VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)

VH usage and somatic hypermutation in peripheral blood B cells of patients withrheumatoid arthritis (RA)

S.-C. HUANG*, R. JIANG, W. O. HUFNAGLE†, D. E. FURST‡, K. R. WILSKE‡ & E. C. B. MILNER*Virginia Mason Research Center,*Department of Immunology, University of Washington,†PathoGenesis Corporation and

‡Rheumatology and Immunology, Virginia Mason Medical Center, Seattle, WA, USA

(Accepted for publication 22 January 1998)

SUMMARY

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that isconserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to bepreferentially used for encoding autoantibodies. To determine if such preferential use of VH genes inautoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH

composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested byhybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients,but no evidence of a distortion in the bias was observed compared with healthy controls. However, theoccurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequenceanalysis) was strikingly different between patients and healthy subjects. The number of VH3rearrangements that had accumulated somatic mutations and the number of mutations per rearrangementwere significantly elevated in three of the four RA patients. A slight but not significant elevation inmutations among rearranged VH4 genes was also observed in these patients. These data suggest thatalthough usage of individual VH genes among peripheral blood B cells is not affected by the disease, theautoimmune process may involve a significant fraction of the B cell compartment.

Keywords rheumatoid arthritis immunoglobulin heavy chain variable region somatichypermutation

INTRODUCTION

RA is an autoimmune disease which results in severe polyarticularinflammation and damage. Multiple pathologic anti-self antibodies(autoantibodies) have been found associated with RA [1–7]. It hasbeen hypothesized that the production of these pathologic auto-antibodies may result either from antigen-driven processes [8–10]or from polyclonal B cell activation [11–16]. However, the exactmechanisms involved in the pathologic autoantibody response inRA remain unclear. Studies of antibody structure may help todecipher the aetiology of a potentially pathologic autoantibodyresponse in RA [8–10,17–22].

Antibodies consist of immunoglobulin heavy and light chains,and are encoded resulting from rearrangements of V genes, Dsegments (for heavy chains), and J segments. The usage ofindividual V genes in peripheral B cells for encoding antibodies

has been the subject of a number of studies. Contrary to the con-ventional notion that adult V gene usage is normalized with respectto family complexity, our laboratory and others have observed anover-representation of a small group of immunoglobulin heavychain V genes (VH) in the human B cell repertoire [23–29]. It isknown that VH family representation in autoimmune diseasesreflects the normal B cell repertoire in humans [17,30,31], andthat VH genes used in rheumatoid factors (RF) are those preferen-tially expressed or utilized during fetal development [8,17,18, 32].It is not clear, however, if the preferential use of individual VH

genes in autoantibodies is associated with a distortion in the VH

repertoire in peripheral B cells of RA patients.Here we report the results of analysis of VH gene usage and

somatic mutation in peripheral B cells of RA patients. AlthoughDNA sequence analysis is the most definitive method for ascertain-ing the identity of any particular rearranged V gene segment, thenumber of rearrangements that can be analysed is limited by theconstraints inherent to sequence analysis itself. We have used analternative approach in which individual VH gene segments are

Clin Exp Immunol 1998;112:516–527

516 q 1998 Blackwell Science

Correspondence: Eric C. B. Milner PhD, Virginia Mason ResearchCentre, 1000 Seneca Street, Seattle, WA 98101, USA.

identified by hybridization of the cloned rearrangements to motif-specific oligonucleotide probes [23,24]. In addition, differentialhybridization with multiple motif-specific probes corresponding todifferent regions of the same V segment has allowed the estimationof somatic mutations among a large number of the rearrangements[33]. In this study, VH3 genes and VH4 genes were analysed by thisapproach for their contribution to the rearranged VH repertoire andfor the accumulation of somatic mutations among peripheral bloodB cells from RA patients and controls. The results demonstrate thatutilization of individual VH segments was similar between RApatients and normal subjects, but that the accumulation of somaticmutations in these genes was significantly elevated in three of fourRA patients, suggesting that the autoimmune process in some RApatients may result in the antigen-driven activation of a significantfraction of the B cell compartment.

MATERIALS AND METHODS

Subjects and DNAPeripheral blood samples were obtained from four patients withactive RA and from four age- and sex-matched normal subjects,under Internal Review Board-approved informed consent. Allpatients and normal subjects were Caucasian. All patients wereRFþ, had erosive chronic disease in excess of 5 years, and met theAmerican College of Rheumatology (ACR) criteria for RA. Eachpatient had been treated with a variety of non-steroidal anti-inflammatory drugs, low-dose prednisone, and one or more slowacting anti-rheumatic drugs. Purification of leucocytes and extrac-tion of genomic DNA was as described [23]. Genomic DNA wasalso obtained from a lymphoblastoid B cell line, L1, which carrieda rearrangement of the VH3 gene, V3-30 [34]. DNA from this Bcell line was used as a control in estimating the amount of artefactin the measurement of CDR1 or CDR2 somatic mutation. In someexperiments, mononuclear cells were stained with PE-conjugatedanti-CD19 and FITC-conjugated anti-IgD and sorted on a CoulterEpics 750 cell sorter (Coulter Electronics, Hialeah, FL). The purity

of the sorted IgDþ B cells was 95%. CD19þ IgDþ sorted B cellswere lysed in lysis buffer as described [23] and the lysate wasused for polymerase chain reaction (PCR) amplification of VH

rearrangements as described below.

VH rearrangement and germ-line library constructionRearranged and germ-line VH genes were quantitatively amplifiedfrom genomic DNA using primers corresponding to VH family-specific 50 leader sequences and 30 consensus JH or a conservedFR3 sequence as described [23,24]. The PCR products werecloned and libraries containing rearranged or germ-line VH3 andVH4 genes were made as described [23,24]. VH3 rearrangementlibraries were also made in duplicate from 1×104 IgDþ B cells perPCR reaction from one RA patient (RA4) and one normal subject(N2). Phages containing single-stranded DNA were rescued andmultiple replicate filters were prepared by dot blotting 10ml ofsupernatant per well on the filter from each library [23,24].

