Institute of Zoology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan, ROC.
Fish Shellfish Immunol. 2011 Jun;30(6):1283-93. doi: 10.1016/j.fsi.2011.03.018. Epub 2011 Apr 2.
CD8 is a membrane glycoprotein found primarily on the surface of T lymphocytes such as cytotoxic T lymphocytes (CTL), natural killer cells (NK) and γδ T lymphocytes. It helps T lymphocytes to kill the infected cells that presents microbial antigen on the cell surface. However, analysis of fish cellular immunity has been limited because of the lack of CD8 antibodies in grouper. In this present study, we cloned full-length CD8α cDNAs from orange-spotted grouper (Epinephelus coioides), an important fish species economically. The deduced protein of CD8α contained 227 amino acid residues in length and included one signal peptide, Ig superfamily V domain, hinge region, transmembrane domain, cytoplasmic tail and conserved binding motif associated with tyrosine kinase p56(lck). The molecular weight of the mature protein was estimated at 22.5 kDa and pI at 9.55. Phylogenetically, the predicted grouper CD8α protein was similar to CD8α from other marine fish species in which the identity was 50-60%. Real-time PCR revealed that CD8α transcript was constitutively expressed in thymus, head kidney, gill, spleen, gut and peripheral blood leucocyte (PBL); and the highest expression in thymus. CD8α transcript in the spleen of fish injected with nervous necrosis virus (NNV) was significantly up-regulated at 4 days post-injection compared to the untreated fish. Rabbit antiserum prepared against recombinant CD8α protein was able to recognize specifically the subset lymphocytes which have a diameter of 7 μm, a high nucleus/cytoplasm ratio and a ring-shaped cytoplasm. The cytotoxicity of CD8α(+) lymphocytes at one-week post-NNV infection was enhanced significantly against NNV-infected autologous fin cells in comparison with NNV-infected allogeneic or RSIV-infected autologous fin cells. Flow cytometry analysis revealed that both the number and mean fluorescence intensity (MFI) of CD8α(+) PBL were significantly increased at 7 days post-NNV infection. The specific cytotoxicity and MHC class I restriction of the lymphocytes sorted by rCD8α antibody are properties that can be attributed to CTL. In addition, low level of cytotoxicity was found in PBL against allogeneic targets as well as CD8α(+) effectors killed autologous targets nonspecifically, implicated presence of cytotoxic T subsets, possibly nonspecific cytotoxic cells (NCC) and γδ T lymphocytes, without MHC class I restriction. In conclusion, grouper cytotoxic CD8α(+) PBL have heterogeneous features in specific antigen recognition and class I MHC restriction.
CD8 是一种主要存在于 T 淋巴细胞表面的膜糖蛋白，如细胞毒性 T 淋巴细胞 (CTL)、自然杀伤细胞 (NK) 和 γδ T 淋巴细胞。它帮助 T 淋巴细胞杀死表面呈现微生物抗原的感染细胞。然而，由于石斑鱼缺乏 CD8 抗体，鱼类细胞免疫的分析一直受到限制。在本研究中，我们从经济上重要的鱼类物种——鞍带石斑鱼 (Epinephelus coioides) 中克隆了全长 CD8α cDNA。CD8α 的推导蛋白包含 227 个氨基酸残基，包括一个信号肽、Ig 超家族 V 结构域、铰链区、跨膜区、胞质尾和与酪氨酸激酶 p56(lck) 相关的保守结合基序。成熟蛋白的分子量估计为 22.5 kDa，等电点为 9.55。系统进化分析表明，预测的石斑鱼 CD8α 蛋白与其他海洋鱼类的 CD8α 蛋白相似，其同一性为 50-60%。实时 PCR 显示，CD8α 转录本在胸腺、头肾、鳃、脾、肠和外周血白细胞 (PBL) 中持续表达; 在胸腺中表达最高。与未处理的鱼相比，注射神经坏死病毒 (NNV) 后的 4 天，鱼脾脏中的 CD8α 转录本显著上调。用重组 CD8α 蛋白制备的兔抗血清能够特异性识别直径为 7μm、核质比高且呈环形细胞质的亚群淋巴细胞。与 NNV 感染的同种异体或 RSIV 感染的自体鳍细胞相比，NNV 感染后一周 CD8α(+)淋巴细胞对 NNV 感染的自体鳍细胞的细胞毒性显著增强。流式细胞术分析显示，NNV 感染后 7 天，PBL 的 CD8α(+)数量和平均荧光强度 (MFI) 均显著增加。用 rCD8α 抗体分选的淋巴细胞具有特异性细胞毒性和 MHC Ⅰ类限制，这是 CTL 的特性。此外，PBL 对同种异体靶标表现出低水平的细胞毒性，并且 CD8α(+)效应物非特异性地杀死自体靶标，暗示存在细胞毒性 T 亚群，可能是非特异性细胞毒性细胞 (NCC) 和 γδ T 淋巴细胞，而没有 MHC Ⅰ类限制。总之，石斑鱼细胞毒性 CD8α(+) PBL 在特异性抗原识别和 I 类 MHC 限制方面具有异质性特征。
