Antibody Repertoire Development in Fetal and Neonatal Piglets. XI. The Relationship of Variable Heavy Chain Gene Usage and the Genomic Organization of the Variable Heavy This information is current as Chain Locus of September 27, 2021. Tomoko Eguchi-Ogawa, Nancy Wertz, Xiu-Zhu Sun, Francois Puimi, Hirohide Uenishi, Kevin Wells, Patrick Chardon, Gregory J. Tobin and John E. Butler J Immunol published online 5 March 2010 Downloaded from http://www.jimmunol.org/content/early/2010/03/05/jimmun ol.0903616 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/03/05/jimmunol.090361 Material 6.DC1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/190/3/1383.full.pdf The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 5, 2010, doi:10.4049/jimmunol.0903616 The Journal of Immunology Antibody Repertoire Development in Fetal and Neonatal Piglets. XI. The Relationship of Variable Heavy Chain Gene Usage and the Genomic Organization of the Variable Heavy Chain Locus Tomoko Eguchi-Ogawa,* Nancy Wertz,† Xiu-Zhu Sun,† Francois Puimi,‡ Hirohide Uenishi,* Kevin Wells,x Patrick Chardon,‡ Gregory J. Tobin,{ and John E. Butler† In this study, we have mapped the 39 H chain V region (VH) genes and those in the H chain diversity, H chain joining, and 59 portion of the H chain constant locus. We show that swine possess only two functional H chain diversity segments and only one functional H chain Downloaded from joining segment. These data help to explain more than a decade of observations on the preimmune repertoire of this species and reveal the vulnerability of swine to natural or designed mutational events. The results are consistent with earlier studies on the region containing Enh, Cm, and Cd while revealing that the ancestral IgG3 is the most 59 Cg gene. We also observed a recent duplication (∼1.6 million years ago) in the VH locus that contains six of the seven VH genes that comprise 75% of the preimmune repertoire. Because there are no known transfers of immune regulators or Ags that cross the placenta as in mice and humans, fetal VH usage must be intrinsically regulated. Therefore, we quantified VH usage in fetal piglets and demonstrated that usage is independent of the http://www.jimmunol.org/ position of VH genes in the genome; the most 39 functional VH gene (IGHV2) is rarely used, whereas certain upstream genes (IGHV14 and IGHV15) are predominately used early in fetal liver but seldom thereafter. Similar to previous studies, three VH genes account for 40% of the repertoire and six for ∼70%. This limited combinatorial diversity of the porcine VH repertoire further emphasizes the dependence on CDR3 diversity for generating the preimmune Ab repertoire of this species. The Journal of Immunology, 2010, 184: 000–000. he H chain Ig variable locus is organized similarly in all functional, whereas 27 DH segments can be expressed, but only six of by guest on September 27, 2021 mammals that have been studied. As few as 12 to .1000 H the nine JH segments are functional (2, 4). The VH genes of verte- T chain V region (VH) genes are thought to be arrayed 59 of brates belong to different families that have been grouped into three multiple H chain diversity (DH) segments that in turn are found 59 of clans based on sequence homologies (5). There are large differences up to nine H chain joining (JH) segments (1, 2). VH genes charac- in the number of VH genes and VH gene families among species. In terized to date include many pseudogenes (3), such as those with swine, ,30 genes have been described, all belonging to the ancestral nonconsensus signal sequences, stop codons, frameshift mutations, VH3 family (6, 7) and have identical or nearly identical framework or the absence of functional leader sequences. In humans, for ex- regions but differ in their CDR1 and CDR2 regions (8, 9). However, ample, only 38–46 of the .100 VH genes (haplotype dependent) are CDR regions are often shared among different porcine VH genes, suggesting they could be a consequence of gene duplication com- bined with a type of genomic gene conversion (10, 11). † *National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan; Department The distribution of V gene segment usage in fetal and newborn of Microbiology, University of Iowa, Iowa City, IA 52242; ‡National Institute for Agro- H nomic Research, Institute of Molecular Biology, Jouy-en-Josas, France; xDivision of mice, humans, rabbits, and swine is nonrandom, and usage patterns { Animal Sciences, University of Missouri, Columbia, MO 