Oligonucleotide probesA panel of diagnostic oligonucleotide probes which identifyindividual VH3 or VH4 gene segments was used to hybridize thereplicate filters (Table 1). Hybridization was conducted by over-night incubation of the filters with each32P-labelled probe asdescribed [23,24,33,35]. These probes were specially designed totarget unique sequences of FRs or CDRs of individual VH genes.The specificity of each oligonucleotide probe has been tested byhybridization to genomic DNA to verify the ability of the probe todetect uniquely, individual germ-line gene segments [34,36–38].Some oligonucleotide probes have been previously described[23,24,34,36–38]. Additional oligonucleotide probes were: E35,AGTGACTACTACATGAGCTGG; E36, AGTGGTAGTACCA-TATACTAC; E41, GAAATCAATCATAGTGGAAGC; E57,GAAATCTATCATAGTGGGAGC; E58, TACATCTATTACA-GTGGGAGC; E86, TCAGCTATTAGTAGTAATGGG; E87,TCAGCTATTAGTGGTAGTGGT; E127, ATTAATAGTGAT-GGGAGTAGC; E149, CGCTGTCTATGGTGGGTCCTT; E150,

Immunoglobulin VH gene usage and somatic hypermutation in RA 517

q 1998 Blackwell Science Ltd,Clinical and Experimental Immunology, 112:516–527

Table 1. Diagnostic criteria for identification of VH genes and for estimation of CDR1 and CDR2mutations by oligonucleotide probes

Minimal profile for Detection of Detection ofVH gene gene identification* CDR1 mutation† CDR2 mutation†

V3–23 M18þ or M8þ or E87þ M8¹ E87¹

V3–33 E122þ or M76þ M16¹ M76¹

V3–30‡ E182þ or M93þ, E122¹ H138¹ or M16¹ E182¹

V3–11 E125þ or E35þ or E36þ, E186þ E35¹ E36¹ or E186¹

V3–74 E126þ or M84þ or E127þ M84¹ E127¹

V3–15 E123þ or H110þ None§ NoneV3–20 M91þ or M94þ, E126¹ None NoneV3–64 E114þ or E86þ None NoneV4–34 E149þ or M109þ or E41þ M109¹ E41¹

V4–31 E151þ or M98þ or E58þ M98¹ E58¹

V4–4 M110þ or E150þ or E57þ E150¹ E57¹

* For some genes such as V3–23 there are multiple unique probes, any one of which can be usedfor gene identification.

† Minimal profile for gene identification plus indicated profile.‡ V3–30 includes 1.9III and its allele, V3–30b (hv3005) as well as the insertion element V3–

30.4 (56p1) [24,61].§ No suitable combination of probes for reliable detection of mutations.

AGTAGTAACTGGTGGACTTGG; E151, GCCAGCACCCA-GGGAAGGGCC; E166, AGTAGCTTTGGCATGCACTGG;E167, GGAAGAAATAAATACTATGCA; E182, GCAGTTATA-TCATATGATGGA; E186, AGTAGTAGTTACACAAACTAC.

Library screening and detection of somatic mutations in CDR1and CDR2 by oligonucleotide probesVH3þ or VH4þ clones were identified by hybridizing the dot-blotted filter to full-length VH3 or VH4 family-specific probes, aspreviously described [23,24]. Individual VH3 and VH4 gene seg-ments were identified by hybridizing the replicate filters with theirFR- and CDR-specific oligonucleotide probes as described [23,24](Table 1). For the VH3 gene V3–15, only oligonucleotide probestargeted at CDR1 and CDR2 were used because there was nounique FR-specific probe available for this gene. The frequency ofoccurrence of each VH gene segment was calculated by dividingthe number of clones hybridizing with diagnostic oligonucleotideprobes by the total number of clones hybridizing with the family-specific probe [23,24]. Somatic mutation was detected by differ-ential motif-specific hybridization as previously described [33].

DNA sequence analysisSupernatant from each well (5ml) containing single-stranded DNAwas used for PCR amplification using M13-forward and M13-reverse primers. The PCR products were purified using the QIAquick-spin PCR purification Kit (Qiagen Inc., Santa Valencia, CA)and sequences were determined using the DyeDeoxy Terminatorcycle sequencing Kit (Perkin Elmer, Foster City, CA) on theApplied Biosystems Model 373A DNA automated sequenatorusing T3 and T7 primers. Comparison and alignment of DNAwere performed using the Lasergene program (DNASTAR, Inc.,Madison, WI). D segment utilization was determined as described[24].

Statistical analysisStudent’st-test was used to test the significance of differences inthe rearrangement frequency of individual VH gene segments, andof differences in somatic mutations between RA patients andnormal subjects.

RESULTS

Representation of individual VH genes in peripheral bloodB cellsTo determine if the VH repertoire in RA patients was different fromhealthy subjects, phagemid libraries containing rearranged VH3and VH4 genes were made from peripheral blood B cells derivedfrom four RA patients and four age- and sex-matched controls.The germ-line origin of individual genes among VH3 or VH4rearrangements was identified by hybridization with a set of motif-specific oligonucleotide probes. We observed that, with oneexception, no significant differences in the rearrangement fre-quency of tested individual VH3 and VH4 genes were foundbetween RA patients and normal subjects (Table 2). Similar resultswere observed in the repeated experiments on one normal subjectand two RA patients from two independent PCR reactions. Theeight VH3 genes analysed accounted for 66% of all VH3 rearrange-ments and the three VH4 genes represented 60% of all VH4rearrangements. The rearranged VH repertoire in peripheral Bcells was biased toward some individual VH genes. For instance,V3–23 was the most commonly rearranged VH3 gene, comprising

20–33% of the total VH3 rearrangements in both RA patients andnormal controls. On the other hand, the VH3 gene V3–64 wasrarely found in the peripheral blood of either RA patients or normalcontrols (Table 2), although this gene was present in the germ-lineof all tested subjects. In addition, the rearrangement frequency ofindividual VH genes varied among patients and normal subjects.For example, V3–74 rearrangement was not found in the RApatient RA3 although this gene was present in the germ-line,whereas in other subjects, V3–74 comprised 2·3–7·7% of allVH3 rearrangements.

