Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations

The highly polymorphic swine leucocyte antigen ( SLA ) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation success. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to type alleles at the three classical SLA class I loci ( SLA-1 , SLA-3 and SLA-2 ) using the PCR-sequence-specific primer (PCR-SSP) strategy. This typing system relies on 47 discriminatory PCR primer pairs designed to amplify the SLA class I alleles by groups that have similar sequence motifs. We applied this low-resolution group-specific typing method to characterize the SLA class I alleles present in three outbred pig populations ( n =  202). Alleles from 24 class I allele groups corresponding to 56 class I genotypes were detected. We also identified 23 low-resolution SLA class I haplotypes in these pigs and found haplotypes Lr-1.0 ( SLA-1 *01XX- SLA-3 *01XX- SLA-2 *01XX) and Lr-4.0 ( SLA-1 *04XX- SLA-3 *04XX- SLA-2 *04XX) in all three pig populations with a high prevalence. Over 80% of the pigs examined ( n  =   162) were found to bear at least one of these haplotypes, resulting in a combined haplotype frequency of nearly 50%. This PCR-SSP-based typing system demonstrates a reliable and unambiguous detection of SLA class I alleles, and can be used to effectively investigate the SLA diversity in outbred pig populations. It will help to identify the role of SLA antigens in disease-resistant pigs and may facilitate the development of effective vaccines.     

Investigation of SLA class I and II haplotypes in the NIH miniature pigs

Xenotransplantation involves the transplantation of organs, tissues and cells from one species to another. A major barrier to successful xenotransplantation is the rejection of the donor tissue by the recipient immune system. Swine leukocyte antigens (SLA) are important molecules within the immune system and play an essential role in fighting infectious diseases and viruses. The present study investigated three SLA class I (SLA-1, SLA-3 and SLA-2) and three SLA class II (DRB1, DQB1 and DQA) alleles in 60 NIH miniature pigs using PCR with sequence-specific primers (PCR-SSP). As the results, nine combinations of SLA class I and II haplotypes, comprising of three homozygous and six heterozygous haplotypes, were examined. The SLA homozygous haplotype Lr-2.4/2.4 was the most prevalent, with an overall frequency of 28.3% (17/60) and heterozygous haplotype Lr-2.2/4.4 was the second most common (20.0%; 12/60), followed by haplotype Lr-4.2/4.2 (16.7%; 10/60), Lr-2.2/2.4 (15.0%; 9/60), Lr-2.2/2.2 (5.0%; 3/60), Lr-2.2/4.2 (5.0%; 3/60), Lr-2.4/4.4 (5.0%; 3/60) and Lr-2.2/3.3 (3.3%; 2/60), Lr-4.2/4.4 (1.7%; 1/60), respectively. These results provide useful information that can be used to establish highly inbred pig lines with fixed SLA homozygous alleles and haplotypes.     

Rapid assignment of swine leukocyte antigen haplotypes in pedigreed herds using a polymerase chain reaction-based assay

We present a simple assay to determine the swine leukocyte antigen (SLA) haplotypes of animals within two experimental populations of MHC defined miniature pigs. The Yucatan miniature pigs have four founder haplotypes ( w, x, y, z) and one recombinant haplotype ( q). The NIH miniature pigs have three founder haplotypes ( a, c, d) and two recombinant haplotypes ( f, g). Because most crossovers occur between the class I and class II regions, haplotypes can be assigned by typing one class I locus and one class II locus for practical purposes. We have previously characterized these seven founder haplotypes by sequencing the cDNA of three SLA class I loci, designated as SLA-1, SLA-3 and SLA-2 and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1 and SLA-DRB1. These sequences were used to design allele-specific primers to amplify one MHC class I and one MHC class II gene for each haplotype. Primers were tested for specificity in homozygous and heterozygous animals. Positive control primers were also designed to amplify a portion of the E-selectin or alpha-actin gene and multiplexed with the allele-specific primers to check for false negatives. This combination of allele-specific and positive control primers produced specific and robust PCR-site-specific primer assays for assigning SLA haplotypes in the two populations.     

