ELSEVIER Journal of Neuroimmunology 51 (1994) 45-52
Journal of Neuroimmunology
Somatically mutated member of the human VAVII I gene family encodes anti-myelin-associated glycoprotein (MAG) activity
G r a c e L e e ,,a R a n d l e R. W a r e h N o r m a n L a t o v a
a Department of Neurology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA b Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA
(Received 24 August 1993; revision received and accepted 10 December 1993)
Abstract
A highly conserved small family of human Va genes was identified by DNA homology to a Va gene isolated from a patient with demyelinating peripheral neuropathy, and which encodes an autoantibody with anti-MAG activity. Comparison of the genes indicates that the patient Vx gene was derived from one of the germline genes. Together with published analyses of other anti-MAG IgM antibodies, which also appear to be mutated in comparison to known germline V genes, these results suggest that development of these pathogenic antibodies may reflect an antigen-driven, T cell-dependent process.
Key words: Myelin-associated glycoprotein; Human lambda light chain; Immunoglobulin variable region gene; Somatic mutation; Autoantibody; Peripheral neuropathy
I. Introduction
High titers of auto-antibodies which recognize a glucuronyl sulfate epitope shared by myelin-associated glycoprotein (MAG), the Po glycoprotein and two gly- cosphingolipids in peripheral nerve occur in some pa- tients with demyelinating peripheral neuropathy (re- viewed in Latov et al., 1988; Tatum, 1993). These antibodies, which often result from monoclonal gam- mopathies, are typically IgMs and, similar to a number of other autoreactive antibodies, are produced by CD5 ÷ B cells (Hayakawa and Hardy, 1988; Lee et al., 1991). The significance of the antibodies as a mediator of neuropathy is indicated by the ability of passively trans- ferred antibodies to reproduce the specific pathologies in experimental animals, and clinical improvement in patients following reduction in antibody concentrations (Hays et al., 1987; Willison et al., 1988; Tatum, 1993).
Analysis of the variable gene (V) regions of anti- MAG antibodies show that although members of the large Vnm family are preferentially used, the set of Vr~ and V L genes encoding the IgMs is diverse, and corn-
parison with known germline V genes suggests that the anti-MAG antibodies are hyperrnutated (Mihaesco et al., 1989, Ayadi et al., 1992; Spatz et al., 1992a). This is in contrast to cold agglutinins and IgM anti-DNA antibodies which frequently derive from a restricted number of V genes, and to most IgM rheumatoid factors which use diverse V genes but show little or no somatic mutation (Dersimonian et al., 1987, 1989; Pas- cual et al., 1992, 1993; Randen et al., 1992). The extent of somatic mutation has bearing on the question of whether an autoreactive antibody may have arisen from polyclonal B cell activation or an antigen-driven, T cell-dependent immune response during which somatic mutation contributes to evolution of antibodies with higher affinities and specificities (Griffiths et al., 1984; Kocks and Rajewsky, 1989; Berek, 1992; Zoudi, 1992). However, since the possibility exists that not all germline V genes have been identified, the degree of somatic mutation, if any, in a given antibody can be difficult to assess.
The variable region of a A light chain (ARMVL) associated with an IgM anti-MAG was identified (Spatz et al., 1992a) which had less than 72% DNA sequence homology to any of the seven previously known germline V A gene families (Kabat et al., 1987; Chuchana et al., 1990) and, as such, may be heavily mutated or
46 G. Lee et al. /Journal at' Neuroimmunology 51 (1994) 45-52
derive from an as yet undefined V gene subgroup. To address the question of whether this anti-MAG speci- ficity is germline-encoded or arose from somatic muta- tion, we sought the germline counterpart of the V A and present evidence that it is a mutated form of a new V~ gene which has been reported (Winkler et al., 1992) and categorized as subgroup VIII since the initiation of this study. In addition, we identified a germline variant of this gene as well as a new member of V~v m by DNA homology to ARMVL.
