Abstract
To analyze the immunoglobulin repertoire of human IgM+ B cells and the CD5(+) and CD5(-) subsets, individual CD19(+)/ IgM+/CD5(+) or CD5(-) B cells were sorted and non-productive as well as productive VH gene rearrangements were amplified from genomic DNA and sequenced. In both subsets, the VH3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members. In the CD5(+) B cell subset, all other VH families were found at a frequency expected from random usage, whereas in the CD5(-) population, VH4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the VH1 family was significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used VH gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells. Evidence for selection based on the D segment and the JH gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity. Somatically hypermutated VHDJH rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM+ B cells, indicating that IgM+ memory B cells were more frequent in the CD5(-) B cell population. Of note, the frequency of specific VH genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulation imposed additional biases on the repertoire of IgM+ B cells. These results indicate that the expressed repertoire of IgM+ B cell subsets is shaped by recombinational bias, as well as selection before and after antigen exposure. Moreover, the influences on the repertoires of CD5(+) and CD5(-) B cells are significantly different, suggesting that human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, respectively.
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- Alt F. W., Blackwell T. K., Yancopoulos G. D. Development of the primary antibody repertoire. Science. 1987 Nov 20;238(4830):1079–1087. doi: 10.1126/science.3317825. [DOI] [PubMed] [Google Scholar]
- Braun J., King L. Unique V gene usage by B-Ly1 cell lines, and a discordance between isotype switch commitment and variable region hypermutation. J Mol Cell Immunol. 1989;4(3):121–128. [PubMed] [Google Scholar]
- Brezinschek H. P., Brezinschek R. I., Lipsky P. E. Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J Immunol. 1995 Jul 1;155(1):190–202. [PubMed] [Google Scholar]
- Buluwela L., Albertson D. G., Sherrington P., Rabbitts P. H., Spurr N., Rabbitts T. H. The use of chromosomal translocations to study human immunoglobulin gene organization: mapping DH segments within 35 kb of the C mu gene and identification of a new DH locus. EMBO J. 1988 Jul;7(7):2003–2010. doi: 10.1002/j.1460-2075.1988.tb03039.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casali P., Burastero S. E., Nakamura M., Inghirami G., Notkins A. L. Human lymphocytes making rheumatoid factor and antibody to ssDNA belong to Leu-1+ B-cell subset. Science. 1987 Apr 3;236(4797):77–81. doi: 10.1126/science.3105056. [DOI] [PubMed] [Google Scholar]
- Chu Y. W., Marin E., Fuleihan R., Ramesh N., Rosen F. S., Geha R. S., Insel R. A. Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome. J Clin Invest. 1995 Mar;95(3):1389–1393. doi: 10.1172/JCI117791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Decker D. J., Linton P. J., Zaharevitz S., Biery M., Gingeras T. R., Klinman N. R. Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro. Immunity. 1995 Feb;2(2):195–203. doi: 10.1016/s1074-7613(95)80092-1. [DOI] [PubMed] [Google Scholar]
- Ebeling S. B., Schutte M. E., Logtenberg T. Peripheral human CD5+ and CD5- B cells may express somatically mutated VH5- and VH6-encoded IgM receptors. J Immunol. 1993 Dec 15;151(12):6891–6899. [PubMed] [Google Scholar]
- Ebeling S. B., Schutte M. E., Logtenberg T. The majority of human tonsillar CD5+ B cells express somatically mutated V kappa 4 genes. Eur J Immunol. 1993 Jun;23(6):1405–1408. doi: 10.1002/eji.1830230636. [DOI] [PubMed] [Google Scholar]
