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Variable Region Gene Analysis of Pathologic Human Autoantibodies to the
Related i and I Red Blood Cell Antigens
By L.E. Silberstein, L.C. Jefferies, J. Goldman, D. Friedman, J.S. Moore, P.C. Nowell, D. Roelcke, W. Pruzanski, J. Roudier,
and G.J. Silverman
To investigate the molecular basis of the autoimmune response t o the related i and I carbohydrate antigens, we
studied cold agglutinins (CA) from B-cell clones and from the
peripheral circulation of patients with lymphoproliferative
syndromes. Sequence analyses of expressed variable region
genes indicate that both anti-i and anti-I specificities from
B-cell clones from two patients are encoded by the V4.21 or
a very closely related V,,4 heavy chain gene, whereas the
expressed light chain genes differed. The anti-i-secreting
B-cells express unmutated germline-encoded Vd.21 and V,l
gene sequences. The V, region gene encoding anti-I has the
closest homology (97%) t o the V,,4.21 germline gene and
differs at the protein level by only three amino acids. In
contrast, while the V, region gene encoding anti-I is most
homologous (96%) t o the VJI, kv328 germline gene, there
are seven amino acid differences due t o nonrandom replace-
ment mutations, which suggests a role for antigen-mediated
selection in the anti-I response of this individual. These
studies were extended by a structural survey of 20 additional
serum CA using antipeptide antibodies specific for determinants in V, and V, regions. All anti-I and anti-i CA were shown
t o express V,,4 heavy chains, and 14 of 17 CA expressed a
previously described V,,4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I
CA used VJl light chains, while the anti-i CA used light
chains from at least three V, families. Taken together, the
data show that anti-i and anti-I CA probably both derive from
the V,,4.21 gene (or a closely related gene). Furthermore, the
restricted V, and different V, gene use in anti-i and anti-I CA
may reflect the close structural relationship of the i and I
antigens.
0 1991by The American Society of Hematology.
T
nantly i-expressing) versus adult (predominantly I-expressing) RBCs. In normal adult sera, the specificity of CA is
almost always anti-I, while the rare autoanti-i specificity is
usually associated with certain disease states (B-cell neoplasia, Epstein-Barr virus [EBV] infection).
The pathologic counterparts of the anti-I autoantibodies
are generally derived from clonal B-cell expansions that
may progress to a frank lymphoma. Limited NH,-terminal
amino acid sequence analyses of the anti-I response in
particular have shown a restricted V,III US^.^'^ Heavy
chain-associated cross-reactive idiotypes (CRI) have been
described:’ but the structural basis of these markers has
not been examined. However, by use of primary sequencedependent antibodies to variable (V) region determinants
we have recently shown that anti-I CA derive from the V,4
family, and that most are also recognized by an antibody
directed against a specific V, second complementarity
determining region (CDR) sequence.’ Because the i and I
antigens are structurally related, we hypothesized that the
corresponding anti-i and anti-I autoantibodies might be
encoded by the same or similar Ig V region genes.
To analyze the structural basis of anti-i and anti-I
binding, we first studied two patients, one with anti-i and
the other with anti-I CA-induced hemolysis. Both individuals have idiopathic CA disease, ie, chronic relapsing autoimmune hemolysis caused by a monoclonal anti-erythrocyte
autoantibody but without evidence of an underlying B-cell
neoplasm. In one individual, a chromosomally aberrant
clone could be identified in peripheral blood (PB), which is
a feature typical of CA disease?.” We generated lymphoblastoid B-cell lines from both patients and compared the
clones and their supernatants with PB mononuclear cells
(PBMC) and serum from the corresponding patient. The
serologic, DNA hybridization, and karyotype analyses established that the secreted CA are closely related to the
pathogenic serum autoantibodies. The nucleic acid sequences were then determined to examine the diversity of
the expressed V region genes encoding these antibody
specificities and to seek evidence for antigen-mediated
HE i AND I red blood cell (RBC) antigens are
oligosaccharide determinants composed of repeating
N-acetyl lactosamine units attached to either protein or
ceramide.’ Previous studies have shown that umbilical cord
RBCs contain mostly i and small amounts of I determinants. During the first 18 months after birth, however, the
proportion of i antigen determinants continues to decrease,
coinciding with an increase in I antigen expression. This
transition is due presumably to a branching enzyme that
attaches sugars by pl-6 glycosidic linkage to galactose
residues of the unbranched structure (i antigen) to form the
complex branched structure (I antigen).’
The corresponding cold reactive anti-i and anti-I antibodies are also known as cold agglutinins (CA). They comprise
a heterogeneous group of binding specificities that recognize different determinants of the oligosaccharide chain.23
Serologically, the anti-i and anti-I specificities are broadly
defined by their differential reactivities to cord (predomiFrom the Department of Pathology and Laboratory Medicine,
University of Pennsylvania, School of Medicine, Philadelphia, PA; the
Immunology Institute, University of Heidelberg, Germany; the Department of Medicine, the University of Toronto, Ontario, Canada; and
the Department of Medicine, University of Califomia, La Jolla.
Submitted Februaly 20, 1991; cccepted June 25, 1991.
Supported by grants from the National Institutes of Health: Nos.
R29Dk3906.5-01, AM25443, Mol-RR833, AI00866; and from the
American Cancer Society: No. PRTF125; and from the University of
Pennsylvania Biomedical Research Support Grant.
Presented in part at the 1990 annual meeting of the American
Society of Hematology, Boston, MA.
Address reprint requests to Leslie E. Silberstein, MD, Department of
Pathology & Laboratory Medicine, Hospital of the University of
Pennsylvania, 3400 Spruce St, F.6.079 Founders Pavilion, Philadelphia, PA 19104-4283.
The publication costs of this article were defrayed in part by page
c h q e payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by TheAmerican Society of Hematology.
0006-4971191 17809-0009$3.0010
2372
Blood, Vol78, No 9 (November 1). 1991: pp 2372-2386
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2373
VARIABLE REGION GENES OF ANTI-I/I AUTOANTIBODIES
clonal selection. We next extended our studies to a panel of
20 additional serum anti-i and anti-I CA to further assess
heavy (H) and light (L) chain usage in CA disease.
MATERIALS AND METHODS
Patient Selection
circulating cells, and the sequence has been reported.” The L16
lymphoblastoid cell line produces a polyreactive autoantibody that
includes anti-DNA activity and was the kind gift of Dr T.
Logtenberg (Utrecht, The Netherlands). This IgM is directly
encoded by the germline V,6 gene.I6 The C6B2 hybridoma produces an IgM antibody with anti-DNA, rheumatoid factor, and
antibacterial polysaccharide activity and was donated by Dr S.
Hoch (Boston, MA).” The Ab26 cell line produces an IgM
polyreactive antibody and was the gift of Dr J.D. Capra (Dallas,
TX).” Deduced V, sequences are listed in Table 1. In addition,
monoclonal R F and other purified monoclonal Ig from patients
with lymphoproliferative syndromes have been previously described.”
Subjects were selected based on referral to a central blood
bank/immunoserology laboratory. Binding specificity was performed by standard clinical laboratory techniques to detect high
titer IgM cold agglutinins to the i and I related series of determinants.” Subjects were omitted if subsequent evaluation indicated
the presence of a postinfectious syndrome, eg, acute infectious
mononucleosis or recent Mycoplasma pneumoniae infection.
Thereby, patients were identified with monoclonal and high-titer
CA associated with chronic CA syndrome, and secondary to B-cell
lymphoma, well differentiated lymphoma, and Waldenstrom’s
macroglobulinemia.’z Samples were designated either anti-i- or
anti-I-binding specificities based on the predominance of cold
reactive agglutination with O+ umbilical cord cells or adult O+
RBCs, respectively. Test erythrocytes were also treated with ficin
to show that binding was not decreased by protease treatment.”
Cytofluorimetry was performed using a FACSCAN analyzer
(Becton Dickinson, Mountain View, CA) equipped with a 488 mm
argon laser, as previously described.” In the karyotype studies, 20
to 40 metaphases were counted, and at least three analyses were
performed on each specimen. Additional karyotypic analyses were
performed to characterize chromosomally abnormal EBV-transformed clones.w
Establishment of EBV-Transformed Clones
Southem Blot Analysis
EBV transformation of PB lymphocytes (PBL) was accomplished as previously p~blished.~
Briefly, lo7 mononuclear cells
were suspended in 4 mL RPMI 1640 medium, supplemented with
15% fetal bovine serum (FBS) and gentamycin (10 pg/mL). The
EBV-containing supernatant of the B95-8 Marmoset cell line was
freeze-thawed and filtered. One milliliter of this supernatant was
added to the lymphocyte suspension. The lymphocytes were then
cultured for 24 hours at 37°C in 5% CO, in 95% humidified air. The
transformed cells were washed in phosphate-buffered saline (PBS),
pH 7.4, and subcloned by limiting dilution in 96-well plates. Wells
were screened for CA activity as previously p~b1ished.l~
Cell lines
that were considered clonal based on statistical analyses were also
karyotyped and the secreted CA purified from supernatant for
further studies (see below).
Genomic DNA ( 5 to 10 pg) was extracted from PBL and the
EBV-transformed B-cell clones digested with EcoRI, HindIII, and
BamHI restriction enzymes.*’The digested DNA was then separated electrophoretically on 0.5% agarose gels, blotted onto nylon
membranes, and hybridized to various DNA probes. The probes
were cloned genomic DNA fragments for the human H chain
joining region (JH), as well as a probe specific for terminal repeat
The probes were labeled with
fragments of linear EBV
32Pby random priming using T4DNA polymerase (Stratagene, San
Diego, CA). Hybridization was performed in 7% sodium dodecyl
sulfate (SDS)/O.5 mol/L NaHPO,, pH 7.2, at 42°C; membranes
were washed with decreasing salt concentrations with a final wash
in 0.1% SDS/O.lX SSC at 65°C.
