Developmental and Comparative Immunology 123 (2021) 104141 Available online 24 May 2021 0145-305X/© 2021 Elsevier Ltd. All rights reserved. Full Length Article Developmental and comparative immunology single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica) Andrea L. Schraven a , Victoria L. Hansen b , Kimberly A. Morrissey b , Hayley J. Stannard c , Oselyne TW. Ong d , Daniel C. Douek e , Robert D. Miller b , Julie M. Old a, * a School of Science and Health, Hawkesbury Campus, Western Sydney University, Locked Bag 1797, Penrith, NSW, 2751, Australia b Center for Evolutionary and Theoretical Immunology, Department of Biology, University of New Mexico Albuquerque, New Mexico, USA c Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga, NSW, 2678, Australia d Children’s Medical Research Institute, Westmead, NSW, 2145, Australia e Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA A R T I C L E INFO Keywords: Marsupial B-cells IgH Igλ Igκ ABSTRACT B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specifc antibodies in the presence of pathogens. The marsupial immuno- globulin (Ig) heavy (H) chain locus encodes four constant region isotypes, IgA, IgG, IgM and IgE, but no IgD, and there are two light (L) chain isotypes, lambda (Igλ) and kappa (Igκ). To gain an understanding of the marsupial humoral immune system, B-cell transcriptomes generated by single-cell RNA sequencing from gray short-tailed opossum (Monodelphis domestica) splenocytes, and peripheral blood mononuclear cells were analysed. The cells used were from a single unimmunized animal and the majority of B-cells were transcribing IgM heavy chains. The ratio of Ig light chain use was roughly 2:1, Igλ:Igκ in this individual. This was not predicted due to Igκ being the more complex of the two L chain loci. The variable (V) gene segment pairs used in individual B-cells confrm greater diversity provided by the L chain V. This study is the frst to report on using single cell analysis to investigate Ig repertoires in a marsupial and confrms a number of prior hypothesis, as well as revealing some surprises. 1. Introduction Typically, Ig molecules comprise two identical IgH and two identical IgL chains, which are paired together on the surface of the B-cell. Both of these chains have a variable (V) domain responsible for binding to an antigen (Alt et al., 1992), and a constant (C) domain which in the heavy chain determines the effector function of the Ig molecule, however no defnitive function has been determined for the C domains of the light chain (Belov et al., 2001). Ig isotypes are determined by their C H domain, where four of the fve mammalian IgH isotypes have been confrmed in the gray short-tailed opossum (Monodelphis domestica). Previous investigations have isolated IgM, IgG, IgA, and IgE heavy chain isotypes (Miller et al., 1998; Aveskogh and Hellman, 1998; Belov et al., 1998, 1999a, 1999b, 1999c; Aveskogh et al., 1999); however, unlike eutherians and monotremes, IgD has not been found in any marsupial species to date. The genomic region predicted to encode the IgD constant domain in the opossum appears to be replaced with insertions of large, duplicated regions of repetitive DNA (Wang et al., 2009). It is not known whether these insertions are typical for all marsupials, or just the opossum. Although, it is clear the absence of IgD is due to a gene loss in the opossum, the presence of IgD in both eutherian and monotreme species (and some non-mammalian vertebrates) is consistent with IgD being ancient in vertebrates (Ohta and Flajnik, 2006; Wang et al., 2009). Like all mammals, marsupials have two IgL isotypes; kappa (Igκ) and lambda (Igλ) (Lucero et al., 1998). In the opossum Igκ, the V and joining (J) gene segments are present in different clusters, upstream of a single C κ domain (Miller et al., 1999), whereas Igλ has multiple J λ -C λ pairs (Lucero et al., 1998; Wang et al., 2009). The specifcity of individual antibodies is determined by the com- bination of V domains located on the N-terminal ends of the IgH and IgL * Corresponding author. E-mail address: j.old@westernsydney.edu.au (J.M. Old). Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/devcompimm https://doi.org/10.1016/j.dci.2021.104141 Received 9 October 2020; Received in revised form 18 May 2021; Accepted 19 May 2021