- Gu H., Tarlinton D., Müller W., Rajewsky K., Förster I. Most peripheral B cells in mice are ligand selected. J Exp Med. 1991 Jun 1;173(6):1357–1371. doi: 10.1084/jem.173.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guigou V., Cuisinier A. M., Tonnelle C., Moinier D., Fougereau M., Fumoux F. Human immunoglobulin VH and VK repertoire revealed by in situ hybridization. Mol Immunol. 1990 Sep;27(9):935–940. doi: 10.1016/0161-5890(90)90161-r. [DOI] [PubMed] [Google Scholar]
- Hardy R. R., Hayakawa K. CD5 B cells, a fetal B cell lineage. Adv Immunol. 1994;55:297–339. doi: 10.1016/s0065-2776(08)60512-x. [DOI] [PubMed] [Google Scholar]
- Huang C., Stollar B. D. A majority of Ig H chain cDNA of normal human adult blood lymphocytes resembles cDNA for fetal Ig and natural autoantibodies. J Immunol. 1993 Nov 15;151(10):5290–5300. [PubMed] [Google Scholar]
- Ichihara Y., Abe M., Yasui H., Matsuoka H., Kurosawa Y. At least five DH genes of human immunoglobulin heavy chains are encoded in 9-kilobase DNA fragments. Eur J Immunol. 1988 Apr;18(4):649–652. doi: 10.1002/eji.1830180426. [DOI] [PubMed] [Google Scholar]
- Ichihara Y., Matsuoka H., Kurosawa Y. Organization of human immunoglobulin heavy chain diversity gene loci. EMBO J. 1988 Dec 20;7(13):4141–4150. doi: 10.1002/j.1460-2075.1988.tb03309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacob J., Przylepa J., Miller C., Kelsoe G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J Exp Med. 1993 Oct 1;178(4):1293–1307. doi: 10.1084/jem.178.4.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jelinek D. F., Lipsky P. E. Comparative activation requirements of human peripheral blood, spleen, and lymph node B cells. J Immunol. 1987 Aug 15;139(4):1005–1013. [PubMed] [Google Scholar]
- Kasaian M. T., Ikematsu H., Casali P. Identification and analysis of a novel human surface CD5- B lymphocyte subset producing natural antibodies. J Immunol. 1992 May 1;148(9):2690–2702. [PMC free article] [PubMed] [Google Scholar]
- Kelsoe G. Life and death in germinal centers (redux). Immunity. 1996 Feb;4(2):107–111. doi: 10.1016/s1074-7613(00)80675-5. [DOI] [PubMed] [Google Scholar]
- Kipps T. J., Duffy S. F. Relationship of the CD5 B cell to human tonsillar lymphocytes that express autoantibody-associated cross-reactive idiotypes. J Clin Invest. 1991 Jun;87(6):2087–2096. doi: 10.1172/JCI115239. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kipps T. J. The CD5 B cell. Adv Immunol. 1989;47:117–185. doi: 10.1016/s0065-2776(08)60663-x. [DOI] [PubMed] [Google Scholar]
- Kirkham P. M., Mortari F., Newton J. A., Schroeder H. W., Jr Immunoglobulin VH clan and family identity predicts variable domain structure and may influence antigen binding. EMBO J. 1992 Feb;11(2):603–609. doi: 10.1002/j.1460-2075.1992.tb05092.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kiyoi H., Naito K., Ohno R., Naoe T. Comparable profiles of the immunoglobulin heavy chain complementarity determining region (CDR)-3 in CD5+ and CD5- human cord blood B lymphocytes. Immunology. 1995 Jun;85(2):236–240. [PMC free article] [PubMed] [Google Scholar]
- Klein U., Küppers R., Rajewsky K. Human IgM+IgD+ B cells, the major B cell subset in the peripheral blood, express V kappa genes with no or little somatic mutation throughout life. Eur J Immunol. 1993 Dec;23(12):3272–3277. doi: 10.1002/eji.1830231232. [DOI] [PubMed] [Google Scholar]
- Klein U., Küppers R., Rajewsky K. Variable region gene analysis of B cell subsets derived from a 4-year-old child: somatically mutated memory B cells accumulate in the peripheral blood already at young age. J Exp Med. 1994 Oct 1;180(4):1383–1393. doi: 10.1084/jem.180.4.1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klinman N. R. In vitro analysis of the generation and propagation of memory B cells. Immunol Rev. 1996 Apr;150:91–111. doi: 10.1111/j.1600-065x.1996.tb00697.x. [DOI] [PubMed] [Google Scholar]