Purification of Cold Agglutinating Antibodies
CA were purified from serum by repetitive cold-warm-cold
incubation with packed RBCs followed by heat elution at 3PC.9.”
CA with anti-I fine-binding specificity were isolated from serum
with adult cells, and CA with anti-i activity were purified on
umbilical cord blood cells. In certain cases, large-scale purifications
were performed by removal of IgG with a protein G-sepharose 4B
column before affinity purification of CA on a desialatedorosomucoid-sepharose 4B column, as described.“ Purified IgM
proteins were assayed for CA activity, and all CA were without
rheumatoid factor (RF) or antinuclear binding activity. IgM CA
from culture supematant and from the heat eluate were purified by
passage over antihuman IgM (p-chain-specific)-cyanogen bromide
(CNBr)-activated Sepharose columns (Cooper Biomedical, Malvern, PA, Pharmacia, Piscataway, NJ). The bound proteins were
eluted with 0.2 mol/L glycine, pH 2.5, rapidly neutralized, and then
dialyzed against PBS. The isotype of eluted antibody was then
evaluated by the Ouchterlony method (Cooper Biomedical) and by
agarose electrophoresis followed by immunotixation.
Monoclonal ZgM Proteins
The LES, monoclonal IgM-RF, was isolated from the peripheral
circulation of a patient with a B-cell chronic lymphocytic leukemia
that is surface phenotype negative for CD5. The V, gene encoding
this autoantibody has been isolated from a genomic library of
Cytojluorimehyand Karyotype Analysis
Sequencing and cDNA Cloning of V Region Genes
The experimental approach for the cloning and sequencing of
the expressed Ig V genes has previously been described.u Briefly,
first-strand cDNA synthesis from 1 pg total cellular RNA was
performed with reverse transcriptase (Life Sciences, St Petersburg,
FL) and an oligonucleotide primer specific for the p chain constant
region (Cp): 5’-CGA GGG GGA AAA GGG m G GGC C-3’. For
the cell line 15B from CAP, we used a 21-bp oligonucleotide primer
corresponding to the Ig diversity-joining region (D-J) junction of
clone 15A (patient CAP) (see Fig 2). Second-strand synthesis was
performed by polymerase chain reaction (PCR) amplification with
Amplitaq recombinant stable Tuq polymerase (Cetus PerkinsElmer, Nonvalk, CT), the same C p or D-J primer, and a 5’ primer
complementary to the conserved portion of published leader
sequences of the V,4 gene family: 5’-ATG AAA CAC CTG TGG
‘ITC TT-3’. The same approach was used to isolate the V, gene
sequences. The primers corresponded to K chain constant region
and leader sequences of the V,I and VJII families (indicated in
Figs 3 and 4). For the isolation of the V, germline sequences (V,I
and kv328) from granulocyte DNA, we constructed oligonucleotide
primers corresponding to sequences in the leader and CDR3
regions that were shared among the expressed V, genes and the
published relevant (closest homology) germline gene sequences
(indicated in Figs 3 and 4). Thirty cycles were performed in a
programmable thermocycler (Coy Laboratory Products, Inc, Ann
Arbor, MI), with soak temperatures of 94T, 55T, and 72°C for 1
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2374
SILBERSTEIN ET AL
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8
Immunodominant portions of the first framework region (FRl)
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the second CDR of all reported germline V& genes?’9 Certain
sequences of these synthetic peptides are compiled in Table 1.
Antisera were also used that identify sequences from the first
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fine-bindingspecificitycertain antipeptide antisera were first preadsorbed with a related peptide at 0.1
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specificity was documented if anti-Ig reactivity of an antipeptide
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minute each followed by 9 minutes at 72°C. Blunt-ended doublestranded amplified DNA was then ligated with T4 ligase (International Biotechnologies, Inc, New Haven, CT)into the phosphatased Sma I site of pBS M13- (Stratagene) and transfected by
electroporation (Biorad Laboratories, Rockville Center, NY)into
the XL-1 blue strain of Escherichia coli.= Clones were screened by
blue/white selection and restriction analysis of plasmid DNA.
Clones containing the expected 450-bp size insert were sequenced
by dideoynucleotide termination method with Sequenase (United
States Biochemical Corp, Cleveland, OH) using both “-40” and
“reverse” universal primers.” Human p and K oligonucleotide
primers were synthesized by an oligonucleotide synthesizer (Applied Biosystems, Inc, Foster City, CA) using phosphotriester
chemistry.
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Monoclonal Anti-IdiotypicReactivity
The IgG2a rat antibody, 9G4, was produced by a rat-mouse
heterohybridoma after immunization of a Lou rat with the IgM-A,
SA. Binding activity of the 9G4 antibody with paraproteins and
inhibition of red blood cold agglutination has been described,’ but
the structural basis of the expression of the 9G4 idiotype has not
been investigated. For assays of binding activity of the monoclonal
anti-idiotype, polystyrene microtiter plates were precoated overnight at 4°C with affinity-purified polyclonal goat antihuman IgM
(Boehringer Mannheim, Indianapolis, IN) at 10 kg/mL in phosphate-buffered saline (PBS), pH 7.0. For the study of the RID CA,
the wells were precoated with either antihuman K or A (Boehringer
Mannheim). Plates were quenched for 1hour at room temperature
(RT) with 1% bovine serum albumin (BSA)-PBS, then serial
dilutions of purified IgM proteins in BSA-PBS were added,
beginning at 2 pg/mL. After incubation at RT for 4 hours, plates
were washed with 0.05% Tween 20-PBS, then wells were incubated
with an anti-idiotypic antibody for 4 hours at RT. After washing,
plates were incubated with either alkaline phosphatase (A€’)labeled affinity-purified F(ab’)2goat antimouse IgG (Calbiochem,
La Jolla, CA) or AP-labeled affinity-purified F(ab’)2goat antirat
IgG. Replicate wells without anti-idiotype or isotype control were
incubated with AP-labeled affinity-purified goat antihuman IgM.
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VARIABLE REGION GENES OF ANTI-III AUTOANTIBODIES
Plates were subsequently developed with 50 mmol/L sodium
carbonate, pH 9.8, containing p-nitrophenyl phosphate and 1
mmol/L magnesium chloride. The IgM-A, SA, and the IgM-K,
VOG, were used as positive controls for the 9G4assays.
StatisticalAnalyses
Statistical detection of positive selection in the CDR was
performed using a previously reported binomial probability
Binding reactivity of the monoclonal anti-idiotype was
designated positive if the value for an Ig protein was greater than 4
SD above the mean A405 of negative proteins. Statistical significance was determined by the two-tailed Fisher exact test.
RESULTS
Establishment of CA-Secreting B-Cell Clones
PBL exposed to EBV were seeded at 1 to 3 x lo4
cells/well of 96-well microtiter plates. After 5 weeks of
tissue culture, the supernatants were assayed for CA
a~tivity.’~
CA activity was detected in only 2 of 418 wells
from different plates, suggesting that these wells likely
contained the derivatives of a single CA-secreting, transformed B cell. Subcloning at less than 10 cells/well was not
feasible for the two cell lines derived from patient CAP
(15A and 15B). Limiting dilution of cell line 15A at 10
cells/well yielded 10 of 235 wells with growth; 6 of 10 also
tested positive for CA activity. This distribution suggested
that these sublines were clonal.
For patient VOG, approximately 64% of wells plated
showed colony growth, of which 56% had CA containing
supernatant. Two clones were established by repeated
limiting dilution at 0.1 cell/well; these two clones are
referred to as 20A and 20C. An additional cell line was
clonable at 10 cells/well and is referred to as 20B.
The three cell lines derived from patient VOG (20A,
20B, and 20C) are all stable Ig-producing clones ( = 5
pg/mL). To prove that these clones represent independent
transformations of different B cells, we performed DNA
hybridization studies with a probe specific for the terminal
repeat segments of EBV.= When this probe is hybridized to
the EcoRI-BamHI-digested DNA from independent EBVtransformed B-cell clones, restriction fragments of different
size are generated,= because the infecting EBV virus fuses
to form a plasmid whose size is determined uniquely at the
time of the fusion process. The three B-cell sublines from
the patient VOG were found to be independent clones by
this method (data not shown).
Relationship of EBV-Transfoimed B-Cell Clones to Patient
Material
Serology of CA. The binding specificity of the RBC
autoantibodies both from serum and clonal supernatants
was determined by their differential reactivity against adult
and cord test R B C S . ’ ~The
~ ~ CA derived from serum and
clonal supernatant of patient CAP reacted more strongly
with cord and I-negative RBCs and was therefore classified
as anti-i; in contrast, the CA from serum and clonal
supernatant of patient VOG preferentially reacted with
I-positive RBCs and was classified as anti-I. For example,
the CAP CA isolated from serum (heat eluate)/clonal
2375
supernatant reacted with adult (I) versus cord (i) erythrocytes at titers of 1:32/1:64 versus 1:64/1:512, respectively.
In contrast, the VOG CA isolated from serum (heat
eluate)/clonal supematant reacted with adult (I) versus
cord (i) erythrocytes at titers of 1:1024/1:128 versus 1:128/
1:4, respectively. For patient VOG, the IgM,K CAwas easily
detectable as a homogeneous band by agarose gel electrophoresis followed by immunofuration, but for the patient
CAP, demonstration of a homogeneous IgM,K band required the isolation of the CA from serum (not shown).
Furthermore, only in patient VOG was a B-cell clone
readily detected in PBL by karyotype and Southern blot
analysis (see Fig 1). These findings indicate that both
patients had evidence of monoclonal CA, although in
patient CAP, the size of the circulating B-cell clone
(CA-secreting) was too small for detection by chromosome
or DNA hybridization analysis.
