Abstract
Background
In rheumatoid arthritis (RA), B-lineage cells in the synovial membrane secrete large amounts of immunoglobulin that contribute to tissue destruction. The CDR3 of an immunoglobulin light chain is formed by rearrangements of VL and JL gene segments. Addition of non-germline-encoded (N) nucleotides at V(D)J joins by the enzyme terminal deoxynucleotidyl transferase (TdT) enhances antibody diversity. TdT was previously thought to be active in B cells only during heavy chain rearrangement, but we and others reported unexpectedly high levels of N addition in kappa light chains. We also found clonally related kappa chains bearing unusually long CDR3 intervals in RA synovium, suggesting oligoclonal expansion of a set of atypical B lymphocytes. In this study, we analyzed lambda light chain expression to determine if N addition occurs throughout immunoglobulin gene rearrangement and to compare CDR3 lengths of lambda and kappa light chains in RA patients and normal individuals.
Materials and Methods
Reverse transcription-polymerase chain reaction (RT-PCR) amplification of VλIII transcripts was performed on RA synovia and peripheral blood lymphocytes (PBL) and normal PBL for which kappa repertoires were previously analyzed. Representative λ+ PCR products were cloned and sequenced.
Results
Analysis of 161 cDNA clones revealed that N addition occurs in lambda light chains of RA patients and normal controls. The lambda light chain repertoires in RA were enriched for long CDR3 intervals. In both RA and controls, CDR3 lengths were strongly influenced by which Vλ gene segment was present in the rearrangement. Five sets of clonally related sequences were found in RA synovia and PBL; one set was found in normal PBL.
Conclusions
In humans, unlike mice, N addition enhances antibody diversity at all stages of immunoglobulin assembly, and the structural diversity of lambda CDR3 intervals is greater than that of kappa light chains. Clonally related Vλ gene segments in RA support an antigen-driven B-cell response.
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References
Harris ED Jr. (1990) Rheumatoid arthritis: pathophysiology and implications for treatment. N. Engl. J. Med. 322: 1277–1289.
Zvaifler NJ. (1973) Immunopathology of joint inflammation in rheumatoid arthritis. Adv. Immunol. 16: 265–336.
Arend WP. (1997) The pathophysiology and treatment of rheumatoid arthritis. Arthritis Rheum. 40: 595–597.
Mellors RC, Heimer R, Corcos J, Korngold L. (1959) Cellular origin of rheumatoid factor. J. Exp. Med. 110: 875–886.
Smiley JD, Sachs C, Ziff M. (1968) In vitro synthesis of immunoglobulin by rheumatoid synovial membrane. J. Clin. Invest. 47: 624–632.
Dreyer WJ, Bennett JC. (1965) The molecular basis of antibody formation: a paradox. Proc. Natl. Acad. Sci. U.S.A. 54: 864–869.
Sanz I. (1991) Multiple mechanisms participate in the generation of diversity of human H chain CDR3 regions. J. Immunol. 147: 1720–1729.
Tonegawa S. (1983) Somatic generation of antibody diversity. Nature 302: 575–581.
Max EE. (1993) Immunoglobulins: molecular genetics. In: Paul WE (ed). Fundamental Immunology, 3rd ed. Raven Press, New York, pp. 315–382.
Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C. (1991) Sequences of Proteins of Immunological Interest, 5th ed. NIH Publ #91-3242. U.S. Department of Health and Human Services, Bethesda, MD.
Desiderio SV, Yancopoulos GD, Paskind M, et al. (1984) Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature 311: 752–755.
Klein U, Küppers R, Rajewsky K. (1993) Human IgM+ IgD+ B cells, the major B cell subset in the peripheral blood, express Vκ genes with no or little somatic mutation throughout life. Eur. J. Immunol. 23: 3272–3277.
Weber J-C, Blaison G, Martin T, Knapp A-M, Pasquali J-L. (1994) Evidence that the VκIII gene usage is nonstochastic in both adult and newborn peripheral B cells and that peripheral CD5+adult B cells are oligoclonal. J. Clin. Invest. 93: 2093–2105.
Klein R, Jaenichen R, Zachau HG. (1993) Expressed human immunoglobulin kappa genes and their hypermutation. Eur. J. Immunol. 23: 3248–3262.
Victor KD, Capra JD. (1994) An apparently common mechanism of generating antibody diversity: length variation of the VL-JL junction. Mol. Immunol. 31: 39–46.
