Miyazawa Ryuichiro, Matsuura Yuta, Shibasaki Yasuhiro, Imamura Shintaro, Nakanishi Teruyuki
Department of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa 252-0880, Japan.
National Research Institute of Fisheries Science, 2-12-4 Fukuura, Yokohama, Kanagawa 236-8648, Japan.
Dev Comp Immunol. 2018 Mar;80:15-23. doi: 10.1016/j.dci.2016.12.002. Epub 2016 Dec 11.
We have monoclonal antibodies (mAbs) against CD4-1 (6D1) and CD8α (2C3) in ginbuna crucian carp Carassius auratus langsdorfii. In our previous studies we showed that 2C3 mAb positive cells are the primary cell type showing specific cytotoxicity against allogeneic targets, suggesting that CD8α lymphocytes in ginbuna are equivalent to cytotoxic T lymphocytes (CTLs) in mammals. We further demonstrated the helper T cell function of 6D1 mAb positive cells by studying mixed leukocyte culture (MLC) and hapten/carrier effects. Here, we report that our mAbs cross-react with zebrafish lymphocytes. First, mAbs 6D1 and 2C3 recognized 7-11% of zebrafish lymphocytes that were ZAP-70 positive and had the typical morphology of lymphocytes. Second, to verify the cell types reacting with the 6D1 and 2C3 mAbs we examined the expression profiles of zebrafish lymphocyte surface markers in FACS-sorted lymphocytes from kidney. cd4-1 (cd8a) and tcrac but not iglc transcripts were detected in 6D1(2C3) lymphocytes, whereas cd4-1 (cd8a) transcripts were not found in 6D1 (2C3) lymphocytes. Third, we further confirmed that 6D1 reacted with zebrafish CD4-1 but not CD4-2, and 2C3 recognized zebrafish CD8α expressed on HEK293T cells. Collectively, these findings suggest that 6D1 and 2C3 lymphocytes in zebrafish are equivalent to CD4 and CD8α T lymphocytes in mammals, respectively. Furthermore, we found the cross-reactivity of our 6D1 and 2C3 mAbs with other cyprinid species including goldfish, common carp and grass carp.
我们拥有针对银鲫（Carassius auratus langsdorfii）中CD4-1（6D1）和CD8α（2C3）的单克隆抗体（mAb）。在我们之前的研究中，我们表明2C3单克隆抗体阳性细胞是对同种异体靶标表现出特异性细胞毒性的主要细胞类型，这表明银鲫中的CD8α淋巴细胞等同于哺乳动物中的细胞毒性T淋巴细胞（CTL）。我们通过研究混合淋巴细胞培养（MLC）和半抗原/载体效应，进一步证明了6D1单克隆抗体阳性细胞的辅助性T细胞功能。在此，我们报告我们的单克隆抗体与斑马鱼淋巴细胞发生交叉反应。首先，6D1和2C3单克隆抗体识别7%-11%的斑马鱼淋巴细胞，这些细胞ZAP-70呈阳性且具有典型的淋巴细胞形态。其次，为了验证与6D1和2C3单克隆抗体发生反应的细胞类型，我们检测了来自肾脏的荧光激活细胞分选（FACS）淋巴细胞中斑马鱼淋巴细胞表面标志物的表达谱。在6D1（2C3）淋巴细胞中检测到cd4-1（cd8a）和tcrac转录本，但未检测到iglc转录本，而在6D1（2C3）淋巴细胞中未发现cd4-1（cd8a）转录本。第三，我们进一步证实6D1与斑马鱼CD4-1反应，但不与CD4-2反应，并且2C3识别在人胚肾293T细胞（HEK293T）上表达的斑马鱼CD8α。总的来说，这些发现表明斑马鱼中的6D1和2C3淋巴细胞分别等同于哺乳动物中的CD4和CD8α T淋巴细胞。此外，我们发现我们的6D1和2C3单克隆抗体与包括金鱼、鲤鱼和草鱼在内的其他鲤科物种存在交叉反应。