The one exception observed in this study was V3–30. Thefrequency of V3–30 rearrangements was significantly higher innormal subjects than in RA patients (means of 15·77% and 4·46%,respectively, Table 2). Subsequent analysis by gel hybridization ofgenomic DNA showed that this difference was probably due todifferences in gene dose between patients and controls. One patient(RA4), from whom no V3–30 rearrangements were observed,appeared to lack the V3–30 gene as V3–30 was not obtainedfrom a germ-line library. This apparent germ-line deletion wasconfirmed by hybridization of genomic DNA with gene-specificoligonucleotide probes (data not shown). Results from similaranalysis indicated that V3–30b (hv3005) was absent in the otherthree patients but that one or more V3–30 elements were present ineach of the controls. High frequency of deletion of V3–30b in RApatients has been shown by others [39].

Detection of somatic mutations by hybridizationTo test if the occurrence of somatically mutated rearrangementswas different between RA patients and normal subjects, wedesigned unique motif-specific oligonucleotide probes targeted atthe CDR1, CDR2 and FR of five VH3 genes and three VH4 genes.By comparison of hybridization profiles of multiple, non-over-lapping gene-specific oligonucleotide probes, the presence ofsomatic mutations in the rearranged genes was revealed by theloss of concordance (relative to the hybridization profile on germ-line clones) of one or more probes (Table 1). We have previouslyused this approach to study the accumulation of somatic mutationsin rearrangements of the V3–23 gene [33]. These probes weretested in germ-line libraries and were found to correlate with over95% of the targeted genes, indicating a background error of# 5%.

Additional system background not detectable by analysis ofgerm-line libraries was estimated from control experiments inwhich a rearranged VH3 library was made from the L1 B lympho-blastoid cell line [34,37] in parallel with the libraries from RApatients and controls. To approximate more closely the conditionsof amplification of VH rearrangements from leucocytes (about 10%B cells), L1 DNA was mixed with autologous granulocyte genomicDNA at a ratio of 1:10 prior to the amplification of VH3 rearrange-ments. Hybridization analysis of 373 clones of the rearrangedheavy chains from this cell line showed concordance between FRand CDR probes 90% of the time. These results together with thegerm-line library data suggested that the combined errors from allsources (PCR artefact, cloning artefact, hybridization analysis)contributed a system error incidence of< 10%. Because eachanalysis assays 42 bp, this is equivalent to a mutation incidence of< 0·3% per bp, or approximately one substitution per rearrange-ment, which is close to the observed PCR error for V generearrangements estimated by others [40,41].

Somatic mutation in VH3 rearrangementsApproximately 2400 rearrangements among the five frequently

518 S.-C. Huanget al.




Tab

le2.

Rea

rran

gem

ent

ofin

divi

dual

V Hge

nes

inpe

riphe

ralb

lood

Bce

llsof

RA

patie

nts

and

cont

rols

VH

3V

H4

V3

–23

V3

–33

V3

–30

V3

–74

V3

–11

V3

–15

V3

–20

3–

64T

otal

V4

–34

V4

–31

V4

–4

Tot

al

IDA

geS

exn

%n

%

RA

150

M33

721

·95·

38·

65·

37·

17·

13.

30

58.8

382

29·8

27·2

16.2

73.2

RA

2*47

M89

929

·710

·13·

93·

37·

54·

92.

40.

262

.222

622

·627

·44.

454

.4R

A3

64M

407

26·8

15·2

7·4

012

·33·

74.

20

69.5

207

30·0

27·5

4.8

62.3

RA

435

F46

922

·415

·7D

†7·

74·

06·

23.

00

59.1

411

20·7

22·1

8.3

51.1

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n25

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·66·

64·

17·

75·

53.

20.

162

.425

·826

·18.

460

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d.3·

24·

22·

02·

83·

01·

30.

70.

14.

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22·

34.

78.

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148

M34

620

·29·

215

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24·

32·

92.

90

60.1

406

10·6

34·0

2.0

46.6

N2*

46M

913

28·3

9·1

11·5

2·9

10·0

9·1

5.1

0.3

76.5

447

38·3

12·3

19.7

70.3

N3

69M

441

19·3

22·5

18·8

2·3

10·2

9·7

1.4

0.2

84.4

261

31·4

25·7

9.6

66.7

N4

35F

461

20·8

6·5

18·2

2·8

10·6

2·8

00.

462

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015

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·16.

144

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ean

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40.

270

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357

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32·

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63·

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90.

110

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patie

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(RA

1–

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–N

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3–

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und

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amin

edby

olig

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ide

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ehy

brid

izat

ion

onge

nom

icD

NA

.‡

Mea

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gnifi

cant

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gher

than

that

from

RA

patie

nts

(P<

0·05

).

used VH3 genes were analysed by hybridization for the occurrenceof somatic mutations. Figure 1 shows the frequency of occurrenceof somatic mutations among the VH3 rearrangements detected byCDR1 (Fig. 1a) and CDR2 (Fig. 1b) probes. The occurrence ofsomatic mutations ranged from 42% to 83% detected by CDR1probes and from 29% to 97% detected by CDR2 probes among theVH3 rearrangements in RA patients. In the controls, however, theoccurrence of somatic mutations among VH3 rearrangements wasmuch lower, ranging from 6% to 42% for CDR1 probes, and from7% to 55% for CDR2 probes. The mean occurrence of somaticmutations detected by CDR1 and CDR2 probes was significantlygreater in VH3 rearrangements from RA patients than from con-trols. For instance, among the V3–23 rearrangements the averageoccurrence of somatic mutations detected was 61% in RA patientscompared with 27% in controls in CDR1 (P< 0·006), and almost70% for RA patients compared with 36% for controls in CDR2(P< 0·003). Similarly, significant elevation of somatic mutationswas found in CDR1 and CDR2 of rearrangements from other VH3genes (Fig. 1a,b). The occurrence of somatic mutations wassignificantly higher in RA patients than in controls even whenthe background (10%) was subtracted from the observed values.However, there was variation in the somatic mutation eventsamong patients and healthy subjects. For instance, the occurrenceof somatic mutations in CDR1 and CDR2 of the five rearrangedVH3 genes from three RA patients (RA1, RA2, RA4) was higherthan that seen in all normal subjects (Fig. 1a,b). In patient RA3,however, the occurrence of somatic mutations was elevated onlyin CDR2 of V3–23 rearrangements and in CDR1 and CDR2 of

V3–11 rearrangements, but was similar to controls in CDR1 andCDR2 of V3–33 and V3–30 rearrangements and in CDR1 ofV3–23 rearrangements.