Bridges SL Jr, Lee SK, Johnson ML, et al. (1995) Somatic mutation and CDR3 lengths of immunoglobulin κ light chains expressed in patients with rheumatoid arthritis and normal individuals. J. Clin. Invest. 96: 831–841.
Lee SK, Bridges SL Jr, Koopman WJ, Schroeder HW. (1992) The immunoglobulin kappa light chain repertoire expressed in the synovium of a patient with rheumatoid arthritis. Arthritis Rheum. 35: 905–913.
Martin T, Blaison G, Levallois H, Pasquali JL. (1992) Molecular analysis of the VκIII-Jκ junctional diversity of polyclonal rheumatoid factors during rheumatoid arthritis frequently reveals N addition. Eur. J. Immunol. 22: 1773–1779.
Chuchana P, Blancher A, Brockly F, Alexandre D, Lefranc G, Lefranc M-P. (1990) Definition of the human immunoglobulin variable lambda (IGLV) gene subgroups. Eur. J. Immunol. 20: 1317–1325.
Williams SC, Frippiat JP, Tomlinson IM, Ignatovich O, Lefranc MP, Winter G. (1996) Sequence and evolution of the human germline V lambda repertoire. J. Mol. Biol. 264: 220–232.
Kawasaki K, Minoshima S, Nakato E, et al. (1997) One-megabase sequence analysis of the human immunoglobulin lambda gene locus. Genome Res. 7: 250–261.
Bridges SL Jr, Lavelle JC, Lee SK, Byer S, Schroeder HW Jr. (1997) CDR3 fingerprinting of immunoglobulin kappa light chains expressed in rheumatoid arthritis: evidence of antigenic selection or dysregulation of gene rearrangement in B cells. Ann. N.Y. Acad. Sci. 815: 423–426.
Ermel RW, Kenny TP, Wong A, Solomon A, Chen PP, Robbins DL. (1994) Preferential utilization of a novel V lambda 3 gene in monoclonal rheumatoid factors derived from the synovial cells of rheumatoid arthritis patients. Arthritis Rheum. 37: 860–868.
Ermel RW, Kenny TP, Chen PP, Robbins DL. (1993) Molecular analysis of rheumatoid factors derived from rheumatoid synovium suggests an antigen-driven response in inflamed joints. Arthritis Rheum. 36: 380–388.
Harindranath N, Goldfarb IS, Ikematsu H, et al. (1991) Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient. Int. Immunol. 3: 865–875.
Fang Q, Kannapell CC, Gaskin F, Solomon A, Koopman WJ, Fu SM. (1994) Human rheumatoid factors with restrictive specificity for rabbit immunoglobulin G: auto- and multi-reactivity, diverse VH gene segment usage and preferential usage of VλIIIb. J. Exp. Med. 179: 1445–1456.
Lee SK, Bridges SL Jr, Kirkham PM, Koopman WJ, Schroeder HW Jr. (1994) Evidence of antigen receptor-influenced oligoclonal B lymphocyte expansion in the synovium of a patient with longstanding rheumatoid arthritis. J. Clin. Invest. 93: 361–370.
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294.
Daley MD, Peng HQ, Misener V, Liu XY, Chen PP, Siminovitch KA. (1992) Molecular analysis of human immunoglobulin V lambda germline genes: subgroups V lambda III and V lambda IV. Mol. Immunol. 29: 1515–1518.
Combriato G, Klobeck HG. (1991) V lambda and J lambda-C lambda gene segments of the human immunoglobulin lambda light chain locus are separated by 14 kb and rearrange by a deletion mechanism. Eur. J. Immunol. 21: 1513–1522.
Blomberg BB, Glozak MA, Donohoe ME. (1995) Regulation of human lambda light chain gene expression. Ann. N.Y. Acad. Sci. 764: 84–98.
English D, Andersen BR. (1974) Single-step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. J. Immunol. Methods 5: 249–252.
Madsen M, Johnsen HE, Hansen PW, Christiansen SE. (1980) Isolation of human T and B lymphocytes by E-rosette gradient centrifugation. Characterization of the isolated subpopulations. J. Immunol. Methods 33: 323–336.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al., eds. (1997) Preparation and analysis of DNA. In: Current Protocols in Molecular Biology. John Wiley & Sons, New York, pp. 2.1.1–2.1.2.