2. Materials and methods Fig. 1. Primers used to sequence both strands of A R M Va-related genes.
2.1. Cells
The hybridoma, A R M / B C l l , which secretes an IgM,h anti-MAG antibody was produced by fusion of patient ARM B cells with the human cell line UC729-6 as previously described (Spatz et al., 1987). An ex- panded pool of purified T cells from the same patient was obtained as described by Kumagai et al. (1987). Briefly, PBL enriched for T cells by centrifugation through Ficoll-Hypaque and E-rosetting were stimu- lated for 6 h at 37°C in DMEM medium (supple- mented with 10% human AB serum, glutamine, and penicillin-streptomycin) with 10 ng/ml phorbol dibu- tyrate (Sigma, St. Louis, MO) and 0.4 gM ionomycin (Sigma). Washed cells were resuspended and cultured in medium containing 50 U/ml of IL-2 (Proleukin, EuroCetus, Germany). Cells were harvested for DNA extraction after 6 days incubation. B cells were not detectable by fluorescence-activated flow cytometry (FACS) after cell surface labeling with fluorescein isothiocyanate (FITC)-conjugated anti-Leu 16 (Becton- Dickinson, San Jose, CA) and > 98% of the cells were CD2 + T cells as determined by labeling with FITC- anti-CD2 antibody (Becton-Dickinson).
The human cell lines LAN5 (neuroblastoma; Mena et al., 1989), UC729-6 (B lymphoblast; Glassy et al., 1983), HeLa (epithelial-like; Gey et al., 1952) or mouse liver were used as controls in some experiments.
2.2. DNA extraction and Southern blot analysis
DNA isolation, restriction enzyme digestion, gel electrophoresis, blotting, and hybridization procedures were performed according to standard protocols (Sambrook et al., 1989). Hybridizations were done at moderate stringency (80% homology) in 50% for- mamide, 5 × SSCPE (0.6 M NaC1, 75 mM Na citrate, 50 mM KH2PO4, 5 mM EDTA, pH 7.2), 5 x Denhart's, 0.5 mg/ml salmon sperm DNA, 0.1% SDS at 42°C for 12-18 h, followed by 3-4 washes at low stringency in 2 × SSC (0.3 M NaCI, 30 mM Na citrate), 0.1% SDS at 68°C. Additional DNA samples from lung or kidney
tissue of various individuals were donated by Dr. B. Tycko (Columbia University).
2.3. Probes and primers
The VJCh2 of patient ARM was previously sub- cloned from a cDNA library (Spatz et al., 1992a) and a 220-bp probe (ARM Va) spanning CDR1-CDR3 was obtained by PCR amplification of this region using the 5' primer 5'-TTGACGTCTGGCTCAGTCTC-3' and 3' primer 5'-CTTCCCACAAACAGCACACA-3'. Addi- tional primers used for DNA sequencing of ARM Va-related genes are shown in Fig. 1. A Ca probe which contains a 3.5-kb EcoRI/HindIII fragment from germline CA2 was provided by Dr. P. Leder (Harvard University).
2.4. Genomic DNA libraries
A library of partially digested human fibroblast ge- nomic DNA fragments cloned into A-FIX II was ob- tained from Stratagene (LaJolla, CA). To construct a genomic DNA library from patient ARM T cells or ARM B cell hybridoma, EcoRI-digested DNA frag- ments of 3-10 kb were agarose gel-purified and ligated into dephosphorylated, EcoRI-digested A-ZAP II arms (Stratagene). The libraries were screened, under the conditions described for Southern blots, with the ARM V A probe labeled with [a-32P]dATP by random hex- amer priming (Boehringer Mannheim kit, Indianapolis, IN), and positive plaques were purified. Relevent in- serts in h-FIX II were removed by EcoRI digestion and subcloned into pBluescript phagemid, while those from the A-ZAP library were excised with helper phage and recircularized following Stratagene protocols to generate subclones in pBluescript.
