Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan.
PLoS One. 2020 Nov 25;15(11):e0242572. doi: 10.1371/journal.pone.0242572. eCollection 2020.
Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.
白细胞表面表达的分化群 4（CD4）分子被认为是辅助性 T 细胞上的 II 类主要组织相容性复合体（MHC）与 T 细胞受体（TCR）结合的共受体。我们之前基于核苷酸测序和基因序列的 PCR 检测，在一个小型猪群体中鉴定出两种 CD4 等位基因（CD4.A 和 CD4.B）。然而，由于缺乏针对 CD4.B 表位的反应性抗体，我们没有检查 CD4.B 蛋白的表达。在这项研究中，我们针对 CD4.B 分子产生了两种猪特异性单克隆抗体（mAb），一种只识别 CD4.B（b1D7），另一种识别 CD4.A 和 CD4.B 等位基因（x1E10），并可用于通过流式细胞术和免疫组织化学区分 CD4 T 细胞亚群。使用这两种 mAb，我们在 CD4.A（+/+）和 CD4.B（+/+）T 细胞表面鉴定出 CD4.A 和 CD4.B 等位基因特异性蛋白，其表达水平相似。此外，体外用毒性休克综合征毒素-1（TSST-1）刺激来自 CD4.A（+/+）和 CD4.B（+/+）猪的外周血单核细胞（PBMC），同样激活了两组细胞，使 CD4/CD8 双阳性（DP）细胞比例略有增加。来自等位基因 CD4.A（+/+）和 CD4.B（+/+）组的 DP 细胞的大部分增强了总 CD4 和 I 类猪白细胞抗原（SLA）的表达。x1E10 mAb 延迟并减少了 TSST-1 诱导的 CD4 T 细胞激活。因此，CD4.B 似乎是一种功能性蛋白，其在激活的 T 细胞上的表达类似于 CD4.A。
