Flies Andrew S, Blackburn Nicholas B, Lyons Alan Bruce, Hayball John D, Woods Gregory M
Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia.
Department of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia.
Front Immunol. 2017 May 3;8:513. doi: 10.3389/fimmu.2017.00513. eCollection 2017.
Immune checkpoint molecules function as a system of checks and balances that enhance or inhibit immune responses to infectious agents, foreign tissues, and cancerous cells. Immunotherapies that target immune checkpoint molecules, particularly the inhibitory molecules programmed cell death 1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), have revolutionized human oncology in recent years, yet little is known about these key immune signaling molecules in species other than primates and rodents. The Tasmanian devil facial tumor disease is caused by transmissible cancers that have resulted in a massive decline in the wild Tasmanian devil population. We have recently demonstrated that the inhibitory checkpoint molecule PD-L1 is upregulated on Tasmanian devil () facial tumor cells in response to the interferon-gamma cytokine. As this could play a role in immune evasion by tumor cells, we performed a thorough comparative analysis of checkpoint molecule protein sequences among Tasmanian devils and eight other species. We report that many of the key signaling motifs and ligand-binding sites in the checkpoint molecules are highly conserved across the estimated 162 million years of evolution since the last common ancestor of placental and non-placental mammals. Specifically, we discovered that the CTLA-4 (MYPPPY) ligand-binding motif and the CTLA-4 (GVYVKM) inhibitory domain are completely conserved across all nine species used in our comparative analysis, suggesting that the function of CTLA-4 is likely conserved in these species. We also found that cysteine residues for intra- and intermolecular disulfide bonds were also highly conserved. For instance, all 20 cysteine residues involved in disulfide bonds in the human 4-1BB molecule were also present in devil 4-1BB. Although many key sequences were conserved, we have also identified immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs) in genes and protein domains that have not been previously reported in any species. This checkpoint molecule analysis and review of salient features for each of the molecules presented here can serve as road map for the development of a Tasmanian devil facial tumor disease immunotherapy. Finally, the strategies can be used as a guide for veterinarians, ecologists, and other researchers willing to venture into the nascent field of wild immunology.
免疫检查点分子作为一种制衡系统，可增强或抑制对传染原、外来组织和癌细胞的免疫反应。近年来，针对免疫检查点分子，特别是程序性细胞死亡1和细胞毒性T淋巴细胞相关蛋白4（CTLA-4）等抑制性分子的免疫疗法彻底改变了人类肿瘤学，但除灵长类动物和啮齿动物外，人们对其他物种中的这些关键免疫信号分子知之甚少。袋獾面部肿瘤疾病是由可传播的癌症引起的，导致野生袋獾种群数量大幅下降。我们最近证明，抑制性检查点分子PD-L1在袋獾面部肿瘤细胞上因γ干扰素细胞因子而上调。由于这可能在肿瘤细胞的免疫逃逸中起作用，我们对袋獾和其他八个物种的检查点分子蛋白质序列进行了全面的比较分析。我们报告说，自胎盘类和非胎盘类哺乳动物的最后一个共同祖先以来，在大约1.62亿年的进化过程中，检查点分子中的许多关键信号基序和配体结合位点高度保守。具体而言，我们发现CTLA-4（MYPPPY）配体结合基序和CTLA-4（GVYVKM）抑制域在我们比较分析中使用的所有九个物种中完全保守，这表明CTLA-4的功能在这些物种中可能是保守的。我们还发现，分子内和分子间二硫键的半胱氨酸残基也高度保守。例如，人类4-1BB分子中参与二硫键的所有20个半胱氨酸残基在袋獾4-1BB中也存在。尽管许多关键序列是保守的，但我们也在基因和蛋白质结构域中鉴定出了基于免疫受体酪氨酸的抑制基序（ITIM）和基于免疫受体酪氨酸的开关基序（ITSM），这些在任何物种中以前都没有报道过。这里对每个分子的检查点分子分析和显著特征综述可作为袋獾面部肿瘤疾病免疫疗法开发的路线图。最后，这些策略可作为兽医、生态学家和其他愿意涉足新兴野生免疫学领域的研究人员的指南。