Difference in number of loci of swine leukocyte antigen classical class I genes among haplotypes

The structure of the entire genomic region of swine leukocyte antigen (SLA)-the porcine major histocompatibility complex--was recently elucidated in a particular haplotype named Hp-1.0 (H01). However, it has been suggested that there are differences in the number of loci of SLA genes, particularly classical class I genes, among haplotypes. To clarify the between-haplotype copy number variance in genes of the SLA region, we sequenced the genomic region carrying SLA classical class I genes on two different haplotypes, revealing increments of up to six in the number of classical class I genes in a single haplotype. All of the SLA-1(-like) (SLA-1 and newly designated SLA-12) and SLA-3 genes detected in the haplotypes thus analyzed were transcribed in the individual. The process by which duplication of SLA classical class I genes was likely to have occurred was interpreted from an analysis of repetitive sequences adjacent to the duplicated class I genes.     

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line,Microminipig

Microminipigs are extremely small‐sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo‐ and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA‐defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA‐1, SLA‐2 and SLA‐3) and two class II (SLA‐DRB1 and SLA‐DQB1) genes of 14 parental Microminipigs using a high‐resolution nucleotide sequence‐based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp‐31.0 (SLA‐1*1502–SLA‐3*070102–SLA‐2*1601) and Hp‐0.37 (SLA‐DRB1*0701–SLA‐DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.     

Effect of swine lymphocyte antigen haplotypes on birth and weaning weights in pigs

Birth weights of 708 live piglets and weaning weights of 566 piglets were used to investigate the effect of the swine lymphocyte antigen (SLA) complex on these traits in Large White pigs. Piglets were from litters of a long-term selection experiment to measure response for selection to increase litter size. SLA haplotypes were determined using conventional class I antisera. A total of 14 haplotypes were detected. The effect of SLA haplotype on birth and weaning weights was investigated using a statistical model that included the effects of experimental group, sire, dam, sex and SLA haplotype. Results indicated that SLA class I haplotype 13.1.3 increased birth weights (P less than 0.10) and significantly increased weaning weights (P less than 0.01). This effect of haplotype 13.1.3 on weaning weight was 605 +/- 215 g (0.3 standard deviations). SLA class I homozygosity did not appear to affect birth and weaning weights. These results suggest that the SLA complex plays an important role in early growth in the pig and that further study of SLA effects on growth and reproduction are warranted.     

Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs

The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F ₂ descendants of F ₁ Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA‐1 , SLA‐2 , SLA‐3 ) and class II ( SLA‐DRB1 , SLA‐DQB1 , SLA‐DQA ) genes of 27 purebred Pietrain pigs using a combination of the high‐resolution sequence‐based typing (SBT) method and a low‐resolution (Lr) PCR‐based method using allele‐group, sequence‐specific primers (PCR‐SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr‐43.0 ( SLA‐1 *11XX– SLA‐3 *04XX– SLA‐2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr‐0.14 [ SLA‐DRB1 *0901– SLA‐DQB1 *0801– SLA‐DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr‐Pie‐0.1 [ SLA‐DRB1 *01XX/be01/ha04– SLA‐DQB1 *05XX– SLA ‐ DQA*blank] and Lr‐Pie‐0.2 [ SLA‐DRB1 *06XX– SLA‐DQB1 *03XX– SLA‐DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR‐SSP‐based assays represents a rapid and cost‐effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease‐resistant pigs in the context of SLA antigens to improve overall swine health.     

Marked genetic polymorphism of the swine steroid 21-hydroxylase gene, and its location between the SLA class I and class II regions

Restriction fragment length polymorphism (RFLP) analysis of the swine 21-hydroxylase (CYP21) region was conducted on 31 unrelated SLA class I typed pigs, mainly Large Whites, including 15 haplotypes. Ten haplotypes were from SLA genotypic homozygotes and five were from SLA class I phenotypic homozygotes. DNA digestion with Hin dIII, TaqI and PstI, and hybridization to a 4.5-kb swine CYP21 genomic probe yielded respectively two, four and three RFLP patterns. Six patterns were identified with combined RFLP. In addition, analysis of the CYP21 region in families comprising several SLA recombinants demonstrated that the CYP21 gene lies in the DNA segment between the SLA class I and class II regions. These overall results reinforce our previous conclusion about the existence in the pig of a single 21-hydroxylase gene. The characterization of at least six CYP21 allelic patterns provides a new tool for studying the associations between the SLA region and zootechnical traits.     

