Abstract
Primary porcine alveolar macrophages (PAM) are useful for studying viral infections and immune response in pigs; however, long-term use of these cells is limited by the cells’ short lifespan. We immortalized primary PAMs by transfecting them with both hTERT and SV40LT 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 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; SLA-DQA*0201/0303, DQB1*0201/0901, DRB1*0201/1301) and iPAM303 (73.3% ± 5.4; SLA DQA*0106/0201, DQB1*0202/0701, DRB1*0402/0602). The difference in peptide binding between the two iPAMs was likely due to the allelic differences between the SLA class II molecules that were expressed. The development of an immortal PAM cell panel harboring diverse SLA haplotypes and the use of an established method in this study can become a valuable tool for evaluating the interaction between antigenic peptides and SLA molecules and is important for many applications in veterinary medicine including vaccine development.
Highlights
Peptide presentation to T helper (CD4+) and cytotoxic T lymphocytes (CTLs, CD8+) by major histocompatibility complexes (MHCs) is essential for adaptive immunity in vertebrates [1,2,3]
Generation of immortalized porcine alveolar macrophages (PAMs) We evaluated the phenotypes of the PAMs transfected with hTERT and simian vacuolating virus 40 large T (SV40LT) at passage 35
The patterns are similar among all compared cells except 3D4/21 which showed much faster proliferation, supporting consistent characteristics between early stage primary PAMs and immortalized PAMs (iPAMs) (Figure 2)
Summary
Peptide presentation to T helper (CD4+) and cytotoxic T lymphocytes (CTLs, CD8+) by major histocompatibility complexes (MHCs) is essential for adaptive immunity in vertebrates [1,2,3]. Various bioinformatic tools and resources that can predict the binding affinity of peptides to MHC molecules have been reported, including MHC-PPM, NetMHCpan-3.0, NetMHCII, and NetMHCIIpan [11,12,13]. The predicted interactions and their effects on the immune system need to be experimentally validated [14, 15]. For this purpose, several methods have been developed, including cell-free systems using purified MHC molecules [16, 17], artificial antigen processing systems by transformation of E. coli or yeast with recombinant MHC
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