Skip to main content
SpringerLink
Log in

Llama peripheral B-cell populations producing conventional and heavy chain-only IgG subtypes are phenotypically indistinguishable but immunogenetically distinct

  • Original Article
  • Published:
Immunogenetics Aims and scope Submit manuscript

A Correction to this article was published on 13 February 2023

This article has been updated

Abstract

Camelid ungulates produce homodimeric heavy chain-only antibodies (HCAbs) in addition to conventional antibodies consisting of paired heavy and light chains. In the llama, HCAbs are made up by at least two subclasses (long-hinge IgG2b and short-hinge IgG2c HCAbs vs. conventional heterotetrameric IgG1s). Here, we generated murine monoclonal antibodies (mAbs) specific for the hinge-CH2 boundary of llama IgG2b (mAb 1C10) and the Fc of llama IgG2c HCAbs (mAb 5E4). Flow cytometric analysis of llama peripheral blood lymphocytes revealed that IgG1+, IgG2b+ and IgG2c+ B cells could be distinguished using mAbs 1C10/5E4 but had equivalent expression of three other cell-surface markers. MiSeq sequencing of the peripheral B cell repertoires of three llamas showed that (i) IgG2b and IgG2c HCAbs were present in similar proportions in the repertoire, (ii) a subset of IgG2b and IgG2c HCAbs, but not IgG1s, entirely lacked a hinge exon and showed direct VHH-CH2 splicing; these “hingeless” HCAbs were clonally expanded, somatically mutated and derived from hinged HCAb precursors, (iii) substantial repertoire overlap existed between IgG subclasses, especially between IgG2b and IgG2c HCAbs, (iv) the complementarity-determining region (CDR)-H3 length distributions of IgG2b and IgG2c HCAbs were broader and biased towards longer lengths compared with IgG1s due to increased N-nucleotide addition, (v) IgG2b and IgG2c HCAbs used a more restricted set of IGHV genes compared with IgG1s, and (vi) IgG2b and IgG2c HCAbs had elevated somatic mutations rates of both CDRs and framework regions (FRs) compared with IgG1s, especially of CDR-H1 and FR3. The distinct molecular features of llama IgG1, IgG2b and IgG2c antibodies imply that these subclasses may have divergent immunological functions and suggest that specific mechanisms operate to diversify HCAb repertoires in the absence of a light chain.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Change history

Notes

  1. Requests for the anti-isotypic mAbs specific for camelid IgG subtypes described here should be directed to the corresponding author. The mAbs and/or their sequences should be available for non-commercial use under MTA.

References

  • Achour I, Cavelier P, Tichit M, Bouchier C, Lafaye P, Rougeon F (2008) Tetrameric and homodimeric camelid IgGs originate from the same IgH locus. J Immunol 181:2001–2009

    Article  CAS  PubMed  Google Scholar 

  • Alamyar E, Duroux P, Lefranc MP, Giudicelli V (2012) IMGT(®) tools for the nucleotide analysis of immunoglobulin (IG) and T cell receptor (TR) V-(D)-J repertoires, polymorphisms, and IG mutations: IMGT/V-QUEST and IMGT/HighV-QUEST for NGS. Methods Mol Biol 882:569–604

    Article  CAS  PubMed  Google Scholar 

  • Baral TN, MacKenzie R, Arbabi Ghahroudi M (2013) Single-domain antibodies and their utility. Curr Protoc Immunol 103:Unit 2:17

  • Bodenhofer U, Bonatesta E, Horejs-Kainrath C, Hochreiter S (2015) msa: an R package for multiple sequence alignment. Bioinformatics 31:3997–3999

    CAS  PubMed  Google Scholar 

  • Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brooks CL, Rossotti MA, Henry KA (2018) Immunological functions and evolutionary emergence of heavy-chain antibodies. Trends Immunol 39:956–960

    Article  CAS  PubMed  Google Scholar 

  • Conrath KE, Wernery U, Muyldermans S, Nguyen VK (2003) Emergence and evolution of functional heavy-chain antibodies in Camelidae. Dev Comp Immunol 27:87–103

    Article  CAS  PubMed  Google Scholar 

  • Cox AD, St Michael F, Aubry A, Cairns CM, Strong PC, Hayes AC, Logan SM (2013) Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection. Glycoconj J 30:843–855