Cytofzuorimetry and chromosome analysis. For the clone
isolated from patient CAP, the surface phenotypes as
determined by cytofluorimetry were p, 6 , K,HLA-DR( +),
CD22( +), CD5( -), whereas the three clones from patient
VOG were all p,, K, HLA-DR(+), CD22(+), CD5(-). In
addition, homogeneous staining of cell populations (clone
15A and 20A) was found with the 9G4 rat monoclonal
anti-idiotype (see below). Neither patient had a monotypic
B-cell population in PB discernible by cytofluorimetry.
Karyotype analysis of PBL and EBV-transformed clones
from patient CAP showed only karyotypically normal cells.
In contrast, both the PBL and the B-cell clones from patient
VOG showed the same aberrant karyotype 48,XX,+3,+ 12
(data not shown)?’
DNA hybridization analyses. Southern blot analyses were
performed on DNA from patients’ PBL as well as theestablished B-cell clones. In patient CAP (Fig lA), DNA
hybridization with the J, probe failed to identify a clonal
B-cell population in PBL and showed a germline band only.
For 15A, the EcoRI digest shows one rearranged band (9.4
kb), whereas the HindIII digest shows two H chain gene
rearrangements (9.6 and 3.0 kb); the latter finding suggests
that the restriction fragments in the EcoRI digest are of
similar size. The same restriction fragments were also
present in DNA from cell line 15B. Additional Ig restriction
fragments, however, were seen for 15B, indicating that this
cell line was not homogeneous (not shown). In patient
VOG, identical Ig gene rearrangements were observed for
all clones (20A, 20B, and 20C) as well as for PBL (which
also showed the germline band). The hybridization experiments for patient VOG are illustrated in Fig 1B and C using
the J, probe, in which the clones have rearranged both H
chain alleles. In the HindIII digest, the J, hybridizing bands
are 9.6 and 4.7 kb (Fig 1A); in the EcoRI digest the
restriction fragments are 28 kb and 9.4 kb.
MolecularAnalyses of Expressed VRegion Genes Encoding
Anti-i and Anti-I
Nucleotide sequencing was performed to determine a
genetic basis for the related anti-i and anti-I specificities.
To control for sequencing and cloning artifacts, at least two
DNA clones were isolated and sequenced in both direc-
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2376
SiLBERSTElN ET AL
A
C
B
EcoRI-JH
EcoR I- JH
Hind 111-JH
Hindm-JH
l
l
-
-m
E
0
m
a
0
u
0
m
0
m
m
E
a
o
0
0
23.1
23.1-
-
0
0
u
0
~
Z c u c u c u
Z c u c u c V N
-
c
23.1
G-
G9.4-
c
9.4-
6.6-
6.6-
6.64.3-
I-
c
2.34.3-
4.3-
Fig 1. DNA hybridizationstudies of patient material and B-cell clones. Southern blot analysis of PBL (15PB and 20PB) and B-ceil clones (154
20A. and 208, and 2OC) isolatedfrom patient CAP (A) and patient VOG (B and C). DNA was also isolatedfrom PBL of a healthy individual (normal)
to determinethe size of the germline band. In panel B there is an additionalcross-hybridizingband of 3.6 kbthat is commonlyseen with this probe
in Hhdlll digests of genomic DNA. The molecular size standards and the positions of the restrictionfragmentsare shown beside the illustrations.
G, germline bands; arrows, rearrangedbands.
tions. Furthermore, first- and second-strand synthesis was
performed twice on each of the EBV clones. We did not
detect sequence variation due to infidelity of reverse
transcription or of the amplification procedure. Sequence
variation due to Taq infidelity has been reported in the
range of 1 error per 5,000 to 9,000 bases am~lified."~'
The error rate observed in our sequence analyses is likely to
be even lower. In sequencing the expressed V, and V,
region genes of the B-cell clones, no discrepancies were
seen in over 5,000 bases amplified and sequenced.
The anti-i- and anti-I-specific B-cell clones erpres similar
V ,genes. For clones 15A, 20A, 20B, and 20C, we used
the V,4 leader+ primer pair for reverse transcription and
PCR amplification. No amplification was observed with
primers corresponding to the other V, family leader sequences, thus supporting the idea that these are clonal cell
lines. Because 15B was not a true clone but did contain a
B-cell population related to 15A (see DNA hybridization
studies above), we sequenced the 15A clone first and
synthesized a 21-mer corresponding to the D-J region of
15A. A V, clone was then isolated from 15B DNA by direct
PCR using the V,4 leader-DJ primer pair. As can be seen
in Fig 2, the V,-D-J sequences of these two clones (15A
and 15B) from patient CAP are identical to each other and
the V, is also identical to the recently reported human
V4.21 germline gene."
The V, sequences of clones 20A, 20B, and 20C are
identical (Fig 2). The perfect sequence homology among
the three clones, particularly in the highly variable CDR3
region, along with the identical Ig gene rearrangements and
same abnormal karyotype, all confirm the clonal relatedness of these lines. When compared with known V,4
germline sequences these rearranged V, sequences are
most homologous (97%) to the published V4.21 germline
gene (Fig 2A and C)." Although the assignment of a
precursor germline gene cannot be made with certainty, we
propose that the V4.21 gene is the likely precursor
germline sequence of the expressed V, genes based on the
following arguments. The complexity of the V,4 gene
family has recently been studied by others; it is one of the
smaller human V, families and has been reported to display
little polymorphism."." In the current study, we have also
isolated a rearranged Vd.21 gene (15A and 15B) from an
unrelated individual (patient CAP)that has a nucleotide
sequence identical to the V,4.21 germline sequence, thus
confirming the lack of polymorphism. Furthermore, the
DNA hybridization studies with a J, probe, shown in Fig 1
for EcoRI and Hind111 digests, show that clones 15A, 20A,
20B, and 20C share a rearranged band of the same size
using three different restriction enzymes (BamHI not
shown). Because all of these clones also use the same J,4,
the findings strongly suggest that these clones have rear-
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2377
VARIABLE REGION GENES OF ANTI-III AUTOANTIBODIES
A
<-----------------------------
158
Met Lys His Leu T r p Phe Phe Leu Leu Leu V a l A l a A l a P r o Arg T r p
GTGAGTGTCTCA GGAATGCGGATA TGAAGATATGAG ATGCTGCCTCTG ATCCCAGGGCTC ACTGTGGGTT
ATG AAA CAC CTG TGG TTC TTC CTC CTC CTG GTG GCA GCT CCC AGA T
___
___
___
___
15A
2 OA
200
20c
___ ___ ___ ___
___ ___ ___ ___
___ ___ ___ ___
___ ___ ___ ___
val
m4.21
150
=---------------------------
Leade
<_________________----___________IntrOn------------------------------------
___ ___ --- --- --- -____---- ___ --- --- --- --- -__--- --- ___ __---- --- --- -_____ --- ___ _-- --- --- --- -___-- --- -
1
2
3
4
5
6
7
8
9 1 0 11 1 2 13 14 15 16 17 18 19 20 2 1 22 23 24 25 26 27
Ser G l n V a l G l n L e u G l n G l n T r p G l y A l a G l y L e u Leu L y a P r o See G l u T h r L e u Ser L e u T h r C y 0 A l a V a l T y r G l y G l y
LBY
CAG GTG CAG CTA CAG CAG TGG GGC GCA GGA CTG TTG AAG CCT TCG GAG ACC CTG TCC CTC ACC TGC GCT GTC TAT GGT GGG
__-Leader--->
GG GTC CTG TCC ___
__ ___ ___ ___ ___
__ ___ -a- ___ ___
__ ___ -a- ___ ___
__ ___ -a- ___ ___
---Intra"--->
TCTCTGTTpJpAG
15A
2OA
200
2 oc
___ ___
___ ___
___ ___
___ ___
___ ___
___ ___
___ ___
___ ___
___ ___
___
___ ___ ___ ___
___ ___
___ ___
___
___ ___ ___
___ ___ ___
___
___ __---- --- --- --_ -_- --- --- --- --- --- --- --- --- --- --- --- --___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ --- --- --- --- ___ _-- --- --___
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ As!?
-AT ___ --c ___ ___ ___ ___
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ -AT ___ --c ___ ___ ___ ___
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ -AT ___ --c ___ ___ ___ ___
I-------->
<---------------------CDR
2-------------------31 32 33 34 35 36 37 38 39 40 41 I 2 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
S e r P h e Ser G l y T y r T y r T r p Ser T r p Ile A r q G l n Pro Pro G l y L y s G l y Leu G l u T r p I l e G l y G l u I l e A s n H i s Ser G l y Ser T h r A s n T y r A o n P r o Ser
TCC TTC AGT GGT TAC TAC TGG AGC TGG ATC CGC CAG CCC CCA GGG AAG GGG CTG GAG TGG ATT GGG GAA ATC AAT CAT AGT GGA AGC ACC AAC TAC AAC CCG TCC
<--------CDR
28
V84.21
150
15A
ZOA
200
2oc
VH4.21
150
15A
ZOA
20B
ZOC
B
150
1511
20A
200
2oc
C
VR4.21
15A.B
20A,B,C
29
30
___ ___ ___ ___ ___
___ ___ ___ ___ ___
___
___ ___ ___ ___
___ ___ ___ ___ ___
___ ___ ___ ___ ___
________-_________
___ ___ ___ ___
___ ___ ___ ___
___
___ ___ ___
___
___ ___ ___
___ ___ ___ ___
___ ___
___ ___
___ ___
___
___
___ ___
___ ___
___ ___
___ AG--l a
___ G-___ G--
___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___ ___
___ --a ___ ___ t-- ___
___ --a ___ ___ t-- ___
___ --a ___ ___ t-- ___
___ _-- --_ ___ ___ ___ __---- --- --- --- --- --- --- --- --___ _-- --_ ___ ___ ___ ___ _-- --- --- --- --- _-- --- --- --___ __--_____ ___ ___ ___ _-- --- --- --- --- ___ --- --- --___ --- --_ ___ ___ ___ ___ --- --- --- --- --- __---- --- --___ __---_ ___ ___ ___ ___ --- --- --- --- --- __---- --- ---
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
L e u L y s Ser A r g V a l T h r I l e S e r V a l A s p T h r Ser L y s A s n G l n P h e Ser L e u L y n Leu Ser Ser V a l T h r A l a A l a A s p T h r A l a V a l T y r T y r C y 8 A l a A r g
CTC AAG AGT CGA GTC ACC ATA TCA GTA GAC ACG TCC AAG AAC CAG TTC TCC CTG AAG CTG AGC TCT GTG ACC GCC GCG GAC ACG GCT GTG TAT TAC TGT GCG AGA
___ ___ ___ ___
___ ___ ___ ___
Thr
___ -c- ___ ___
___ -c- ___ ___
___ -c- ___ ___
___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ __-___ ___ ___
___ ___ --t ___ ___ ___ ___ ___
___ ___ --t ___ ___ ___ ___ ___
___ ___ --t ___ ___ ___ ___ ___
<. . . . . . . . . . . . . . . . . . . . . D Region
___
___
___
___
___
**,
....................
