Fluorescence signals from the peptide-SLA class II complexes was quantified by FACScalibur (BD Bioscience, San Jose, CA, USA) with CellQuest software. fluorescence levels from SLA-peptide complexes of PAM61 and PAM303 differ with and and established two immortalized cell lines (iPAMs) actively proliferating even after 35 passages. These cells possessed the characteristics of primary PAMs, including strong expression of swine leukocyte antigen (SLA) class II genes and the inability to Ubiquinone-1 grow anchorage-independently. We characterized their SLA genes and subsequently performed peptide-SLA binding assays using a peptide from porcine circovirus type 2 open reading frame 2 to experimentally measure the binding affinity of the peptide to SLA class II. The number of peptides bound to cells measured by fluorescence was very low for PK15 cells (7.0%??1.5), which are not antigen-presenting cells, unlike iPAM61 (33.7%??3.4; or yeast with recombinant MHC class II and chains [18C20], and mammalian cell systems using native MHC molecules on the cell surface [21, 22]. Studies to address the interaction between MHCs and epitopes have been limited for nonhuman species such as pigs. The development Ccna2 of disease resistance and effective vaccines against major infectious diseases in pigs is a critical issue in large-scale pig farming as diseases cause significant losses in productivity [23, 24]. However, interactions between swine leukocyte antigens (SLAs) and the epitopes of viral pathogens have not been extensively studied. To improve their capacity for antigen presentation, MHC genes have evolved to expand their diversity [9]. For example, 2165 alleles are currently reported for human leukocyte antigens (HLAs) [25]. On the other hand, 99 alleles are currently reported for SLAs [26], suggesting that further studies are needed to improve our understanding of the immune system of domestic animals. Monocytes and macrophages play critical roles in the immune response through phagocytosis, antigen presentation, and cytokine secretion [27, 28]. Simultaneously, macrophages serve as target cells for the replication of major pathogenic viruses in pigs, such as porcine respiratory and reproductive syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine circovirus 1 (PVC-1), and porcine circovirus 2 (PCV-2) [29C32]. A pig alveolar macrophage (PAM) cell line showed the ability to support the replication of porcine adenovirus (PAV), vaccine virus (VV), bovine adenovirus (BAV), parainfluenza virus, herpes simplex virus (HSV), swine poxvirus, African swine fever virus (ASFV), classical swine fever virus (CSFV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV) [33]; therefore, it can be a good model for studying virus-host interactions, including the formation of peptide-SLA complexes. However, because of difficulties involving cell preparation, limited lifespan, and experimental variation from different source animals, stable cell lines with the phenotypic characteristics of primary PAMs are being considered as potential alternatives. The use of simian vacuolating virus 40 large T (SV40LT) antigen and human telomerase (hTERT) has been proven to be a simple and reliable method to immortalize primary cells [34]. In this study, we established two stable PAM cell lines with known MHC haplotype information and used them to measure the binding affinity between MHC class II molecules and a peptide from PCV2. Our strategy can be used to evaluate the binding affinity of various pathogenic peptides to SLA class II molecules, which can be useful for immunogenetic applications in pigs. Materials and methods Collection of porcine alveolar macrophages and cell culture PAMs were isolated from ten 10-week-old clinically healthy Yorkshire pigs raised at a local pig farm. Animals were humanely euthanized and Ubiquinone-1 the lungs were collected for cell isolation. Phosphate buffer was used for bronchoalveolar lavage through a conventional method [35]; the bronchoalveolar lavage fluid was subsequently filtered. PAMs were collected by centrifugation at 400?for 5?min and were cultured in six-well plates in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS) Ubiquinone-1 and a 1% penicillinCstreptomycin-gentamycin antibiotic mixture (Gibco, NY, USA) at 37?C and 5% Ubiquinone-1 CO2 in an incubator. The adherent alveolar macrophages were cultured for 48?h and frozen in cell freezing media (RPMI.