Immunology and Cell Biology (2001) 79, 222–230
Research Article
In vitro and in vivo expression of a nephritogenic Ig heavy chain
determinant: Pathogenic autoreactivity requires permissive light
chains
B R E N DA G C O O P E R S TO N E , 1 * M O H A M M E D M R A H M A N, 2 † E A R L H RU D O L P H 2 ‡ a n d
M A RY H F O S T E R 2 ‡
Departments of 1Paediatrics and 2Medicine, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania, USA
Summary Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders
marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor
modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation.
The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus
anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several
autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease
progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated
genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a
9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete
12 kb construct, termed 238H-Cµ, resulted in secretion of intact Ig pairing 238H-Cµ with a lambda L chain;
however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cµ H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cµ,
however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of
238H-Cµ with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen
binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains
to specify autoimmunity. The 238H-Cµ transgenic model should prove useful in dissecting the in vivo fate of
238H-Cµ-L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted
interventions.
Key words: autoimmunity, immunoglobulin heavy chain variable region, immunoglobulin transgene, nephritogenic
immunoglobulin.
Introduction
The gene rearrangements and somatic mechanisms that
generate B- and T-cell receptors create unique clonal markers
that are feasible targets for cell-specific manipulations in
autoimmunity and other disorders, for example, certain
infections and lymphoid malignancies, characterized by
clonal expansion or restricted antigen receptor expression.
Biased TCR and Ig gene expression is described among
Correspondence: Dr MH Foster, Department of Medicine,
Division of Nephrology, Box 3014, Duke University Medical
Center, Durham, NC 27710, USA. Email: [email protected]
*Present address: BG Cooperstone, Wyeth-Ayerst Pharmaceuticals, Wayne, PA, USA.
†
Present address: MM Rahman, Montefiore Medical Center, New
York, NY, USA.
‡
Present address: EH Rudolph and MH Foster, Department of
Medicine, Duke University Medical Center, Durham, NC, USA.
Received 5 September 2000; accepted 8 January 2001.
pathogenic lymphocytes in several autoimmune diseases,
including murine and human lupus. These form the basis of
idiotype- and peptide-based therapies that have been used
successfully in experimental autoimmunity and in some cases
have proven safe in early clinical trials in humans.1 However,
variable outcomes in murine lupus after experimental manipulation of disease-associated Ig idiotypes2,3 indicate that these
lymphocyte determinants participate in complex regulatory
interactions in vivo.4 It is clear that a better understanding of
their relative roles in direct disease pathogenesis versus
immune regulation will facilitate application of receptortargeted modulation, including interventions aimed at
eliminating or inactivating autoreactive cells or re-establishing
tolerance to relevant self-antigen.
The Ig H chain epitopes encoded by the VHB/W16 gene5 are
of particular interest in murine lupus because this gene is
expressed recurrently among anti-DNA antibodies derived
from different lupus-prone mouse strains as reported in the
literature and databases.6–8 Five of these clonally unrelated Ig,
termed Ab52, H241, H161, H238 and H8, have been shown
Expression of a nephritogenic Ig heavy chain
to induce renal disease manifest as massive proteinuria,
mesangial immune deposition and/or glomerulonephritis.8–10
These latter Ig express the same VH gene, but use diverse
kappa L chains and disparate H chain CDR3 sequences,
suggesting that the common VHB/W16 gene is itself a major
determinant of pathogenicity. However, it remains unclear
if specificity for nucleic acid is important to confer pathogenicity to VHB/W16-encoded Ig or in determining the in vivo
fate and regulation of the corresponding B cell. Gangemi
et al. found that although five IgG VHB/W16-encoded autoantibodies contained arginine residues in their H chain CDR3,
the presence of these residues did not correlate with binding
to dsDNA or with pathogenicity.7 Several of the pathogenic
anti-DNA also bind directly to kidney extract and/or matrix
proteins in vitro or to glomeruli ex vivo,8–10 and at least one
VHB/W16-encoded anti-DNA IgG does not form renal deposits
upon passive transfer.7 Notably, the VHB/W16 gene is present
only in mice with the j haplotype for the J558 gene family,7
which includes several of the autoimmune strains. Its absence
in commonly studied non-autoimmune strains circumvents
ready analysis of its recruitment during induced immune
responses to foreign or self-antigen in these non-disease
prone genetic backgrounds.