t**
___ ___ ___ ___ ___ ___ ___ __--_____
___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___ --a _-- --- -_____
___ ___ ___ ___ ___ --a ___ __--_____
___ ___ ___ ___ ___ --a ___ ___ -_____
ttt t t t
t*f
***
> <__________________-_____ 54 Region
___ ___
___ ___
___ ___
___ ___
___ ___
___ ___ -_- --- --- --- ___ ___
___ ___ ___ ___ _-- --- ___ ___
___ ___ -_---- --- --- ___ ___
___ ___ ___ ___ _----_ ___ ___
___ ___ -_- ___ --- --- ___ ___
________________________ >
<
--- ---
__----
--- ---
___ ---
--- ---
_________ c Regia"-------->
Ala L e u M e t T h r A l a T y r T y r P h e A s p T y r T r p G l y G l n G l y T h r L e u V a l T h r Val S e r Ser G l y Ser A r g Ser A l a P r o T h r
GCC CTT ATG ACG GCT TAC TAC TTT GAC TAC
___ ___ _-_--- --- -_____ ___ ___ ___ TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA GGG AGT GCA TCC
et
___ ___ ___ ___ ___ ___ A-A-m M
A-- ___ ___ ___ ___ ___ ___ ___ ___ ___
__-_-- --- ___ ___ ___ ___ ___ ___ ___ ___ _-- ___
___ ___ ___ ___ ___ ___ -A- A-- ___ ___ ___ ___ ___ ___ ___ ___ ___ GCC CCA ACC
--- --- --- ___ ___ ___ ___ ___ -_---- --- --- -__
___ ___ ___ ___ ___ ___ -A- A-- ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
G l y P h e A s p L e u G l y P r o T h r A s n Ile T h r Ser A r q P r o
GGC TTC GAT TTA GGT CCC ACT AAT ATT ACA TCT CGT CCG
E
[ CDRl 1
QVQLQQWGAG
LLKPSETLSL
TCAVYGGSFS
GYYWSWIRQP
---------- __________ _____-____ ---------__-------_-_-_______
N-----------------A
COR2
1
PGKGLEWIGE
INHSGSTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAADT AWYCAR
__________ __________
__________ __________ __________ _______
__________ __________ ---T-----__________ __________ _______
Fig 2. ExpressedV, sequences encoding a n t i and anti-I. (A) Nucleotide sequences of the expressed V, region genesfrom anti-i (15A and 158)
and anti-I (20A. 2OB. and 2OC) EBV clones; (B) the D and J, region gene sequences. To isolatethe V, region gene of 158. a 20-bp oligonucleotide
sequence (indicated with an asterisk) correspondingto the D-J junction of clone 15A (I'-CCA GTA GTC M A GTA GTA AGC-3') was usedto prime
cDNA and in subsequent PCR amplification. Amino acid translation is given above nucleotide sequence and numbered accordingto Sanz et el."
CDRs (CDR1. CDR2. and CDR3) and D and J,,4 gene segments are Indicated. Homology of the nucleotide with the germline sequence on the top
line is shown as a dash. Differences (in clones 2OA, 20B. and 2 x 1 from the published V,4.21 germline sequence are indicated in both nucleotide
and amino acid sequences. (C) The translated amino acid sequences of the published V"4.21 germline and expressed V, genes of the anti-i- and
anti-kpecific B a l l clones.
ranged and express the same V,4.21 or a closely related
gene.
Although the V, genes encoding anti-i and anti-I are
highly homologous, there is no similarity in the CDR3
regions (Fig 2B). A comparison of each of the D genes with
published germline D gene families did not identify a homologous D segment, probably because of the large number of
somatic changes occurring in the generation of D segment~:'*~' The J, regions were quite similar to each other
and to the J,4 germline sequence. The J, region of patient
CAP had one silent difference from the germline and was
thus identical in amino acid composition to the germline
J& sequence. The J, region of patient VOG, however, did
show two differences resulting in amino acid replacements,
compared with the published J& germline ~equence.4~
Isolation of the VKgemline gene encoding the 15A anti-i.
While the expressed V, gene of the anti-i secreting cell line
15A is 92% homologous to a previously reported V,I
germline gene, a much better match (greater than 98%
homology) is found with a rearranged V,I gene expressed
by the Walker cell line derived from a Burkitt's lymphoma.""' This finding suggests that the expressed V, genes
of the 15A cell line and the Walker cell line may originate
from a different, closely related V,I germline gene. To test
this hypothesis, we sought to isolate the relevant precursor
V,I germline gene from patient CAP using PCR and
cloning. The amplification primers were designed to limit
the number of amplified V, genes to those closely related to
the expressed V,I gene of cell line 15A. The leader (sense)
primer sequence was homologous to several previously
published V,I genes including the Walker cell line. The
antisense primer corresponded to the distal CDR3 region
sequence (Fig 3). A more distal (3') primer that would be
fairly specific for this new V,I gene could not be readily
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SILBERSTEIN ET AL
2370
***
15A
15.G2 ,G1 ,G4 ,G6,GI0
15.G3
15.G5
15.G7
15.G8
15.G9
LSGVKl
20GVK1
*I* t t l
**t
It*
*
<----------------------------------Leade=-__------------------------------Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu leu Leu Trp Leu Arg Gly
ATG GAC ATG AGG GTC CCC GCT CAG CTC CTG GGG CTC CTG CTA CTC TGG CTC CGA G
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
- --- --_ ___ --- ___
__---_ ___ ___ ___ ___
Pro
--- --- --- --- --- _-- --- ___ ___ ___ ___ ___ -c- ___
--- --- --- --- --- --- --- --- __---_ ___ ___ ___ ___
--- --- --- --- --- ___ --- ___
--- --- --- --- --- --- --- --- -_- --- ___ ___ ___ ___
--- --- --- --- --- _-- --- _-____ - _ ____ ___ ___ ___
--_ ___ ---
--- --- --- --- --- --- --- ---
___
___
___
___
___
___
___
___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
<--------------Intron-------_-----
-
GTAAGGATGGA GAACACTAGG AATTTACTCAG
-----______
--________
___________
---------__-------___
___--______
-----______
---_______
___________
-
___________ ______-___
___________
--------___------____
___---_____
---________
-_________ ___________
--_________
__________ ___________
-
____ Leader--->
----------------------------------------------I"~ro"----------------_------------------------------>
15A
15.G2,Gl.G4,G6,GlO
15.G3
15.G5
15.G7
15.G8
15.G9
LSGVK1
2 OGVKl
1
2
Ala Arg Cys Asp Ile
GT GCC AGA TGT GAC ATC
CCAGTGTGCT CAGTACTGAC TGGAACTTCA GGGAAGTTCT CTGATAACAT GATTAATAGTA AGAATATTTG TTTTTATGTT TCCAATCTCAG
-------------------------------------
---------- ---------- ---------- ---_------ ______---__
______-___
_____-____
___________
---------- ---------- ---------- -___-----______---__
__________ __________ ___________
---------- ---------- ---------- ____------_____---__
__________ __________ ___________
---------- ---------- ---------- ____-----_
-------G--______-___
__________ ___________
---------- ---------- ---------_ ____----_______---__
____-_____
---------- ---------- ---------- ---------- ____-----_______--__
__________
---------- ---------- ---------- -_-------- ____-----_______--__ __________
------c---
-- --- --- --- --- --__ ___ ___ ___ ___ ___
__ ___ ___ ___ ___ ___
__ _ _ _ ___ ___ ___ ___
__ ___ ___ ___ ___ ___
__________ ___________ __ ___ LYS
-A- ___ ___ ___
__________ ___________ __ ___ ___ ___ ___ ___
__________ ___________ __ ___ ___ ___ ___ ___
<--------------CDR
I------_-------4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Gln Met Thr Gln Ser Pro Ser Ser Leu Sei Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu
CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTA GGA GAC AGA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT AGC AGC TAT TTA
3
15A
15 6 2 G1 64 6 6 GI0
15.63
15.G5
. , , . ,
15.G7
15.G8
15.G9
LSGVKl
20GvXl
15A
15.GZ,G1,G4,G6,G10
15.63
15.G5
15.G7
15.G8
15.G9
LSGVK1
20GVKl
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
--- --- --- --- --- --- --- --- --- --- --- -__--- --- ___ __---_ ___ ___ --- ___ ___ --- ___ ___
--- -__--- --- --_ _-- --- -_____ --- ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
Pro
--- --- --- --- --- --- --- --- -c- --- --- -__--- --- ___ __--_____ ___ --- ___ ___ --- ___ ___
--_ ___ --- --- ___ _-- --- -_____ --- ___ ___ ___ ___ __-___ ___ _-____ ___ ___ ___ ___ ___ ___
--- --- --- --- --- --- --- --- --_ --- --- ___ --- --- ___ __--_____ ___ --- ___ ___ --_ ___ ___
Thr
--- -c- --- --- --- --- --- --- ___ --- --_ ___ --- --_ ___ __-___ ___ ___ --- ___ ___ --- -_____
--- --_ --- --- --- _-- --- --- --_ --- --_ ___ --- --_ ___ __-___ ___ ___ --- -_____ --- --_ ___
___ ___ ___ ___
___ ___ ___ ___
___ ___ ___ ___
__--_____ ___
___ ___ ___ ___
__---_ ___ ___
__---- ___ ___
__---- -_____
--- --___ ___
--- -_--- ----- ---
-->
<-----..----CDR
2---------->
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 5 0 5 1 52 53 54 5 5 56 51 58 59 60 61 62 63 64
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lyo Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC TAT GCT GCA TCC AGT TTG CAA AGT GGG GTC CCA TCA AGG TTC AGT GGC
___
___
___
___
___
___
___
___
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ __-___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ -_____ ___ __-___ ___ ___ --_ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ --- -_____ __---_ ___ ___ --- --___
___ ___ ___ --x ___ ___ --x ___ ___ x-- ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ __---_ ___ ___ --- --___ --- --_ ___ _-- --- ___ ___ --- -_____ --- ___ ___ __---_ ___ ___ --- ___ --- --- --- __---- --- -__--- --___
___
-_____
--_ ___
___ _-- --- ___
___ _-- -_____
___ ___ ___ ___ __-___ ___
___ ___ ___ ___ _-- ___ ___
___ --- ___
___ --- ___
___ -_____
___ --- ___
___ --- ___ ___ __---_ ___
___ --- _-____ __---_ ___
___ --- ___ ___ __-___ ___
___ --- ___ ___ __-___ ___
_-_
___ --- - _ ____
___ --- --_ ___
___ --- ___ ___
___ ___ ___ ___
---------
--- --- __---- ----- --- --- --- ----- --- __---- ----- -____---- --** ***
15A
15.G2 ,G1 ,G4 ,G6 ,GI0
___ ___
-_____
___ ___
--- --- ----- --- ----- --- ----_ --- --If.
