD21lowCD27� B-cell population, the mutational fre-quency varied significantly among individual cells. Except forsubject 389, each HCV�MC� subject from whom Igswere cloned had several Igs with significantly scarce R muta-tions in their FR regions, (pR(FR) 0.05), indicative ofselection based on structural constraint for antigen-Ig binding.However, only 3 of these Igs had a significant excess ofR mutations in CDR, (pR(CDR) 0.05). Despite VH SHM,CDR3 sequences were strikingly conserved among clonalpopulations, suggestive of a positive selection. Most V� wereless hypermutated than their corresponding VH, reflective of thelower mutation rate of Ig� seen in the general population.49 Incontrast to VH, more V� had an excess of R(CDR) than they hada scarcity of R(FR).

Igs from HCV�MC� subjects demonstrate features of clonalevolution

We constructed phylogenetic trees for VH and V� from subject1432’s IgM��CD21lowCD27� B cells (Figure 2). When com-bined with the Ig mutation rates from Table 7, these treesdemonstrate that for both VH and V�, mutations are sharedamong different B cells. A pattern of clonal evolution isapparent, as the more hypermutated clones also contain muta-tions present in the less hypermutated clones. Interestingly,6 B cells (D2, D4, D6, D9, E3, and F6) that had highly evolvedIgH also had significant scarce R(FR), further indication that astructural constraint in FR provides selective pressure for thesecells. The VH and V� phylogenies correlate well with oneanother. Cells with relatively large VH mutational frequencieshave correspondingly large V� mutational frequencies. This fact,combined with similar patterns of shared VH and V� mutationsamong these cells, provides strong evidence that the observedmutations are not PCR artifacts. Similar results were obtainedon phylogenetic analysis of Ig clones from subjects 773, 1308,and 1403 (data not shown). Taken together, these data suggestthat the expanded IgM��CD21lowCD27� B cells in certainHCV�MC� subjects undergo successive rounds of SHM.However, the relatively low mutation rates and theoccasional presence of unmutated B-cell clonal expansionssuggest that affinity maturation is not an absolute requirementfor these cells.

Discussion

A major finding of our study is the identification of clonallyexpanded IgM��IgDlow/�CD21lowCD27� B cells expressinghypermutated RF-like Ig in a subgroup of HCV�MC� patients.In addition, we describe for the first time cloning of VH and V�

from single B cells obtained from HCV�MC� patients. Ourcloning strategy was unbiased in its amplification of Ig genesegments; the gene segment usage we observed in our healthyvolunteers corresponds to that previously reported in the general

Table 6. Ig� gene segment usages, nucleotides insertions, andCDR3 sequences in IgM��CD21lowCD27� B cells

Subject no./status V� J� VJ (P,N) Ig� CDR3 sequence

LDU 077/HCV Ab�

Clones, no.

1/8 4-1*01 2*01 0,20 CHQYYAFPHTFGO

1/8 2-28*01 4*01 1,2 CMQALQTPLTFGG

1/8 1-39*01 2*01 1,10 CQQSYSTPSHTFGQ

1/8 1-33*01 2*01 0,0 CQQYDNLPPYTFGQ

1/8 3-20*01 1*01 0,15 CQHYRSSLWTFGQ

1/8 2-29*01 1*01 1,17 CMQGLHLPPTFGQ

1/8 1-8*01 1*01 2,0 CQQYYSYPRTFGQ

LDU 095/HCV Ab�

Clones, no.

1/2 4-1*01 4*01 0,0 HQYYRTPLTFGG

1/2 1-39*01 2*01 0,0 CQQSYRTPHTFGQ

773/HCV�MC�

Clones, no.

7/12* 3-20*01 1*01 0,1 CQQYGSSPQTFGQ

4/12* 3-20*01 1*01 0,1 CQQYGSSPQTFGQ

1/12 3-20*01 1*01 1,1 CQQYGSSRTFGQ

1403/HCV�MC�

Clones, no.