2.5. PCR and DNA sequencing
Genomic (1 /.Lg) or plasmid (1 ng) DNA was ampli- fied by PCR in a Perkin Elmer DNA thermal cycler
G. Lee et aL /Journal of Neuroimmunology 51 (1994) 45-52 47
with 20 pm each of the CDR1-3 primer pair, 400/~M each dNTP, 0.5 /.d VENT polymerase (New England Bio-Labs, Beverly, MA) in 100/zl buffer (10 mM KCI, 20 mM Tris. HCI, pH 8.8, 10 mM ( N H 4 ) 2 S O 4 , 2 mM M g S O 4 , 0.1% Triton X-100, 100 /zg/ml BSA). Nor- mally, 30-40 cycles of denaturation (94°C, 1 rain), annealing (50°C, 1 rain), and extension (72°C, 1 rain) were performed with a final extension step at 72°C for 15 rain. The PCR products were electrophoresed on 2% ultra-pure agarose gels and bands were electro- eluted, phenol- and ether-extracted, and ethanol-pre- cipitated. Kinased genomic DNA PCR fragments were blunt-end ligated into dephosphorylated EcoRV-di- gested pBluescript vector. XL-1 colonies transformed with the ligated products were screened with the 32p. labeled ARM V A probe.
All plasmids containing inserts of interest were ex- tracted and purified on Qiagen columns (Qiagen, Inc., Studio City, CA) for DNA sequencing using the T3, T7
or internal primers in an automated Applied Biosys- terns 370A DNA sequencer.
3. Results
To assess the heterogeneity of ARM Vx-related genes, DNA from various human cell lines, lung or kidney tissue from different individuals, and the A-pro- ducing ARM hybridoma (BCll) were digested with restriction enzymes and hybridized, after Southern blotting, to the 32p-labeled ARM V a probe at moder- ate stringency to minimize binding to distantly related genes. Following washes at low stringency, two bands of approximately 4 and 8 kb in size were evident in all DNA samples cut with EcoRI (Fig. 2A). Digestion with BamHI (Fig. 2A) and combinations of enzymes shown in Fig. 2B yielded 2-3 bands that were common to all samples. The results suggest that the ARM V~ is
A EcoRI BamHI
I
9.4kb- ~ . . . . . . . . 6.6- 4 . 4 -
B
9.4kb- 6.6-
4.4-
EcoRI EcoRI EcoRl EcoRl EcoRl EcnRI BamHI P~t I Hin¢l l Pvu I I H i n d l l l
I I I t !
T B UC T B UC T B UC T B UC T B T B
2 . 3 -
2.0 - ~
1.35- i ~ " 1 . 0 8 -
0.87 -
0.60-
0.31 -
C
Aat l l / A p a I
LAN5
23.1kb- 4.4-
2.3- 2.0-
UC T B
587 bp- !ii i
184 . . . .
124
Fig. 2. Hybridization of genomic DNA to ARM V~ probe. Southern blots of genomic DNA (10/zg (A,B) or 15/~g (C) per sample) digested with the restriction enzymes shown were hybridized with the ARM "CA probe. (A). ML, mouse liver; UC, human lymphoblastoid cell line UC 729-6 (fusion partner in ARM B cell hybridoma); LAN5, human neuroblastoma cell line; remaining lanes, kidney or lung samples from unrelated individuals. (B) Lane T, T cells from patient ARM; B, ARM B cell hybridoma (BCll); UC, hybridoma fusion partner. The band marked with an asterisk in lane B of each set of digests corresponds to the functionally rearranged ARM A gene. (C) Lanes LAN5, UC, T and B are as described for (A) and (B). A unique 164-bp band in lane B is generated by an AatlI site and ApaI site in the CDR1 and FR3 regions, respectively, of ARM V a.
48 G. Lee et al. /Journal of Neuroimmunolo~' 51 (1994) 45-52
derived from a very small gene family with little or no polymorphism.
In addition to the c o m m o n bands, a unique frag- ment (marked with an asterisk) is evident in all digests of A R M B cell hybridoma D N A . This band also hy- bridized to the 32p-labeled CA2 probe, indicating that it contains the functionally rearranged A R M A-light chain gene (data not shown).
A A-FIX II library of partially digested genomic D N A from a human fibroblast cell line was screened with the A R M Va probe to isolate it's germline equiva- lent. A m o n g 600000 pfu screened, 10 positive clones were identified and each contained an EcoRI frag- ment of either 4 kb (2 clones) or 8 kb (8 clones) which hybridized to A R M V A. The positive EcoRI band from each phage clone was isolated and subcloned into pBluescript. Restriction enzyme mapping showed that the subclones (designated F L I - 1 0 ) within the 4-kb or 8-kb groups were identical or very similar to each other
(data not shown). Comparison of the A R M V A c D N A sequence (Spatz et al., 1992a) with those obtained from small PstI or Sac I fragments from one representative of each group, FL6 (8 kb) and FL7 (4 kb), subcloned into M 1 3 m p l 8 or M 1 3 m p l 9 showed the three Va genes to be highly homologous , but not identical, Primers were constructed to conserved areas in both strands of the leader, framework 2 (FR2) and framework 3 (FR3) to obtain complete sequences of the FL6 and FL7 genes, including the 5' and 3' flanking regions (Fig. 3). A m o n g the other eight EcoRI subclones sequenced, FL3 was identical to FL7, and the remainder identical to FL6 (data not shown).