To further investigate the roles of VHB/W16-encoded V
regions in autoimmunity and in the absence of a clearly
established identity of the relevant pathogenic self-antigen
or tissue targets, we constructed an IgM H chain, termed
238-Cµ, that expresses this gene. The approach takes advantage of previously isolated genomic fragments containing Ig
regulatory (promoter and enhancer) and signal sequences
that were linked to the PCR-amplified VDJ gene of Ig 238
by unique restriction sites generated via site-directed mutagenesis. The IgM 238 was chosen for study because it is
derived from a lupus-prone MRL-lpr/lpr mouse11 and
expresses the unmutated VHB/W16 gene,8 has specificity for
intrinsic renal antigens and DNA, and is capable of inducing
significant glomerular injury and proteinuria in vivo.12
Generation of 238-Cµ H chain transfectant and transgenic Ig
permits examination of its reactivity after in vitro and in vivo
combination with novel L chains.
Materials and Methods
Production of a construct containing the 238H VDJ cDNA
and H50-derived regulatory sequences
To produce a DNA construct encoding the nephritogenic 238H V
region with appropriate tissue-specific expression of secreted and
transmembrane IgM, we took advantage of previously isolated DNA
fragments containing Ig regulatory and signal sequences. We previously described cloning of a 5.1 kb genomic fragment containing the
rearranged antilaminin IgG H50 VDJ gene, termed LamH, and Ig
promoter and Cµ enhancer regulatory sequences from a subgenomic
library in lambda gt10.13 Taking advantage of unique restriction sites,
the 5′ and 3′ regulatory and signal sequences from LamH were
isolated and ligated to a modified 238H VDJ cDNA to generate a
complete functionally rearranged Ig H chain gene (Fig. 1a). Briefly,
the 238H VDJ cDNA was amplified using site-directed mutagenesis
to insert a unique Sfu I (Asu II) TTCGAA restriction site at the signal
peptide-VH junction. An oligonucleotide containing this restriction
site, 5′-TTCGAAATCCAGCTGCAGCAGTCTG-3′, was synthesized and used as a 5′ primer for PCR. The 3′ primer was an
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oligonucleotide homologous to JH2, 5′-GACTGTGAGAGTGGTGC-3′. The 238 hybridoma RNA was extracted and cDNA
synthesized as described.13 The 238H cDNA was amplified in a
50 µL reaction containing 2 µL cDNA reaction as template, 0.5 U of
Amplitaq DNA polymerase [Perkin Elmer Cetus (PEC), Norwalk,
CT, USA], 5 µL of 10X PCR buffer (PEC), 0.24 mmol/L of each
deoxynucleotide triphosphate, 15 pmol of each primer, denaturation
at 92°C for 8 min and 35 cycles of denaturation (92°C, 1 min),
annealing (52°C, 1 min) and primer extension (72°C, 2 min),
followed by 7 min at 72°C in an automated thermal cycling heat
block (PEC). The PCR product was cloned into the pCRII vector
following manufacturer’s directions (Invitrogen, San Diego, CA,
USA).
The 5.1 kb LamH EcoRI fragment13 was used as a template to
amplify 700 bp of 5′ flanking DNA, including Ig promoter, transcription initiation site and leader sequence. Site-directed mutagenesis was used to insert the unique Sfu I restriction site at the leader-VH
junction at the 3′ end of this construct. For this purpose, an oligonucleotide, 5′-TTCGAAGAGGACACCTGTGA-3′, was synthesized
as a 3′ PCR primer. The 5′ primer, 5′-GCCTTCTTCCTTATCAACA3′, was synthesized based on sequences conserved within the
5′-flanking region of H50 and previously published VH genes.14
Template DNA (0.5–1 µg) was amplified in a 100 µL reaction as
described, with these modifications: 40 cycles with anneal temperature 41°C. The PCR product was ligated into the pGEM-T vector
following manufacturer’s directions (Promega, Madison, WI, USA).
A Not I/Sfu I 700 bp fragment containing the 5′ flanking and regulatory sequences was then isolated and ligated to Not I/Sfu I digested,
dephosphorylated 350 bp VDJ fragment and cloned into EcoRI/Not
I digested plasmid Bluescript.
Generation of the final 3 kb construct containing the rearranged
238H VDJ and regulatory sequences took advantage of the fact that
both the LamH and 238H V regions use the JH2 gene, containing a
Sau I restriction site. A 2 kb fragment containing JH genes and the
Ig enhancer was isolated from the 5.1 kb LamH construct by partial
digestion with Sau I and EcoRI, then ligated with the Sau I/EcoRIdigested plasmid containing the 1 kb fragment including a 5′-flanking
sequence and 238H-VDJ. A complete 238H VDJ-Cµ H chain
construct (Fig. 1b) was generated as previously described with slight
modification.15 The 3 kb fragment containing the rearranged 238H
VDJ and regulatory sequences was ligated to an EcoRI/Not
I-restricted, dephosphorylated 9 kb genomic fragment carried in
plasmid Bluescript and encoding the IgM constant region, including
transmembrane exons, 3′ untranslated sequences and a polyadenylation site. The latter construct was generously provided as part of an
approximately 45 kb Cµ-δ genomic fragment cloned in the pNNL
cosmid by Dr David Nemazee (The National Jewish Center for
Immunology and Respiratory Medicine, Denver, CO, USA).16 For
use in transfection experiments, plasmid DNA bearing the complete
238H-Cµ construct was linearized with Not I and purified.