9/18* 3-20*01 4*01 0,0 CQQYGSSPTFGG

5/18* 3-20*01 2*01 1,0 CQQYGSSRYTFGQ

1/18 3-20*01 2*01 1,0 CQQYGSSPYTFGQ

1/18 3-20*01 4*01 2,0 CQQYGSSSLTFGG

1/18 1-27*01 4*01 0,0 CQKYNSAPLTFGG

1/18 1-6*01 2*01 0,0 CLQDYNYPYTFGQ

1308/HCV�MC�

Clones, no.: 15/15* 3-15*01 1*01 0,0 CQQYNNWPPWTFGQ

1432/HCV�MC�

Clones, no.: 22/22* 3-20*01 2*02 1,1 CQQYGSSPRTFGQ

VJ (P,N) indicates the number of palindromic (P) and nontemplated (N)nucleotides inserted at the VJ join. The bold type in the Ig� CDR3 sequence indicatesaa residues between position 106 and the conserved phenylalanine in all J�

segments.* in column 1 indicates clonal populations.





population.58 It is also unlikely that we overestimated muta-tional frequencies because of potential Taq polymerase errors.We performed duplicate VH PCR reactions on reverse-transcribed cDNA from 10 different B cells, and we noted only2 discordant nucleotides (2 stage VH PCR error rate: 1 of 1860nucleotides). Moreover, we routinely did not detect mutations inCD27� cells, and mutational frequencies among subjects’ VH

and V� were highly correlated.The finding that a subset of HCV�MC� patients have

clonally expanded, hypermutated, Ig gene segment–restricted,memory-like B cells suggests that specific antigenic stimulationis responsible for the B-cell proliferation in HCV MC. Ourfindings contrast with an earlier claim that polyclonal activationof naive B cells occurs during chronic HCV infection.27 Of note,we did not detect any increased CD69 or CD25 expressionamong any subjects’ B cells. However, this latter result is notsurprising, given the role of CD69 in retaining activated B cellsin lymph nodes.59 It is possible that any recently activatedB cells may leave the blood and not be detected. Important, notall HCV�MC� patients have noticeably expanded B-cell subsets.The finding of clonal VH1-69/JH4 B cells in subject1446 (HCV�MC� and without a detectable clonal CDR3 band)suggests that these clonal expansions may be occurring subclini-cally in some patients. That we did not recover clonal Ig in theIgM��CD21lowCD27� B cells of patient 389 and in theIgM��CD27� B cells of patient 1073 (both of whomwere HCV�MC� with clonal CDR3, yet without detect-able B-cell expansion) suggests that either clonal Ig is present atlow frequencies or is present in different B-cell subsets inthese patients. Our results, which are consistent with2 prior studies, lend support to the hypothesis that chronic,specific antigenic stimulation of IgM� B cells predisposestoward neoplastic transformation. In one study, regression ofWA RF-bearing IgM��IgD�CD5� B cells occurred concomi-tantly with the decline of HCV RNA during interferon-�treatment.57 The CDR3 of the cells in the cited study57 is highlyhomologous to that of subject 1446’s clonal IgM��CD27�CD5�

B cells. In another study, splenic marginal zone lymphomaregressed in patients whose HCV was successfully treated withinterferon �-2b.60

IgM�CD27� cells are important in defending against bacte-rial pathogens in a T cell–independent manner, and it has beenproposed that such cells are the circulating form of splenicmarginal zone (MZ) B cells.61,62 MZ B cells are thought to bedistinct from GC or post-GC B cells,63 and SHM in these cellsdoes not require accessory molecules such as CD40L or CD40.64

In healthy humans, IgM� memory B cells have been demon-strated to comprise approximately 10% to 25% of peripheralB cells64,65 and to have lower SHM rates than class-switchedmemory B cells. We have found that IgM�CD27� cells com-prise up to 75% of circulating B cells in a subset of HCV�MC�

patients and that these cells have low levels of SHM. Moreover,these IgM� memory B cells are IgDlow/�, which distinguishesthem from previously reported hypermutated IgM�IgD�CD27�

B cells in healthy adults.49 Our findings are consistent with thefact that HCV is associated with both splenic66,67 and extras-plenic, nodal MZ lymphomas, the latter of which have beenfound to be made up of B cells frequently harboring mutatedVH1-69 gene segments.68

Our finding that in certain HCV�MC� subjects, IgM� memory-like B cells have reduced expression of CD21 contrasts with thecharacterization of MZ cells as being CD21high.62 Interestingly,

reduced CD21 expression was reported on B cells from patientswith systemic lupus erythematosis69 and HIV.70 We have previouslyobserved reduced CD21 expression on HCV� patients’ B cells.33

Down-regulation of CD21 may serve to dampen B-cell responsive-ness to BCR-mediated activation and proliferation. However, it isalso possible that reduced CD21 is a normal feature of B cells thathave been stimulated by antigen,71 or it may be emblematic ofapoptotic cells.72

Our analysis of CD5�IgM��CD27� and CD5�IgM��CD27�

B cells, although conducted on only 3 subjects and 1 subject,respectively, suggests that the clonal, VH1-69/JH4-restricted B cellsseen in some HCV�MC� patients are CD5�. This is consistent withprior reports of CD5� MZ-like B-cell lymphoproliferation in someHCV�MC� patients,57,60 and it suggests that CD5 expression is nota universal feature of the expanded B-cell subsets seen in HCVMC. We are currently in the process of sorting CD5�IgM��CD27�

and CD5�IgM��CD27� B cells from additional, newly identifiedHCV�MC� patients with clonal IgM��IgDlow/�CD21lowCD27�

B-cell expansions.An interesting finding is that, whereas 3 HCV�MC� subjects

had hypermutated clonal B cells, one patient’s clonal B cells wereof germ line sequence, despite being CD27�. Our studies areconsistent with the findings of Ivanovski et al,19 who foundrestricted, hypemutated VH and VL genes in PBMCs isolated fromHCV patients with immunocytomas. It is unclear if SHM correlateswith autoreactivity in HCV MC. We are currently defining theautoreactive profiles of Ig cloned from our HCV�MC� subjects.Whether autoreactivity is inversely correlated with the presence ofSHM is of extreme interest.

It is not known how these Ig-restricted memory-like B cellsare stimulated to expand in some HCV�MC� patients. Onepossibility is that the Ig on these B cells reacts with an HCVantigen and/or cross-reacts with a self-molecule, such as IgG,which may be bound to HCV. The finding that the clonal B-cellpopulation of one of our HCV�MC� patients expresses a CDR3that is exactly identical to the CDR3 from a patient with salivarygland lymphoma is intriguing in light of a report that a particularantigen selects Igs bearing identical CDR3 in different per-sons.73 Persistent inflammation is a common feature of HCVinfection, Sjogren syndrome, and H pylori infection. All of thesesyndromes are associated with B-NHL. In these diseases,antigen-containing immune complexes may bind to low-affinityautoreactive BCR. Concomitant stimulation of toll-like recep-tors (TLR), coupled with (or in lieu of) cognate T-cell help, maylead to B-cell activation and proliferation. It has been shown invitro that IgG-bound Borrelia burgdorferi is capable of breakingRF-expressing B cells’ state of immunologic tolerance bybinding to both BCR and TLR.74 Moreover, it has beenshown that chromatin-IgG complexes can lead to activation ofRF� B cells by engagement of IgM and TLR9.75 We arecurrently testing the hypothesis that immune complexedHCV induces expansion of RF� B cells by dual engagement ofBCR and TLR7.