FL7 is identical to VL8, a recently reported germline Vz gene constituting subgroup VIII (Winkler et al., 1992) except for an extra C immediately before the recombination heptamer in our sequence. FL6 and FL7 share 91% homology over the 353-bp coding re- gion and have very similar leader intron and recombi-
Fig. 3. Comparison of ARM V~ with the most homologous germline and cDNA genes. Dots indicate gaps in sequences to facilitate alignment and dashes indicate identity to the FL7 or TL7 sequence. Nucleotides of coding regions are shown in capital letters and the leader peptide, framework (FR1-3) and complementarity determining regions (CDR1-3) are denoted. The heptamer and nonamer recombination signal sequences in the germline genes are underlined. FL6, FL7; TL6, TL7; and BL6, BLT, BL7T, ARM were isolated from genomic DNA libraries constructed from human fibroblasts, patient ARM T cells, and ARM BCll B cell hybridoma cells, respectively. The sequences of BL6 and BL7 (not shown) are identical to those of FL6, TL6 and FL7, TL7, respectively, over the entire region shown above. In addition, the 5' and 3' flanking regions of BL7 x are identical to those of FLT, TL7 and BL7 and are also not shown. Sequence data for TLT, TL6, BL7x, ARM, FL7 and FL6 are available from Genbank/EMBL/DDBJ under accession numbers UO3635-UO3640, respectively.
G. Lee et al. /Journal of Neuroirnrnunology 51 (1994) 45-52 49
nation/23-bp spacer sequences, indicating that FL6 is another member of this V gene subgroup (Fig. 3). These genes are < 75% homologous to the other known V~ families (EMBL/Genbank database; Kabat et al., 1987; Chuchana et al., 1990; Williams and Win- ter, 1993). The ARM V A cDNA is 87% and 95% homologous to the coding regions of FL6 and FL7, respectively, and a comparison of the sequences sug- gests that ARM V~ may have arisen from FL7 rather than FL6 by somatic mutation.
To explore the possibility that the differences be- tween ARM V~ and FL7 are not due to somatic muta- tion but to genetic polymorphism among individuals, a genomic DNA library was made from EcoRI-digested, autologous ARM T cells and screened for ARM V~-re- lated genes. Similarly, a library was constructed from the ARM BCll B cell hybridoma to obtain a full-length genomic ARM VA gene containing the leader intron and 5' flanking region for better comparisons.
A total of 500000 pfu from each library were screened with the ARM V~ probe. Three positive T cell-derived clones (2 with a 4-kb insert; 1 with an 8-kb insert) and eight positive B cell-derived clones (3, 4 and 1 clones with 3.5-kb, 4-kb and 8-kb inserts, respec- tively) were isolated and sequenced. The 4-kb and 8-kb EcoRI fragments from the T cells contain genes (shown as TL7 and TL6 in Fig. 3) identical to FL7 and FL6, respectively, over the regions sequenced. No V genes identical to ARM V~ were found in ARM T cells. In contrast, the 3.5-kb B cell-derived fragment contained the full length genomic rearranged ARM VJC~2 gene (ARM in Fig. 3).
The 8-kb clone (BL6) from the ARM hybridoma has the same sequence as FL6/TL6, while the four clones with 4-kb inserts (BL7 in Fig. 3) were identical to FL7 except for 1 (BL7T) which differed from BL7 by a single nucleotide in FR3 (Fig. 3), resulting in a substi- tution of cysteine for arginine in the predicted amino acid sequence. That this difference is not due to se- quencing or PCR artifact was confirmed by the detec- tion of this substitution in 1/5 and 2 /5 sequenced FL7-1ike clones generated by two independent PCR amplifications of ARM hybridoma DNA, and among similarly obtained clones from the hybridoma fusion partner and HeLa cells (data not shown). This variant was not found among 5 FL7-1ike PCR-derived clones from ARM T cells or 2 similar clones from LAN5 cells.