ttt
+**
<-_--------CDR 3----------65 66 67 68 69 70 71 72 73 14 75 76 77 78 79 80 81 82 83 84 85 86 81 88 89 90 91 92 93 94 95
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro
AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG CAA CCT GAA GAT TTT GCA ACT TAC TAC TGT CAR CAG AGT TAC AGT ACC CCA
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ __---- ___ ___ --- ---
15.G3
15.G5
15.G7
15.G8
15.G9
LSGVKl
2OGVKl
___
___
___
___
___
___
___
___
15A
<---------------__-Jl
Region-----_------------>
96 97 98 99 100 101 102 103 104 105 106 101
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
TGG ACG TTC GGC CAA GGG ACC RAG GTG GAA ATC AAA
___
___ ___ ___ ___ ___ ___ Phe
T-- ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ __-___ ___ __---- --- _-- --- --___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ _-- ___ ___ __---- --- --- --- --___ ___ ___ ___ ___ ___ ___ _ _ _ ___ ___ ___ _-- -_____ __---- ___ ___ --- --- --- --- --- --- --- --- --- --- --- --___ ___ ___ ___ ___ ___ ___ ___ --_ ___ ___ --- --_ ___ __---- ___ ___ --- --- --_ --- --- --- --- --- --- --- --- --___ ___ ___ ___ ___ ___ --c ___ ___ ___ ___ ___ ___ ___ ___ ___ _ _ _ ___ ___ ___ ___ _-- --_ ___ __---- --- --- --- --___ ___ ___ ___ ___ ___ ___ ___ -_____ ___
---
___ ___
__---_ ___
___
--- --- -_- --- --- --- --- --- --- --- --- ---
___ ___ ___ ___ __-___ ___ ___ --_ -_____ --- --- ___ __---- ___ ___ --- --- --- --- --- --- --- --- --- --- --- --<-----------------------c
KBppa_---_------------------>
108 109 110 111 112 113 114 115 116 117 118 119 120 121
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
CGA ACT GTG GCT GCA CCA TCT GTC TTC ATC TIC CCG CCA TCT
Fig 3. Sequence analyses of V,l encoding anti-i. Nucleotide sequence analysis of the expressed Vel region gene from the 15A anti4 clone is
shown on the top line. The VJ germline precursor sequencesisolatedfrom patient CAP granulocyte DNA are indicated as 1%-2, -3, -4, -5, -7. -8. -9.
-18, and -27. Isolate 1565 is a pseudogene; X denotes nucleotide deletion. Sequences LSGV,I and 2oGV,I represent relevant V,l germline
sequences isolatedfrom two unrelated individuals using the same primers. Homology of the nucleotide with the germline sequence on the top
line is shown as a dash. (*) The oligonucleotide sequences used as flanking (5' and 3') primers for amplification of the V,l germline gene from
patients' granulocyte DNA. The 5' primer corresponds to the V,l leader sequence [5'-ATG GAC ATG AG(GA)G TCC 3'1. The 3' primer corresponds
to the V.I-CDR3 region of 15A: 5'-TGT GGG GTA CTG TAA C-3'.
determined, because the untranslated intron region of this
gene is not known. However, to control for contaminating
lymphocytes related to clone 15A, we used the D-J junction
primer described in the legend of Fig 2. This primer in
conjunction with the leader primer consistently did not
amplify granulocyte DNA. Together, these results imply
that amplificationof Ig genes of contaminatinglymphocytes
related to clone 15A was unlikely.
We cloned and sequenced a total of 11 isolates from
patient CAP. One isolate (15G5) is a pseudogene as a result
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2379
VARIABLE REGION GENES OF ANTI41 AUTOANTIBODIES
of three separate nucleotide deletions causing a frame shift.
Of the remainder, 5 of 10 are identical to the rearranged
V,I of clone 15A, the other five sequences differ from each
other and the rearranged V, gene by a single base difference. The isolates from two unrelated individuals showed
similar results: one isolate (20GVJ) is also identical to the
rearranged V,I of 15A, while the other (LSGVJ) differs
only by one nucleotide. Thus, of the 12 functional germline
genes isolated from granulocyte DNA, six are identical and
the other six differ only by one (although not the same) base
pair. These single base-pair changes are likely to be the
result of one or a combination of the following possibilities:
(1) Taq infidelity; (2) allele-specific differences; or (3)
multiple separate but greater than 99% homologous V,I
genes. Based on the low sequence variation observed,
theoretically, all five base-pair differences can be ascribed
to Taq infidelity.
Our method involved repeated PCR amplification of
genomic granulocyte DNA, cloning of the PCR product,
and sequencing multiple independent isolates. In addition,
the same procedures were performed on granulocyte DNA
from two unrelated individuals to gather support for the
existence of a yet-unreported new V,I germline sequence.
Subsequent to the experiments described here, this V,I
sequence has been isolated from a chronic lymphocytic
leukemia (CLL) patient in our laboratory as well as from
another CLL patient studied in the laboratory of Dr T.
Kipps (La Jolla, CA, personal communication). The observed homology between the genes isolated from granulocyte DNA and the expressed V,I genes from different
individuals, as well as the repeated occurrence of this
sequence in the sample of clones analyzed, strongly suggests that the V, expressed by clone 15A is identical to a
new V,I germline gene, which we designate 15AVKI.
Isolation of the V, germline gene encoding the 20A anti-I.
The V,L chains of clones 20A, 20B, and 20C are identical to
each other, and share most homology (96%) to a previously
published V,III (kv328) germline gene (Fig 4).&
In patient VOG we sought to isolate the relevant germline VJII sequence to determine the degree of somatic
diversity. Genomic granulocyte DNA from patient VOG
was amplified, cloned, and sequenced on three independent occasions to control for possible Taq infidelity. A total
of nine isolates were sequenced, and we found that six of
nine are homologous to kv328h5 and the other three to
kv328 (Fig 4). The germline sequences kv328h5 and kv328
are very homologous, differing only by one nucleotide in the
coding region (silent thymidine to cytidine change in codon
5~7):~These homologous VJII genes represent either
alleles of a single V, gene or different but almost identical
V, genes in the human gen0me.4~Of the six isolated genes
homologous to kv328h5, four are identical to each other
and to the published germline sequence, while the remaining two differ only by one nucleotide in the untranslated
intron region. Of the other three genes homologous to
kv328, one is identical to the published germline sequence,
whereas the other two also differ by a single base pair only.
Given the very low sequence variation observed, the single
base-pair differences noted in some of the sequences can be
ascribed to Taq infidelity.
A polymorphic allele of the kv328 gene may still exist in
this individual. However, because all kv328-related genes
(germline as well as the rearranged 20A V, gene) could be
amplified equally well by the primer pair used (not shown),
the isolation of the kv328 germline sequence from nine of
nine randomly selected bacterial clones argues against
missing an alternate VJII gene. Furthermore, based on
Southern blotting experiments, the kv328 gene (or closely
related gene) is likely to be present as a single copy (or very
few copies) in the germline with little heterogeneity at the
nucleotide level among different individuals studied to
date .“6,a
Collectively,the findings of previously reported studies as
well as our repeated isolation of a VJII gene that has the
highest homology of all known V,III germline genes to the
expressed 20A anti-I CA strongly suggest that kv328 is the
most likely germline precursor of the 20A anti-I CA.
Compared with the kv328 germline sequence isolated from
patient VOG, a total of 10 basepair changes are noted in
the expressed V, gene. In the CDR there are a total of five
replacement mutations and one silent mutation, whereas
the FR have two replacement mutations and one silent
mutation. We have conservatively counted the base-pair
changes at positions 151 and 152 in CDR2 as a single
replacement mutation because they affect the same codon.