In conclusion, we have characterized in detail a subset ofHCV�MC� patients with gene segment-restricted, clonallyexpanded IgM��IgDlow/�CD21lowCD27� B cells. We empha-size that our studies are of peripheral B cells. It is unclear howrepresentative these cells are of the B cells in the hepaticmicroenvironment. Importantly, clonal intrahepatic RF-produc-ing B cells have been shown in HCV�MC� patients.17 Theidentification of such highly restricted, potentially autoreactive,Igs in a subset of HCV�MC� patients gives us hope that





Table 7. Analysis of mutations in IgM��CD21lowCD27� B cells

Subject no., B cell

Heavy chain Light chain

VH mutations(%) aa �

R/S(FR)

R/S(CDR)

p(R)FR

p(R)CDR

V� mutations(%) aa �

R/S(FR)

R/S(CDR)

p(R)FR

p(R)CDR

LDU 077

F1 23 (8.32) 17 12/5 5/1 0.163 0.131 7 (2.81) 4 1/0 3/2 0.019 0.025

G1 0 (0.00) 0 0/0 0/0 — — 0 (0.00) 0 0/0 0/0 — —

G4 23 (8.32) 11 3/8 8/4 0.000 0.006 7 (2.85) 3 1/1 3/2 0.008 0.041

G5 7 (2.59) 4 4/2 0/1 0.388 0.817 6 (2.60) 4 3/0 2/1 0.292 0.131

G6 1 (0.37) 0 0/1 0/0 0.180 0.567 0 (0.00) 0 0/0 0/0 — —

H3 23 (8.32) 16 8/5 9/1 0.004 0.001 9 (3.85) 8 3/0 6/0 0.052 0.000

H6 16 (5.93) 8 4/3 7/2 0.002 0.002 12 (4.8) 9 8/3 1/0 0.820 0.700

H7 0 (0) 0 0/0 0/0 — — 0 (0.00) 0 0/0 0/0 — —

LDU 095

B3 5 (1.94) 4 3/1 1/0 0.450 0.324 2 (0.80) 2 1/0 1/0 — —

C4 8 (3.14) 4 4/3 0/1 0.246 0.841 4 (1.73) 3 0/1 3/0 0.338 0.083

LDU 389

A1 1 (0.37) 1 1/0 0/0 0.686 0.569 — — — — — —

A4 0 (0.00) 0 0/0 0/0 — — — — — — — —

B3 3 (1.12) 1 1/2 0/0 0.182 0.679 — — — — — —

B7 0 (0.00) 0 0/0 0/0 — — — — — — — —

C10 0 (0.00) 0 0/0 0/0 — — — — — — — —

C12 0 (0.00) 0 0/0 0/0 — — — — — — — —

D5 0 (0.00) 0 0/0 0/0 — — — — — — — —

D9 1 (0.36) 1 1/0 0/0 0.686 0.569 — — — — — —

E2 0 (0.00) 0 0/0 0/0 — — — — — — — —

E8 6 (2.22) 4 3/2 1/0 0.271 0.392 — — — — — —

2F1 3 (1.12) 2 2/1 0/0 0.533 0.679 — — — — — —

F2 6 (2.27) 5 2/1 3/0 0.086 0.021 — — — — — —

G9 2 (0.74) 0 0/0 0/2 0.069 0.628 — — — — — —

LDU 773

D7* 6 (2.22) 3 1/3 2/0 0.017 0.110 5 (2.14) 3 2/1 1/1 0.132 0.206

D10* 6 (2.22) 3 1/2 2/1 0.017 0.110 2 (0.85) 2 1/0 1/0 0.338 0.083

E3* 7 (2.59) 1 1/5 0/1 0.007 0.817 5 (2.14) 4 2/1 2/0 0.132 0.032

E5* 5 (1.85) 4 2/1 2/0 0.174 0.077 6 (2.56) 5 2/1 3/0 0.062 0.005

