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.

识别淋巴细胞上分化簇（CD）分子的单克隆抗体（mAb）是研究流式细胞仪（FCM）分析中不同淋巴细胞亚群的有用工具。CD4是一种糖蛋白，存在于辅助T细胞，单核细胞，巨噬细胞和树突状细胞的表面。在这项研究中，我们描述了一个农场的日本黑（JB）小牛，该农场的外周血单核细胞（PBMC）不与CD4特异性mAb反应。为了鉴定具有低mAb反应性的PBMC的牛犊，使用两种不同的牛CD4 mAb（克隆＃CC8和＃CACT138A）检查了来自同一农场的21头JB犊牛（1–12个月龄）的PBMC。FCM分析表明，根据与两种mAb的反应性，小牛分为两组，即双阳性（DP）小牛，其PBMC被两个mAb克隆均识别，和单阳性（SP）犊牛，其PBMC仅被＃CACT138A识别。来自七个犊牛的PBMC未被＃CC8识别，尽管它们与另一个mAb＃CACT138A具有正常的反应性。与DP组相比，这些牛犊中CD4基因的序列分析显示SP组的编码区有四个核苷酸取代（G918 T，A930C，G970A和G1074A）。四个突变中的三个与氨基酸取代相关（Q306H，K310 N和A324 T）。在A324 T处的取代位于CD4基因的D4结构域中。基于氨基酸序列的同源性建模显示，该部分分子的表面结构在SP和DP组之间显着不同。因此，＃CC8 CD4 mAb识别的表位因这种遗传突变而在犊牛中发生了改变，这导致来自＃CC8 mAb的SP组小牛的PBMC的反应性较低。总之，这是鉴定JB牛CD4变异的第一项研究。我们证实了这些变体不会影响淋巴细胞功能，如促分裂原刺激或脂多糖诱导的细胞因子基因表达。