Frippiat JP, Lefranc MP. (1994) Genomic organisation of 34 kb of the human immunoglobulin lambda locus (IGLV): restriction map and sequences of new V lambda III genes. Mol. Immunol. 31: 657–670.
Sanger F, Nicklen S, Coulson AR. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463–5467.
Elgavish RA, Schroeder HW Jr. (1993) SAW: A graphical user interface for the analysis of immunoglobulin variable domain sequences. Biotechniques 15: 1066–1071.
Lafaille JJ, DeCloux A, Bonneville M, Takagaki Y, Tonegawa S. (1989) Junctional sequences of T cell receptor gamma delta genes: implications for gamma delta T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 59: 859–870.
Lewis SM. (1994) P nucleotide insertions and the resolution of hairpin DNA structures in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 91: 1332–1336.
Frippiat JP, Williams SC, Tomlinson IM, et al. (1995) Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum. Mol. Genet. 4: 983–991.
Frippiat JP, Dard P, Marsh S, Winter G, Lefranc MP. (1997) Immunoglobulin lambda light chain Orphons on human chromosome 8q11.2. Eur. J. Immunol. 27: 1260–1265.
Daley MD, Olee T, Peng HQ, Soto-Gil RW, Chen PP, Siminovitch KA. (1992) Molecular characterization of the human immunoglobulin V lambda I germline gene repertoire. Mol. Immunol. 29: 1031–1042.
Williams SC, Winter G. (1993) Cloning and sequencing of human immunoglobulin V lambda gene segments. Eur. J. Immunol. 23: 1456–1461.
Ch’ang LY, Schnell MG, Ringelberg CS, Weiss D, Solomon A. (1995) Molecular characterization of a human VλVIII germline gene. Mol. Immunol. 32: 49–55.
Deftos M, Soto-Gil R, Quan M, Olee T, Chen PP. (1994) Utilization of a potentially universal downstream primer in the rapid identification and characterization of V lambda genes from two new human V lambda gene families. Scand. J. Immunol. 39: 95–103.
Ch’ang LY, Yen CP, Besl L, Schell M, Solomon A. (1994) Identification and characterization of a functional human Ig VλVI germline gene. Mol. Immunol. 31: 531–536.
Ignatovich O, Tomlinson IM, Jones PT, Winter G. (1997) The creation of diversity in the human immunoglobulin Vλ repertoire. J. Mol. Biol. 268: 69–77.
Blomberg BB, Solomon A. (1997) The murine and human lambda light chain immunoglobulin loci: organization and expression. In: Herzenberg LA, Weir DM, Herzenberg LA, Blackwell C (eds). Handbook of Experimental Immunology. Blackwell Science, Inc., Boston. Fifth Edition.
Bauer TR Jr, Blomberg B. (1991) The human lambda L chain Ig locus. Recharacterization of JC lambda 6 and identification of a functional JC lambda 7. J. Immunol. 146: 2813–2820.
Vasicek TJ, Leder P. (1990) Structure and expression of the human immunoglobulin lambda genes. J. Exp. Med. 172: 609–620.
Niewold TA, Murphy CL, Weiss DT, Solomon A. (1996) Characterization of a light chain product of the human JC lambda 7 gene complex. J. Immunol. 157: 4474–4477.
Dariavach P, Lefranc G, Lefranc MP. (1987) Human immunoglobulin C lambda 6 gene encodes the Kern+Oz− lambda chain and C lambda 4 and C lambda 5 are pseudogenes. Proc. Natl. Acad. Sci. U.S.A. 84: 9074–9078.
Udey JA, Blomberg B. (1987) Human lambda light chain locus: organization and DNA sequences of three genomic J regions. Immunogenetics 25: 63–70.
Ishikawa H, Ziff M. (1976) Electron microscopic observations of immunoreactive cells in the rheumatoid synovial membrane. Arthritis Rheum. 19: 1–14.
Brown KA, Perry ME, Mustafa Y, et al. (1995) The distribution and abnormal morphology of plasma cells in rheumatoid synovium. Scand. J. Immunol. 41: 509–517.
Kelley DE, Perry RP. (1986) Transcriptional and posttranscriptional control of immunoglobulin mRNA production during B lymphocyte development. Nucl. Acids Res. 14: 5431–5447.
