Graduated School of Science, Technology and Innovation, Kobe University, 7-1-49 Minatojimaminami-machi, Chuo-ku, Kobe, 650-0047, Japan.
School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, 069-8501, Japan.
Vet Immunol Immunopathol. 2021 Feb;232:110167. doi: 10.1016/j.vetimm.2020.110167. Epub 2020 Dec 7.
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.
单克隆抗体（mAbs）能够识别淋巴细胞表面的分化群（CD）分子，是流式细胞术（FCM）分析中研究不同淋巴细胞亚群的有用工具。CD4 是一种存在于辅助性 T 细胞、单核细胞、巨噬细胞和树突状细胞表面的糖蛋白。在这项研究中，我们描述了一个农场中的日本黑（JB）牛，其外周血单个核细胞（PBMC）与 CD4 特异性 mAb 无反应。为了鉴定 PBMC 对 mAb 反应性低的小牛，我们使用两种不同的牛 CD4 mAb（克隆 #CC8 和 #CACT138A）检测了同一农场 21 头 JB 小牛（1-12 月龄）的 PBMC。FCM 分析表明，根据对两种 mAb 的反应性，小牛可分为两组，即双阳性（DP）小牛，其 PBMC 被两种 mAb 克隆识别，以及单阳性（SP）小牛，其 PBMC 仅被 #CACT138A 识别。尽管 7 头小牛的 PBMC 对另一种 mAb #CACT138A 有正常反应，但它们不能被 #CC8 识别。对这些小牛的 CD4 基因进行测序分析发现，与 DP 组相比，SP 组的编码区有 4 个核苷酸替换（G918→T、A930→C、G970→A 和 G1074→A）。这 4 个突变中有 3 个与氨基酸替换有关（Q306→H、K310→N 和 A324→T）。A324→T 替换位于 CD4 基因的 D4 结构域。基于氨基酸序列的同源建模显示，该分子这部分的表面结构在 SP 组和 DP 组之间存在显著差异。因此，#CC8 CD4 mAb 识别的表位在具有这种遗传突变的小牛中发生改变，导致 SP 组小牛的 PBMC 对 #CC8 mAb 的低反应性。总之，这是首次在 JB 牛中鉴定出 CD4 变体。我们证实，这些变体不影响淋巴细胞功能，如丝裂原刺激或脂多糖诱导的细胞因子基因表达。