    Article  CAS  PubMed  Google Scholar 

  • Daley LP, Gagliardo LF, Duffy MS, Smith MC, Appleton JA (2005) Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids. Clin Diagn Lab Immunol 12:380–386

    CAS  PubMed  PubMed Central  Google Scholar 

  • de Jong BG, H IJ, Marques L, van der Burg M, van Dongen JJ, Loos BG, van Zelm MC (2017) Human IgG2- and IgG4-expressing memory B cells display enhanced molecular and phenotypic signs of maturity and accumulate with age. Immunol Cell Biol 95:744–752

    Article  PubMed  PubMed Central  Google Scholar 

  • Deschacht N, De Groeve K, Vincke C, Raes G, De Baetselier P, Muyldermans S (2010) A novel promiscuous class of camelid single-domain antibody contributes to the antigen-binding repertoire. J Immunol 184:5696–5704

    Article  CAS  PubMed  Google Scholar 

  • Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30:E9–E99

  • Feige MJ, Grawert MA, Marcinowski M, Hennig J, Behnke J, Auslander D, Herold EM, Peschek J, Castro CD, Flajnik M, Hendershot LM, Sattler M, Groll M, Buchner J (2014) The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins. Proc Natl Acad Sci U S A 111:8155–8160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173

    Article  CAS  PubMed  Google Scholar 

  • Griffin LM, Snowden JR, Lawson AD, Wernery U, Kinne J, Baker TS (2014) Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species. J Immunol Methods 405:35–46

    Article  CAS  PubMed  Google Scholar 

  • Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448

    Article  CAS  PubMed  Google Scholar 

  • Harmsen MM, Ruuls RC, Nijman IJ, Niewold TA, Frenken LG, de Geus B (2000) Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features. Mol Immunol 37:579–590

    Article  CAS  PubMed  Google Scholar 

  • Henry KA (2018) Next-generation DNA sequencing of VH/VL repertoires: a primer and guide to applications in single-domain antibody discovery. Methods Mol Biol 1701:425–446

    Article  CAS  PubMed  Google Scholar 

  • Henry KA, Hussack G, Collins C, Zwaagstra JC, Tanha J, MacKenzie CR (2016a) Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing. Protein Eng Des Sel 29:439–443

    Article  CAS  PubMed  Google Scholar 

  • Henry KA, MacKenzie CR (2018) Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs 10:815–826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Henry KA, Sulea T, van Faassen H, Hussack G, Purisima EO, MacKenzie CR, Arbabi-Ghahroudi M (2016b) A rational engineering strategy for designing protein A-binding camelid single-domain antibodies. PLoS One 11:e0163113

    Article  PubMed  PubMed Central  Google Scholar 

  • Henry KA, Tanha J, Hussack G (2015) Identification of cross-reactive single-domain antibodies against serum albumin using next-generation DNA sequencing. Protein Eng Des Sel 28:379–383

    Article  CAS  PubMed  Google Scholar 

  • Holzlohner P, Butze M, Maier N, Hebel N, Schliebs E, Micheel B, Funer J, Heidicke G, Hanack K (2018) Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3. Vet Immunol Immunopathol 197:1–6

    Article  PubMed  Google Scholar 

  • Klarenbeek A, El Mazouari K, Desmyter A, Blanchetot C, Hultberg A, de Jonge N, Roovers RC, Cambillau C, Spinelli S, Del-Favero J, Verrips T, de Haard HJ, Achour I (2015) Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform. MAbs 7:693–706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–1645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Holzer W, De Genst E, Wyns L, Muyldermans S (1998) Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J 17:3512–3520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li X, Duan X, Yang K, Zhang W, Zhang C, Fu L, Ren Z, Wang C, Wu J, Lu R, Ye Y, He M, Nie C, Yang N, Wang J, Yang H, Liu X, Tan W (2016) Comparative analysis of immune repertoires between Bactrian camel's conventional and heavy-chain antibodies. PLoS One 11:e0161801

    Article  PubMed  PubMed Central  Google Scholar 

  • Maass DR, Sepulveda J, Pernthaner A, Shoemaker CB (2007) Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J Immunol Methods 324:13–25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Magoc T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Manceur AP, Zou W, Marcil A, Paquet E, Gadoury C, Jaentschke B, Li X, Petiot E, Durocher Y, Baardsnes J, Rosa-Calatrava M, Ansorge S, Kamen AA (2017) Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza. PLoS One 12:e0180314