Ribonucleic acid isolation and sequence analysis
Total RNA was extracted from transfectant or hybridoma cell lines
using TRIzol reagent (Life Technologies, Rockville, MD, USA). For
sequence analysis, the Ig H chain V region cDNA was amplified
essentially as described in a 100 µL reaction containing 40 pmol/L
of each oligonucleotide primer: 5′-GAGGTCCAGCTGCAACA-3′
(VHLamH/238H, 5′) and 5′-ATTTGGGAAGGACTGAC-3′ (Cµ, 3′),
with denaturation at 92°C for 8 min and 35–40 cycles of denaturation (92°C, 1 min), annealing (50°C, 1.5 min) and primer extension
(72°C, 2 min), followed by 7 min at 72°C in an automated thermal
cycling heat block. The approximately 330 bp PCR product was then
cloned using the TA Cloning Kit (Invitrogen, San Diego, CA, USA)
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BG Cooperstone et al.
Figure 1 (a) Generation of the
238H heavy chain variable region
DNA construct. P1, 5′ PCR primer
complementary to the LamH-Cµ
5′ flank region; P2 and P3, 3′ and
5′ PCR primers, respectively, containing Sfu I sites for directed
mutagenesis at the Leader-VH
gene junction; P4, 3′ PCR primer
complementary to JH2; *, the Sfu
I restriction site; P, Ig promoter; L,
leader sequence; E, Ig enhancer;
R, EcoRI; Sau, Sau I (Bsu 361);
Sfu, Sfu I (Asu II). (b) Linear map
of the complete 238H VDJ-Cµ
heavy chain DNA construct. Solid
boxes depict exons; EcoRI, R;
XhoI, X; N, Not 1; pBS, plasmid
Bluescript. The 3.0 kb NotI-EcoRI
restriction fragment containing
the rearranged VH-D-JH gene and
leader (L) sequences also contains
the Ig transcriptional control
elements (promoter, P, and Cµ
enhancer, E).
and plasmid insert sequences determined by the University of
Pennsylvania Core Sequencing Facility. Sequence analysis of
endogenous L chains was determined as described;17 cDNA was
PCR-amplified using a 3′ primer complementary to Cκ and three
5′ primers designed to complement disparate Vκ families.
Production of transfectants expressing the complete
238H-Cµ chain
Heavy chain-loss variant myeloma J558L cells (L-chain-only) were
cotransfected by electroporation using a Gene Pulsar apparatus
(BioRad, Hercules, CA, USA) in Ham’s F12 nutrient medium
(Life Technologies) in the presence of a 10:1 molar ratio of the Not
I-linearized 12 kb 238H VDJ-Cµ H chain construct and the mammalian expression vector, pGEM7(KJ1)-neo,18 containing as a
selectable marker aminoglycoside phosphotransferase. Transfected
cells were selected in medium containing 400 µg/mL of the aminoglycoside G-418 (Life Technologies), screened for secreted IgM by
ELISA as described below and subcloned twice by limiting dilution.
Transfectant 238H/238L was similarly derived by cotransfection of
the 238L L chain-only cell line, using the Lipofectin reagent (Life
Technologies) following the manufacturer’s directions.
Cell lines
The origins of cell lines have been described previously: The
BALB/c-derived J558L line is a light chain-only derivative of an
antidextran myeloma;19 238L is an L chain-only derivative of the
MAb 238 hybridoma;13 MRL-lpr/lpr-derived antilaminin hybridoma
H50;20 LamH-Cµ transfectant E2313 and LamH-Cµ transgenederived antilaminin hybridoma A10C.15
Animals
Balb/c, C57BL/6 and CB6F1/J [(Balb/cXC57BL6)F1] mice were
obtained from The Jackson Laboratory (Bar Harbor, ME, USA).