Crystal structure of swine major histocompatibility complex class I SLA-1 0401 and identification of 2009 pandemic swine-origin influenza A H1N1 virus cytotoxic T lymphocyte epitope peptides

The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1 0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIV(NW9); NSDTVGWSW) or Ebola virus (Ebola(AY9); ATAAATEAY) were determined in this study. The overall peptide-SLA-1 0401 structures resemble, as expected, the general conformations of other structure-solved peptide major histocompatibility complexes (pMHC). The major distinction of SLA-1 0401 is that Arg(156) has a "one-ballot veto" function in peptide binding, due to its flexible side chain. S-OIV(NW9) and Ebola(AY9) bind SLA-1 0401 with similar conformations but employ different water molecules to stabilize their binding. The side chain of P7 residues in both peptides is exposed, indicating that the epitopes are "featured" peptides presented by this SLA. Further analyses showed that SLA-1 0401 and human leukocyte antigen (HLA) class I HLA-A 0101 can present the same peptides, but in different conformations, demonstrating cross-species epitope presentation. CTL epitope peptides derived from 2009 pandemic S-OIV were screened and evaluated by the in vitro refolding method. Three peptides were identified as potential cross-species influenza virus (IV) CTL epitopes. The binding motif of SLA-1 0401 was proposed, and thermostabilities of key peptide-SLA-1 0401 complexes were analyzed by circular dichroism spectra. Our results not only provide the structural basis of peptide presentation by SLA I but also identify some IV CTL epitope peptides. These results will benefit both vaccine development and swine organ-based xenotransplantation.     

Microsatellite diversity and crossover regions within homozygous and heterozygous SLA haplotypes of different pig breeds

Our aim was to investigate microsatellite (MS) diversity and find crossover regions at 42 polymorphic MS loci in the swine leukocyte antigen (SLA) genomic region of 72 pigs with different well-defined homozygous and heterozygous SLA haplotypes. We analyzed the genetic polymorphisms of 42 MS markers in 23 SLA homozygous-heterozygous, common pig breeds with 12 SLA serological haplotypes and 49 National Institutes of Health (NIH) and Clawn homozygous-heterozygous miniature pigs with nine SLA serological or genotyped haplotypes including four recombinant haplotypes. In comparing the same and different haplotypes, both haplospecific patterns and allelic variations were observed at the MS loci. Some of the shared haplotype blocks extended over 2 Mb suggesting the existence of strong linkage disequilibrium (LD) in the entire SLA region. Crossover regions were easily defined by the MS markers within the class I and/or III region in the NIH and Clawn recombinant haplotypes. The present haplotype comparison shows that our set of MS markers provides a fast and cost-efficient alternative, or complementary, method to the serological or sequence-based determination of the SLA alleles for the characterization of SLA haplotypes and/or the crossover regions between different haplotypes.     

Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation

To lay background for studying rejection mechanisms in xenotransplantation and developing the strate-gies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced. The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors. All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their ac-cession numbers AY102467- AY102472 from GenBank. This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A^*0201) are better than those between pigs and mice (H-2D^b/H-2K^b). The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules. The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted. A functional test indicated that the human T cells specific for the prokaryotically expressed SLA Plprotein could respond quite well in vitro to the class I-positive swine chon-drocytes and PBMCs in presence of human APCs. This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens.     

