Antibody Repertoire Development in Fetal and Neonatal Pigs. VII. Characterization of the Preimmune κ Light Chain Repertoire

Abstract

Combinatorial diversity is highly restricted in the preimmune porcine H chain repertoire compared with that in humans and mice. This raised the question of whether similar restriction characterized the preimmune L chain repertoire. In this study we present evidence that >90% of all expressed Vκ genes in the porcine preimmune repertoire belong to three subfamilies of Vκ genes that share 87% sequence similarity with human IGKV2. This porcine Vκ family also shares sequence similarity with some, but not all, Vκ genes from sheep. Hybridization with sperm DNA and sequence analyses of polynucleotides from overlapping bacterial artificial chromosome clones suggest swine possess ∼60 IGVK2 genes. The latter method also revealed that certain IGKV2 subfamilies are not expressed in the preimmune repertoire. Six members of an IGVK1 family were also expressed as part of the preimmune repertoire, and these shared 87% sequence similarity with human IGVK1. Five Jκ segments, complete with recombination signal sequences and separated by ∼300 nt, were identified ∼3 kb upstream of a single Cκ. Surprisingly, Jκ2 accounted for >90% of all framework region 4 sequences in the preimmune repertoire. These findings show that swine use ∼10 IGVK2 genes from three of six subfamilies and preferentially one Jκ segment to generate their preimmune κ repertoire. These studies, like those of porcine Ig constant regions and MHC genes, also indicate unexpected high sequence similarity with their human counterparts despite differences in phylogeny and the mechanism of repertoire diversification.

Targeted disruption of the porcine immunoglobulin kappa light chain locus

“…Thus, the single C region provides an ''Achilles heel'' ( Fig. 1; Butler et al 2004) that could be targeted to produce a null allele. To inactivate the j locus we deleted the Cj region, using a conventional gene targeting strategy that did not employ any enrichment strategies, following positive G418 antibiotic selection.…”

“…The size of the pig j Ig locus is in the mega-base range and is very complex comprising multiple families of variable (Vj) genes, of which only two have been identified (Vj1 and Vj2, [50 genes each), 5 joining (Jj) regions and a single constant (Cj) gene (Butler et al 2004;Butler et al 2005). However, the physical layout of its various segments is similar to that of all other placental mammals (Butler et al 2004). To inactivate the pig j locus, the single Cj gene offered the best option, as this strategy had proven successful in the mouse Takeda et al 1993).…”

Development of the neonatal B and T cell repertoire in swine: implications for comparative and veterinary immunology

“…The preimmune V-KAPPA (IGKV-J) repertoire is dominated by the use of IGKV genes of a subgroup with homology to the human IGKV2 subgroup and a single IGKJ gene, i.e. the pre-immune V-KAPPA repertoire is also highly restricted [19]. The exact degree of restriction in V-LAMBDA (IGLV-J) expression has not yet been established but the same tendency for restricted expression is observed 3 .…”

“…The swine kappa locus contains ~ 80 IGKV genes of which 60 are of a subgroup with homology to the human IGKV2 subgroup [19]. The latter subgroup is preferentially used in the pre-immune repertoire [19,21]. Expressed porcine IGLV genes comprise predominately two subgroups with homology to human IGLV3 and IGLV8 subgroups 3 .…”

“…The lambda locus contains multiple tandem IGLJ-IGLC genes [12,24]. The swine kappa locus contains ~ 80 IGKV genes of which 60 are of a subgroup with homology to the human IGKV2 subgroup [19]. The latter subgroup is preferentially used in the pre-immune repertoire [19,21].…”