Definition of immunoglobulin germline genes by next generation sequencing for studies of antigen-specific B cell responses

Immunoglobulins play a critical role in the adaptive immune system, existing as cell surface-expressed B cell receptors and secreted antibodies. Circulating antibodies are the main correlate of protective immunity for most vaccines. An improved understanding of the germline genes that rearrange to encode the vast repertoire of antibodies is therefore of central interest. Despite this, current databases of immunoglobulin germline gene variation are incomplete, both for humans and research animal models, limiting studies of antigen-specific B cell responses.

In Paper I, we developed a computational tool, IgDiscover, which infers germline immunoglobulin V alleles from the repertoire of expressed antibodies in a given individual. We validated IgDiscover for the identification of human, mouse and rhesus macaque IGHV alleles and described novel IGHV alleles in all three species. Our results highlighted a high degree of inter-individual allelic diversity in rhesus macaques. In Paper II, we optimized and compared two major immunoglobulin library production methods based on 5′RACE and 5′multiplex PCR, respectively. We observed that, despite 5′RACE being unbiased in terms of amplification and having the advantage of not requiring 5′ end IGHV genomic information, current limitations on high-throughput sequence read length resulted in the 5′ multiplex method delivering a higher quality output due to its shorter amplicon size. In Paper III, we inferred germline immunoglobulin alleles in 45 macaques from four sub-populations of the two most common species used in biomedical research, rhesus and cynomolgus macaques. We confirmed and extended our observations concerning high inter-individual diversity, demonstrating that it was highest among Indonesian cynomolgus macaques and lowest among Mauritian cynomolgus macaques in the sub-populations studied. We compiled comprehensive IGHV, D and J allele databases and used several methods to independently validate novel alleles.

In conclusion, the work presented in this thesis establishes a road map to generate individualized immunoglobulin germline gene databases from diverse species, even if genomic immunoglobulin loci information is limited. This thesis also examines the advantages and disadvantages of commonly used next generation sequencing library preparation methods. Finally, it reports novel inferred immunoglobulin alleles in humans and macaques and illustrates a high degree of inter-individual immunoglobulin allelic diversity in primates, underlining the utility of generating individualized immunoglobulin databases for studies of immune repertoires.

List of scientific papers

I. Martin M. Corcoran, Ganesh E. Phad, Néstor Vázquez Bernat, Christiane Stahl-Hennig, Noriyuki Sumida, Mats A.A. Persson, Marcel Martin, and Gunilla B. Karlsson Hedestam. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity. Nature Communications. 2016 Dec; 7:13642.
https://doi.org/10.1038/ncomms13642

II. Néstor Vázquez Bernat, Martin M. Corcoran, Uta Hardt, Mateusz Kaduk, Ganesh E. Phad, Marcel Martin and Gunilla B. Karlsson Hedestam. High-quality library preparation for NGS-based immunoglobulin germline gene inference and repertoire expression analysis. Frontiers in Immunology. 2019 Apr; 10:660.
https://doi.org/10.3389/fimmu.2019.00660

III. Néstor Vázquez Bernat, Izabela Nowak, Mateusz Kaduk, David Spencer, Nancy Haigwood, Pauline Maisonasse, Roger Le Grand, Martin M. Corcoran and Gunilla B. Karlsson Hedestam. High inter-individual diversity among rhesus and cynomolgus macaques revealed by immunoglobulin germline gene inference. [Manuscript]