Comparative analysis of swine leukocyte antigen gene diversity in Göttingen Minipigs.

Worldwide, pigs represent economically important farm animals, also representing a preferred preclinical large animal model for biomedical studies. The need for swine leukocyte antigen (SLA) typing is increasing with the expanded use of pigs in translational research, infection studies, and for veterinary vaccine design. Göttingen Minipigs (GMP) attract increasing attention as valuable model for pharmacological studies and transplantation research. This study represents a first-time assessment of the SLA gene diversity in Göttingen Minipigs in combination with a comparative metadata analysis with commercial pig lines. As Göttingen Minipigs could harbor private as well as potential novel SLA allele combinations, future research projects would benefit from the characterization of their SLA background. In 209 Göttingen Minipigs, SLA class I (SLA-1, SLA-2, SLA-3) and class II (DRB1, DQB1, DQA) genes were characterized by PCR-based low-resolution (Lr) haplotyping. Criteria and nomenclature used for SLA haplotyping were proposed by the ISAG/IUIS-VIC SLA Nomenclature Committee. Haplotypes were assigned based on the comparison with already known breed or farm-specific allele group combinations. In total, 14 SLA class I and five SLA class II haplotypes were identified in the studied cohort, to manifest in 26 SLA class I but only seven SLA class II genotypes. The most common SLA class I haplotypes Lr-24.0 (SLA-1*15XX or Blank-SLA-3*04:04-SLA-2*06:01~02) and Lr-GMP-3.0 (SLA-1*16:02-SLA-3*03:04-SLA-2*17:01) occurred at frequencies of 23.44 and 18.66%, respectively. For SLA class II, the most prevalent haplotypes Lr-0.21 (DRB1*01XX-DQB1*05XX-DQA*04XX) and Lr-0.03 (DRB1*03:02-DQB1*03:01-DQA*01XX) occurred at frequencies of 38.28 and 30.38%. The comparative metadata analysis revealed that Göttingen Minipigs only share six SLA class I and two SLA class II haplotypes with commercial pig lines. More importantly, despite the limited number of SLA class I haplotypes, the high genotype diversity being observed necessitates pre-experimental SLA background assessment of Göttingen Minipigs in regenerative medicine, allo-transplantation, and xenograft research.

Identification of MHC-I-Presented Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) Peptides Reveals Immunogenic Epitopes within Several Non-Structural Proteins Recognized by CD8+ T Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most relevant porcine pathogens worldwide. Active control of the disease relies on modified live virus vaccines (MLVs), as most inactivated vaccines provide very limited protection. Neutralizing antibodies occur late in infection; therefore, CD8+ T cells are considered important correlates of protection and are a frequent focus of investigation. Our aim was to identify viral peptides naturally bound by the class I major histocompatibility complex (MHC-I) and to confirm their ability to stimulate CD8+ T cells. For this purpose, we immunoprecipitated MHC-I/peptide complexes of PRRSV (strain AUT15-33) -infected cells (SLA-I Lr-Hp 35.0/24 mod) to isolate the viral epitopes and analyzed them with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Furthermore, we employed these identified peptides to stimulate peripheral blood mononuclear cells (PBMCs) of previously PRRSV-infected pigs and measured the PRRSV-specific CD8+ T-cell response with an intracellular cytokine staining (ICS). Our data revealed that PRRSV non-structural proteins (NSPs), encoded in open reading frame 1a and 1b (ORF1), present the major source of MHC-I-presented peptides. Additionally, we show that our identified epitopes are able to trigger IFNγ responses in vitro. These findings are a basis for understanding the proteasomal degradation of PRRSV proteins, the cellular ability to display them via MHC-I, and their potential to restimulate CD8+ T cells.

Revisiting lymphocyte clonality testing in feline B-cell lymphoma.

Differentiation between resident mature lymphocyte populations and small-cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. Although primer coverage of potential IG gene rearrangements in feline B-cell neoplasms constantly improves, the possibility of false negative and false positive test results still poses a problem. In this retrospective study, we assessed diagnostic sensitivity and specificity of a novel developed multiplex PCR assay for routine diagnosis of B-cell clonality. Therefore, 24 feline patients were subjected to comparative clonality testing by using different primer sets. Feline lymphoma cell lines and confirmed patient material served as positive control. Compared to previous studies, this novel developed multiplex PCR assay showed positive effects on diagnostic sensitivity, specificity, accuracy, and positive predictive value accompanied by a slight impairment of negative predictive value. Notably, none of the primer sets was superior; hence, we recommend the combined application of the herein tested primer sets in routine diagnostics. However, a more in-depth-evaluation of the dynamic of assay specific parameters in dependency on primer set usage requires prospective studies on larger cohorts of feline patients.