Transgenic mice bearing the 238H-Cµ IgM H chain construct were
produced by standard techniques in the University of Pennsylvania
Expression of a nephritogenic Ig heavy chain
Transgenic Core Facility as previously described for the LamH-Cµ
construct.15 Offspring were genotyped at 2–3 weeks of age initially
by Southern blot and later by PCR analysis of tail DNA and three
founders identified. Initial breeders were established on the C57BL/6
(hereafter, B6) background obtained from The Jackson Laboratory.
Mice were reared under conventional conditions in the University of
Pennsylvania animal facilities. The care and use of all experimental
animals was in accordance with institutional guidelines.
Immunoglobulin purification and binding specificity
Immunoglobulin M was purified from hybridoma or transfectant
culture supernatants by ammonium sulfate precipitation followed by
extensive dialysis against PBS. Purified Ig were stored before use at
–20°C. Selected IgM were concentrated in an ultrafree-4 centrifugal
filter unit (Millipore Corporation, Bedford, MA, USA). Immunoglobulin G were purified from supernatants by elution from a Protein
G-sepharose column and concentrated in a Centriprep-30 ultrafiltration unit (Amicon, Beverly, MA, USA). Anti-DNA and antilaminin
Ig activities were determined by direct binding ELISA as previously
described,20 with the following modification: 3% BSA was used as a
blocking agent for ssDNA coated plates. Goat antimouse isotypespecific reagents were purchased from Boehringer Mannheim (Indianapolis, IN, USA). Co-expression of the H and L chains in a single
antibody molecule in transfectant supernatant was determined by a
capture ELISA, as previously described.13 Briefly, Immulon II plates
(Dynatech Laboratories, Alexandria, VA, USA) were coated with
goat antiserum specific for mouse IgM+G or IgM only H chains
(Boehringer Mannheim) diluted 1/2000 in 0.05 mol/L sodium
borate, pH 8.6 at 4°C overnight. After blocking with 3% BSA/PBS,
culture supernatants or purified Ig diluted in 0.1% BSA/PBS were
applied for 1 h at room temperature. Bound Ig was detected with
alkaline phosphatase-conjugated goat antisera specific for mouse
IgM+G H chains or kappa or lambda light chain (Southern Biotechnology Associates, Birmingham, AL, USA) diluted 1/2000 in 0.1%
BSA/PBS. Reactions were monitored at 405 nm. For assays of the
inhibition of antigen binding by competitive ELISA, the MAb dilution that gave 50% maximal binding to ssDNA was determined by
direct-binding ELISA; this dilution was then preincubated with
varying concentrations of inhibitor (soluble ssDNA) for 1 h at 37°C
before incubation in ssDNA-coated wells. Assays were then developed as described and percentage binding calculated as (OD405 with
inhibitor/OD405 without inhibitor) × 100.
In vivo evaluation of Ig
Ten days after i.p. priming with 1.0 mL pristane (2,6,10,14tetramethylpentadecane), 5 × 105 transfectant cells were injected into
the peritoneum of 6–8-week-old histocompatible normal Balb/c
mice. One kidney from each animal was analysed for IgM deposits
using a Zeiss fluorescence microscope as previously described.13
Kidneys from mice injected with non-transfected J558L myeloma
cells or the LamH-Cµ-transgene-derived antilaminin A10C IgM
hybridoma15 were used as negative and positive controls, respectively, for the staining procedure.
Flow cytometry
Splenocytes were separated from lymphoid tissue by gentle
maceration between the frosted edges of sterile microscope slides in
Dulbecco’s modified Eagle’s medium, depleted of RBC using trisammonium chloride, counted, volumes adjusted and subsequently
maintained on ice for all steps. Cells (106) were incubated with the
appropriate concentrations of biotinylated or fluoresceinated Abs
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in FACS buffer (0.5% BSA in 1X Hank’s balanced salt solution, Life
Technologies), incubated 20–30 min, and washed twice with FACS
buffer. Binding of biotinylated reagents was revealed with phycoerythrin (PE) or with FITC-streptavidin (Pharmingen, San Diego,
CA, USA). Stained cells were studied immediately or fixed in 1%
paraformaldehyde/PBS and stored at 4°C. Flow cytometric analysis
was performed with a FACScan (Becton Dickinson, University of
Pennsylvania Cancer Center). List mode data were collected with
live gating on small lymphocytes (by light scatter) on 10 000–20 000
cellular events and were analysed with CELLQuest software (Becton
Dickinson).
Antibodies
Reagents for flow cytometry (FITC-conjugated anti-IgM-a, PEconjugated anti-B220, biotin-conjugated anti-IgM-a and avidinFITC) were obtained from Pharmingen (San Diego, CA, USA).