However, if these nucleotide substitutions occurred asynchronously, they would have generated two different, independent replacement mutations.
Hand L Chain Usage ty Serum Anti-i and Anti-I CA
Immunoblot analysis of V, structural diversity. To determine the structural diversity of the L chains of the circulating CA, analyses were performed with primary sequencedependent antibodies. The reactivities of these reagents
with replicate blots of the separated L chains of polyclonal
IgM, the anti-I CA ODO, and seven anti-i CA are shown in
Fig 5. One of the anti-i CA (RID) is shown to have both K
(top panel) and A L chains (bottom panel), but both types of
L chains were associated with similar H chains (see below).
The O D 0 sample is characteristic of anti-I CA because it
has only VJII-derived L chains. Similarly, four anti-i CA
(GRO, PRO, TM, and FA) also have only VJII-derived L
chains. In contrast, the anti-i CA, LG, has V,I-derived L
chains. By this method we also confirmed, equivalent to
their respective cell lines, that the affinity-purified serum
CA from CAP expressed only V,I L chains, and VOG
expressed only VJII L chains (not shown). The lack of
reactivity of the L chains of PER* and RID with the V,
family-specificreagents may have been due to the presence
of variant V,-FRl region sequences of the known V,
families not identified by the antipeptide antibodies, which
has previously been observed in two VJII L chains.’’
Control studies suggested that it was unlikely that nonreactivity was due to inadequate amounts of the L chains to
detect subsets. In summary, these studies show that 13 of 14
anti-I CA use VJII-derived L chains, while only four of
eight anti-i CA use V,III-derived L chains. The L chains of
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
SILBERSTEIN ET AL
2380
f".
t l l *** +*+ f * * *+N f l t
*.+
<-------------------------------Lead~=-----------------------------
20.64
20.G2
20.G3
20.65
20.66
20 .G9
20A
20B
20c
*
*
Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp l e u Pro Asp
ATG GAA ACC CCA GCC CAG CTT CTC TTC CTC CTG CTA CTC TGG CTC CCA G
**
**
-__
--_ ___ --- ---
___
___ g-- - _ ____
___ ___ g-- ___ ___
___ ___ ___ ___
___ ___ ___ ___
___ ___ ___ ___ ___
*
', Gl*,G7**, G8**
*
*
*
20.G4
2O.GZ
20.G3
20.G5 **
20 G6 **
20.G9 ',Gl*,G7**,G8**
___ --- -_____ __---_ ___ ___
___ --_ ___ ___ _ _ -___ ___ ___
___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___ ___ ___ _ _ _
___ ___ ___ ___ ___ ___ ___ ___
*
*
*
______
____---_______
______-_______
____-_-________
______________
______________
ZOA
200
20c
____ Leader--->
1
2
3
Thr Thr Gly G1u Ile Val
AT ACC ACT GGA GAA ATA GTG
GTGGGTTTGATTTT GATTACATGAGTGC ATTTCTGTTTTATT TCCAATITCAG
______________ ____----_-____
_____---______
____------______________ ____-----_____
_____----_____
____------____-_________
____------____
____-----_____
____------______________ ____----_-____
____----______
____------______________ _____--_______
_____-________
____----_--
__
__
__
__
__
__
__
*
*
*
20.G3
20.G5 * *
20.G6 **
20.G9 S,Gl*,G7**,G8**
ZOA
20B
20c
___ ___ _-- --- -_____
___ ___ _-- --- --- --_
___ ___ _-- --- --- _-_
___ ___ _-- --- --- --___ ___ _-- --- --- --___ ___ ___ ___ ___ ___
___ ___ _-- --- --- --___ ___ ___ ___ ___ ___
<------------------CDR
I------------------>
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 2 9 30 31 32 33 34
4
5
Met Thr Gln ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cya Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala
ATG ACG CAG TCT CCA GCC ACC CTG TCI GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC AGG GCC ACT CAG AGT GTT AGC AGC AAC TTA GCC
___
___
___
___
___
___
___
___
___ ___ ___
___ ___ ___
___ ___ _ _ _
___ ___ ___
___ ___ _ _ _
___ _ _ _ ___
___ ___ ___
___ _ _ _ ___
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___ ___ ___ ___ ___ _ _ _ ___
___ ___ ___ ___ ___ ___ ___ ___ ___ -_____
___ ___ _ _ _ ___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ _ _ _ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___ ___ ___ _ _ _ ___ ___ ___ ___ ___ _ _ _ _ _ _
___
___
___
___
___ ___ ___ ___ --- ___ ___ __---- --- --- --- --- --___ ___ ___ ___ --- _-____ __---- --- --- --- --- --- --___ ___ ___ ___ --- ___ ___ __---- --- --- --- --- --- --___ ___ ___ ___ --- --_ ___ __---- --- --- --- --- --- --___ ___ ___ ___ --- --- --_ __---- --- --- --- --- --- --Leu Asn
_-____ ___ ___ ___ ___ _-_--_ ___ C-- -A- _-- --- --- --___
- _ __-- -_- - _ ____ ___ __---- C-- -A- --- --- --- --___ ___ ___ ___ ___ ___ ___ --_ ___ C-- -A- _-- --- --- --.
A
<----------CDR 2---------->
37 3 8 39 40 41 42 43 44 45 46 47 40 49 50 51 5 2 53 54 55 56 57 58 59 60 61 62 63 64 65
Trp Tyr Gln Gln LYS Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser
TGG TAC CAG CAG AAA CCT GGC CAG GCT CCC AGG CTC CTC ATC TAT GGT GCA TCC ACC AGG GCC ACT GGC ATC CCA GCC AGG TTC AGT GGC AGT
--- --- --- --- --- --- --- --- --- --- --- - _ _ --_
___ --*
Ala
--- --- --- --- --- --- --- --- --- --- --- -__ --- - _ _
G-___
----- --- --- --- --- --- ----- --_
--- - _ _
--T
___
--- --- --- --- --_ --A --- - _ _ --_
___--__
___
___
___ ___
--- --- --- --- ----- ----__--T
___
___
Met
Asp Ile
--- --- --- --- --- --- --- --- --- --- --- _-_ --G -__-A- AT- --t
_-_
___
_ _ _ --a
___
--- --- --- --- --- --- --- --- --- --- ----G
-A- AT- --t
--..
--a
___
--- --- --- --- --- --- --- --- --- --- --- ----G --- -A- AT- --t
--___
--a
35
20.G4
20.62
___-----_________
____-_--______
____-----_____
____---_______
____---_-______
___---________
___---_-______
___---_-______
___------___-____
____-_________
____---_______
____-_-_______
______-________
___-__________
-------------A
___---________
___------_-______
__________ ____---_-_____
____--________
_______________ ___---________
____----______
___-----_-_-__
___------------__
______________ ______________ ____--________
_______________ ___--_________
___---_-______
___-------_-__
___--------------
__
*
*
*
36
___ ___
___
--___
__- ___ ___ ___ ___
__- ___ ___ ___ ___ ___
_-_-_--
---
---
___
_-_----
___ ___
___
_-____
___
___ ___
___
___ ___ ___
___
___ ___ ___ ___ ___ ___
___ ___ ___ ___ ___
___ ___ ___ ___ ___
___ ___ ___ ___ ___
___ ___ ___ ___
___ ___ ___ ___ ___
___ ___ ___ ___
-_____ ___ ___ ___ ___
___ ___ ___ ___ ___
___ ___ ___ ___ ___
___ ___ ___
___ ___ ___ ___ ___
___
___ ___
___ ___ ___ ___
___ ___ ___ ___ ___
*
*
*
*
20.G4
20.G2
20.G3
20.G5 **
20.G6 **
20.G9 S,G1t,G7*",G8**
20A
206
2oc
ZOA
ZOB
20c
-
<----------------------------------------------------------I"~=~"----------------------------------------------------------
200
20c
20.64
20.62
20.63
20.65 * *
20.66 **
20.G9 ',Gl*,G7**,G8**
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
GTGAGGGGAATATG AGGTGGTTTTGCAC ATCAGTGAAAACTC CTGCCACCTCTGCTC AGCAAGAAATATAA TTAAAATTCRRTGT AGATCAACAATTTT GGCTCTACTTAAAGACA
--G-----------
-------------------------IntTOn------------------------>
20.64
20.62
20.G3
20.G5 **
20.G6 **
20.G9 S,Glt,G7**,G8**
20A
_-- - _ ____ ___ ___ -
_-_
ff*
___
___
___
___
___
___
___
*** * * * + * *
<--------CDR 3-------->
66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 8 3 84 85 86 87 8 8 89 90 91 92 93 94 95 96
Gly Ser Gly Thr G1u Phe Thr Leu Ihr Ile Ser Ser Leu Gln Ser G1U Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr A m A m Trp Pro Pro
GGG TCT GGG ACA GAG TTC ACT CTC ACC ATC AGC AGC CTG CAG TCT GAA GAT TTT GCA GTT TAT TAC TGT CAG CAG TAT AAT AAC TGG CCT CCG
-__--- --- --_
--- --___
___
___
___
___
___ __-_-____
___ ___
___ ___
___ ___ ___
__---- ___ ___
__---_ ___ ___
__-- _ ____ ___
__---_ ___ ___
___ ___ ___ ___
___ ___
--- --- --- --- --_ ___
--_ ___ ___ -_____ _ _ _
--- --- ___ --- --_ ___
--- --- -_- --- --_ ___
--- --_ __---- ___ ___
--- --_ __---- -_____
--- --_ ___ ___ -_____ ___
___ ___ ___ ___ ___
___ ___ -_____
___ ___ ___ ___ ___ ___
_-____ ___ ___ --_ ___
___ ___ ___ ___ --_ ___
___ ___ ___ ___ - _ ____
-_____ ___ ___ --_ ___
___ ___ ___ ___ ___ ___
_-____
--- --- --- --- --_ _ ---- --- _-____
--- --- --- --- ----- --- --- --- --ASP
--- --- --- --- --c
--- --- --- --- --c
___ --- -_____ --c
-------------
<------------------J4
97 98 99 100 101
