The porcine antibody repertoire: variations on the textbook theme

Abstract

The genes encoding the heavy and light chains of swine antibodies are organized in the same manner as in other eutherian mammals. There are ∼30 VH genes, two functional DH genes and one functional JH gene, 14-60 Vκ genes, 5 Jκ segments, 12-13 functional Vλ genes, and two functional Jλ genes. The heavy chain constant regions encode the same repertoire of isotypes common to other eutherian mammals. The piglet models offers advantage over rodent models since the fetal repertoire develops without maternal influences and the precocial nature of their multiple offspring allows the experimenter to control the influences of environmental and maternal factors on repertoire development postnatally. B cell lymphogenesis in swine begins in the fetal yolk sac at 20 days of gestation (DG), moves to the fetal liver at 30 DG and eventually to the bone marrow which dominates until birth (114 DG) and to at least 5 weeks postpartum. There is no evidence that the ileal Peyers patches are a site of B cell lymphogenesis or are required for B cell maintenance. Unlike rodents and humans, light chain rearrangement begins first in the lambda locus; kappa rearrangements are not seen until late gestation. Dissimilar to lab rodents and more in the direction of the rabbit, swine utilize a small number of VH genes to form >90% of their pre-immune repertoire. Diversification in response to environmental antigen does not alter this pattern and is achieved by somatic hypermutation (SHM) of the same small number of VH genes. The situation for light chains is less well studied, but certain Vκ and Jκ and Vλ and Jλ are dominant in transcripts and in contrast to rearranged heavy chains, there is little junctional diversity, less SHM, and mutations are not concentrated in CDR regions. The transcribed and secreted pre-immune antibodies of the fetus include mainly IgM, IgA, and IgG3; this last isotype may provide a type of first responder mucosal immunity. Development of functional adaptive immunity is dependent on bacterial MAMPs or MAMPs provided by viral infections, indicating the importance of innate immunity for development of adaptive immunity. The structural analysis of Ig genes of this species indicate that especially the VH and Cγ gene are the result of tandem gene duplication in the context of genomic gene conversion. Since only a few of these duplicated VH genes substantially contribute to the antibody repertoire, polygeny may be a vestige from a time before somatic processes became prominently evolved to generate the antibody repertoire. In swine we believe such duplications within the genome have very limited functional significance and their occurrence is therefore overrated.

Keywords: antibody repertoire; development; gene duplication; swine.

Figure 1

The genomic repertoire of heavy and light chain genes of swine. (A) The light chain and heavy chain variable gene loci compared to humans. Duplicons are illustrated in parentheses with the actual number given in the mini-table above designated as “n.” This includes duplicons of cassettes as in the lambda locus. In the swine only two DH and one JH gene segments are functional (given in parenthesis). (B) Variation in the organization of the heavy chain constant region among human, swine, and rabbit. Modified from Butler et al. (2011c).

Figure 2

The critical window of immunological development. The factor affecting events in the “window” are indicated. There are a number of cases of superimposing events; e.g., Passive immunity (red) superimposed on innate immunity (green) produces yellow. The “lined section” prepartum applies only to species, e.g., mice and humans, in which passive immunity also takes place in utero. In Ungulates like swine there is no transfer of passive antibodies in utero. Modified from Butler et al. (2006b).

Figure 3

B cell lymphogenesis in swine. Heavy and light chain rearrangements and signal joint circles (SJC) recovered from. YS, yolk sac; FL, fetal liver, BM, bone marrow; DG, day of gestation; N.D., not detected.

Figure 4

The proportional usage of 11 porcine VH genes during fetal life. VHB and VHB* (IGHV6 and IGHV12) are considered as a group in this analysis. Both the familiar and IMGT nomenclature (if available) are given. Data are based on >5500 VDJ clones and were analyzed using a computer modeling program. The horizontal bars and numbers above each VH gene correspond to the frequency of usage; 1 = highest frequency. For VHN and VHC, the order changes during development. UNK includes VH genes other than those shown and mutated versions of those shown. From Butler et al. (2011a).

Figure 5

Diversification of the porcine antibody repertoire during fetal and postnatal life expressed as a repertoire diversification index [RDI; (A)] or as the frequency of somatic hypermutation [SHM; (B)]. DG, days of gestation. Values for 95 DG are pooled from clone frequencies recovered from spleen, IPP, and MLN since there were no tissue differences. GF, germfree isolator piglets; Col S-FLU, colonized or S-FLU infected isolator piglets; PIC, parasite-infected young adults reared conventionally. From Butler et al. (2011a).

Figure 6

The proportion of VDJ clones from fetal and postnatal piglets that hybridize with cocktails that contain probes for the CDR1 and CDR2 regions of the major seven VH genes used to form the pre-immune repertoire (see Figure 4). Legend for hybridization/non-hybridization is on the figure. Failure to hybridize means that either other VH genes are used or the major seven VH have been mutated to the extent that they no long hybridize with the CDR-specific probes. The number within the bar representing non-hybridizing VH genes indicates the proportion that are the major VH genes as determined by sequence analysis. This means, e.g., that in C/V piglets, 85% are a mutated version of the seven major genes. GF, germfree; C/V, colonized and/or virus infected; PIC, parasite-infected conventional pigs.

Figure 7

Adaptive changes to the antibody repertoire as a result of postnatal infection with S-FLU. (A) Effect on IgG3 transcription in the tracheal-bronchial lymph node (TBLN). NB, newborn; GF, 5-week germfree isolator piglets; S-FLU, 5-week S-FLU infected isolator piglets. (B) The repertoire diversification index (RDI) for VH genes transcribed with IgM, IgG3 and downstream Cγ transcripts (called: “other”) in GF and S-FLU infected piglets. The boxed values are the number of clones examined. Data indicate that the IgM and IgG3 repertoire does not diversify in S-FLU infection.

Figure 8

Evidence in support of duplication of VH genes in swine. Diagrammatic comparison of 16 of 32 VH3 family genes described for swine. The sequences are drawn to approximate scale. CDR sequences are identified by capital letter. Only the first fifteen 3′ VH genes have been mapped; pseudogenes are not shown. Those VH genes with the same A,B, etc. CDR designations share the same CDR1 or CDR2 regions. In the case where small differences (genomic point mutations) in FR and CDR regions are involved, vertical lines are drawn.

Figure 9

The gene structure of 11 porcine Cγ genes representing the six expressed IgG subclasses and allotypes of swine. Different marking patterns are used to identify domain sequences. Domains with the same pattern indicate they are shared among Cγ genes. Note that no domains are shared with IgG3.