MOPC 104E (IgM,λ control) was obtained from Sigma (St Louis,
MO, USA). Monoclonal antibodies 66 and 54 are antilaminin IgM
derived from a LamH-Cµ transgenic mouse.17
Monoclonal antibodies from lipopolysaccharidestimulated splenic cells
Hybridomas were prepared by fusion of LPS-stimulated spleen cells
from transgenic mice as previously described15 using the Sp2/mIL-6
myeloma (ATCC, Rockville, MD, USA) or NSObcl-2, a fusion
partner genetically altered to constitutively express bcl-221 and
generously provided by Dr Betty Diamond (Albert Einstein College
of Medicine, NY, USA). Culture supernatants were initially screened
for secreted transgenic IgM by ELISA using both isotype and
a-allotype-specific reagents; selected clones were subcloned twice
by limiting dilution.
Results
Production and expression of an IgM construct expressing
the nephritogenic 238H Ig heavy chain
We devised a method to efficiently generate the novel H
chain construct by ligating PCR-amplified cDNA encoding
the H chain V region of Ig 238 to previously cloned genomic
fragments containing Ig transcriptional control elements
(promoter and Cµ enhancer) (Fig. 1a). This was made possible by the introduction via site-directed mutagenesis of a rare
Sfu I restriction site (TTCGAA) in the leader sequences to
facilitate ligation of 5′ regulatory sequences while preserving
the integrity of the amino acid sequence (phenylalanineglutamic acid) at the critical leader-VH junction. 3′-flanking
DNA containing a compatible Sau I restriction site was
amenable to partial digestion. Correct identity and orientation of the complete construct (Fig. 1b) were confirmed by
restriction and partial sequence analyses (data not shown).
To confirm expression of the intact 12 kb construct, we
examined both in vitro secretion and in vivo B-cell surface
expression. The J558L H chain-loss variant myeloma cell line
that produces a J558L (Vλ1,Jλ1) L chain was transfected
with linearized IgM 238H-Cµ DNA construct. Secretion of
intact IgM, termed BGC, requires pairing of 238H-Cµ with
lambda L chains. This was confirmed by capture ELISA
using isotype-specific antisera to demonstrate both the presence and physical association of H and L chains in transfectant culture supernatants (Fig. 2a).
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BG Cooperstone et al.
Figure 2 (a) Capture ELISA demonstrating physical association of H and L chains of secreted transfectant Ig. Supernatants
from the L-chain-only variant cell line before (J558L) and after
(BGC) transfection with the 238H VDJ-Cµ heavy chain DNA
construct were assayed for the presence of Ig chains. Antisera that
recognize specific Ig isotypes were used to coat the assay plate
(C, capture) or to detect bound Ig (D, detection), as indicated:
M = IgM (µ) H chain; M + G = both M + G heavy chains. The
mean values of triplicate determinations are shown. Control purified IgG, kappa (H241) at 10 µg/mL is indicated. (b,c) In vitro
autoantigen binding of (b) transfectant BGC and controls
(10 µg/mL), and (c) transgenic mAb and control anti-DNA Ig
H241 and antilaminin mAb 54 (5 µg/mL). Antigen-coated
microplates were incubated with concentration-matched mAb, as
described in the Materials and Methods section. Results are
expressed as mean OD405 × 103 on antigen coated wells minus
mean OD405 × 103 on diluent-coated plate (SHAM)-wells based
on duplicate samples. Values for Ig sampled at 10 µg/mL on
SHAM wells were: 98 (H241), 36 (Ab 66), 655 (H50), 33
(MOPC), and 62 (E23), 59 (BGC on ssDNA) and 67 (BGC on
laminin); values for all transgenic mAb on SHAM wells were
<0.012. Controls are H50 (antilaminin IgG), Ab 66 and 54 (antilaminin IgM), H241 (anti-DNA IgG), MOPC (MOPC 104E,
IgM,λ) and E23 (nonautoreactive LamH-Cµ IgM,λ transfectant).
(d) Inhibition of mAb binding to ssDNA by soluble ssDNA. The
dilution of mAb that gave 50% of maximal binding to ssDNA was
preincubated with varying concentrations of ssDNA before
incubation with ssDNA-coated wells. Results for parental IgM
238 (238,), transfectant 238H/238L (Tx238H/L,) and H241
() IgG are shown.
