- Email: [email protected]
Highly related immunoglobulin light chain sequences in different multiple sclerosis patients
Journal of Neuroimmunology 100 Ž1999. 98–101 www.elsevier.comrlocaterjneuroim
Highly related immunoglobulin light chain sequences in different multiple sclerosis patients J. Edwin Blalock a
a,e,)
, Shan-Ren Zhou b, Curtis C. Maier John N. Whitaker a,d,e,f
a,c
, F. Shawn Galin a ,
Department of Physiology and Biophysics, UniÕersity of Alabama at Birmingham (UAB), 1918 UniÕersity BlÕd. (MCLM 896), McCallum Building, Birmingham, AL 35294-0005, USA b Department of Neurology, Vanderbilt UniÕersity, NashÕille, TN 32713, USA c SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA d Department of Neurology, The UniÕersity of Alabama at Birmingham, Birmingham, AL, USA e Center for Neuroimmunology, The UniÕersity of Alabama at Birmingham, Birmingham, AL, USA f Neurology and Research SerÕices, Birmingham Veterans Medical Center, Birmingham, AL 35294, USA Received 30 August 1999; accepted 17 September 1999
Abstract Although immunoglobulin G and free light ŽL. chains of oligoclonal origin in cerebrospinal fluid ŽCSF. are the most common immunologic abnormalities in multiple sclerosis ŽMS., it is unknown whether homologous CSF L chain sequences are present in different individuals with MS. Using Southern blotting, a particular kappa Žk . L chain variable region ŽV. probe was recently found to hybridize to VK cDNA from CSF B cells from almost one half of the MS patients tested but only 10% of normal or other neurologic disease controls wZhou, S.-R., Maier, C.C., Mitchell, G.W., LaGanke, C.C., Blalock, J.E., Whitaker, J.N., 1998. A cross-reactive idiotope in cerebrospinal fluid cells in multiple sclerosis: further evidence for the role of myelin basic protein. Neurology 50, 411–417.x Here, we report that this likely results from remarkable sequence similarity in certain Vk from CSF B cells from different individuals with MS. The high degree of sequence homology even extended to all three complementarity determining regions ŽCDR. which in part form an antibody combining site. In addition, marked sequence homology was observed between the light chains from the MS patients and those from certain mouse antibodies against myelin basic protein ŽMBP.. The results establish, in principle, that the same or very similar k light chain variable regions can be shared between CSF B lymphocytes from different individuals with MS as well as with certain antibodies against MBP. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Immunoglobulin; Idiotype; Variable region; Myelin basic protein; Autoimmunity
1. Introduction Multiple sclerosis ŽMS. is an inflammatory demyelinating disease of the central nervous system, which is characterized by multifocal lesions ŽAdams, 1983.. T and B lymphocytes, together with macrophages, are present within the foci of inflammation. Although certain models of MS, experimental allergic encephalomyelitis ŽEAE., are clearly
) Corresponding author. Department of Physiology and Biophysics, University of Alabama at Birmingham ŽUAB., 1918 University Blvd. ŽMCLM 896., 896 McCallum Building, Birmingham, AL 35294-0005, USA. Tel.: q1-205-934-6439; fax: q1-205-934-1446; e-mail: [email protected]
T cell-mediated disorders and MS itself is known to have a prominent T cell component, antibody and B cells play a regulatory and perhaps active role in the disease process ŽGenain et al., 1999; Wekerle, 1999.. Indeed, increased cerebrospinal fluid ŽCSF. levels of immunoglobulin ŽIg.G and free light ŽL. chains of restricted heterogeneity or oligoclonality are the most common and undisputed immunologic abnormality in individuals with MS ŽRudick, 1987; Whitaker and Benveniste, 1996.. In MS patients, the oligoclonal IgG and free L chains are synthesized within the central nervous system ŽCNS. which contains large numbers of plasma cells ŽPrineas, 1985., particularly in the later phases of disease ŽOzawa et al., 1994.. Whether there is a relatednes of the structure or sequence for the CSF IgG or L chains of one MS patient to those of another is
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 8 8 - 5
J.E. Blalock et al.r Journal of Neuroimmunology 100 (1999) 98–101
unknown at present but is clearly important to an eventual molecular understanding of the disease. We have previously found indirect evidence of related L chains in different MS patients. Specifically, a particular k L chain variable ŽV. region cDNA probe Žtermed 845D3. was found to hybridize to Vk cDNA from CSF B lymphocyte from 14 of 31 MS patients, but only one of the nine patients with other neurologic diseases and one of the 10 non-neurologic patients ŽZhou et al., 1998.. This was even more intriguing when one considers that the 845D3 cDNA probe represents the Vk of a murine mAb which is reactive with myelin basic protein ŽMBP.. In this study, we have sought direct evidence for a sharing of L chains among different MS patients by sequencing the Vk cDNA from three of the aforementioned MS patients. Here, we report that there can be remarkable similarity in certain Vk sequences from CSF of different individuals with MS.