Leu Thr Phe Gly Gly
CTC ACT TTC GGC GGA
_-___
Region------------------,
<-------------------Ck
102 103 104 105 106 107 108 109 110 111 112 113
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
GGG ACC AAG GTG GAG ATC AAA CGA ACT GTG GCT GCA
-__
___
CGA ACT GTG GCT GCA
--- ---
___
___
___ ___
___ ___
--- ---
___
___ ___
___ ___
___
___
__----
___
___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___
___ ___ ___ ___
___ _ _ _ ___ ___ ___
_ _ _ ___ ___ ___ _ _ _
___ ___ ___ ___ ___
___ ___ __-___ ___
___ ___ Thr
-c- ___ ___ ___
___ ___ -c- ___ ___ ___
_ _ _ ___ -c- ___ ___ ___
Regio"---------------------,
114 115 116 117 118 119 120 121
Pro S a Val Phe Ile Phe Pro Pro
CCA
CCA TCI GTC TTC ATC TIC CCG CCA
Fig 4. Sequence analyses of VJl encoding anti-I. Nucleotide sequences of VJl region gene from the anti-l-swreting clones are indicated as
20A. 208. and MC; the precursor germllne sequences isolatedfrom granulocyte DNA are indicated as M G . l through 9. Amino acid translation Is
given above nucleotide sequence and numbered according to reference. Homology of the nucleotide wRh the germllne sequence on the top line is
shown as a dash. (+) The ollgonucleotide sequences used as flanking (5' and 3') primers for amplification of the VJl germline gene from
granulocyte DNA. The 5' primer corresponds to the leader sequence of V.111 W-ATG GAA ACC CCA GC(ACGT)CAG C l T CTC 3'1; the 3' primer
shares homology with the CDR3 region of the 2OA light chain as well as to the CDR3-intron region of the published kv328 germllne sequence
(5'-CAC TGT GGG AGG CCA G l T ATT A-3').* The symbols (*, ++, and T) next to the sequence numbers indicate different amplification
experiments.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2381
VARIABLE REGION GENES OF ANTI-III AUTOANTIBODIES
~JMODOGROPROPER*TM
l
i
i
i
i
FA LG RID
i
i
i
I
anti-CK
anti-VK1-FR1
-
.-
anti-VK2-FR 1
anti-VK3-FR1
anti-VK4-FR1
Flg 5. V, diversity in CA autoantibodies. Displayed am mplicate immunoblots of the L chains of
polyclonal IgM, one anti4 CA, ODO, and seven anti-i
CA. In the top row, the anti-& antibody is shown t o
react with L chains of all of the IgM proteins. At the
bottom row, the anti-CA antibody recognizes the L
chains of the polyclonal IgM and the anti-i CA, RID.
The L chains of the CA, ODO, GRO, PRO, TM, and FA
ere shown t o derive from the V,IIi family. The anti-i
CA. LG, is shown t o use V,I L chains. L chains from the
V.lV family were not detected in any of the CA.
anti- C i
the anti-i CA derive from at least three V gene families.
There is a significant difference in the usage of non-VJI L
chains in anti-I and anti-i CA (P = .039), and the data are
compiled in Table 2.
The immunoblot finding of both K and A L chains in the
RID CA specimen was unexpected, because only a monoclonal IgM-A was detected by immunoelectrophoresis (not
shown). Therefore, enzyme-linked immunosorbent assay
(ELISA) studies were performed with wells precoated with
IgM ODOGROPROPER*TM
FA LG
RID
either antihuman K or antihuman A antibodies, which
showed that the RID CA contains both K and A Ig that are
associated with H chains with the 9G4 idiotype (see below).
This finding suggests that the RID CA may be biclonal,
similar to a previously reported case of a biclonal anti-I
CA.4'
Immunoblot analysis of VHstructural diversiq. V, diversity was analyzed with antipeptide antibodies to FRl
determinants that are specific for the six known V, fami-
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SILBERSTEIN ET AL
2382
IgG IgM C6B2 SA LES CAPGROPROODOHIG
RF
i
i
i
I
I
Table 2 Structural and Idlotypic Dhreniy
L Chain
Name
Anti-I CA
1. JOH(AJ)
2. BAT
3. BON
4. HIG
5. KAU
6. LEA
7. NAD
8. OD0
9. PER
10. POS
11. RIC
12. SOC
13. TRI
14. VOG
Anti4 CA
1. CAP
2. FA
3. GRO
4. LG
5. PER.
6. PRO
7. RID
8. TM
Non-CA
1. LES
2. SA
3. GRE
Sequence
H Chain
VL
V.
V&FRla
V&HV2e
964
anti-p
VJI
V"4
V,III
V"4
V,Iil
V&
V,lll
V&
V,lil
V&
V,lIl
V&
V,II
V&
VJl
V&
VJl
Vd
V,Ill
V&
VJl
V&
V,lll
V"4
VJl
V&
V,lll
V&
(V.111) 13/14 14/14
V"l
VJII
VJl
V&
V&
V"4
V&
V&
NT
-
NT
+
+
+
+
+
NT
NT
+
+
NT
+
+
-+
NT
+
+
+
+
-
+
+
+
NT
NT
NT
+
+
NT
NT
NT
NT
NT
719
819
919
+
+
NT
+
+
+
+
+
+
-
+
+
+
+
+
+
VJl
vp
+
v.t/v,
V"4
V&
818
-+
+
+
+
+
+
718
618
718
+
-
+
+
+
v.1
v.t
VJl
(VJI) 418
+
-
+
-
+
anti-VHCFRl
--V
I)
anti-VHCFRl
+
VH4-FRla
peptide
anti-VH4-FR1a
+
V, and V, family was determined by immunoblot analyses with
antipeptide antibodies. The presence of the V&FRla and V&HV2a
sequences was determined by immunoblot analyses after preincubation of antipeptide antibodies with synthetic peptides with related
sequences to improve fine binding specificity. Certain V, sequences
have been determined, and below "Sequence" are designated (+). V,
amino acid sequences were available for the anti-I CA, VOG(2OA) and
KAU, and the anti-i CA, CAP(15A)and the RF, LES (compiled in Table 1).
"Indicates the presence of two different patients with the same initials
PER.
tThese K light chains were unreactive with the V, family-specific
reagents (see text).
lies." All of the eight anti-i CA expressed H chains that
were identified only by the V,,4 family-specific antibody
(examples in Fig 6); all (14 of 14) anti-I CA were identified
by the V,,4 family-specificreagent, as previously reported?
As shown in Fig 6, these proteins were further characterized with reagents specific for variations in V,4 framework
sequences. After preincubationof the anti-VH4-FRI antiserum with the VH4-FRla peptide, reactivity with the four
CA is abolished. In contrast, after preincubation of the
anti-VH4-FRla antiserum with the VHCFRl peptide, the
H chains of four representative CA (GRO, PRO, HIG, and
ODO) are still reactive. In toto, seven of nine anti-I CA and
seven of eight anti-i CA are identified by the pretreated
anti-VH4-FR1a
+
VH4-FR1
peptide
anti-VHGHVPa
+
VH4-HV2c
peptide
IgG IgM C6B2SA LESCAPGRO PROODO HIG
Fig 6. lmmunoblot analyseswith V,&speclRc antipeptide antibodI-. Displayed are replicate lmmunoblota of the H chains of polyclonal
IgG and IgM, the IgM proteins of known sequence (the polyreacthre
antibody, C6B2, the RF, LES, and the anti4 CA, CAP), and the IgM of
unknown sequence (the 964 Immunogen, SA, the anti4 CA, GRO and
PRO, and the anti-I CA, OD0 and HIG). The peptide-induced, VH4
family-specific antibody, anti-VH4-FR1, identifies the H chains of all of
the IgM proteins. After the anti-VH4-FR1 was preincubated with the
VH4-FRla peptide, only the C6B2 H chain that expresses the exact
VH4-FRl sequence, SA, and the polyclonal IgM and IgG are identified.
The anti-VHCFRla antibody does not identify the C6B2 or SA H
chains, but the anti-i and anti-I CA are recognized. After preincubation
of the anti-VHdHV2a reagent with the VH4-HV2c peptide, the exact
VH4-HV2a sequence in the H chain of C6B2 Is identified, but not the
LES H chain, which varies by two residues in the amino terminal
aspect of this sequence. In this panel the anti-i CA, CAP and GRO, and
the anti-I, OD0 and HIG, are also identified (peptide sequences listed
in Table 1).
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2383
VARIABLE REGION GENES OF ANTI-III AUTOANTIBODIES
anti-VH4-FRla reagent (compiled in Table 2). These data
suggest that almost all of the CA share a variant V 4 first
framework sequence, VH4-FRla.
The H chains of the anti-i CA were also studied with a
reagent that is specific for a VJ-CDR2 sequence, VH4HV2a, which is present in a subset of germline V,4 genes
reported thus far?,& We have previously shown that after
preincubation of the anti-VH4-HV2a antibody with a
related peptide that varied by only a single amino acid, only
V, regions that express the exact cognate sequence were
identified? At the bottom of Fig 6, as predicted from the V,
sequence after preincubation, the anti-VH4-HV2a reagent
identified the V 4 of known sequence, C6B2 and CAP, but
the LES R F was not identified. Of the eight anti-i CA,
seven were identified by the anti-VH4-HV2a reagent. In
related studies we also showed that the CA from serum and
clonal supernatant from the patients VOG and CAP both
expressed the same V gene families and unique VH4-HV2a
determinant.