Somatic mutation in VH 4 rearrangementsSimilar analyses of< 1500 rearrangements of the three VH4 genesrevealed that there were fewer mutations in VH4 rearrangementsthan in VH3 rearrangements among RA patients, and that amongcontrols the occurrence of somatic mutations in VH4 rearrange-ments did not differ from the occurrence of somatic mutations inVH3 rearrangements (Fig. 2). A marginal elevation of somaticmutations in CDR2 of V4–34 rearrangements was observed in theRA patients compared with controls (P¼ 0·057). Somatic muta-tions in CDR2 in the rearrangements of the other two VH4 genesand in CDR1 in the rearrangements of the three VH4 genes fromRA patients were slightly, albeit not significantly, higher than thosefrom normal subjects (P> 0·08, Fig. 2).

Comparison of VH3 and VH 4 mutationsThe mean incidence of somatic mutations was calculated bycombining data from the five VH3 genes or from the three VH4genes. Each mean value from each subject was treated as a singlepoint in statistical analysis. As shown in Fig. 3, the occurrenceof somatic mutations in VH3 rearrangements was significantlyhigher in RA patients than in controls (P¼ 0·0006). However,the occurrence of somatic mutations in VH4 rearrangements wasnot statistically different between RA patients and controls(P¼ 0·054). Among RA patients, the occurrence of somaticmutations in VH3 rearrangements was significantly greater thanin VH4 rearrangements (P¼ 0·027), but among controls, mutationin VH3 was not different from that in VH4 (P¼ 0·277).



20

40

60

80

100(a)

(b)

0

Pe

rce

nt

mu

tate

d

V3–23 V3–33

VH segment

V3–30 V3–74 V3–11

V3–23 V3–33 V3–30 V3–74 V3–11

N1

N2

N3

N4

RA1

RA2

RA3

RA4

20

40

60

80

100

0

Fig. 1.Somatic mutations among rearrangements of five VH3 genes derivedfrom peripheral blood B cells of four RA patients and four normal controls.Somatic hypermutation events were estimated by hybridizing VH3 librarieswith motif-specific oligonucleotide probes targeted at CDR1 or CDR2sequences of each gene as described [33]. Filled symbols, RA patients;open symbols, normal controls. Paired samples are indicated by shape ofsymbol. (a) Percent of rearrangements with somatic mutations in CDR1. (b)Percent of rearrangements with somatic mutations in CDR2.

20

40

60

80

100 (a)

0V4–34

Pe

rce

nt

mu

tate

d

V4–31 V4–4

20

40

60

80

100(b)

0

VH segment

V4–31V4–34 V4–4

N1

N2

N3

N4

RA1

RA2

RA3

RA4

Fig. 2.Somatic mutations among rearrangements of three VH4 genes in theblood samples of four RA patients and four healthy subjects. Somatichypermutation events were estimated by hybridizing VH4 libraries withmotif-specific oligonucleotide probes targeted at CDR1 or CDR2 sequencesof each gene as described [33]. Filled symbols, RA patients; open symbols,normal controls. Paired samples are indicated by shape of symbol. (a)Percent of rearrangements with somatic mutations in CDR1. (b) Percent ofrearrangements with somatic mutations in CDR2.

Sequence analysis of rearrangementsThe hybridization assay indirectly measures somatic mutations bydetermining the loss of hybridization to motif-specific probes. Tocorrelate results obtained by this assay with actual mutations, thecomplete nucleotide sequences of randomly selected rearrange-ments and of germ-line clones were determined. This analysisfocused exclusively on the V3–23 gene from each subject. Eachsubject carried at least one germ-line V3–23 allele that was foundto be identical to the known V3–23 sequence (not shown) [42]. Inaddition, patients RA1, RA2 and RA4 as well as control N4 carrieda second V3–23 allele in which a G for a T was substituted in thefirst position of codon 5, resulting in substitution of valine withleucine. This variant V3–23 germ-line sequence was not identicalto any of the alleles identified by Sassoet al. [43] and thereforerepresents a new allele of V3–23. DNA sequences of V3–23rearrangements from each individual were compared with therespective germ-line sequences, which allowed a precise identifi-cation of substitutions.

DNA sequence analysis was performed on 61 V3–23þ

rearrangements from the four RA patients and 62 V3–23 rearrange-ments from the four controls. Among the V3–23 rearrangementsfrom the RA patients, two were found to result from PCR crossing-over of V3–23 genes with other genes, and were excluded fromfurther analysis. One V3–23 rearrangement from patient RA2contained 3 base insertion (GCC) at codon position 14, whichwas treated as a single mutation. Figure 4 shows a comparison ofrearrangements that had acquired two or fewer substitutions(representing unmutated rearrangements and accounting forsystem background) with rearrangements that had acquired threeor more substitutions (definite somatic mutations). As shown, more

than twice as many rearrangements had acquired mutations in RApatients than in controls. In conjunction with Fig. 5, which showsthe results expressed as the averaged number of substitutions per100 nucleotides for each region of the V segment, the results showthat more rearrangements were mutated and there were moremutations per rearrangement in RA patients compared with con-trols. The results from Fig. 5 also fully corroborate the hybridization



20

40

60

80

100

0

Pe

rce

nt

mu

tate

d

RA

VH

3 VH

4

N RA N

P =0.0541

P =0.2765

P =0.0006

P =0.0265

Fig. 3. Somatic mutations in VH3 versusVH4 rearrangements amongpatients and controls. The mean incidence of somatic mutations wascalculated from each subject from the data in Figs 1 and 2. Each meanvalue from each subject was treated as a single point in statistical analysis(pair-wise Student’st-test).

