Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs

<div><p>The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen <i>(SLA)-1</i>*<i>0401</i> after the stimulation of peripheral blood mononuclear cells (PBMCs) by <i>Staphylococcus aureus</i> superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only <i>SLA-1</i>*<i>0401</i>, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from <i>SLA-1</i>*<i>0401</i> homozygous pigs were stimulated, the <i>SLA-1</i>*<i>0401</i> mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the <i>SLA-1</i>*<i>0401</i> mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.</p></div

Class I SLA-related mRNA expression after TSST-1 stimulation.

<p>The PBMCs of two pigs with the Hp-16.0 haplotype (individuals #965 and #1938) were examined for classical class I SLA (A) and related mRNA (B) expression after stimulation. Closed squares with a solid line show TSST-1-stimulated PBMCs, open squares with a broken line show IFN-γ stimulation, and closed squares with a dotted line show the negative control.</p

Tertiary structure of X2F6 mAb and the predicted antibody epitope.

<p>(A) Amino acid sequences of heavy and light chains of the X2F6 variable region. The database sequence PDB ID 3V7A is shown as the control sequence. (B) The predicted tertiary structure of the X2F6 mAb. (C) The tertiary structures of the YLL set in SLA-1*0501, which reacts with X2F6 with high reactivity (left panel), and the DVF set in SLA-1*1104, which cannot react with X2F6 (right panel), are shown. Pink (hydrophobic) and green (hydrophilic) colors represent the amino acid character. The structure is largely different, and the binding affinity is predicted to be different.</p

Primer sequences.

<p>Primer sequences.</p

Specificity of the X2F6 mAb.

<p>(A) <i>SLA-1</i>*<i>0401</i>, <i>SLA-2</i>*<i>0901</i>, and <i>SLA-3</i>*<i>0602</i>, which are the classical class-I SLA alleles of Haplotype Hp-16.0, and <i>SLA-6</i>*<i>0101</i>, which is a non-classical class-I SLA allele of Hp-16.0, were transfected into HEK293 parent cells, and the reactivity of X2F6 (right panels) was examined by flow cytometry (FCM). Propidium iodide (PI) positive-dead cells were avoided for the gating. PT-85A, the pan-specific MHC class-1 antibody, was used for the positive control (middle panels). (B) The species specificity was examined using swine (Hp-16.0), human, and common marmoset PBMCs. Lymphoid gate was used for the analysis. The percentages shown above the panels are the double-positive cell percentages.</p

Amino acid alignment of each classical class I allele in five SLA class I haplotypes.

<p>The amino acid sequence alignment of the alleles of each SLA locus is shown. For the haplotypes with a specific set of amino acids (Y102, L103, L109; the YLL set), in which each allele reacted with X2F6, the number of YLL sets determined the level of reactivity. The MFI for X2F6 reactivity was highest in the Hp-10.0 PBMCs that possessed three YLL sets in the SLA-1, SLA-2 and SLA-3 loci.</p

SLA class I genotypes and haplotypes deduced from SBT and sequence-specific primer (SSP) methods and MFI scores for Microminipigs.

<p>SLA class I genotypes and haplotypes deduced from SBT and sequence-specific primer (SSP) methods and MFI scores for Microminipigs.</p

Haplotype-specific reactivity of the X2F6 mAb.

<p>(A) Microminipig PBMCs of each haplotype were stained with X2F6 or PT85A followed by anti-mouse IgG-FITC and analyzed by FCM. Lymphoid gate was used for the analysis. Solid lines represent the X2F6-stained patterns. Broken lines represent PT85A-stained patterns. Filled lines represent the isotype-control-stained patterns. MFI scores for each haplotype are shown in the panels. The YLL type-allele number is also shown. (B) The MFIs of Hp-10.0 and Hp-43.0 haplotypes were selected and the homozygous pig PBMCs and heterozygous pig PBMCs were compared. The upper panels are the data from FACSCalibur, and the MFI score is significantly lower than in the lower panels, whereas the relative levels of the surface protein are comparable.</p