- Kraj P., Friedman D. F., Stevenson F., Silberstein L. E. Evidence for the overexpression of the VH4-34 (VH4.21) Ig gene segment in the normal adult human peripheral blood B cell repertoire. J Immunol. 1995 Jun 15;154(12):6406–6420. [PubMed] [Google Scholar]
- Kristiansen S. V., Pascual V., Lipsky P. E. Staphylococcal protein A induces biased production of Ig by VH3-expressing B lymphocytes. J Immunol. 1994 Oct 1;153(7):2974–2982. [PubMed] [Google Scholar]
- Kurosawa Y., Tonegawa S. Organization, structure, and assembly of immunoglobulin heavy chain diversity DNA segments. J Exp Med. 1982 Jan 1;155(1):201–218. doi: 10.1084/jem.155.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis S., Gifford A., Baltimore D. DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes. Science. 1985 May 10;228(4700):677–685. doi: 10.1126/science.3158075. [DOI] [PubMed] [Google Scholar]
- Logtenberg T., Schutte M. E., Ebeling S. B., Gmelig-Meyling F. H., van Es J. H. Molecular approaches to the study of human B-cell and (auto)antibody repertoire generation and selection. Immunol Rev. 1992 Aug;128:23–47. doi: 10.1111/j.1600-065x.1992.tb00831.x. [DOI] [PubMed] [Google Scholar]
- Lozano F., Maertzdorf B., Pannell R., Milstein C. Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing. EMBO J. 1994 Oct 3;13(19):4617–4622. doi: 10.1002/j.1460-2075.1994.tb06783.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melchers F., Rolink A., Grawunder U., Winkler T. H., Karasuyama H., Ghia P., Andersson J. Positive and negative selection events during B lymphopoiesis. Curr Opin Immunol. 1995 Apr;7(2):214–227. doi: 10.1016/0952-7915(95)80006-9. [DOI] [PubMed] [Google Scholar]
- Milili M., Schiff C., Fougereau M., Tonnelle C. The VDJ repertoire expressed in human preB cells reflects the selection of bona fide heavy chains. Eur J Immunol. 1996 Jan;26(1):63–69. doi: 10.1002/eji.1830260110. [DOI] [PubMed] [Google Scholar]
- Neuberger M. S., Milstein C. Somatic hypermutation. Curr Opin Immunol. 1995 Apr;7(2):248–254. doi: 10.1016/0952-7915(95)80010-7. [DOI] [PubMed] [Google Scholar]
- Nuñez C., Nishimoto N., Gartland G. L., Billips L. G., Burrows P. D., Kubagawa H., Cooper M. D. B cells are generated throughout life in humans. J Immunol. 1996 Jan 15;156(2):866–872. [PubMed] [Google Scholar]
- Pascual V., Capra J. D. Human immunoglobulin heavy-chain variable region genes: organization, polymorphism, and expression. Adv Immunol. 1991;49:1–74. doi: 10.1016/s0065-2776(08)60774-9. [DOI] [PubMed] [Google Scholar]
- Potter K. N., Li Y., Pascual V., Williams R. C., Jr, Byres L. C., Spellerberg M., Stevenson F. K., Capra J. D. Molecular characterization of a cross-reactive idiotope on human immunoglobulins utilizing the VH4-21 gene segment. J Exp Med. 1993 Oct 1;178(4):1419–1428. doi: 10.1084/jem.178.4.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Qian L., Vu M. N., Carter M. S., Doskow J., Wilkinson M. F. T cell receptor-beta mRNA splicing during thymic maturation in vivo and in an inducible T cell clone in vitro. J Immunol. 1993 Dec 15;151(12):6801–6814. [PubMed] [Google Scholar]
- Ravetch J. V., Siebenlist U., Korsmeyer S., Waldmann T., Leder P. Structure of the human immunoglobulin mu locus: characterization of embryonic and rearranged J and D genes. Cell. 1981 Dec;27(3 Pt 2):583–591. doi: 10.1016/0092-8674(81)90400-1. [DOI] [PubMed] [Google Scholar]
- Rettig M. B., Vescio R. A., Cao J., Wu C. H., Lee J. C., Han E., DerDanielian M., Newman R., Hong C., Lichtenstein A. K. VH gene usage is multiple myeloma: complete absence of the VH4.21 (VH4-34) gene. Blood. 1996 Apr 1;87(7):2846–2852. [PubMed] [Google Scholar]
- Sanz I. Multiple mechanisms participate in the generation of diversity of human H chain CDR3 regions. J Immunol. 1991 Sep 1;147(5):1720–1729. [PubMed] [Google Scholar]