25

50

75

100

0

Pe

rce

nt

of

rea

rra

ng

em

en

ts

0–2

Number of substitutions

3+

Fig. 4.The fraction of mutated rearrangements is larger in RA patients thancontrols. Rearrangements that had 0–2 substitutions (unmutated and systembackground) are compared with rearrangements that had three or moresubstitutions (mutated).B, RA patients;A, normal controls.

Su

bsti

tuti

on

s p

er

10

0

nu

cle

oti

de

s

FR1 CDR1 FR2 CDR2 FR3

5

10

15

20

25

30

0

N1

N2

N3

N4

RA1

RA2

RA3

RA4

Fig. 5.Nucleotide substitutions in FRs and CDRs of V3–23 rearrangementsfrom RA patients and healthy subjects. A total of 59 V3–23 rearrangementsfrom four RA patients and 62 V3–23 rearrangements from four healthysubjects were randomly selected for DNA sequence analysis. Filledsymbols, RA patients; open symbols, normal controls. Paired samples areindicated by shape of symbol. Data are presented as number of nucleotidesubstitutions per 100 nucleotides among the respective regions. FR andCDR are defined according to Kabatet al. [62].

data shown in Fig. 1. In addition, comparison of the nucleotidesequence with the hybridization data confirmed the hybridizationresults in all cases. The number of substitutions and their effect oncoding amino acids (i.e. replacement or silent) in different regionsof V segment of V3–23 rearrangements from RA patients andhealthy controls are shown in Table 3. The total number ofnucleotide substitutions in FRs and CDRs of V3–23 rearrange-ments from RA patients was greater than that from healthycontrols. The ratio of replacement to silent substitutions (R/S) inboth FRs and CDRs varied among subjects. The total R/S ratio inCDRs was greater than that in FRs, regardless of disease status.However, the total R/S ratios in the CDRs as well as in the FRs ofV3–23 rearrangements from RA patients were lower comparedwith that from normal subjects (Table 3).

CDR3 and usage of D segments in rearrangementsTo determine independence of rearrangements and to compareutilization of D segments between RA patients and controls, CDR3sequences were analysed. Table 4 shows the CDR3 sequences fromrandomly selected V3–23þ rearrangements from RA patients andnormal subjects. We observed 50 unique CDR3 among 59rearrangements from RA patients and 59 unique CDR3 among62 rearrangements from controls. Among 18 V3–23 rearrange-ments from RA3, a repeated CDR3 was observed in six rearrange-ments and another repeated CDR3 was found in threerearrangements (Table 4). Such highly repeated CDR3 whichindicate clonal expansion were not observed among the normalcontrols or the other RA patients. Figure 6 shows the comparison ofD segment usage between RA patients and controls. D–D joinswere found in two CDR3 sequences from N1 and in one CDR3sequence from N4. The usage of individual D segments variedbetween subjects regardless of status, and some D segmentsseemed to be used more frequently than others (Fig. 6).

Somatic mutations in pre-immune B cells (IgDþ B cells)To determine if the pre-immune B cell compartment contributed tothe observed elevation in somatic mutation in RA patients, the VH



Table 3. Distribution of nucleotide substitutions in V3–23 rearrangements from RA patients (RA) and healthy subjects (N)

FR1 CDR1 FR2 CDR2 FR3

Subject n* R† S R S R S R S R S

N1 13 13 10 8 4 4 5 38 8 27 15N2 12 5 1 5 0 10 0 5 1 6 3N3 20 8 2 3 0 1 0 7 9 3 1N4 17 15 13 11 1 5 4 33 4 23 17Total 62 41 26 27 5 20 9 83 22 59 36R/S‡ 1?50 5?40 2?22 3?77 1?64RA1 10 15 15 20 5 4 7 44 11 21 21RA2 12 30 28 20 8 10 9 49 17 28 34RA3 18 8 19 7 1 4 2 36 6 32 15RA4 19 25 40 34 5 18 4 74 31 63 30Total 59 78 102 81 19 36 22 203 65 144 100R/S‡ 0?77 4?26 1?64 3?12 1?44

* n, Number of sequences.† R, Replacement substitution; S, silent substitution.‡ R/S ratio was calculated by dividing the total number of replacement substitutions by the total number of silent substitutions in each V3–23 region from

RA patients or from controls.

DH22/12

DLR1

DLR2

DLR3

DLR4

DLR5

DK1

DK4

DN1

DN4

DM2

DA1

DNEW

DIR1

DIR2

DIR4

DIR5

Unknown

DH21/10

DH21/9

DH21/7

DX'P1

DXP4

DXP1

Incidence

Normal

D s

eg

me

nt

0 2 4 6 8 10 12

RA

Fig. 6. D segment usage in RA patients and in controls. Individual Dsegments were identified as described [24]. Total number of each Dsegment from four RA patients or from four controls is presented.



Table 4. DNA sequences of CDR3 of V3–23 rearrangements from RA patients and normal subjects (N)