2. Materials and methods CSF specimens were obtained for clinically indicated reasons from three non-related, patients with clinically definite MS as previously described ŽZhou et al., 1998.. All studies were approved by the Institutional Review Board. Four to six milliliters of fresh CSF from each of the MS patients were centrifuged at 1500 rpm for 15 min at 48C. Cells from CSF were then washed twice with cold PBS and were pelleted by centrifugation at 5000 rpm for 1 min. Cell pellets then underwent a rapid reverse transcriptasepolymerase chain reaction ŽRT-PCR. for cDNA synthesis and amplication ŽLeBoeuf et al., 1989. that has been
99
modified for small numbers of normal lymphocytes ŽMaier and Blalock, 1995.. The sequence of Vk of mAb F23 has been previously published ŽMaier et al., 1993.. The sequence of 845D3 VK and human Vk clones were determined by using the same cloning and sequencing method ŽLeBoeuf et al., 1989; Maier et al., 1993; Maier and Blalock, 1995.. In this method, the amplified PCR products from 845D3 hybridoma cells or MS B cells were extracted with phenolrchloroform and digested with 10 U SalI at 378C for 2 h. The restriction digestion mix was loaded onto a 5% Nusieve GTG low-melting point agarose gel ŽFMC. in TBE buffer. The DNA band at the desired molecular weight was excised from the gel and purified with Gelase ŽEpicenter Technologies, Madison, WI. according to the manufacturer’s protocol. The digestion product was then ligated into the SalI site of M13mp18 bacteriophage and recombinant clones were selected. The selected clones were subsequently sequenced using US Biochemical ŽCleveland, OH. Sequenase Version 2.0 kit and protocol. The reaction products were electrophoresed on both 4% and 6% acrylamide wedge gels in order to allow the entire variable region sequences to be clearly visualized.
3. Results To determine the sequence relationship of light chain V regions from 845D3 positive MS patients, VK cDNA from the CSF B cells of three such MS patients Ždenoted A, B, or C. were cloned and sequenced. Three independent clones Ždenoted 1, 2, or 3. from each of the MS patients were analyzed. All of the MS patients had at least one VK
Fig. 1. Alignment and identity Žusing the GenePro 5.0 alignment program. of the inferred amino acids of Vk from two anti-MBP monoclonal Ab Ž845D3 and F23C6. with nine human Vk sequences from MS patients. Patients are denoted A, B or C and clones are denoted 1, 2, or 3. Within a group, identical sequences are in black and non-identical are red.
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J.E. Blalock et al.r Journal of Neuroimmunology 100 (1999) 98–101
Fig. 2. Ža. Inter-MS patient and intra-MS patient homologies of the sequences from Fig. 1. Bold numbers indicate when comparisons are of two sequences from the same subgroup. Analysis was with the Wisconsin GCG package ŽVersion 8.0.. Žb. Comparison of the amino acid identities for the CDRs of subgroup IV and I human Vk sequences from Fig. 1.
