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Identification and characterization of two CD4 alleles in Microminipigs
Tatsuya Matsubara1,2, Naohito Nishii3,4, Satoshi Takashima2
1United Graduate School of Veterinary Sciences, Gifu University, Gifu, 501-1193, Japan.
Background:
We previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies.
Results:
We identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation.
Conclusions:
We identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis.
Insights
Genetic variants in CD4 alleles (CD4.A and CD4.B) explain differing reactivity of Microminipig cells to anti-pig CD4 antibodies. Amino acid substitutions in CD4.B, particularly in domain 1, cause this reactivity loss, enabling new diagnostic methods.
Area of Science:
- Immunology
- Genetics
Background:
- Previous flow cytometry identified two CD4+ cell phenotypes in Microminipigs based on reactivity to anti-pig CD4 monoclonal antibodies.
- This study aimed to identify the genetic basis for these differing cell reactivities.
Purpose of the Study:
- To analyze CD4 coding sequences and CD4 molecule expression.
- To identify genetic variants responsible for positive and negative peripheral blood mononuclear cell (PBMC) reactivity to anti-pig CD4 monoclonal antibodies.
Main Methods:
- Nucleotide sequencing of PCR products using CD4-specific primers.
- Comparison of identified CD4 alleles (CD4.A and CD4.B) with swine CD4 sequences.
- Restriction Fragment Length Polymorphism (RFLP) analysis using BseRI to characterize CD4 alleles.
- HeLa cell transfection with FLAG-tagged CD4.A and CD4.B vectors.
- Analysis of amino acid substitutions in relation to antibody reactivity.
Main Results:
- Two CD4 alleles, CD4.A (antibody positive) and CD4.B (antibody negative), were identified.
- CD4.A showed seven amino acid substitutions, while CD4.B had fifteen compared to swine CD4.
- CD4.B domain 1 sequences were identical to a previously reported modified swine CD4.2.
- RFLP analysis characterized homozygous and heterozygous alleles.
- Failure of CD4.B cells to react with anti-CD4 antibodies was linked to amino acid substitutions in domain 1 and/or joining region 3.
- Defective exon 8 in some alleles led to loss of the transmembrane domain, suggesting secreted CD4 proteins.
Conclusions:
- Amino acid substitutions in domain 1 of CD4.B cause the failure of some CD4-expressing cells to react with specific anti-pig CD4 monoclonal antibodies.
- A PCR-RFLP method was developed for simple identification of CD4 variants and PBMC reactivity, eliminating the need for flow cytometry.