Uterine MHC class I molecules and beta 2-microglobulin are regulated by progesterone and conceptus interferons during pig pregnancy

MHC class I molecules and beta(2)-microglobulin (beta(2)m) are membrane glycoproteins that present peptide Ags to TCRs, and bind to inhibitory and activating receptors on NK cells and other leukocytes. They are involved in the discrimination of self from non-self. Modification of these molecules in the placenta benefits pregnancy, but little is known about their genes in the uterus. We examined the classical class I swine leukocyte Ags (SLA) genes SLA-1, SLA-2, and SLA-3, the nonclassical SLA-6, SLA-7, and SLA-8 genes, and the beta(2)m gene in pig uterus during pregnancy. Uterine SLA and beta(2)m increased in luminal epithelium between days 5 and 9, then decreased between days 15 and 20. By day 15 of pregnancy, SLA and beta(2)m increased in stroma and remained detectable through day 40. To determine effects of estrogens, which are secreted by conceptuses to prevent corpus luteum regression, nonpregnant pigs were treated with estradiol benzoate, which did not affect the SLA or beta(2)m genes. In contrast, progesterone, which is secreted by corpora lutea, increased SLA and beta(2)m in luminal epithelium, whereas a progesterone receptor antagonist (ZK137,316) ablated this up-regulation. To determine effects of conceptus secretory proteins (CSP) containing IFN-delta and IFN-gamma, nonpregnant pigs were implanted with mini-osmotic pumps that delivered CSP to uterine horns. CSP increased SLA and beta(2)m in stroma. Cell-type specific regulation of SLA and beta(2)m genes by progesterone and IFNs suggests that placental secretions control expression of immune regulatory molecules on uterine cells to provide an immunologically favorable environment for survival of the fetal-placental semiallograft.     

RFLP analysis of SLA haplotypes in Swiss large white and American Hampshire pigs using SLA class I and class II probes

Summary

Restriction fragment length polymorphism (RFLP) analyses of swine leukocyte antigen (SLA) class I and class II genes from Swiss Large White and American Hampshire families were performed using porcine DNA probes. Class I and class II RFLPs associated with the serologically‐defined haplotypes SLA H1, H8, H16 and H24 and with serotypes SLA 15, 16; SLA 14; and SLA 6, SB 19, were identified. Seven allelic class I RFLP patterns were observed. For genes in the SLA class II region, six allelic RFLP patterns of DQA and DQB; five allelic RFLP patterns of DRA; and seven allelic RFLP patterns of DRB were observed. The serologically‐defined H8 haplotype was subtyped based on differences in class II RFLPs.     

Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations

The highly polymorphic swine leucocyte antigen (SLA) genes are among the most important determinants of swine immune responses to disease and vaccines. Accurate and effective SLA genotyping methods are required to understand how SLA gene polymorphisms affect immunity, especially in outbred pigs with diverse genetic backgrounds. In this study, we present a simple and rapid molecular‐based typing system for characterizing SLA class II alleles of the DRB1, DQB1 and DQA loci. This system utilizes a set of 47 sequence‐specific PCR primers developed to differentiate alleles by groups that share similar sequence motifs. We applied this typing method to investigate the SLA class II diversity in four populations of outbred pigs (n = 206) and characterized a total of 19 SLA class II haplotypes, six of which were shared by at least three of the sampled pig populations. We found that Lr‐0.1 (DRB1*01XX–DQB1*01XX–DQA*01XX) was the most prevalent haplotype with a combined frequency of 16.0%, followed by Lr‐0.2 (DRB1*02XX–DQB1*02XX–DQA*02XX) with 14.6% and Lr‐0.15b (DRB1*04XX–DQB1*0202–DQA*02XX) with 14.1%. Over 70% of the pigs (n = 147) had at least one copy of one of these three haplotypes. The PCR‐based typing system described in this study demonstrates a reliable and unambiguous detection method for SLA class II alleles. It will be a valuable tool for studying the influence of SLA diversity on various immunological, pathological and physiological traits in outbred pigs.     

