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Characterization of a variant CD4 molecule in Japanese Black cattle
Yuko Kato-Mori1, Taku Okamura2, Norihito Kawashita3
1Graduated School of Science, Technology and Innovation, Kobe University, 7-1-49 Minatojimaminami-machi, Chuo-ku, Kobe, 650-0047, Japan.
Insights
Researchers identified genetic CD4 variants in Japanese Black calves. These variants alter the epitope recognized by a specific monoclonal antibody, affecting flow cytometry analysis but not lymphocyte function.
Area of Science:
- Veterinary Immunology
- Molecular Genetics
- Flow Cytometry
Background:
- Monoclonal antibodies (mAbs) targeting cluster of differentiation (CD) molecules are crucial for lymphocyte subset analysis via flow cytometry (FCM).
- CD4, a glycoprotein on various immune cells, is a common target for such studies.
- Previous research has not identified CD4 variants in Japanese Black cattle.
Purpose of the Study:
- To investigate the cause of low reactivity of peripheral blood mononuclear cells (PBMCs) from certain Japanese Black calves to a CD4-specific monoclonal antibody.
- To identify genetic variations in the CD4 gene associated with this altered antibody binding.
- To assess the functional impact of these CD4 variants on lymphocyte activity.
Main Methods:
- Flow cytometry (FCM) analysis of PBMCs from 21 Japanese Black calves using two different bovine CD4 mAbs (clones #CC8 and #CACT138A).
- Sequencing of the CD4 gene in calves exhibiting differential mAb reactivity.
- Homology modeling to analyze the structural impact of identified genetic mutations.
Main Results:
- Calves were categorized into double-positive (DP) and single-positive (SP) groups based on reactivity to the CD4 mAbs.
- Seven calves showed PBMCs not recognized by the #CC8 mAb but reactive to #CACT138A.
- CD4 gene sequencing revealed four nucleotide substitutions in SP calves, three causing amino acid changes, notably in the D4 domain, altering the epitope for the #CC8 mAb.
- Homology modeling indicated significant surface structure differences in the D4 domain between SP and DP groups.
Conclusions:
- This study is the first to identify CD4 variants in Japanese Black cattle.
- A specific genetic mutation in the CD4 gene alters the epitope recognized by the #CC8 mAb, leading to reduced binding.
- These identified CD4 variants do not impair essential lymphocyte functions, including responses to mitogens and lipopolysaccharide stimulation.
Abstract:
Monoclonal antibodies (mAbs) that recognize cluster of differentiation (CD) molecules on lymphocytes are useful tools for the study of different lymphocyte subsets in flow cytometry (FCM) analysis. CD4 is a glycoprotein found on the surfaces of helper T cells, monocytes, macrophages, and dendritic cells. In this study, we describe Japanese Black (JB) calves in a farm whose peripheral blood mononuclear cells (PBMCs) did not react with a CD4-specific mAb. To identify calves with PBMCs with low mAb reactivity, PBMCs from 21 JB calves (1-12 months of age) bred at the same farm were examined using two different bovine CD4 mAbs (clones #CC8 and #CACT138A). FCM analysis indicated that the calves fell into two groups based on reactivity against the two mAbs, i.e., double-positive (DP) calves, whose PBMCs were recognized by both mAbs clones, and single-positive (SP) calves, whose PBMCs were only recognized by #CACT138A. PBMCs from seven calves were not recognized by #CC8, although they had normal reactivity with another mAb, #CACT138A. Sequencing analysis of the CD4 gene in these calves revealed four nucleotide substitutions (G918 T, A930C, G970A, and G1074A) in the coding region in the SP group when compared to the DP group. Three of the four mutations were associated with amino acid substitution (Q306H, K310 N, and A324 T). The substitution at A324 T was located in the D4 domain of CD4 gene. Homology modeling based on the amino acid sequences revealed that the surface structure of this part of the molecule was significantly different between the SP and the DP groups. Therefore, the epitope recognized by the #CC8 CD4 mAb was altered in calves with this genetic mutation, and this led to the low reactivity of the PBMCs from calves in the SP group aginst the #CC8 mAb. In conclusion, this is the first study to identify CD4 variants in JB cattle. We confirmed that the variants did not affect lymphocyte functions, such as mitogen stimulation or lipopolysaccharide-induced cytokine gene expression.
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