The genomic DNA sequence of ARM V A shows that, in addition to the high degree of homology in the coding regions, its leader intron is identical to that of TL7 except for a single nucleotide and the 5' flanking regions of the two genes share 100% homology for >300 bp (Fig. 3). The ARM V~ has 97% overall homology to TL7 over 744 bp. Excluding differences near the VJ junction which may have been generated during recombination, nine of 16 nucleotide changes
Leader ) ~RI I CDRI
FL6, TL6 --C ................. S ....... L- G .......... A ........ FL7,TL7 I~__W~__LLL~__L LA_Y_GS_.G_VDS_. Q T~_ TQE_pSF_S VS_PGGTVTLT C GLS_SGS_VS_T
BL7 T ARM ........................................... T ..... S
V--D--II ......... N FL7FL6 ,' TL6TL7 ADDESDYYC V LYMGSG I
BL7~ ................. ARM -E ....... I- -FV--- pPTWV/Jxz
Fig. 4. Comparison of the ARM V~ amino acid sequence with peptides predicted from germline V genes shown in Fig. 3. Dashes indicate homology to the FL7 or TL7 sequence shown.
were noted in the coding regions which results in seven amino acid substitutions. In particular, six of the seven replacements occur in the CDR regions (Fig. 4). We were unable to detect the ARM V A or BL7 T sequences in the ARM T cell or fibroblast genomic DNA li- braries, although both genes were readily isolated from ARM B cell hybridoma DNA. Furthermore, the re- placement of 'GC' in the third codon of the germline TL7 CDR1 by 'CG' in the ARM V~ sequence gener- ates an A a t l I restriction enzyme site which, together with a common ApaI site in FR3, would yield a 164-bp band upon double digestion with these enzymes. Fig. 2C shows that this unique band is present in genomic DNA digests of the ARM B cell hybridoma, but not in autologous ARM T cells or other control human cell lines. Together, these results strongly indicate that the ARM V A gene encoding anti-MAG activity arose from somatic mutation of the germline TL7 (VL8) gene.
4. Discussion
In this study, members of the recently defined VAVII I subgroup were revealed by homology to the gene en- coding the A light chain of an anti-MAG antibody from a patient with demyelinating peripheral neuropathy. Two germline genes, TL6 and TL7, located on EcoRI fragments of approx. 8 kb and 4 kb, respectively, were identified from autologous T cells. TL7 is equivalent to the single reported V~w n gene, VL8. TL6 and TL7 share 91% homology in the coding regions, have identi- cal heptomer/nonamer recombination recognition se- quences and very similar leader, leader intron and immediate 5' /3 ' flanking regions, are < 75% homolo- gous to the other known V~ gene subgroups and, thus, are considered to be related members of the same V~ family. These genes appear to be highly conserved as restriction fragment length polymorphism was not ob- served in Southern blots of DNA from different indi- viduals, and identical coding, intron and immediate flanking regions for each gene were sequenced from
50 G. Lee et al. / Journal of Neuroimmunology 51 (1994) 45-52
ARM cells and commercial fibroblasts. Preliminary data from partial sequencing of clones derived from the ARM hybridoma fusion partner, as well as HeLa and LAN5, suggests that identical forms of TL6 or TL7 are present in these cells as well. These findings are consistent with reports that some V genes, particularly members of smaller families, display little or no poly- morphism (Anderson et al., 1984; Chen et al., 1988, Sanz et ah, 1989; Logtenberg et ah, 1992).
A third germline gene, BL7T, located on a 4-kb EcoRI fragment and which differs from TL7 by a single nucleotide in FR3, was also isolated from the ARM B cell hybridoma. Gene duplication has been suggested to be a significant mechanism in the evolu- tion of immunoglobulin genes, given the high degree of homology among groups of these genes, and some germline sequences display only a few or no differ- ences (Chen et al., 1990; Rubenstein et al., 1993). Since we were able to detect BL7T in a genomic library or PCR clones constructed from the ARM hybridoma and the fusion partner but not from autologous ARM T cells, it is likely that BL7 T is an allelic form of TL7 present in the hybridoma fusion partner. However, we cannot exclude the possibility that our sample size was not large enough to detect this gene in the ARM T cells. A more detailed study of the relative distribution and chromosomal location is required to formally show whether these genes are indeed alleles or are present at different loci on the same chromosome and, in addition, if variations of TL6 exist.