65211; and Biological Mim- vary during ontogeny. In the fetal liver of mice, members of the VH5 etics, Frederick, MD 21702 and VH2 families (especially 7183 and Q52, respectively) are Received for publication November 11, 2009. Accepted for publication February 1, preferentially used (12, 13), but after birth, splenic B cells use the 2010. VH1 family (J558) (14). The VH2 and VH5 family genes are the most This work was supported by National Science Foundation-Integrative Organismal Systems Grant 15203800 and Grant 58-1265-5-053 from the National Pork Board 39 in the locus, whereas the J558 family is midway (more 59) in the and subaward C080090-UI from Biological Mimetics. locus, suggesting that VH usage in the preimmune repertoire favors The sequences presented in this article have been submitted to GenBank under 39 VH genes but that usage shifts upstream, presumably due to ex- accession numbers AB513624 and AB513625. posure to environmental Ag because this shift does not occur in Address correspondence and reprint requests to Dr. John E. Butler, Interdisciplinary germ-free mice (14, 15). A preferential usage of the most JH prox- Immunology Program, Department of Microbiology, University of Iowa, 51 Newton imal V 3 family genes has also been reported in humans (16, 17). Road, Iowa City, IA 52242. E-mail address: [email protected] H Early V usage in rabbits favors the most 39 V gene (V 1) in 90% The online version of this article contains supplemental material. H H H of VDJ rearrangements, but this preferential usage declines during Abbreviations used in this paper: BAC, bacterial artificial chromosome; CH, H chain development and with exposure to environmental Ag (18). How- constant; DG, day of gestation; DH, H chain diversity; FR, framework region; IMGT, ImMunoGeneTics; IPP, ileal Peyer’s patches; JH, H chain joining; LINE, long in- ever, developmental repertoire changes in rabbits are compounded terspersed nucleotide element; RSS, recombination signal sequence; VH, H chain V by somatic gene conversion, a phenomenon rarely seen in mice (19). region. The pattern of preferential usage of 39 VH genes is not supported in Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 all studies. In humans, V3-23 and V3-30 are preferentially used in the www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903616 2 VH USAGE AND GENOME ORGANIZATION preimmune repertoire, but these map to the middle of the locus (20). region of the H chain locus that includes 15 DH proximal VH genes Alternatives to the locus position concept include: 1) negative se- and the 59 portion of the H chain constant (CH) genome. Our map lection for B cells utilizing VH genes that recognize certain self-Ags; shows that swine possess only one functional JH gene segment, ap- 2) positive selection for B cells by stromal ligands based on features parently two functional DH segments, and that preferential VH usage of their BCR; and 3) the evolutionary conservation of BCRs bearing in primary B cell tissues of fetal animals cannot be explained as VH genes that encode binding sites that recognize bacteria that a stochastic process dependent on the location of VH genes in the threaten the newborn. The basis for 2) and 3) above could be the same. locus. In contrast, the dominant early expression of porcine IgG3 in For example, human V3-23, mouse VH283 (a VH2 family gene), and late-term fetuses in an organ believed to develop an Ag-independent shark VH genes share .80% sequence similarity at the protein level repertoire appears correlated with its position in the H chain C region (7, 21) and encode BCRs that are polyreactive and recognize bacterial of the locus. polysaccharides (22). In humans and mice, such Abs are often as- sociated with IgG3 (23, 24) and class switch that occurs in the ab- Materials and Methods sence of environmental Ag (25, 26) and in CD40/CD40L-deficient Nomenclature mice (27). In swine, IgG3 expression accounts for .60% of the total The porcine VH genes were originally named according to their order of IgG expression in the ileal Peyer’s patches (IPP) of fetal piglets discovery and given vernacular names like VHA and VHB (Table I) (9). (28). The IPP of swine is the anatomical homolog of the IPP of sheep, The intention was that once their location in the genome was known, the nomenclature would be changed to the familiar VH1, VH2, etc., system or in which Ab repertoire development is considered to be Ag-in- the ImMunoGeneTics (IMGT) system (IGHV1, IGHV2).