Because exclusively kappa L chains are reported among
the anti-DNA antibodies that use the VHB/W16 gene, we
performed binding studies with BGC to determine if the
V region determinants generated by the 238H VDJ gene
contributed to autoantigen binding and/or pathogenicity
independent of a kappa L chain (Fig. 2b). The results indicate
that the novel BGC IgM (238H/J558L) has minimal to no
activity against laminin or ssDNA (Fig. 2b). The novel BGC
Ig also failed to form significant renal deposits or proteinuria
when injected intraperitoneally as hybridoma cells into histocompatible normal mice (data not shown). However, BGCrecipient mice did achieve elevated serum levels of lambdaencoded Ig (mean 2.4 µg/mL), compared to mice injected
with non-transfected J558L cells (0.72 µg/mL), indicating
in vivo production of BGC. Mice injected with A10C, a
LamH-Cµ transgene-derived antilaminin monoclonal IgM,κ,
had no detectable serum lambda-containing Ig.
To determine if the 238H-Cµ H chain is expressed and
pairs with endogenous L chains in vivo within a nonautoimmune background, we examined its expression on
splenic B cells from C57BL/6 mice rendered transgenic for
238H-Cµ. Injection of the 238H-Cµ H chain construct into
fertilized eggs yielded three founders that carried and
expressed the transgene, as determined by Southern blot and
serologic analysis. Flow cytometric analysis of early generation progeny derived from backcross of these founders with
Expression of a nephritogenic Ig heavy chain
227
soluble ssDNA (Fig. 2d). Thus lack of autoreactivity in the
transgenic mAb is dependent on the associated L chain.
Analysis of the endogenous L chains expressed by these
four mAb shows the use of three different Vκ families and
three Jκ genes (Fig. 4a). Comparison to sequences in the
nucleic acid databases indicates that the L chains used by
these transgenic IgM are minimally mutated. Monoclonal
antibody 139.1 expresses a Vκ gene identical in sequence to
IgVκ aq4 (GenBank accession AJ231222), a germline Vκ
gene cloned from C57BL/6 liver cosmid library.26 Monoclonal antibodies 142.3 and 150.4, derived from different
transgenic mice, use different genes of the V14 family
(IMGT designation; VK9B by conventional nomenclature).23
Monoclonal antibody 142.3 shares 99% nucleotide identity
with the B6 germline IgVκ ba9 gene.26 The MAb 150.4 Vκ
sequence is identical, with the exception of residue 290 at the
Vκ-Jκ junction, to that of several previously reported IgM
rearranged Vκ genes (not shown), and therefore likely represents unmutated germline sequence. Monoclonal antibody
20.6 has 98% nucleotide identity to several rearranged V5
family genes; all differences result from ambiguous residues
in the published sequences.
Figure 3 Flow cytometric analysis of splenic B cells. Spleen
cells were stained with FITC-anti-IgM-a and phycoerythrin
(PE)-anti-B220 and analysed by FACS as described in the
Materials and Methods section. Shown are representative dot
plots of log fluorescence data for two-colour staining of unstimulated spleen cells from 238H-Cµ transgenic and non-transgenic
littermates gated on small lymphocytes on the basis of forward
and side-scatter. The percentage of positively staining cells in
each quadrant is indicated.
C57BL/6 breeders showed that the bulk of splenic B cells
(B220+) from transgenic mice express transgene-encoded
IgM-a on their surface (Fig. 3). Non-transgenic littermates
express only the endogenous IgM b-allotype (not shown).
Attempts to recover transgene-encoded Ig met with
limited success, despite the abundance of B cells expressing
surface transgenic receptors. Few mAb were recovered
despite fusion by standard techniques with LPS-stimulated
splenocytes and using robust myeloma fusion partners,
suggesting that these cells are refractory to recovery by this
approach. Eight clones producing transgene a-allotype IgM
were initially recovered from five 238-Cµ transgenic mice
derived from two different founders. None of the eight clones
bound ssDNA on initial screening; absence of DNA or
laminin binding reactivity was confirmed for the four mAb
subsequently recovered by subcloning (Fig. 2c). Sequence
analysis of H chain V region cDNA from two transgenic mAb
confirmed its origin from the transgene (not shown). To
confirm that the 238H-Cµ construct could encode autoreactivity when recombined with an appropriate L chain,
this construct was used to transfect an H-chain-loss variant
cell line carrying the original 238L chain. The newly generated 238H/238L transfectant IgM yielded high OD in direct
binding ELISA to ssDNA; however, because diluent-coated
plate (SHAM) binding was also significant, and similar to
promiscuous binding observed with the parent 238 IgM,
specificity for ssDNA was confirmed by inhibition with
Discussion
We used transfectant and transgenic approaches to determine