Clone CDR3 sequence

N1 1 GGATTACTCCGAGGCTCAGGGAATTATCCCGCCTCTTACTGGGACTGCN1 2 GGTTACGGTATGGATGTCN1 3 GTGAGGGGGATTACGTTTGGGGGAGGCCCCGTGCCGTTGACTACN1 4 GATCACTATACAAGTGGCTGGTCATACTTTGACTGCN1 5*a TTGAAGGAGGAGGGTGGCTGGTACTACTTTGACTACN1 6 GAGGGTGGTATAGTGGGATTACCTCTCTTGGACCACN1 7*a TTGAAGGAGGAGGGTGGCTGGTACTACTTTGACTACN1 8 GCCCTCTACGGCTATGACTACN1 9 AGCAGTGGTCGTGACTTCN1 10 GCGCTGACTATGATAGTAGTCCCCGACTACN1 11 GAATTAAATAACAGTGGCTGGTACCACCTTGACCACN1 12 GATATTTTCTCAGGGGGCGATGTCCCGGTTCCGGCGTACTTTGACTACN1 13 GAGGGTGGTATAGTGGGATTACCTCTCTTGGACCACN2 1 GATGGGTTTAAGTTTGACTGGTTATTACTGGGGGGGTACTTTGACTACN2 2 GATCGCCCCGTAGGATATTGTAGTGGTGGTAGCTGCTACTCAGCCACGTACTACTACTACGGTATGGACGTCN2 3 GATCTGCGGCAGTGGCTGGTGATTTTCCAGCACN2 4 GGGGAGGGGGATTACGATATTTTGACTGGTTATTATTTAGACAACTGGTTCGACCCCN2 5 GATAGGTACGGGATCTTTGACTACN2 6 GATCGCGGGGTGCCACGGGACTACN2 7 GGTCAGCAGTGGCTGGTACGGGGCTACN2 8 GAAGGAGTGGCTGGTACGACGTGGTGGCAAGGAATTTCTCACTTTGACTACN2 9* GATCGAGACGACTACGGTGACTACCCCAACTGGTTCGACCCCN2 10 GAGGTTAGATATTGTAGTAGTACCAGCTGCCATTACTACTACTACTACGGTATGGACGTCN2 11 GGCGGACTAGTGGACTACN2 12 GGGTTCGGTACTATGATAGTAGTGGTAAGGGTTGACTACN3 1 GCGGGCTGGTGTAGTAGTACCAGCTGTCATGGGTTCTTTGACTACN3 2 GCCCCTCGCCCGGGTTACGATATTTTGACTGGCCAACAGGACTACN3 3 GCTCATCTCTCATCCCCGGACTACGGTGACTACGTTAGGCACTACTACGGTATGGACGTCN3 4 GAAAAGCAGTGGCTGGTTTCTTTTGACTACN3 5 GGGGGCTACGGTGACTACCGAAATGACTACN3 6 GATCGGAGGGGAGCTACGGGGTTTGACTACN3 7 TTTAGAAGCTTTTTACCAGCTGCTAATTCTTACTACN3 8 GGGGAACGTATAGCAGTGGCGCTACGAGGTAACTACTACTACGGTATGGACGTCN3 9 GATGGCGATATTTTGAGTGTTTATTATAATCACTTTGACTACN3 10 CCTATAGGAGGTAGTTTGATGCGGGGAGCCTACTACTACGGTGTGGACGTCN3 11 ATGGGGATATTGTAGTGGTGGTAGCTGCTATGACGATACN3 12 GTGGGGCCACATATTGTGGTGGTGACTGCTATTCGTTCGGGTTTTGACTACN3 13 GATGACTACGGTGATCTTACCCCCTATGGCTACN3 14 GATCTTGGCAGTGGCTGGCGAAACGAATACTACTTTAACTATN3 15* GATCAGAGACTACGGTGACTACGTTTAGGGCTACN3 16* CCCCGGGGGAGTTGAAAAGCTATGATAGTAGTGGTTATACCCCAAGCCCCTATTTTTGACTACN3 17 GAGCGCCCGTATTACGATATTTTGACTGGTTATTATTCGGCGGCGTTTGACTACN3 18 GATGAGGGGGTTAAATACGGTGACTACCTATTTGACTACN3 19* GAGGGTCTATTGGCCTACATTCTATCGAGTATAGCAGCTCGTCCGGTGTTGGTGTCGGATATGTAGGGGCTTCGAGT

CCTCGCCCCTGAAGAGATACTACTTTGACTACN3 20* GTGGGGATTACGATTTTTGGAGTGGTTATTATTTCAATTTTGACTACN4 1b GAAGAGGCGGGTTTTTATTGTAGTGGTGGTAGCTGCTCCTTCCAGCACN4 2 GGGCGTATACAGCTATGGCCACAACACTACGGTATGGACGTCN4 3 TCGCTACGGTGGTTTGACTACN4 4 GGAGGGCATGATACTAGTGCTTATTACTACGGAACGCCTCTTAAGAACTTTGACTACN4 5 GTGGGAGTGGGACATAGTACTTTTTACTTTGACTTGN4 6c GTTTCCAAGTGGGAGCTCACCTACTTTGGCTACN4 7 GCGGGATTACCATGGTTCGGGGAGTCCACCTCAGACGACGACTTTTACTACN4 8 GTTCGGGTCCTTGGGAACTACTACTACTACTACGGTCTGGAAGTCN4 9c GCCCCCACGGTGGTAACTCCGGGGCAAGGGTACTACTTTGACTACN4 10 CAAAGAGGGAGCAGTGTCTACACTGAGGGCCACTACN4 11 GTTTCCAAGTGGGAGCTCACCTACTTTGACTACN4 12 TTAGGGGAGGGGGCTTTCCGGCTCGACCCCN4 13 GGGGGACACCCCTACN4 14* TTCCCGGGGAGTTTGCTCAAAACTGGTTCGACCCCN4 15 TTAGGGGAGGGGGCTTTCCGGCTCGACCCCN4 16 GATATGATCGATTGTAGTAGTACCAGCTGCTATGACTACTACTACGGTATGGACGTC