Klein U, Küppers R, Rajewsky K. (1997) Evidence for a large compartment of IgM-expressing memory B cells in humans. Blood 89: 1288–1298.
Clausen BE, Bridges SL Jr, Lavelle JC, et al. (1998) Clonally related immunoglobulin VH domains and nonrandom use of DH gene segments in rheumatoid arthritis synovium. Mol. Med. 4: 240–257.
Campbell MJ, Zelenetz AD, Levy S, Levy R. (1992) Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire. Mol. Immunol. 29: 193–203.
Pääbo S, Higuchi RG, Wilson AC. (1989) Ancient DNA and the polymerase chain reaction. J. Biol. Chem. 264: 9709–9712.
Pääbo S, Irwin DM, Wilson AC. (1990) DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265: 4718–4721.
Lundberg KS, Shoemaker DD, Adams MW, Short JM, Sorge JA, Mathur EJ. (1991) High-fidelity amplification using a thermostable DNA polyermase isolated from Pyrococcus furiosus. Gene 108: 1–6.
Tindall KR, Kunkel TA. (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymorphism. Biochemistry 27: 6008–6013.
Barnes WM. (1992) The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion. Gene 112: 29–35.
Billips LG, Lassoued K, Nunez C, et al. (1995) Human B-cell development. Ann. N.Y. Acad. Sci. 764: 1–8.
Kubagawa H, Cooper MD, Carroll AJ, Burrows PD. (1989) Light-chain gene expression before heavy-chain gene rearrangement in pre-B cells transformed by Epstein-Barr virus. Proc. Natl. Acad. Sci. U.S.A. 86: 2356–2360.
Schroeder J. H. W., Burrows PD, Cooper MD, Kubagawa H. (1992) N region addition and unusual CDR3 length distributions in κ-chain only pre-B cells [abstract]. J. Cell. Biochem. S17B: 235.
Nishimoto N, Kubagawa H, Cooper MD. (1991) Comparison of pre-B cell differentiation in normal and X-linked agammaglobulinemia (XLA) individuals [abstract]. FASEB J. 5: 1346.
Goossens T, Klein U, Küppers R. (1998) Frequent occurrence of deletions and duplications during somatic hypermutation: implications for oncogene translocations and heavy chain disease. Proc. Natl. Acad. Sci. U.S.A. 95: 2463–2468.
Wilson PC, de Bouteiller O, Liu YJ, et al. (1998) Somatic hypermutation introduces insertions and deletions into immunoglobulin V genes. J. Exp. Med. 187: 59–70.
Bridges SL Jr, Lee SK, Koopman WJ, Schroeder HW Jr. (1993) Analysis of immunoglobulin gamma heavy chain expression in synovial tissue of a patient with rheumatoid arthritis. Arthritis Rheum. 36: 631–641.
Ruff-Jamison S, Glenney J. (1993) Requirement for both H and L chain V regions, VH and VK joining amino acids, and the unique H chain D region for the high affinity binding of an anti-phosphotyrosine antibody. J. Immunol. 150: 3389–3396.
Randen I, Brown D, Thompson KM, et al. (1992) Clonally related IgM rheumatoid factors undergo affinity maturation in the rheumatoid synovial tissue. J. Immunol. 148: 3296–3301.
Randen I, Thompson KM, Pascual V, et al. (1992) Rheumatoid factor V genes from patients with rheumatoid arthritis are diverse and show evidence of an antigen-driven response. Immunol. Rev. 128: 49–71.
Martin T, Crouzier R, Blaison G, Levallois H, Pasquali JL. (1993) A minor group of rheumatoid factors isolated from a patient with rheumatoid arthritis is derived from somatically mutated Vk1 genes further evidence that rheumatoid factors during autoimmune diseases undergo an antigen driven maturation. Autoimmunity 15: 163–170.
Olee T, Lu EW, Huang DF, et al. (1992) Genetic analysis of self-associating immunoglobulin G rheumatoid factors from two rheumatoid synovia implicates an antigen-driven response. J. Exp. Med. 175: 831–842.
Schroder ARE, Greiner A, Seyfert C, Berek C. (1996) Differentiation of B cells in the nonlymphoid tissue of the synovial membrane of patients with rheumatoid arthritis. Proc. Natl. Acad. Sci. U.S.A. 93: 221–225.