sequence from Vk subgroups I and III and two had Vk subgroup IV sequences as well ŽFig. 1.. While most of the intra-subgroup sequences had the 80%–85% homology expected within a subgroup ŽKabat et al., 1991., one subgroup I pair between patient A clone 1 Ži.e., A-1. and B-1 and the subgroup IV pair between A-2 and C-2 had very high 96% and 93% homologies, respectively ŽFig. 2a.. When a BLAST search of the non-redundant GenBank database was performed with A-1, only 12 of the 1661 Ž0.7%. human VK sequences had equivalent or greater homology to A-1 as A-1 has to B-1. Similarly, only 29 of the 1661 Ž1.7%. human VK sequences had equivalent or greater homology to C-2 as C-2 has to A-2. Thus, the appearance of shared sequences in two individuals with MS at these levels of homology are unlikely to be random occurrences since by chance this would occur one in 20,408 times for the A-1rB-1 pair and one in 3460 times for the A-2rC-2 pair. The probability that patient A would have one sequence shared with patient B and the other with patient C is exceedingly low Žone in 7.06 = 10 7 .. Not only are the entire VK sequences homologous, there is a high degree of identity in all three complementarity determining regions ŽCDR. for the A-1rB-1 pair and the A-2rC-2 pair Ži.e., CDR1, 88%–100%; CDR2, 86%– 100%; CDR3, 75%–78%. ŽFig. 2b.. For the subgroup I sequences where comparisons are possible between the ‘‘unrelated clones,’’ identities are quite low Ži.e., CDR1, 55%–64%; CDR2, 14%–57%; CDR3, 25%–37%. ŽFig. 2b.. Collectively, these results strongly suggest that certain MS patients can share an overlapping set of VK in their CSF. The Vk sequences from the MS patients also provide an explanation for our earlier finding of hybridization of the 845D3 probe to Vk cDNA from CSF B cells of MS patients ŽZhou et al., 1998.. Specifically, there is marked
sequence homology at the amino acid ŽFig. 1. and nucleotide level Ždata not shown.. This homology also extended to the VL of another murine mAb ŽF23C6. against MBP, which is related in sequence ŽFig. 1. and idiotype to 845D3 ŽZhou and Whitaker, 1990.. Interestingly, 845D3 Vk was most homologous to the A-2rC-2 pair Ž68% and 72%, respectively. while F23C6 Vk was most homologous to the A-1rB-1 pair Ž67% and 69%, respectively.. These homology levels were even higher than 845D3 Vk compared to F23C6 Vk Ž54%. or the 20% to 40% homology one might expect between Vk from different species ŽKabat et al., 1991..
4. Discussion This study provides the first determination of VK sequences from CSF B lymphocytes of MS patients. The results establish proof in principle that the same or very similar VK gene products can be shared between different individuals with MS. Because of the limited number of patients studied, the data do not allow a determination of the prevalence of this finding. This obviously must await the elucidation of VK sequences from additional MS patients. However, when one considers the homology between the 845D3 VK cDNA probe and at least one cloned VK sequence from each of the three MS patients herein reported, the results suggest that almost 50% of MS patients may have overlapping VK sequences since CSF B cell cDNA from 14 out of 31 MS patients were positive by Southern analysis with the 845D3 VK cDNA probe ŽZhou et al., 1998.. Considering the observed hybridization of the aforementioned probe to certain controls, there will undoubtedly be related sequences between L chains from MS patients and individuals not afflicted with MS. This, how-
J.E. Blalock et al.r Journal of Neuroimmunology 100 (1999) 98–101
ever, should be relatively rare since only 10% of other neurologic and non-neurologic disease controls were positive by Southern analysis ŽZhou et al., 1998.. Interestingly, two recent reports have described a similar result for heavy ŽH. chain V regions from MS patients ŽOwens et al., 1998; Qin et al., 1998.. Specifically, there is preferential usage of the VH IV family and thus different MS patients are expressing related VH genes. Although purely speculative at this time, if highly related VK were paired with highly related VH there would be the distinct possibility that different MS patients would share Ab with similar antigen ŽAg. specificity against a myelin component. Alternatively, we have recently demonstrated that a particular L chain alone in certain monoclonal antibodies can determine reactivity with MBP ŽGalin et al., 1996.. Indeed, one or both of these situations could account for the finding that 95% of autoantibody binding to MBP in 11r12 postmortem MS cases were direct to MBP residues 83–97 ŽWucherpfennig et al., 1997.. This suggests that it is perhaps less than coincidence that the shared VK sequences among MS patients are homologous, especially in the CDRs, to 845D3 VK which was derived from a mAb to human MBP 80–89 ŽZhou and Whitaker, 1990..