Table 4. Continued

Clone CDR3 sequence

N4 17b GAAGAGGCGGGTTTTTATTGTAGTGGTGGTAGCTGCTCCTTCCAGCACRA1 1 GGACTGGCCCCCTATTACRA1 2* GATGCTATCGGTGACTAGATCTCACCACRA1 3* CGCGGTCTACTACRA1 4 GAGAATATAGCAGCTCGCCGTGGGTTCTATTTTGACTACRA1 5 TGGAGCGGGTCGTATAGCAGTGGCTGGTACGACTACRA1 6 GCCACTATGGCAGCAGCTGAATCAAGTTATGACTACRA1 7 GATGGGATTTTTGGAGTGGTTATTCCTCCCAAATATTACTACGGTATGGACGTCRA1 8 GAGAGTGAGGATGGTTCGGGGAGTTATTATAACGCCGACTACTTTGACTACRA1 9 TTAAATATTGCGCCGCGATCGGTGCCAGATGCTAGCCTTTATGACTACRA1 10 GATCTATTGGATTGCAGTGATAGTAGTTGCTATTACTACTACTATTACTATGGTTTGGACGTCRA2 1 GATGGTGGTGGTGACCCCTACGGTTTGGACGTCRA2 2 TACTACGGTTCGGGGAGTCTATACRA2 3 GCCGGCTATTGTGGTGGTCACTGCTATTCCGACGATATATACCCGATTGATTACRA2 4 GAGAATATAGCAGCTCGCCGTGGGTTCTATTTTGACTACRA2 5 GATTGGGAGAGCATAGTAGTGGTTAACTACTTTGACTACRA2 6 GACACTAAGGATGGTGCGGGGAGTTGGACCTGGGGGGTCTTTGACTACRA2 7 TACACCTATGGTTATGATGCTTTTGATATCRA2 8 AACCGTGGGGCAGTGGCGCCCCATTATCACTTTGACTACRA2 9* GCACCTTTATACGATATTGTGACTGCAACATGACTACRA2 10 GATTTTTGGGGTACGGGGACCCACCCCTTTGACTACRA2 11 GGTTACGGTATGGACGTCRA2 12 GTTCGACGTGAGGTTGGACTACAGACGCCGTTATCGTTCTTTGACTACRA3 1d TCTCACGGATACAGCTATGGTTACAACTGGTTCGACCCCRA3 2 CCACCAGTGGCTGGTACGGAAGACTACTACCACTCCGGTATGGACGTCRA3 3*e GATGCTATCGGTGACTAGATCTCACCACRA3 4f CTAGTTGATAGTCGACTACCCCATACTRA3 5 CTCGGGGGAGTTATCGTTCTTTTAGGCTACRA3 6*e GATGCTATCGGTGAGTAGATCTCACCACRA3 7 GATCGTCCCGACTACAGTACCCCCTTCTACCACTACTACATGGACGTCRA3 8 TTTCCTATAGCAGTGGCTGGTCTTATTGACTACRA3 9d TCTCACGGATACAGCTATGGTTACAACTGGTTCGACCCCRA3 10 GACGCCGGCAGTGGCTGGGGCGAAATAAACTACTACTACTACGGTATGGACGTCRA3 11f CTAGTTGATAGTCGACTACCCCATACTRA3 12*e GATGCTATCGGTGACTAGATCTCACCACRA3 13*e GATGCTATCGGTGACTAGATCTCACCACRA3 14*e GATGCTATCGGTGACTAGATCTCACCACRA3 15*e GATGCTATCGGTGACTAGATCTCACCACRA3 16 CTTTATAGTGGCTACGATTACTTTGCCTCTGACTACRA3 17 GATAGGGTGGGAGCATCACCGCTCCATGACTACRA3 18f CTAGTTGATGGTCGACTACCCCATACTRA4 1 CGAGGGGATACAGTTATGGATTACTTTGACTATRA4 2 GATTCGTGGTCCACAGCTATGGTTTTCRA4 3 CCGGGAGACGGAATGAGGGGACGACACTTTTGGAGTGGTAAAAGCTACTGGTACTTCGATCTCRA4 4g GGGGGGTGGAAAGGGGACTACTTCGACTTCRA4 5g GGGGGGTGGAAAGGGGACTACTTCGACTTCRA4 6 GGAAGCAGCGCCGTCTCCRA4 7 GAGGGAGTTTCTGCCCCGAGGGATGACTACRA4 8 TCCCGTTGTGCCGGTGACTGTTCCTCCGTCGCCGTGGCGGCGGGGCTCGGTGTGGACGTCRA4 9 GCGTATTACTATGGTTCGGGGAGGTATTATCCTCTTGACTACRA4 10 ATGACAACAGGTGGCGGTAGGGGGGCTTTTGACTACRA4 11 GAGGGAGTGTCTGGCCCCAGGGTTGACTACRA4 12* GGAGGACATTACTGCCGAATCTGACTACRA4 13 GATCTTTGGGCATTCGGCCAACCGGGCGGCCTATTTGACTACRA4 14 GATTCGTGGGCCACAGCTATGGTTRA4 15 GATACTAAGGGATATTGTAGAAGTGGTAGCTGCTCCACCTTTGACTACRA4 16 GGTTCCCCTTCCGGGTCTGACAGTGGCTGGTACATCTATGGCTTTGACTACRA4 17 GGAGGTAACTATGGCRA4 18 GGGGGGGGGGGATACAACTATGGTCCTTATTACTTCRA4 19 GAGGGAGTGTCTTGCCCCAGGGTCGACTAC

*Non-productive rearrangements resulting from frame-shift or stop codons in CDR3.a–gSame letter indicates identical CDR3 among independently isolated rearrangements.

repertoire from IgDþ B cells was studied in one RA patient (RA4)and one normal subject (N2). By flow cytometry analysis, 75% ofcirculating CD19þ B cells were IgDþ in N2 and 60% in RA4. Thepercentage of IgDþ B cells was also analysed in blood samplesfrom the other three RA patients 2 years after the initial VH

repertoire experiment. IgDþ B cells accounted for 81%, 75% and76% of CD19þ cells for RA3, RA2 and RA1, respectively. Inaddition to VH3 rearrangement libraries from sorted IgDþ B cells,new libraries were also made from unsorted B cells from RA4 andN2. Figure 7 shows the results of analyses for somatic mutations inV3–23 rearrangements from unsorted and from sorted (IgDþ) Bcells of RA4 and N2. The incidence of somatic mutations in V3–23rearrangements from unsorted B cells was much higher in the RApatient than in the normal subject, but no significant difference wasobserved in the rearrangements derived from the IgDþ sorted Bcells (Fig. 7), indicating that pre-immune B cells did not contributeto the elevation in somatic mutations seen in RA patients.

DISCUSSION

In this study we have analysed the utilization of eight individualVH3 genes and three individual VH4 genes among VH3 and VH4rearrangements in peripheral blood B cells from four RA patientsand four age- and sex-matched controls. Data from this study areconsistent with earlier observations that immunoglobulin VH geneutilization in peripheral blood B cells from adults exhibits a stablebut non-stochastic pattern [23,24]. No significant differences inindividual VH gene usage in peripheral B cells were observedbetween RA patients and controls, suggesting that although someVH genes such as V4–34, V3–23 and V1–69 have been foundpreferentially used in RF [8–10,17,18], the overall representationof individual genes in the peripheral B cells from these RA patientsmay not be affected.

