6-OR: A comprehensive system for tissue-typing pigs

Abstracts

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6-OR

A COMPREHENSIVE SYSTEM FOR TISSUE-TYPING PIGS. Chak-Sum Ho,1 Jun-Heon Lee,2 Joan K. Lunney,3 Megan H. Franzo-Romain,1 Douglas M. Smith.1 1Pathology, University of Michigan, Ann Arbor, MI, USA; 2Animal Science, Chungnam National University, Daejeon, Korea; 3APDL, BARC, ARS, USDA, Beltsville, MD, USA. Aim: Pigs are important large animal models for transplantation and are being explored as potential xenograft donors. The porcine MHC, or swine leukocyte antigens (SLA), are important targets for both humoral and cell-mediated allo- and xeno-immune recognition. Accurate and effective tissue-typing methods for pigs are thus essential to study the influence of these antigens on transplant outcomes. Methods: We have developed a RT-PCR-, sequence-based typing (SBT) method for characterizing the complete coding sequence of SLA alleles at three classical class I loci (SLA-1, -2, -3), one non-classical class I locus (SLA-6) and four class II loci (DRA, DRB1, DQA, DQB1). Low-resolution PCR-SSP genotyping assays have also been developed as rapid and inexpensive alternatives to tissue-type pigs. Results: Using the SBT method, we have defined the SLA specificities in multiple resource populations of pigs worldwide, including the Hanford, NIH, Sinclair, Westran, Yucatan, the Korean native pigs, and a herd of commercial Meishan pigs. Using the PCR-SSP assays, we have characterized the SLA haplotypes in a herd of outbred pigs that will soon be used as a source for pancreatic islet xenografts in clinical trials. We have also analyzed multiple commercial pig lines and found very limited SLA diversity in current breeding stock populations. Altogether, we have surveyed the SLA diversity in nearly 1100 animals derived from a number of outbred pig herds and characterized 57 class I and 35 class II haplotypes. Conclusions: These SLA typing methods will facilitate the use of pigs, particularly from outbred populations with diverse immunogenetic backgrounds, as effective large animal transplant models.

7-OR

THE MACAQUE MHC-B LOCUS ENCODING Mamu-B*30, Mane-B*15, AND Mafa-B*38 FAMILY ALLELES IS FOUND ON A MINORITY OF HAPLOTYPES AND HAS LIMITED DIVERSITY. J. Wu,1 S. Bassinger,1 B. Holder-Lockyer,1 T.D. Tsewang,1 M.A. Krencicki, Jr,1 J.B. Woods,1 J. Lee,2 T.M. Williams.1 1Pathology, University of New Mexico, Albuquerque, NM, USA; 2One Lambda Inc, Canoga Park, CA, USA. Aim: Macaques are important animal models for understanding infectious disease, vaccine development, and transplantation research. The macaque Class I B region has undergone massive duplication with multiple poorly understood loci and haplotypes. Our goal is to understand the allelic diversity at the rhesus macaque B5 locus (B*30 alleles) and related loci in the pigtail and cynomolgus macaques (B*15 and B*38 alleles, respectively). Methods: PCR primers were designed to amplify Mamu-B*30 alleles from rhesus genomic DNA. Exons 2-4 were directly sequenced with intronic primers. These primers were also used to amplify and sequence B*30-like alleles from pig-tail and cynomolgus genomic DNA. Sequences were analyzed with Assign software (Conexio, Perth, AU). SSP-PCR was used as a secondary method to detect these alleles. Results: We detected B*30/*15/*38 alleles in 81 of 331, 116 of 308, and 50 of 279 rhesus, pigtail, and cynomolgus macaques. In these animals, 3 B*30 alleles were identified in rhesus, 2 B*15 alleles in pigtails, and 10 B*38 alleles in cynomolgus macaques, including 9 novel Mafa-B*38 alleles and one new Mane-B*15 allele. In these 3 species, these alleles were highly conserved, differing at most by 3 expected encoded amino acids from Mamu-B*3002. Many animals may lack this B locus; however, polymorphisms underlying the primers employed cannot be ruled out. Conclusions: While the MHC locus encoding B*30/*15/*38 alleles in rhesus, pigtail, and cynomolgus macaques is highly expressed (Bontrop, 2008), these data indicate that it has limited allelic diversity and is found on less than 20% of haplotypes.