that autoreactivity and pathogenicity attributed to the VHB/W16
Ig H chain determinant are dependent on its recombination
with permissive L chains in concert with a permissive H
chain CDR3. The approach depended on successful generation of an IgM construct termed 238-Cµ containing the
VHB/W16 gene, which is expressed recurrently among nephritisinducing murine lupus autoantibodies reactive with DNA and
glomerular antigens. The absence of DNA reactivity in the
BGC transfectant and among recovered transgenic mAb
expressing 238-Cµ in combination with endogenous kappa
chains indicates that neither expression of the VHB/W16 gene
alone nor the entire V region of the nephritogenic Ig 238
independently dictates specificity for nucleic acid. The
current findings reveal that multiple L chains can abrogate
DNA binding and at least in some cases block the capacity of
238-Cµ to induce renal lesions. As transfectant BGC lacks
both DNA reactivity and nephritogenicity, these experiments
do not necessarily resolve the question of whether DNA
specificity is requisite for immune deposition by Ig expressing this H chain. Because the recipient mice achieve relatively low serum levels of BGC, measured as lambdaencoded Ig, we cannot rule out the possibility that pathogenicity of BGC is dose-dependent. Nonetheless, the lack of
immune deposition in the face of quantifiable circulating
levels indicates a lack of avid nephrotropism by either BGC
or BGC-containing immune complexes, as striking renal
deposits can be observed even in the absence of detectable
serum activity.13
It is notable that there is no overlap in the Vκ genes used
by the five non-DNA binding recombinant Ig (BGC and four
transgenic mAb) reported here and those used by the five
previously reported VHB/W16-encoded nephritogenic anti-DNA
Ig8–10 or by 17 nephrotropic autoantibodies recovered from
mice transgenic for the LamH-Cµ H chain (Table 1; Fig. 4).17
Like the anti-DNA 238H V region, the antilaminin LamH V
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BG Cooperstone et al.
Figure 4 (a) Predicted light chain amino acid sequence and V gene segment use by four 238H-Cµ transgenic mAb. L chains were
sequenced by PCR and assigned to Vκ gene families based upon homology to published sequences22 and using the nomenclature of
the IMGT, the International ImMunoGeneTics database.23 Jκ genes are assigned to known BALB/c Jκ germline gene segments.24 Dots
have been introduced to maximize homology; CDR, complementarity determining region; FR, framework region.25 The 10 5′-proximal
residues are omitted due to their origin from degenerate 30-base oligonucleotide primers used for PCR amplification. The nucleotide
sequences from which these translated sequences were derived are available from GenBank under accession numbers AF294224AF294227. (b) Comparison of the L chain of 238H-Cµ transgenic mAb 139.1 with L chains of three antilaminin LamH-Cµ transgenic
mAb (A10C, A9D and 79) and the L chain of the MRL-lpr-derived parental anti-DNA mAb 238, all of which use the V4 (VK4/5), Jk5
combination. Asterisks indicate amino acid differences between mAb 139.1 and reference sequences (238 or the consensus Vκ sequence
based on the LamH-Cµ transgenic mAb A10C, A9D and 79).
region was derived from an autoantibody that arose spontaneously in a diseased MRL/lpr lupus mouse.11 LamH uses a
J558 VH gene homologous to VHB/W16 and is expressed by
anti-dsDNA Ig derived from autoimmune mice.20 Many of the
LamH-Cµ transgenic Ig selected for laminin binding also
cross-react with ssDNA.17 Notably, two MRL/lpr-derived
anti-DNA Ab expressing the homologous VHB/W16 gene were
shown to cross-react with murine laminin.12,27 Thus, both the
VHB/W16 and LamH V region gene can encode antilaminin and
anti-DNA reactivity. The considerable overlap in Vκ gene
families expressed by these two groups of autoantibodies,
that is, LamH-encoded antilaminin and VHB/W16-encoded antiDNA Ig (Table 1), may reflect selection for VH-VL gene
combinations preferentially expressed among cross-reactive
lupus autoantibodies. This is consistent with the notion that
germline Ig V region CDR1 and CDR2 residues permit considerable antigenic crossreactivity.28 Other VH-VL combinations, such as the VHB/W16–V5 and VHB/W16–V14 combinations
used by the non-autoreactive transgenic mAb 20.6, 142.3 and
150.4 described here, may encode V region conformations
that in the unmutated configuration are unable to engage
DNA and laminin despite the presence of a permissive H
chain CDR3.
Whereas 238H-Cµ transgenic mAb 20.6, 142.3 and 150.4
use Vκ gene families not found among the nephritogenic
or nephrotropic Ig, transgenic mAb 139.1 uses a Vκ-Jκ
combination (V4-Jk5) that is used both by the parental
MRL/lpr-derived anti-DNA Ig 238 and by three nephrotropic
antilaminin LamH-Cµ mAb (Fig. 4b). The mAb 139.1 Vκ is
identical to the IgVκ aq4 germline gene of the V4 family.