F5* 8 (2.96) 1 1/4 0/3 0.003 0.841 3 (1.28) 3 1/0 2/0 0.152 0.010

F10* 8 (2.96) 5 3/3 2/0 0.088 0.185 6 (2.56) 3 2/2 1/1 0.062 0.245

G1* 4 (1.48) 3 2/1 1/0 0.319 0.263 1 (0.43) 1 1/0 0/0 0.669 0.542

C10* 2 (0.74) 2 1/0 1/0 0.377 0.134 4 (1.71) 3 2/0 1/1 0.265 0.166

E7* 18 (6.67%) 6 5/10 1/2 0.002 0.819 6 (2.56) 3 1/2 2/1 0.010 0.046

F8* 3 (1.11) 2 2/1 0/0 0.538 0.675 4 (1.71) 3 2/1 1/0 0.265 0.166

G7* 14 (5.19) 9 7/4 2/1 0.179 0.431 4 (1.71) 2 2/2 0/0 0.265 0.647

C11 9 (3.33) 6 4/2 2/1 0.148 0.226 6 (2.56) 5 5/1 0/0 0.787 0.703

LDU 1403

A5* 0 (0.00) 0 0/0 0/0 — — 3 (1.28) 2 2/1 0/0 0.488 0.615

G3* 0 (0.00) 0 0/0 0/0 — — 3 (1.28) 2 2/1 0/0 0.488 0.615

C6* 0 (0.00) 0 0/0 0/0 — — 3 (1.28) 2 2/1 0/0 0.488 0.615

C7* 0 (0.00) 0 0/0 0/0 — — 3 (1.28) 2 2/1 0/0 0.488 0.615

D3* 0 (0.00) 0 0/0 0/0 — — 3 (1.28) 2 2/1 0/0 0.488 0.615

D9* 0 (0.00) 0 0/0 0/0 — — 5 (2.14) 4 4/1 0/0 0.711 0.676

D11* 0 (0.00) 0 0/0 0/0 — — 3 (1.28) 2 2/1 0/0 0.488 0.615

H9* 0 (0.00) 0 0/0 0/0 — — 4 (1.71) 3 3/1 0/0 0.612 0.647

B2* 0 (0.00) 0 0/0 0/0 — — 3 (1.28) 2 2/1 0/0 0.488 0.615

E1* 9 (3.33) 3 1/4 2/2 0.001 0.226 2 (0.85) 1 0/0 1/1 0.057 0.083

E3* 9 (3.33) 3 1/4 2/2 0.001 0.226 3 (1.28) 2 1/0 1/1 0.152 0.125

H1* 11 (4.07) 4 2/5 2/2 0.002 0.308 4 (1.71) 3 2/0 1/1 0.265 0.166

H4* 9 (3.33) 3 1/4 2/2 0.001 0.226 3 (1.28) 1 0/1 1/1 0.027 0.207

B1* 15 (5.56) 6 3/7 3/2 0.001 0.227 1 (0.43) 0 0/1 0/0 0.169 0.541

F2 14 (5.19) 12 8/2 4/0 0.344 0.068 5 (2.14) 3 2/2 1/0 0.132 0.206

E9 8 (2.96) 6 3/1 3/1 0.088 0.047 3 (1.28) 1 0/2 1/0 0.019 0.125

G6 17 (6.30) 9 7/8 2/0 0.042 0.543 5 (2.03) 5 4/0 1/0 0.711 0.206

H12 12 (4.44% 8 2/2 6/2 0.001 0.001 5 (2.03) 4 5/0 0/0 0.936 0.676

V mutations indicate number of mutations in V; aa , number of aa replacements in V; R/S (FR), ratio of replacement mutations to synonymous mutations in the V FR; R/S(CDR), ratio of replacement mutations to synonymous mutations in the V CDR1 and CDR2; p(R)FR, probability of significant scarcity of R mutations in FR; p(R)CDR, probabilityof significant excess of R mutations in CDR1 and CDR2.

— indicates that the value was not computed.*indicates clonal populations.





elucidation of the precise antigenic stimulus responsible for MCformation will reveal the mechanism of HCV-induced B-cellproliferative disorders.