- Sanz I., Wang S. S., Meneses G., Fischbach M. Molecular characterization of human Ig heavy chain DIR genes. J Immunol. 1994 Apr 15;152(8):3958–3969. [PubMed] [Google Scholar]
- Sasso E. H., Buckner J. H., Suzuki L. A. Ethnic differences of polymorphism of an immunoglobulin VH3 gene. J Clin Invest. 1995 Sep;96(3):1591–1600. doi: 10.1172/JCI118198. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sasso E. H., Johnson T., Kipps T. J. Expression of the immunoglobulin VH gene 51p1 is proportional to its germline gene copy number. J Clin Invest. 1996 May 1;97(9):2074–2080. doi: 10.1172/JCI118644. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sasso E. H., Willems van Dijk K., Bull A. P., Milner E. C. A fetally expressed immunoglobulin VH1 gene belongs to a complex set of alleles. J Clin Invest. 1993 Jun;91(6):2358–2367. doi: 10.1172/JCI116468. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schittek B., Rajewsky K. Maintenance of B-cell memory by long-lived cells generated from proliferating precursors. Nature. 1990 Aug 23;346(6286):749–751. doi: 10.1038/346749a0. [DOI] [PubMed] [Google Scholar]
- Schroeder H. W., Jr, Hillson J. L., Perlmutter R. M. Early restriction of the human antibody repertoire. Science. 1987 Nov 6;238(4828):791–793. doi: 10.1126/science.3118465. [DOI] [PubMed] [Google Scholar]
- Schwartz R. S., Stollar B. D. Heavy-chain directed B-cell maturation: continuous clonal selection beginning at the pre-B cell stage. Immunol Today. 1994 Jan;15(1):27–32. doi: 10.1016/0167-5699(94)90022-1. [DOI] [PubMed] [Google Scholar]
- Silberstein L. E., Jefferies L. C., Goldman J., Friedman D., Moore J. S., Nowell P. C., Roelcke D., Pruzanski W., Roudier J., Silverman G. J. Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens. Blood. 1991 Nov 1;78(9):2372–2386. [PubMed] [Google Scholar]
- Stall A. M., Wells S. M., Lam K. P. B-1 cells: unique origins and functions. Semin Immunol. 1996 Feb;8(1):45–59. doi: 10.1006/smim.1996.0007. [DOI] [PubMed] [Google Scholar]
- Stewart A. K., Huang C., Long A. A., Stollar B. D., Schwartz R. S. VH-gene representation in autoantibodies reflects the normal human B-cell repertoire. Immunol Rev. 1992 Aug;128:101–122. doi: 10.1111/j.1600-065x.1992.tb00834.x. [DOI] [PubMed] [Google Scholar]
- Suzuki I., Pfister L., Glas A., Nottenburg C., Milner E. C. Representation of rearranged VH gene segments in the human adult antibody repertoire. J Immunol. 1995 Apr 15;154(8):3902–3911. [PubMed] [Google Scholar]
- Tarlinton D. M., McLean M., Nossal G. J. B1 and B2 cells differ in their potential to switch immunoglobulin isotype. Eur J Immunol. 1995 Dec;25(12):3388–3393. doi: 10.1002/eji.1830251228. [DOI] [PubMed] [Google Scholar]
- Tornberg U. C., Holmberg D. B-1a, B-1b and B-2 B cells display unique VHDJH repertoires formed at different stages of ontogeny and under different selection pressures. EMBO J. 1995 Apr 18;14(8):1680–1689. doi: 10.1002/j.1460-2075.1995.tb07157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vernino L. A., Pisetsky D. S., Lipsky P. E. Analysis of the expression of CD5 by human B cells and correlation with functional activity. Cell Immunol. 1992 Jan;139(1):185–197. doi: 10.1016/0008-8749(92)90111-2. [DOI] [PubMed] [Google Scholar]
- Yamada M., Wasserman R., Reichard B. A., Shane S., Caton A. J., Rovera G. Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J Exp Med. 1991 Feb 1;173(2):395–407. doi: 10.1084/jem.173.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zouali M. B-cell superantigens: implications for selection of the human antibody repertoire. Immunol Today. 1995 Aug;16(8):399–405. doi: 10.1016/0167-5699(95)80009-3. [DOI] [PubMed] [Google Scholar]
- van Es J. H., Meyling F. H., Logtenberg T. High frequency of somatically mutated IgM molecules in the human adult blood B cell repertoire. Eur J Immunol. 1992 Oct;22(10):2761–2764. doi: 10.1002/eji.1830221046. [DOI] [PubMed] [Google Scholar]