For the following reasons we believe that ARM Va is probably not germline encoded but derive from TL7 by somatic mutation. (i) In addition to 95% coding region homology, their leader introns differ by only one nucleotide and the 5'flanking regions are identical over > 300 bp. (ii) The ARM V A sequence was readily detected and isolated from the ARM B cell hybridoma but not from autologous T cells or human fibroblasts, suggesting that it is not present in the germline. (iii) Furthermore, Southern blots of DNA from ARM T cells and other human cell lines do not show a unique 164-bp AatII/ApaI restriction enzyme band, predicted from the sequence of ARM Vx, which is detectable in digests of ARM B cell hybridoma DNA.
Excluding differences at the VJ junction which may have been generated by recombination rather than somatic mutation, six of seven amino acid replacements in ARM Vx relative to TL7 occur in the CDR regions. While hypervariability in the CDRs relative to the framework regions, even between members of the same V family, pre-exists in the germline (Givol et al., 1981; Loh et al., 1983; Blankenstein et al., 1984; Heinrich et al., 1984; Schiff et al., 1985), further accumulation of replacements in the CDRs is consistent with patterns of somatic mutations which arise as a consequence of antigenic selection (Griffiths et al., 1984, McKean et
al., 1984; Kocks and Rajewski, 1989). In addition, ARM V A utilizes JCA2 but differs from the germline Jx2 by one nucleotide which generates an amino acid substitu- tion (Spatz et al., 1992a). Although this difference may be due to genetic polymorphism among JCx2 genes (Vasicek et al., 1990), it may also have arisen through mutation.
Previous studies have shown that both heavy and light chains of anti-MAG antibodies contribute to anti- gen binding (Spatz et al., 1992b). In similar experi- ments in progress, comparison of the reactivity and binding affinities of the ARM anti-MAG heavy chain co-expressed with either the mutated ARM Va or TL7, the lambda germline counterpart, may demonstrate whether the mutations found on ARM V~ select for a light chain with higher affinity for MAG, and if anti- MAG activity can be germline-encoded. Interestingly, a recent study shows that the multivalent nature of both the MAG antigen and IgM antibody contributes significantly, perhaps even more so than mutations, to enhanced avidity since their monomeric forms display relatively low binding capacity for each other (Ogino et al., submitted).
The developmental biology of autoreactive B cells in patients with peripheral neuropathies and the mecha- nisms by which demyelinating antibodies are elicited remain unclear. IgM antibodies which result from B cell activation by T cell-independent antigens or poly- clonal activators do not isotype switch and bear little or no somatic mutation (Maizels and Bothwell, 1985; Berek, 1992). In contrast, anti-MAG IgMs are typically high affinity antibodies encoded by mutated V genes. Consequently, they may result from an antigen-driven, T cell-dependent process during which immunoglobu- lin in responsive B cells undergo somatic mutation and affinity maturation. While this is generally accompa- nied by class switch, there is increasing evidence that somatic mutation and isotype switching are not causally linked and some IgM antibodies are highly mutated (Griffiths et al., 1984; Rudikoff et al., 1984; Berek, 1992; Logtenberg, 1992; Randen et al., 1992; Varade et al., 1993). T cell involvement in the generation of anti-MAG activity is further suggested by the preferen- tial expression of an HLA-DR class II molecule in patients with anti-MAG (Vrethem et ah, 1993) and the influence of helper or suppressor T cells on antibody secretion (Latov et al., 1985). As the regulatory net- works and the structural requirements of antibody/an- tigen interaction or antigen presentation become known, it may be possible to develop therapeutic agents to block-specific points of T or B cell activation or suppress ongoing responses.
S. Acknowledgements
This work was supported by NIH grant NS25196.
G. Lee et al. /Journal of Neuroimmunology 51 (1994) 45-52 51
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