Acknowledgements The authors thank Diane Weigent for excellent editorial assistance. These studies were supported by an NIH Institutional Training Grant ŽT3207335., and NIH grants ŽNS29719 and AI37670., and the Research Program of the Veterans Administration.
References Adams, C.W.M., 1983. The general pathology of multiple sclerosis: morphological and chemical aspects of the brain. In: Hallpike, J.F., Adams, C.W.M., Tourtellotte, W.W., ŽEds.., Multiple Sclerosis: Pathology, Diagnosis and Management. Williams and Wilkins, Baltimore, pp. 203–240. Galin, F.S., Maier, C.C., Zhou, S.-R., Whitaker, J.N., Blalock, J.E., 1996. Murine Vl x and Vl x-containing antibodies bind human myelin basic protein. J. Clin. Invest. 97, 486–492.
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Genain, C.P., Cannella, B., Hauser, S.L., Raine, C.S., 1999. Identification of autoantibodies associated with myelin damage in multiple sclerosis. Nat. Med. 5, 170–175. Kabat, E.H., Wu, T.T., Perry, H.M., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunologic Interest, Bethesda, US Department of Health and Human Services, Public Health Service, National Institutes of Health. LeBoeuf, R.D., Galin, F.S., Hollinger, S.K., Peiper, S.C., Blalock, J.E., 1989. Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction. Gene 82, 371–377. Maier, C.C., Blalock, J.E., 1995. Cloning and sequencing immunoglobulin and T-cell receptor variable regions involved in neuroimmune disorders. Methods Neurosci. 24, 321–334. Maier, C.C., LeBoeuf, R.D., Zhou, S.R., Whitaker, J.N., Jarpe, M.A., Blalock, J.E., 1993. The structure of a myelin basic protein-associated idiotope. J. Neuroimmunol. 46, 235–243. Owens, G.P., Kraus, H., Burgoon, M.P., Smith-Jensen, T., Devlin, M.E., Gilden, D.H., 1998. Restricted use of VH 4 germline segments in an acute multiple sclerosis brain. Ann. Neurol. 43, 236–243. Ozawa, K., Suchanek, G., Breitschopf, H., Bruck, W., Budka, H., Jellinger, K., Lassmann, H., 1994. Patterns of oligodendroglia pathology in multiple sclerosis. Brain 117, 1311–1322. Prineas, J.W., 1985. The neuropathology of multiple sclerosis. In: Vinken, P.J.B.G.W., Klawans, H.L., Koetsier, J.C., ŽEds... Handbook of Clinical Neurology. Elsevier, Amsterdam, pp. 213–257. Qin, Y., Duquette, P., Zhang, Y., Talbot, P., Poole, R., Antel, J., 1998. Clonal expansion and somatic hypermutation of VH genes of B cells from cerebrospinal fluid in multiple sclerosis. J. Clin. Invest. 102, 1045–1050. Rudick, R.A., 1987. Free light chains of immunoglobulins in multiple sclerosis: a putative index of the intrathecal humoral immune response. In: Lowenthal, A., Raus, J., ŽEds.., Cellular and Humoral Immunological Components of Cerebrospinal Fluid in Multiple Sclerosis. Plenum, New York, pp. 187–200. Wekerle, H., 1999. Remembering MOG: autoantibody mediated demyelination in multiple sclerosis?. Nat. Med. 5, 153. Whitaker, J.N., Benveniste, E.N., 1996. Cerebrospinal fluid. In: Cook, S.D. ŽEd.., Handbook of Multiple Sclerosis, 2nd edn. Marcel Dekker, New York, pp. 295–316. Wucherpfennig, K.W., Catz, I., Hausmann, S., Strominger, J.L., Steinman, L., Warren, K.G., 1997. Recognition of the immunodominant myelin basic protein peptide by autoantibodies and HLA-DR2-restricted T cell clones from multiple sclerosis patients. J. Clin. Invest. 100, 1114–1122. Zhou, S.R., Whitaker, J.N., 1990. An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein. J. Immunol. 145, 2554–2560. Zhou, S.-R., Maier, C.C., Mitchell, G.W., LaGanke, C.C., Blalock, J.E., Whitaker, J.N., 1998. A cross-reactive idiotope in cerebrospinal fluid cells in multiple sclerosis: further evidence for the role of myelin basic protein. Neurology 50, 411–417.
