https://doi.org/10.1007/s00392-025-02625-4
1Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I München, Deutschland; 2LMU Klinikum der Universität München Herzchirurgische Klinik und Poliklinik München, Deutschland; 3Klinikum der LMU Walter Brendel Zentrum München, Deutschland; 4LMU München Lehrstuhl für molekulare Tierzucht und Biotechnologie Oberschleissheim, Deutschland
Background
Swine Leukocyte Antigen (SLA) is the porcine equivalent of the Major Histocompatibility Complex (MHC) in humans. LEA29Y expressing hearts from a published pig line (Bähr et al.,PLos One, 2016) and wild type (WT) hearts were transplanted into wild type recipient pigs in an allogeneic heterotopic abdominal heart transplantation (HAHT) model to evaluate the role of polymorphism in SLA in the survival of LEA29Y expressing hearts.
Methods and results
Clinical transplantation outcome in the LEA29Y group showed a high degree of variability (complete rejection (n=5); functional survival with some signs of rejection n=3). SLA mismatch was identified as a potential cause for the mixed protective outcome the LEA29Y transgene showed on pig heart allografts. To evaluate the polymorphism in the SLA sequence between all recipients and all donors, RT-PCR products from donor and recipient tissues were used for establishment and optimization of PCR assays.
SLA-1 and SLA-2 from class I and DRB-1, DQB1 and DQA from class II were selected as the genes of interest for primer design. We designed primers specific to the exons 2 and 3 of SLA class I genes and exon 2 of SLA class II genes. These exons encode the antigen binding domains in SLA genes and their polymorphic nature is crucial for analysis for co-relation between the SLA mis-match and graft survival. The sets of primers were optimized under various PCR conditions using cDNA samples from the LEA29Y allotransplants. PCR products were sequenced and sets of primers that amplified out regions of interest were selected.
In total, 11 animals from the LEA29Y to WT allotransplantation group and 5 WT-WT allotransplantations were analyzed. cDNA samples from left atrium (LA), left ventricle (LV), right atrium (RA) and right ventricle (RV) from donor animals and spleen and liver from recipient animals were used as templates. The resulting sequences from all donors and recipients were subsequently aligned in BioEdit to locate single nucleotide polymorphisms (SNPs). The cumulative SNPs between donors and recipients were translated to amino acids to study the role of amino acid variations in the varied transplantation outcomes.
Conclusion
We have successfully established SLA PCR assays in pigs to determine the SLA genotypes of individual animals. In addition to histological assessments and cytokine expression analysis, this will allow us to
evaluate the impact of SLA mis-match in transplantation settings further.