Among known V, genes, only the V,4.21 gene expresses
both the VH4-FRla and VH4-HV2a sequences (Table 1).
Collectively, the data suggest that, while anti-i CA and
anti-I CA have differences in V, expression, they both have
a comparable restriction to a subset of V 4 H chains based
on reactivity with the V region-specific reagents (compiled
in Table 2).
Structural correlation with the 9G4 idiotype. We next
assessed the expression of the H chain-associated 9G4
idiotype on the CA. The 9G4 anti-idiotype was expressed by
nine of nine anti-I CA and seven of eight anti-i CA. To
determine whether the 9G4 idiotype was only associated
with CA, we studied a previously described panel of
non-CA monoclonal IgM with 34 R F that include 10 V,4
RF,I9 but only the VJ IgM-RF of known sequence, LES,
was recognized by the 9G4 antibody. Similarly, of 34 other
monoclonal Ig that included eight other V,4 IgM of
unknown binding reactivity, only one additional IgM, GRE,
was found to also express the 9G4 idiotope. The 9G4
antibody did not recognize any of the 18 V,l, one V,2, 29
V,3, two V,5, or two VH6-derivedproteins. In summary, 16
of 17 V,4 CA, 1 of 10 VH4-RF,and one of eight V,4 IgM of
unknown binding activity express the 9G4 idiotope. As
compiled in Table 2, the expression of the 9G4 idiotope was
independent of L chain usage but restricted to a subset of
V,4 H chains. The association of the 9G4 CRI with CA is
highly significant, even in comparison with VH4-derivedRF
autoantibodies (P= .00007) and to VH4-derived IgM of
unknown binding activity (P= .0004). In addition, these
studies indicate that the 9G4 antibody identifies a set of H
chains that are nonoverlapping with the V,4 H chains that
express the RF-associated idiotype, LCl.19
The structural diversity of V regions of known sequence
was also studied for 9G4 idiotope expression. In this
examination, the 9G4 idiotype-reactive IgM include the
LES R F and two CA (VOG and CAP), and they are
compared with three related H chains that do not express
the 9G4 idiotope (C6B2, AB26, and L16). While nearly
identical in the portion encoded by the V gene segment,
these VH regions are diverse in the CDR3 and FR4 that are
the result of VDJ segment joints. By comparison with the
known germline VH4 gene segment sequences, the CA and
RF autoantibodies that express the 9G4 idiotope have the
least sequence variation from the deduced sequence of the
germline V4.21 gene." The recurrent identification of both
the VH4-FRla and VH4-HV2asequences is also consistent
with the impression that most, if not all, monoclonal anti-I
and anti-i CA derive from the V,4.21 gene (or closely
related gene) (Tables 1and 2). By comparison, of the six V,
sequences, expression of the 9G4 idiotope was associated
with certain conserved residues in the FR1 region adjacent
to the first CDR region, and with a few residues at the
amino terminus of the second CDR. Binding of the 9G4
antibody was not inhibited with available synthetic peptides
(data not shown). However, these initial data are inadequate for specifically identifymg the exact idiotopic contact
residues.
DISCUSSION
To understand the genetic basis of the related anti-i and
anti-I specificities, we established B-cell lines from two
individuals and characterized the pathologic antibodies
from an additional 20 patients with anti-i and anti-I
autoantibody-induced hemolysis. We subsequently determined the relationship of the pathologic serum autoantibodies to those derived from the EBV-transformed clones. In
the patient VOG, the close relationship was readily shown
by Southern blot and chromosome analyses. In patient
CAP, however, the autoantibody-secreting clone was not
detectable by these methods. In this case, we would argue
that the anti-i-secreting cell lines are, in fact, representative of the pathologic serum anti-i autoantibodies based on
the isolation of two of two cell lines that shared anti-i
specificity and the VH4-HV2a primary-sequence-dependent marker. These findings are also representative of our
panel of circulating CA, as eight of nine anti-I CA and
seven of eight anti-i CA also shared this marker.
Sequence analyses of the V region genes expressed by the
CA-producing cell lines showed that both anti-i and anti-I
antibodies were encoded by the same V,4.21 gene. In
contrast, the anti-i antibody used V,I and the anti-I antibody uses VJII. Taken together, the structural characterization of the 14 autoanti-I and eight autoanti-i CA suggests
that the anti-i and anti-I autoimmune responses are highly
restricted in H chain expression and may derive from a
single V,4 gene. We have also shown that the 9G4 monoclonal anti-idiotype probably identifies CA due to the
expression of these V,4 H chains.
The expressed V region genes were also examined for the
number and pattern of somatic mutations to evaluate the
role of antigen-mediated selection. We determined that
both the V, and V, genes encoding the anti-i antibody are
identical to germline sequences, whereas numerous basepair differences were noted in the anti-I response. Compared with its most likely germline precursor, VH4.21, the
V, gene encoding anti-I has only three amino acid differences (Fig 2C). Two of these amino acid substitutions are
located in FR regions, and one is in a CDR. In contrast, the
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2384
V, sequence encoding anti-I has a relatively high number of
amino acid substitutions (relative to the total number of
base-pair changes) when compared with the likely precursor germline sequence (kv328) isolated from autologous
DNA, these amino acid differences are due to a nonrandom
distribution of replacement mutations in the CDRs. We
applied a binomial probability model to the pattern of
mutations in this V, gene as a test for positive ~election.’~
The expected fraction of replacement mutations in CDR
for this V, sequence is 0.20, calculated by multiplying the
relative size of the CDR (0.26) by the fraction of all
mutations in CDR that could result in amino acid replacements (0.77). Using the binomial probability model, we
conservatively calculated that the distribution of five replacement mutations in CDR of a (corrected) total of 11
mutations had a low probability of occurring at random
(P = .05). However, if the two nucleotide substitutions in
codon 51 occurred asynchronously, they would have generated two independent replacement mutations. In the latter
instance, the probability of six replacement mutations
occurring at random of a (corrected) total of 12 mutations
would be even lower (P = .02). Thus, the statistical model
provides evidence that positive selection led to the accumulation of amino acid substitutions in the light chain of this
anti-I antibody. Such positive selection of replacement
mutations in CDR has been observed in murine immune
responses and presumably reflects selection for enhanced
antigen binding.50-52
In summary, we have found a nonrandom distribution of
replacement mutations in the V, encoding the anti-I
antibody, which supports a role for antigen-mediated clonal
selection. However, the selective pressure may have favored conservation of the H chain germline sequence while
favoring mutation of the light chain amino acid sequence.
In this regard, the recently reported amino terminal protein
sequence analysis of an anti-I CA KAU is in agreement
with the conserved V&-V,III use.53This CA also uses the
VH4.21 gene, differing by one amino acid only from the
translated V4.21 sequence; the light chain is most likely
derived from the kv325, V,III gene differing by four amino
acid changes, of which one is located in FR and three are in
CDR.
Based on the fact that the anti-i and anti-I responses use
the same or closely related V, but different V, genes, we
propose a model for the relative contributions of these H
and L chains to antigen binding. The V, sequence would be
required for a global interaction with the i/I carbohydrate
antigen complex. In contrast, the V, sequence would confer
fine specificity to distinct but related determinants of this
complex antigen. The D region genes, which differ between
SILBERSTEIN ET AL
the anti-i and anti-I, may also play a role in determining
specificity. Interestingly, in the immune response to the
influenza virus hemagglutinin, also a complex antigen, an
analogous model of differential contributions of H and L
chains has been postulated based on V region gene sequence analysis.54
The current studies have assessed the genetic basis of
pathogenic autoantibodies to the i and I antigens using both
molecular and serologic methods. The findings suggest a
restricted use of the VH4.21or a closely related gene, which
implies a nonstochastic biologic selection. This observation
could have occurred due to preferential rearrangement of
the VH4.21 gene, but this gene does not appear to be
overrepresented in cell lines described to date. The lack of
somatic mutations in the V region gene sequences encoding
the anti-i (CAP), as opposed to the anti-I cell lines (VOG),
is intriguing and may reflect differential regulation of the
immune responses to these related autoantigens. We hypothesize that the high expression of i antigen on fetal RBCs
may mediate tolerance to i, either by clonal anergy or
deletion of B cells with anti-i spe~ificity?~,~~
The expression
of I antigen occurs much later in development so immunologic tolerance for I may therefore differ from that for i.
This model would explain the low levels of anti-I autoantibodies present in the umbilical cord and adult blood of
healthy individual^,^^*^' and would also explain the absence
of point mutations in the anti-i CA from CAP, as opposed
to the somatic mutation pattern in the VOG cell lines
consistent with antigenic selection. Further studies are
needed to substantiate the proposed model of H and L
chain contributions to antigen binding. We also plan to
explore the structural and regulatory differences that might
exist between physiologic and pathogenic autoimmune
responses with anti-i and anti-I specificity.
NOTE ADDED IN PROOF
Two additional nucleotide sequences of cold agglutinins have
recently been reported. The expressed V, genes are similar to the
V,4.21 gene and both express the 9G4 idiotope. (Pascual V, et al: J
Immunol146:4385,1991.)
ACKNOWLEDGMENT
The authors thank Dr F. Stevenson for her generous gift of the
9G4 antibody.We recognize Dr Mark Shlomchik for his help with
the statistical analyses and for his critical review of the manuscript,
and Eric Rapaport for his expert technical assistance. Additionally,
we are grateful to Dr Sanford Shattil and Dr Albert Fink, as well as
their patients, for their cooperation, and to the General Clinical
Research Center of the Scripps Clinic and Research Foundation
for the handling of clinical specimens.
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1991 78: 2372-2386
Variable region gene analysis of pathologic human autoantibodies to
the related i and I red blood cell antigens
LE Silberstein, LC Jefferies, J Goldman, D Friedman, JS Moore, PC Nowell, D Roelcke, W
Pruzanski, J Roudier and GJ Silverman
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