The frequency of rearrangement of individual genes in periph-eral B cells was estimated using a set of probes specifically targetedat unique FR, CDR1 and CDR2 motifs of each gene. This strategyallowed us to estimate the accumulation of somatic mutations aswell [33]. The FR-specific probes hybridized to more than 90% ofrearrangements of each gene. Among five VH3 genes tested in

normal controls,< 70% of clones that were identified by the FRprobes were concordantly identified by hybridization to CDR1- orCDR2-specific oligonucleotide probes. However, in three of thefour RA patients, only 10–40% were similarly concordant, sug-gesting an elevation of somatic mutations in the CDRs. As shownby control experiments, the excess accumulation of somatic muta-tions was neither due to mutations introduced by PCR nor due toallelic differences in the probe-targeted regions. Furthermore,DNA sequence analysis revealed that there were significantlymore nucleotide substitutions in CDR1 and CDR2 among V3–23rearrangements from these three RA patients than from the normalsubjects (Fig. 5). These observations suggest that in some RApatients, the fraction of B cell repertoire that has been antigen-driven is substantially larger than in normal controls. Observationsby others are consistent with this conclusion [19,21,22].

It is unclear why the occurrence of somatic mutation in therearranged VH3 genes was not elevated in one RA patient (RA3),but a similar finding has been reported [19], suggesting that anelevation in somatic mutation occurs in only a fraction of RApatients. Whether this correlates to a clinically definable subset ofRA remains to be determined.

Several possible explanations for the increase in somaticmutation in RA patients may exist. First, somatic mutation maybe occurring prematurely in B cell development. The population ofB cells in the blood that is newly derived from bone marrow (pre-immune B cells) and has not encountered antigens is positive forCD19 and IgD. It is known that high incidence of somatic hyper-mutations can be observed in the IgD¹ B cell component but rarelyin the IgDþ B cell component in the blood of normal humans[33,40,41]. It is also known that IgDþIgM¹ germinal centre B cellsfrom normal subjects can express a high rate of somatic mutationsin VH genes [44]. Because the percentage of IgDþ B cells in theblood from RA patients either does not differ or is slightly reducedcompared with that from healthy controls [14,45], mutations inblood IgDþ B cells might contribute a component of the overallelevation of somatic mutations in rearranged VH3 genes from RApatients.

To test this possibility, we investigated the VH repertoire inIgDþ B cells. The frequency of rearrangements with mutations inCDR1 and CDR2 of V3–23 among IgDþ B cells was low in boththe tested RA patient and the control subject, but did not differbetween the patient and the control (Fig. 7), suggesting that theexcess mutations in RA are not in the IgDþ B cell compartment,but must be derived from IgD¹ B cells.

Second, RA patients may have a higher percentage of circulat-ing antigen-activated B cells in the peripheral blood. The higherincidence of somatic mutations among VH3 rearrangements in thethree RA patients may be due to an alteration of the peripheral Bcell compartment in these patients. Although the contribution ofindividual VH3 or VH4 genes to the total VH3 or VH4 repertoire issimilar between RA and normal controls, the contribution of eachVH family to the total peripheral B cell repertoire was notdetermined in our experiments. However, it has been shown thatVH3 genes contribute 50–60% to the total B cell repertoire inhealthy adults [46–48]. In this study, we observed that theincidence of somatic mutation in CDRs among the rearrangementsfrom three tested VH4 genes in RA patients was much lower thanfrom the five tested VH3 genes, and was not significantly differentfrom controls. It is possible that, among those three RA patients,the increased number of peripheral B cells carrying mutated VH

genes may be preferentially derived from the VH3-bearing B cells.



10

20

30

40

50

60

0

Pe

rce

nt

mu

tate

d

CDR1 CDR2

Total PBL

CDR1 CDR2

CD19+IgD+

RA4

N2

Fig. 7. Somatic mutations in B cell subsets. VH3 rearrangement librarieswere made from unsorted PBL and from sorted IgDþ/CD19þ B cells. Purityof the sorted IgDþ CD19þ B cells was 95%. V3–23þ clones were identifiedby hybridization with at least one of the V3–23-specific probes, M18, M8,or E87. Somatic hypermutation events in CDR1 and CDR2 were estimatedby loss of concordant hybridization to M8 or E87 probes, respectively.

Such cells could represent a population of polyclonally activated Bcells as well as a population of antigen-driven B cells.

Somatic hypermutation is the extraordinary strategy of theimmune system to produce antibodies of high affinity by intro-ducing large numbers of point mutations into immunoglobulin Vgenes. Several recent studies have shown that VH genes used innatural autoantibodies are those preferentially expressed or utilizedduring fetal development, and rearrangements of these naturalautoantibodies are mostly unmutated [8,17,18,31,32,49]. In con-trast, VH gene usage in IgM RF from peripheral blood of RApatients has been shown to be genetically heterogeneous andsomatically mutated [21,22]. Further, an elevation of somatichypermutation has been observed in CDRs of VH and VL in twoof three IgG RF [20]. Since somatic hypermutations in immuno-globulin V genes have been demonstrated to be responsible forgenerating pathologic autoantibodies in animal and other humanautoimmune diseases [50–59], it would be interesting to know ifthe elevation of somatic mutations is involved in the pathogenesisof RA.

DNA sequence analysis of CDR3 showed that the usage ofindividual D segments varied between subjects regardless ofdisease status, although some D segments (e.g. DN1 and DA1)seemed to be preferentially used in normal subjects (Fig. 5). OneCDR3 was found to be repeated among six clones among the 18randomly selected V3–23 clones from patient RA3, suggesting thepresence of B cell oligoclonality in patient RA3. While it ispossible that this was a result of PCR artefact, no similarly highrepetition of CDR3 was observed in normal controls or in the otherthree RA patients (Table 4). Clonal B cell expansions in RApatients have been observed by others [19,60]. Thus, it is likelythat clonal expansion of B cells due to antigen-driven processes is acommon occurrence in a subset of RA patients.

ACKNOWLEDGMENTS

We would like to thank Patricia A. Breen for collecting blood samples fromRA patients. This work was supported in part by Grant AR39918 from theNational Institutes of Health and a Biomedical Science Grant from theArthritis Foundation.

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VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)