Highly related immunoglobulin light chain sequences in different multiple sclerosis patients J. Edwin Blalock a
a,e,)
, Shan-Ren Zhou b, Curtis C. Maier John N. Whitaker a,d,e,f
a,c
, F. Shawn Galin a ,
Department of Physiology and Biophysics, UniÕersity of Alabama at Birmingham (UAB), 1918 UniÕersity BlÕd. (MCLM 896), McCallum Building, Birmingham, AL 35294-0005, USA b Department of Neurology, Vanderbilt UniÕersity, NashÕille, TN 32713, USA c SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA d Department of Neurology, The UniÕersity of Alabama at Birmingham, Birmingham, AL, USA e Center for Neuroimmunology, The UniÕersity of Alabama at Birmingham, Birmingham, AL, USA f Neurology and Research SerÕices, Birmingham Veterans Medical Center, Birmingham, AL 35294, USA Received 30 August 1999; accepted 17 September 1999
Abstract Although immunoglobulin G and free light ŽL. chains of oligoclonal origin in cerebrospinal fluid ŽCSF. are the most common immunologic abnormalities in multiple sclerosis ŽMS., it is unknown whether homologous CSF L chain sequences are present in different individuals with MS. Using Southern blotting, a particular kappa Žk . L chain variable region ŽV. probe was recently found to hybridize to VK cDNA from CSF B cells from almost one half of the MS patients tested but only 10% of normal or other neurologic disease controls wZhou, S.-R., Maier, C.C., Mitchell, G.W., LaGanke, C.C., Blalock, J.E., Whitaker, J.N., 1998. A cross-reactive idiotope in cerebrospinal fluid cells in multiple sclerosis: further evidence for the role of myelin basic protein. Neurology 50, 411–417.x Here, we report that this likely results from remarkable sequence similarity in certain Vk from CSF B cells from different individuals with MS. The high degree of sequence homology even extended to all three complementarity determining regions ŽCDR. which in part form an antibody combining site. In addition, marked sequence homology was observed between the light chains from the MS patients and those from certain mouse antibodies against myelin basic protein ŽMBP.. The results establish, in principle, that the same or very similar k light chain variable regions can be shared between CSF B lymphocytes from different individuals with MS as well as with certain antibodies against MBP. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Immunoglobulin; Idiotype; Variable region; Myelin basic protein; Autoimmunity
1. Introduction Multiple sclerosis ŽMS. is an inflammatory demyelinating disease of the central nervous system, which is characterized by multifocal lesions ŽAdams, 1983.. T and B lymphocytes, together with macrophages, are present within the foci of inflammation. Although certain models of MS, experimental allergic encephalomyelitis ŽEAE., are clearly
) Corresponding author. Department of Physiology and Biophysics, University of Alabama at Birmingham ŽUAB., 1918 University Blvd. ŽMCLM 896., 896 McCallum Building, Birmingham, AL 35294-0005, USA. Tel.: q1-205-934-6439; fax: q1-205-934-1446; e-mail: [email protected]
T cell-mediated disorders and MS itself is known to have a prominent T cell component, antibody and B cells play a regulatory and perhaps active role in the disease process ŽGenain et al., 1999; Wekerle, 1999.. Indeed, increased cerebrospinal fluid ŽCSF. levels of immunoglobulin ŽIg.G and free light ŽL. chains of restricted heterogeneity or oligoclonality are the most common and undisputed immunologic abnormality in individuals with MS ŽRudick, 1987; Whitaker and Benveniste, 1996.. In MS patients, the oligoclonal IgG and free L chains are synthesized within the central nervous system ŽCNS. which contains large numbers of plasma cells ŽPrineas, 1985., particularly in the later phases of disease ŽOzawa et al., 1994.. Whether there is a relatednes of the structure or sequence for the CSF IgG or L chains of one MS patient to those of another is
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 8 8 - 5
J.E. Blalock et al.r Journal of Neuroimmunology 100 (1999) 98–101
unknown at present but is clearly important to an eventual molecular understanding of the disease. We have previously found indirect evidence of related L chains in different MS patients. Specifically, a particular k L chain variable ŽV. region cDNA probe Žtermed 845D3. was found to hybridize to Vk cDNA from CSF B lymphocyte from 14 of 31 MS patients, but only one of the nine patients with other neurologic diseases and one of the 10 non-neurologic patients ŽZhou et al., 1998.. This was even more intriguing when one considers that the 845D3 cDNA probe represents the Vk of a murine mAb which is reactive with myelin basic protein ŽMBP.. In this study, we have sought direct evidence for a sharing of L chains among different MS patients by sequencing the Vk cDNA from three of the aforementioned MS patients. Here, we report that there can be remarkable similarity in certain Vk sequences from CSF of different individuals with MS.