Acknowledgments

We are grateful for the generosity of the patient volunteers. We thank theRockefeller University Hospital Department of Nursing and PatientCare Services, Gertrudis Soto and Queenie Brown for assistance inpatient enrollment and sample acquisition. Svetlana Mazel, manager ofRockefeller’s Flow Cytometry Research Center, and Peter Lopez,manager of Aaron Diamond AIDS Research Center’s Core Facilities,provided expert assistance with flow cytometry. We acknowledgeRaymond Peterson for database management and Knut Wittkowski forstatistical advice. We also thank Michel Nussenzweig and SergeyYurasov for helpful discussions.

This study was supported in part by the National Institutes ofHealth/National Institute of Allergy and Infectious Diseases grantR01AI60561 (L.B.D.), the Irma T. Hirschl/Monique Weill-Caulier Trust(L.B.D.), General Clinical Research Center grant M01-RR00102 (toRockefeller University Hospital), and Center for Translational ScienceAward grant 1UL1 RR024143-01 (to Rockefeller University Hospital)from the NIH National Center for Research Resources. E.D.C. issupported in part by the Clinical Scholar Program of RockefellerUniversity Hospital.

AuthorshipContribution: E.D.C., L.B.D., R.M.G., and S.M. devised andperformed experiments; E.D.C., G.V.L.-B., A.H.T., and I.M.J.provided patient referrals; E.D.C., L.B.D., C.M.R., S.M., G.V.L.-B., A.H.T., and I.M.J. interpreted results; and E.D.C. and L.B.D.designed the research and wrote the paper.

Table 7. Analysis of mutations in IgM��CD21lowCD27� B cells (Continued)

Subject no. B cell

Heavy chain Light chain

VH mutations(%) aa �

R/S(FR)

R/S(CDR)

p(R)FR

p(R)CDR

V� mutations(%) aa �

R/S(FR)

R/S(CDR)