Anti-DNA Ig 238, donor of the V region for the 238H-Cµ
transgene, rearranges a distinct V4 gene, the translated
sequence of which differs from that of 139.1 by 16 amino
acid residues. It is notable that the 238L chain contains arginine and asparagine residues in its CDR1 region; these two
amino acids are predicted to be particularly important for
protein binding to DNA,29 and may particularly enhance
DNA binding when clustered within CDR1 of L chains
paired with permissive H chains.30 These L chain residues, as
well as a lysine contained within the 238 CDR1, are absent
in CDR1 of mAb 139.1 and may explain the difference in
DNA binding by these two Ig. The V4 gene used by mAb
139.1 is also distinct from the V4 gene coexpressed with the
LamH-Cµ transgenic H chain among DNA-cross-reactive
antilaminin Ig17 (Fig. 4b). The mAb 139.1 L chain differs by
seven amino acids in the CDR1-FR1-CDR2 region from the
Expression of a nephritogenic Ig heavy chain
Table 1
Summary of light chain use by VHB/W16-encoded nephritogenic anti-DNA and by LamH-Cµ and 238H-Cµ transgenic mAb
V kappa family*
V1 (1)
V1 (2)
V3 (21)
V4 (4/5)
V5 (Vκ23)
V8 (8)
V10 (10)
V12 (12/13)
VHB/W16-encoded
nephritogenic anti-DNA
mAb
Jκ
H161
5
H241
Ab52
238
4
1
5
H8
2
LamH-Cµ-encoded
antilaminin Ig†
mAb
Jκ
61
15
123
5
5
2
A10C
A9D
79
5
1
5
68
129
87
131
B8E
1
2
2
1
2
V14 (Vκ9B)
V16 (RF)
V17 (20)
229
54
66
A10D
238H-Cµ-encoded
non-autoreactive Ig
mAb
Jκ
139.1
5
20.6
5
142.3
150.4
1
4
2
2
2
*L chains were assigned to Vκ gene families using the nomenclature of the IMGT, the International ImMunoGeneTics database23 followed
by common use nomenclature in parentheses. †Vκ use by 14 antilaminin mAb derived from LamH-Cµ transgenic mice is shown. Three
additional transgenic Ig with L chain sequences 100% identical to those shown are not included; see Fitzsimons et al. for details.17
consensus V4 sequence used by the cross-reactive Ig. Collectively, the lack of overlap in Vκ gene use by autoreactive
versus non-autoreactive Ig suggests that a restricted subset of
nascent L chains are suited to fold with either 238H-Cµ or
LamH-Cµ to form binding pockets that engage exposed
epitopes of the relevant autoantigen.
Nonetheless, previous work clearly indicates that the
VHB/W16 gene can rearrange with different DH and JH elements and combine with unrelated kappa L chains to specify
activity against DNA. Thus our inability to recover 238H-Cµ
transgenic mAb with DNA reactivity is somewhat striking.
Assuming that Ig gene and chain recombinatorial events are
random during B-cell development in the bone marrow, this
discussion suggests that substantial populations of both DNA
binding and non-DNA binding 238-Cµ transgene-encoded
B cells will be produced. Numerous observations in other
model systems indicate that their subsequent fate and ability
to exit the bone marrow depends on their reactivity with
self-antigen, which is determined in part by antigen structure, accessibility, concentration and binding avidity.31,32.
Our flow cytometric studies indicate that a large population
of 238-Cµ transgenic B cells developing in the nonautoimmune B6 background escape deletion centrally and
appear in peripheral lymphoid tissues. The absence of DNA
reactivity among mAb recovered from this population thus
invites speculation that 238-Cµ transgenic anti-DNA B cells
are refractory to mitogenic stimulation and/or recovery by
fusion, possibly due to antigen-induced anergy or deletion
in vivo. Both fates have been described for anti-DNA B cells,
with high avidity anti-dsDNA binding being most likely
to trigger deletion.33 The cross-reactive nature of previously
characterized VHB/W16-encoded anti-DNA Ig also invites
speculation that other basement membrane or kidney glomerular antigens or idiotypic interactions may determine
their fate in vivo. Future investigations of 238H-C Cµ transgenic mice will hopefully resolve these questions.
Acknowledgements
BGC was the recipient of a Kidney Foundation of Canada
Fellowship Award. This work was supported in part by the
National Institute of Health Grant DK47424. The research
protocol was approved by the Institutional Animal Care and
Use Committee of the University of Pennsylvania.
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