2. Materials and methods CSF specimens were obtained for clinically indicated reasons from three non-related, patients with clinically definite MS as previously described ŽZhou et al., 1998.. All studies were approved by the Institutional Review Board. Four to six milliliters of fresh CSF from each of the MS patients were centrifuged at 1500 rpm for 15 min at 48C. Cells from CSF were then washed twice with cold PBS and were pelleted by centrifugation at 5000 rpm for 1 min. Cell pellets then underwent a rapid reverse transcriptasepolymerase chain reaction ŽRT-PCR. for cDNA synthesis and amplication ŽLeBoeuf et al., 1989. that has been
99
modified for small numbers of normal lymphocytes ŽMaier and Blalock, 1995.. The sequence of Vk of mAb F23 has been previously published ŽMaier et al., 1993.. The sequence of 845D3 VK and human Vk clones were determined by using the same cloning and sequencing method ŽLeBoeuf et al., 1989; Maier et al., 1993; Maier and Blalock, 1995.. In this method, the amplified PCR products from 845D3 hybridoma cells or MS B cells were extracted with phenolrchloroform and digested with 10 U SalI at 378C for 2 h. The restriction digestion mix was loaded onto a 5% Nusieve GTG low-melting point agarose gel ŽFMC. in TBE buffer. The DNA band at the desired molecular weight was excised from the gel and purified with Gelase ŽEpicenter Technologies, Madison, WI. according to the manufacturer’s protocol. The digestion product was then ligated into the SalI site of M13mp18 bacteriophage and recombinant clones were selected. The selected clones were subsequently sequenced using US Biochemical ŽCleveland, OH. Sequenase Version 2.0 kit and protocol. The reaction products were electrophoresed on both 4% and 6% acrylamide wedge gels in order to allow the entire variable region sequences to be clearly visualized.
3. Results To determine the sequence relationship of light chain V regions from 845D3 positive MS patients, VK cDNA from the CSF B cells of three such MS patients Ždenoted A, B, or C. were cloned and sequenced. Three independent clones Ždenoted 1, 2, or 3. from each of the MS patients were analyzed. All of the MS patients had at least one VK
Fig. 1. Alignment and identity Žusing the GenePro 5.0 alignment program. of the inferred amino acids of Vk from two anti-MBP monoclonal Ab Ž845D3 and F23C6. with nine human Vk sequences from MS patients. Patients are denoted A, B or C and clones are denoted 1, 2, or 3. Within a group, identical sequences are in black and non-identical are red.
100
J.E. Blalock et al.r Journal of Neuroimmunology 100 (1999) 98–101
Fig. 2. Ža. Inter-MS patient and intra-MS patient homologies of the sequences from Fig. 1. Bold numbers indicate when comparisons are of two sequences from the same subgroup. Analysis was with the Wisconsin GCG package ŽVersion 8.0.. Žb. Comparison of the amino acid identities for the CDRs of subgroup IV and I human Vk sequences from Fig. 1.
