Analysis of peptide-SLA binding by establishing immortalized porcine alveolar macrophage cells with different SLA class II haplotypes

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.

Figure 1

Morphological comparison of immortalized porcine alveolar macrophages (iPAMs) at different passages. The names of cells with the corresponding passage numbers are indicated on top. Scale bar: 100 µm.

Figure 2

Growth curves of immortalized porcine alveolar macrophages (iPAMs), early stage primary PAMs and 3D4/21. The primary PAM cells at passage 4 and iPAMs at passage 35 are used. Duration of culture is indicated in the x-axis.

Figure 3

Anchorage-independent growth of immortalized porcine alveolar macrophages (iPAMs) in soft agar. The names of cells are indicated on top iPAM61 and 303 are from passage 35 (p35). Arrowheads indicate single cells. Scale bar: 50 µm.

Figure 4

Comparison of SLA-DR expression levels by immunohistochemical analysis. Primary PAMs, PAM61 (A) and PAM303 (B); immortalized PAMs, iPAM61 (A) and iPAM303 (B). Control cells, 3D4/21 (C) and PK15 (C). The first, second, and third columns show the results from DAPI staining with anti-SLA-DR specific antibody. The third column indicates merged images with ×20 magnification. Scale bar: 100 µm.

Figure 5

Comparison of the efficiency of PCV2 ORF2 peptide binding to SLA class II molecules among different cells. The fluorescence levels from SLA-peptide complexes were compared among iPAM61, iPAM303, and PK15. The left (A, C, E) and right (B, D, F) columns indicate groups without and with biotin-labeled peptides, respectively. Fluorescence levels from the SLA-peptide complexes of PAM61 and PAM303 were shown in Additional file 3. Comparison of fluorescence signals from the SLA-peptide complexes of all three types of PAMs were shown (G). Comparisons indicated on top with brackets differ with P < 0.001.