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

  • Kevin A. HenryEmail author
  • Henk van Faassen
  • Doreen Harcus
  • Anne Marcil
  • Jennifer J. Hill
  • Serge Muyldermans
  • C. Roger MacKenzie
Original Article

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.

Keywords

Heavy chain-only antibody Single-domain antibody VHLlama Immunogenetics Next-generation DNA sequencing 

Notes

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 information

This work was funded by the National Research Council Canada.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

251_2018_1102_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1269 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Kevin A. Henry
    • 1
    Email author
  • Henk van Faassen
    • 1
  • Doreen Harcus
    • 2
  • Anne Marcil
    • 2
  • Jennifer J. Hill
    • 1
  • Serge Muyldermans
    • 3
  • C. Roger MacKenzie
    • 1
    • 4
  1. 1.Human Health Therapeutics Research CentreNational Research Council CanadaOttawaCanada
  2. 2.Human Health Therapeutics Research CentreNational Research Council CanadaMontréalCanada
  3. 3.Laboratory of Cellular and Molecular ImmunologyVrije Universiteit BrusselBrusselsBelgium
  4. 4.School of Environmental SciencesUniversity of GuelphGuelphCanada

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