sequence from Vk subgroups I and III and two had Vk subgroup IV sequences as well ŽFig. 1.. While most of the intra-subgroup sequences had the 80%–85% homology expected within a subgroup ŽKabat et al., 1991., one subgroup I pair between patient A clone 1 Ži.e., A-1. and B-1 and the subgroup IV pair between A-2 and C-2 had very high 96% and 93% homologies, respectively ŽFig. 2a.. When a BLAST search of the non-redundant GenBank database was performed with A-1, only 12 of the 1661 Ž0.7%. human VK sequences had equivalent or greater homology to A-1 as A-1 has to B-1. Similarly, only 29 of the 1661 Ž1.7%. human VK sequences had equivalent or greater homology to C-2 as C-2 has to A-2. Thus, the appearance of shared sequences in two individuals with MS at these levels of homology are unlikely to be random occurrences since by chance this would occur one in 20,408 times for the A-1rB-1 pair and one in 3460 times for the A-2rC-2 pair. The probability that patient A would have one sequence shared with patient B and the other with patient C is exceedingly low Žone in 7.06 = 10 7 .. Not only are the entire VK sequences homologous, there is a high degree of identity in all three complementarity determining regions ŽCDR. for the A-1rB-1 pair and the A-2rC-2 pair Ži.e., CDR1, 88%–100%; CDR2, 86%– 100%; CDR3, 75%–78%. ŽFig. 2b.. For the subgroup I sequences where comparisons are possible between the ‘‘unrelated clones,’’ identities are quite low Ži.e., CDR1, 55%–64%; CDR2, 14%–57%; CDR3, 25%–37%. ŽFig. 2b.. Collectively, these results strongly suggest that certain MS patients can share an overlapping set of VK in their CSF. The Vk sequences from the MS patients also provide an explanation for our earlier finding of hybridization of the 845D3 probe to Vk cDNA from CSF B cells of MS patients ŽZhou et al., 1998.. Specifically, there is marked
sequence homology at the amino acid ŽFig. 1. and nucleotide level Ždata not shown.. This homology also extended to the VL of another murine mAb ŽF23C6. against MBP, which is related in sequence ŽFig. 1. and idiotype to 845D3 ŽZhou and Whitaker, 1990.. Interestingly, 845D3 Vk was most homologous to the A-2rC-2 pair Ž68% and 72%, respectively. while F23C6 Vk was most homologous to the A-1rB-1 pair Ž67% and 69%, respectively.. These homology levels were even higher than 845D3 Vk compared to F23C6 Vk Ž54%. or the 20% to 40% homology one might expect between Vk from different species ŽKabat et al., 1991..
4. Discussion This study provides the first determination of VK sequences from CSF B lymphocytes of MS patients. The results establish proof in principle that the same or very similar VK gene products can be shared between different individuals with MS. Because of the limited number of patients studied, the data do not allow a determination of the prevalence of this finding. This obviously must await the elucidation of VK sequences from additional MS patients. However, when one considers the homology between the 845D3 VK cDNA probe and at least one cloned VK sequence from each of the three MS patients herein reported, the results suggest that almost 50% of MS patients may have overlapping VK sequences since CSF B cell cDNA from 14 out of 31 MS patients were positive by Southern analysis with the 845D3 VK cDNA probe ŽZhou et al., 1998.. Considering the observed hybridization of the aforementioned probe to certain controls, there will undoubtedly be related sequences between L chains from MS patients and individuals not afflicted with MS. This, how-
J.E. Blalock et al.r Journal of Neuroimmunology 100 (1999) 98–101
ever, should be relatively rare since only 10% of other neurologic and non-neurologic disease controls were positive by Southern analysis ŽZhou et al., 1998.. Interestingly, two recent reports have described a similar result for heavy ŽH. chain V regions from MS patients ŽOwens et al., 1998; Qin et al., 1998.. Specifically, there is preferential usage of the VH IV family and thus different MS patients are expressing related VH genes. Although purely speculative at this time, if highly related VK were paired with highly related VH there would be the distinct possibility that different MS patients would share Ab with similar antigen ŽAg. specificity against a myelin component. Alternatively, we have recently demonstrated that a particular L chain alone in certain monoclonal antibodies can determine reactivity with MBP ŽGalin et al., 1996.. Indeed, one or both of these situations could account for the finding that 95% of autoantibody binding to MBP in 11r12 postmortem MS cases were direct to MBP residues 83–97 ŽWucherpfennig et al., 1997.. This suggests that it is perhaps less than coincidence that the shared VK sequences among MS patients are homologous, especially in the CDRs, to 845D3 VK which was derived from a mAb to human MBP 80–89 ŽZhou and Whitaker, 1990..
Acknowledgements The authors thank Diane Weigent for excellent editorial assistance. These studies were supported by an NIH Institutional Training Grant ŽT3207335., and NIH grants ŽNS29719 and AI37670., and the Research Program of the Veterans Administration.