p(R)FR

p(R)CDR

LDU 1308

B12* 9 (3.33) 4 1/3 3/2 0.001 0.065 2 (0.87) 2 2/0 0/0 0.781 0.580

C3* 3 (1.11) 2 2/0 0/1 0.538 0.675 5 (2.16) 3 2/1 1/1 0.132 0.206

C4* 8 (2.96) 4 2/2 2/2 0.021 0.185 3 (1.30) 1 0/2 1/0 0.019 0.125

C9* 5 (1.85) 1 1/4 0/0 0.039 0.756 1 (0.43) 1 0/0 1/0 0.169 0.042

C11* 4 (1.48) 2 1/2 1/0 0.087 0.263 2 (0.87) 2 0/0 2/0 0.057 0.003

D1* 0 (0.00) 0 0/0 0/0 — — 1 (0.43) 1 0/0 1/0 0.169 0.042

D4A* 8 (2.96) 5 4/2 1/1 0.246 0.487 5 (2.16) 2 1/3 1/0 0.026 0.206

D4B* 10 (3.70) 6 3/4 3/0 0.024 0.087 7 (3.03) 3 3/3 1/0 0.114 0.283

D5* 8 (2.96) 4 3/2 1/2 0.088 0.487 0 (0.00) 0 0/0 0/0 — —

D11* 6 (2.22) 5 3/1 2/0 0.278 0.110 6 (2.60) 4 3/2 1/0 0.217 0.245

E3* 6 (2.22) 5 4/0 1/1 0.568 0.381 6 (2.60) 4 3/1 2/0 0.217 0.046

H2* 6 (2.22) 4 4/1 0/1 0.568 0.789 4 (1.73) 3 3/1 0/0 0.612 0.647

H7* 5 (1.85) 2 2/2 1/0 0.174 0.324 2 (0.87) 1 0/1 1/0 0.057 0.083

I6* 2 (0.74) 2 1/0 1/0 0.377 0.134 2 (0.87) 1 0/1 1/0 0.057 0.083

LDU 1432

B2* 3 (1.11) 2 2/1 0/0 0.538 0.675 5 (2.14) 2 2/1 0/2 0.132 0.676

B4* 10 (3.47) 6 4/3 2/1 0.085 0.267 3 (1.28) 1 1/1 0/1 0.152 0.615

B5* 3 (1.04) 1 1/2 0/0 0.186 0.675 3 (1.28) 1 1/1 0/1 0.152 0.615

C2* 7 (2.59) 5 4/2 1/0 0.388 0.436 4 (1.71) 4 3/0 1/0 0.012 0.166

C3* 7 (2.59) 4 3/1 1/2 0.161 0.436 5 (2.14) 2 2/2 0/1 0.132 0.676

C4* 5 (1.85) 4 3/1 1/0 0.450 0.323 5 (2.14) 2 1/1 1/2 0.026 0.206

C8* 2 (0.74) 2 1/0 1/0 0.377 0.134 4 (1.71) 3 3/0 0/1 0.612 0.647

D1* 3 (1.11) 2 2/0 0/1 0.538 0.675 2 (0.85) 1 1/0 0/1 0.338 0.580

D2* 12 (4.44) 6 3/4 3/2 0.006 0.137 6 (2.56) 2 2/3 0/1 0.062 0.703

D4* 8 (2.96) 2 2/4 0/2 0.021 0.041 5 (2.14) 2 2/2 0/1 0.132 0.676

D5* 3 (1.11) 2 2/1 0/0 0.538 0.675 3 (1.28) 1 1/1 0/1 0.152 0.615

D6* 11 (4.07) 4 4/3 1/3 0.047 0.619 5 (2.14) 2 2/2 0/1 0.132 0.676

D8* 9 (3.33) 4 4/2 0/3 0.150 0.863 6 (2.56) 3 2/2 1/1 0.132 0.676

D9* 9 (3.33) 4 3/3 1/2 0.047 0.534 7 (2.99) 3 4/2 0/1 0.310 0.728

D11* 8 (2.96) 5 4/2 0/1 0.388 0.817 2 (0.85) 1 0/0 1/1 0.057 0.083

E2* 5 (1.85) 3 2/2 1/0 0.174 0.324 3 (1.28) 2 1/0 1/1 0.152 0.125

E3* 9 (3.33) 3 2/4 1/2 0.010 0.534 6 (2.56) 3 2/2 1/1 0.062 0.245

E5* 5 (1.85) 2 2/3 0/0 0.174 0.756 3 (1.28) 2 2/0 0/1 0.488 0.615

E11* 1 (0.37) 1 1/0 0/0 0.688 0.567 2 (0.85) 1 1/0 0/1 0.338 0.580

F1* 5 (1.85) 2 1/3 1/0 0.039 0.324 4 (1.71) 1 1/1 0/2 0.064 0.647

F3* 7 (2.59) 5 5/0 1/1 0.669 0.436 4 (1.71) 1 1/1 0/2 0.064 0.647

F6* 8 (2.96) 2 2/4 0/2 0.021 0.841 7 (2.99) 4 3/2 1/1 0.114 0.283

V mutations indicate number of mutations in V; aa , number of aa replacements in V; R/S (FR), ratio of replacement mutations to synonymous mutations in the V FR; R/S(CDR), ratio of replacement mutations to synonymous mutations in the V CDR1 and CDR2; p(R)FR, probability of significant scarcity of R mutations in FR; p(R)CDR, probabilityof significant excess of R mutations in CDR1 and CDR2.

— indicates that the value was not computed.*indicates clonal populations.





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, NY10021; e-mail: [email protected].

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online October 17, 2007 originally publisheddoi:10.1182/blood-2007-07-101717

2008 111: 1344-1356

Ira M. Jacobson, Charles M. Rice and Lynn B. DustinEdgar D. Charles, Rashidah M. Green, Svetlana Marukian, Andrew H. Talal, Gerond V. Lake-Bakaar, HCV-associated mixed cryoglobulinemia

B cells in+CD27+Clonal expansion of immunoglobulin M

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Clonal expansion of immunoglobulin M CD27 B cells in HCV

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Transcript of Clonal expansion of immunoglobulin M CD27 B cells in HCV