    Article  PubMed  PubMed Central  Google Scholar 

  • Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797

    Article  CAS  PubMed  Google Scholar 

  • Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7:1129–1135

    Article  CAS  PubMed  Google Scholar 

  • Nguyen VK, Hamers R, Wyns L, Muyldermans S (1999) Loss of splice consensus signal is responsible for the removal of the entire CH1 domain of the functional camel IgG2a heavy-chain antibodies. Mol Immunol 36:515–524

    Article  CAS  PubMed  Google Scholar 

  • Nguyen VK, Hamers R, Wyns L, Muyldermans S (2000) Camel heavy-chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen-binding repertoire. EMBO J 19:921–930

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nguyen VK, Muyldermans S, Hamers R (1998) The specific variable domain of camel heavy-chain antibodies is encoded in the germline. J Mol Biol 275:413–418

    Article  CAS  PubMed  Google Scholar 

  • Nguyen VK, Su C, Muyldermans S, van der Loo W (2002) Heavy-chain antibodies in Camelidae; a case of evolutionary innovation. Immunogenetics 54:39–47

    Article  CAS  PubMed  Google Scholar 

  • Pluciennik A, Stolarczyk M, Bzowka M, Raczynska A, Magdziarz T, Gora A (2018) BALCONY: an R package for MSA and functional compartments of protein variability analysis. BMC Bioinformatics 19:300

    Article  PubMed  PubMed Central  Google Scholar 

  • Rast JP, Amemiya CT, Litman RT, Strong SJ, Litman GW (1998) Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes. Immunogenetics 47:234–245

    Article  CAS  PubMed  Google Scholar 

  • Saccodossi N, De Simone EA, Leoni J (2012) Structural analysis of effector functions related motifs, complement activation and hemagglutinating activities in Lama glama heavy chain antibodies. Vet Immunol Immunopathol 145:323–331

    Article  CAS  PubMed  Google Scholar 

  • Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sun Y, Gadoury C, Hirakawa MP, Bennett RJ, Harcus D, Marcil A, Whiteway M (2016) Deletion of a Yci1 domain protein of Candida albicans allows homothallic mating in MTL heterozygous cells. MBio 7:e00465–e00416

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • van der Linden R, de Geus B, Stok W, Bos W, van Wassenaar D, Verrips T, Frenken L (2000) Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama. J Immunol Methods 240:185–195

    Article  PubMed  Google Scholar 

  • Vu KB, Ghahroudi MA, Wyns L, Muyldermans S (1997) Comparison of llama VH sequences from conventional and heavy chain antibodies. Mol Immunol 34:1121–1131

    Article  CAS  PubMed  Google Scholar 

  • Woolven BP, Frenken LG, van der Logt P, Nicholls PJ (1999) The structure of the llama heavy chain constant genes reveals a mechanism for heavy-chain antibody formation. Immunogenetics 50:98–101

    Article  CAS  PubMed  Google Scholar 

  • Zou X, Osborn MJ, Bolland DJ, Smith JA, Corcos D, Hamon M, Oxley D, Hutchings A, Morgan G, Santos F, Kilshaw PJ, Taussig MJ, Corcoran AE, Bruggemann M (2007) Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice. J Exp Med 204:3271–3283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge the excellent technical assistance of Christine Gadoury and Mike Lowden. We thank Tammy-Lynn Tremblay for preparing LC-MS samples.

Funding

This work was funded by the National Research Council Canada.

Author information

Authors and Affiliations

  1. Human Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, K1A 0R6, Canada

    Kevin A. Henry, Henk van Faassen, Jennifer J. Hill & C. Roger MacKenzie

  2. Human Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, Montréal, QC, H4P 2R2, Canada

    Doreen Harcus & Anne Marcil

  3. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium

    Serge Muyldermans

  4. School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada

    C. Roger MacKenzie

Authors
  1. Kevin A. Henry
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Henk van Faassen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Doreen Harcus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anne Marcil
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jennifer J. Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Serge Muyldermans
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. C. Roger MacKenzie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kevin A. Henry.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 1269 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Henry, K.A., van Faassen, H., Harcus, D. et al. Llama peripheral B-cell populations producing conventional and heavy chain-only IgG subtypes are phenotypically indistinguishable but immunogenetically distinct. Immunogenetics 71, 307–320 (2019). https://doi.org/10.1007/s00251-018-01102-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00251-018-01102-9

Keywords

Search

Navigation