References Adams, C.W.M., 1983. The general pathology of multiple sclerosis: morphological and chemical aspects of the brain. In: Hallpike, J.F., Adams, C.W.M., Tourtellotte, W.W., ŽEds.., Multiple Sclerosis: Pathology, Diagnosis and Management. Williams and Wilkins, Baltimore, pp. 203–240. Galin, F.S., Maier, C.C., Zhou, S.-R., Whitaker, J.N., Blalock, J.E., 1996. Murine Vl x and Vl x-containing antibodies bind human myelin basic protein. J. Clin. Invest. 97, 486–492.
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Genain, C.P., Cannella, B., Hauser, S.L., Raine, C.S., 1999. Identification of autoantibodies associated with myelin damage in multiple sclerosis. Nat. Med. 5, 170–175. Kabat, E.H., Wu, T.T., Perry, H.M., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunologic Interest, Bethesda, US Department of Health and Human Services, Public Health Service, National Institutes of Health. LeBoeuf, R.D., Galin, F.S., Hollinger, S.K., Peiper, S.C., Blalock, J.E., 1989. Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction. Gene 82, 371–377. Maier, C.C., Blalock, J.E., 1995. Cloning and sequencing immunoglobulin and T-cell receptor variable regions involved in neuroimmune disorders. Methods Neurosci. 24, 321–334. Maier, C.C., LeBoeuf, R.D., Zhou, S.R., Whitaker, J.N., Jarpe, M.A., Blalock, J.E., 1993. The structure of a myelin basic protein-associated idiotope. J. Neuroimmunol. 46, 235–243. Owens, G.P., Kraus, H., Burgoon, M.P., Smith-Jensen, T., Devlin, M.E., Gilden, D.H., 1998. Restricted use of VH 4 germline segments in an acute multiple sclerosis brain. Ann. Neurol. 43, 236–243. Ozawa, K., Suchanek, G., Breitschopf, H., Bruck, W., Budka, H., Jellinger, K., Lassmann, H., 1994. Patterns of oligodendroglia pathology in multiple sclerosis. Brain 117, 1311–1322. Prineas, J.W., 1985. The neuropathology of multiple sclerosis. In: Vinken, P.J.B.G.W., Klawans, H.L., Koetsier, J.C., ŽEds... Handbook of Clinical Neurology. Elsevier, Amsterdam, pp. 213–257. Qin, Y., Duquette, P., Zhang, Y., Talbot, P., Poole, R., Antel, J., 1998. Clonal expansion and somatic hypermutation of VH genes of B cells from cerebrospinal fluid in multiple sclerosis. J. Clin. Invest. 102, 1045–1050. Rudick, R.A., 1987. Free light chains of immunoglobulins in multiple sclerosis: a putative index of the intrathecal humoral immune response. In: Lowenthal, A., Raus, J., ŽEds.., Cellular and Humoral Immunological Components of Cerebrospinal Fluid in Multiple Sclerosis. Plenum, New York, pp. 187–200. Wekerle, H., 1999. Remembering MOG: autoantibody mediated demyelination in multiple sclerosis?. Nat. Med. 5, 153. Whitaker, J.N., Benveniste, E.N., 1996. Cerebrospinal fluid. In: Cook, S.D. ŽEd.., Handbook of Multiple Sclerosis, 2nd edn. Marcel Dekker, New York, pp. 295–316. Wucherpfennig, K.W., Catz, I., Hausmann, S., Strominger, J.L., Steinman, L., Warren, K.G., 1997. Recognition of the immunodominant myelin basic protein peptide by autoantibodies and HLA-DR2-restricted T cell clones from multiple sclerosis patients. J. Clin. Invest. 100, 1114–1122. Zhou, S.R., Whitaker, J.N., 1990. An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein. J. Immunol. 145, 2554–2560. Zhou, S.-R., Maier, C.C., Mitchell, G.W., LaGanke, C.C., Blalock, J.E., Whitaker, J.N., 1998. A cross-reactive idiotope in cerebrospinal fluid cells in multiple sclerosis: further evidence for the role of myelin basic protein. Neurology 50, 411–417.