Genetic and other effects on antibody and cell mediated immune response in swine leucocyte antigen (SLA)-defined miniature pigs

Miniature pigs of eight swine leucocyte antigens (SLA) haplotypes were immunized with sheep erythrocytes (SRBC), hen egg white lysozyme (HEWL) and the synthetic peptide (T, G)-A--L to induce antibody. Bacillus Calmette Geurin (BCG) and dinitrochlorobenzene (DNCB) were used to induce cell mediated immune response (CMI). Analysis of variance by least squares was used to assess the effects of SLA haplotype, sire, dam, litter and sex of pig on the magnitude of the primary and secondary antibody response and on dermal delayed type hypersensitivity induced by purified protein derivative of tuberculin (PPD) and DNCB-induced contact hypersensitivity. The statistical model accounted for 43.50-77.30% of the observed variability in antibody and CMI at various times after immunization or challenge. While SLA had a significant effect on both antibody and CMI to some antigens at some, but not all times, sire, dam and litter were more frequently significant and to a greater degree. Haplotypes dd, dg and gg produced more antibody to SRBC and (T, G)-A--L while dg and gg had higher primary, but not secondary antibody response to HEWL. Delayed hypersensitivity to PPD was most marked in pigs of dd, dg and gg haplotypes while contact hypersensitivity to DNCB was expressed least in the dg and gg haplotype pigs. Heritability estimates were high for response to (T, G)-A--L and HEWL indicating feasibility of selective breeding for these traits.     

Restriction fragment length polymorphism of the major histocompatibility complex of the pig

Human HLA cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the SLA major histocompatibility complex in swine. Cellular genomic DNA from 19 SLA homozygous pigs representing 13 different haplotypes was digested with restriction endonucleases Eco RI, Hind III, or Bam H1, separated by electrophoresis, and transferred onto diazobenzyloxymethyl paper by the Southern blot technique. The blots were probed with ³²P-labeled class I or beta-DR class II cDNA. Depending on the haplotypes and the endonucleases used, seven to ten restriction fragments hybridized with the class I probe, and five to seven with the beta-DR probe. Their sizes ranged from 3.4 to 22 kilobase-pairs. Few bands were common to all 13 haplotypes. With all but one haplotype, identical autoradiogram patterns were obtained from unrelated, but phenotypically SLA-identical pigs, suggesting that most of the RFLP revealed were controlled by the SLA region. Further polymorphism was found in a group of seven unrelated pigs which typed serologically as SLA A15 CI B18 homozygotes but could be divided into two subgroups, with five animals in one subgroup and two in the other, when the genomic DNA was hybridized with the class I probe. When the class 11 beta-DR probe was tested on the same seven pigs, another subdivision was seen, and this correlated with MLR data. These results demonstrate that HLA class I and class II probes can be used to identify certain well-established SLA haplotypes and to identify subclasses within at least one SLA haplotype.Abbreviations MHC major histocompatibility complex - MLR mixed lymphocyte reaction - kbp kilobase pair(s) - RFLP restriction fragment length polymorphism     

Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL, and GenBank databases under accession numbers AB158486 and AB158487     

Genetic and other effects on antibody and cell mediated immune response in swine leucocyte antigen (SLA)-defined miniature pigs

Summary. Miniature pigs of eight swine leucocyte antigens (SLA) haplotypes were immunized with sheep erythrocytes (SRBC), hen egg white lysozyme (HEWL) and the synthetic peptide (T, G)-A–L to induce antibody. Bacillus Calmette Geurin (BCG) and dinitrochlorobenzene (DNCB) were used to induce cell mediated immune response (CMI). Analysis of variance by least squares was used to assess the effects of SLA haplotype, sire, dam, litter and sex of pig on the magnitude of the primary and secondary antibody response and on dermal delayed type hypersensitivity induced by purified protein derivative of tuberculin (PPD) and DNCB-induced contact hypersensitivity.
The statistical model accounted for 43.50–77.30% of the observed variability in antibody and CMI at various times after immunization or challenge. While SLA had a significant effect on both antibody and CMI to some antigens at some, but not all times, sire, dam and litter were more frequently significant and to a greater degree.
Haplotypes dd, dg and gg produced more antibody to SRBC and (T, G)-A–L while dg and gg had higher primary, but not secondary antibody response to HEWL. Delayed hypersensitivity to PPD was most marked in pigs of dd, dg and gg haplotypes while contact hypersensitivity to DNCB was expressed least in the dg and gg haplotype pigs.
Heritability estimates were high for response to (T, G)-A–L and HEWL indicating feasibility of selective breeding for these traits.