Bridges SL Jr, Clausen BE, Lavelle JC, Fowler PG, Koopman WJ, Schroeder HW Jr. (1995) Analysis of immunoglobulin gamma heavy chains from rheumatoid arthritis synovium: evidence of antigen-driven selection. Ann. N.Y. Acad. Sci. 764: 450–452.
McBlane JF, van Gent DC, Ramsden DA, et al. (1995) Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83: 387–395.
Gellert M. (1997) Recent advances in understanding V(D)J recombination. Adv. Immunol. 64: 39–64.
Prak EL, Weigert M. (1995) Light chain replacement: a new model for antibody gene rearrangement. J. Exp. Med. 182: 541–548.
Chen C, Prak EL, Weigert M. (1997) Editing disease-associated autoantibodies. Immunity 6: 97–105.
Pelanda R, Schwers S, Sonoda E, Torres RM, Nemazee D, Rajewsky K. (1997) Receptor editing in a transgenic mouse model: site, efficiency, and role in B cell tolerance and antibody diversification. Immunity 7: 765–775.
Han S, Dillon SR, Zheng B, Shimoda M, Schlissel MS, Kelsoe G. (1997) V(D)J recombinase activity in a subset of germinal center B lymphocytes. Science 278: 301–305.
Papavasiliou F, Casellas R, Suh H, et al. (1997) V(D)J recombination in mature B cells: a mechanism for altering antibody responses. Science 278: 298–301.
Han S, Zheng B, Schatz DG, Spanopoulou E, Kelsoe G. (1996) Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells. Science 274: 2094–2097.
Ziff M. (1974) Relation of cellular infiltration of rheumatoid synovial membrane to its immune response. Arthritis Rheum. 17: 313–319.
Iguchi T, Ziff M. (1986) Electron microscopic study of rheumatoid synovial vasculature. Intimate relationship between tall endothelium and lymphoid aggregation. J. Clin. Invest. 77: 355–361.
van Dinther-Janssen AC, Pals ST, Scheper R, Breedveld F, Meijer CJ. (1990) Dendritic cells and high endothelial venules in the rheumatoid synovial membrane. J. Rheumatol. 17: 11–17.
Randen I, Mellbye OJ, Førre O, Natvig JB. (1995) The identification of germinal centres and follicular dendritic networks in rheumatoid synovial tissue. Scand. J. Immunol. 41: 481–486.
Steere AC, Duray PH, Butcher EC. (1988) Spirochetal antigens and lymphoid cell surface markers in Lyme synovitis. Comparison with rheumatoid synovium and tonsillar lymphoid tissue. Arthritis Rheum. 31: 487–495.
Schröder ARE, Sieper J, Berek C. (1997) Antigen-dependent B cell differentiation in the synovial tissue of a patient with reactive arthritis. Mol. Med. 3: 260–272.
Kumon I. (1992) In situ characterization of mononuclear cell phenotype in intrahepatic lymphoid follicles in patients with chronic viral hepatitis. Gastroenterol. Jpn. 27: 638–645.
Freni MA, Artuso D, Gerken G, et al. (1995) Focal lymphocytic aggregates in chronic hepatitis C: occurrence, immunohistochemical characterization, and relation to markers of autoimmunity. Hepatology 22: 389–394.
Mosnier JF, Degott C, Marcellin P, Henin D, Erlinger S, Benhamou JP. (1993) The intraportal lymphoid nodule and its environment in chronic active hepatitis C: an immunohistochemical study. Hepatology 17: 366–371.
Acknowledgments
This work was supported by a VA Merit Review grant and NIH grant AR44243. The technical assistance of Stephanie Byer and Jennifer Collins is greatly appreciated. The author thanks Drs. Ralf Küppers, Peter Burrows, Zhixin Zhang, and Harry W. Schroeder, Jr., for critical review of the manuscript.
This work was published in part in abstract form in Arthritis Rheum. 39(Suppl.): S159, 1996 and Arthritis Rheum. 40(9Suppl.): S246, 1997.
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Bridges, S.L. Frequent N Addition and Clonal Relatedness among Immunoglobulin Lambda Light Chains Expressed in Rheumatoid Arthritis Synovia and PBL, and the Influence of Vλ Gene Segment Utilization on CDR3 Length. Mol Med 4, 525–553 (1998). https://doi.org/10.1007/BF03401757
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DOI: https://doi.org/10.1007/BF03401757