Gene Expression Analysis of Dog Natural Killer Cells as Immunotherapy Target

项目摘要 狗是大型的远系繁殖动物，在免疫系统完整的情况下会自发地患上癌症 以及与人类共享环境暴露的情况下。人类和狗也有一对 进化历史导致犬和人类基因组和微生物组之间有更大的相似性 比老鼠和人类之间的差别更大总之，这些特征使狗成为一个有利的翻译模型， 研究癌症免疫学和癌症免疫疗法。狗的临床试验允许研究复合免疫 本发明的目的是提供治疗期间的相互作用，同时还解决癌症免疫疗法的长期功效和毒性。 然而，免疫解剖需要开发稳健且经验证的测定和试剂，并且需要较高的成本。 在对狗免疫学有更深入的了解之前，使用狗免疫疗法的影响研究还为时过早， 免疫生物标志物，特别是自然杀伤（NK）细胞。这项提案将建立在我们令人兴奋的 表征犬NK细胞的初步数据，包括犬NK过继转移的首次犬中临床试验 细胞和新的细胞因子递送与吸入IL-15。与医学院合作， 兽医，和数据密集型生物实验室，进行跨物种的基因组，转录， 和宏基因组序列分析，我们将检查狗NK差异基因表达1）多个 刺激和稳态条件; 2）来自狗血液和肿瘤生物样本;和3）来自响应 和在研究性狗临床试验中用吸入IL-15治疗的无应答狗。我们将利用多个 应用高通量测序技术（RNASeq和单细胞RNA测序）， 来自离体和体内的静息和活化的狗NK细胞的更稳健和经验证的基因特征 进一步分析狗肉瘤浸润NK细胞的条件。然后我们将这些基因特征 来自鼠和人NK细胞的高通量测序的数据，以弥合物种差异， 便于临床翻译。最终，犬NK细胞在免疫防御和癌症中的作用的知识 目前尚不清楚这是由于物种间的生物学差异还是由于 用于调查的试剂。这项建议的结果将扩展犬研究提供的重要联系 在小鼠临床前研究和人类临床试验之间，一般用于癌症免疫治疗研究， 特别是对于NK研究，其中高影响力的临床翻译仍然是难以捉摸的。

Project Summary Dogs are large, outbred animals that develop cancer spontaneously in the presence of an intact immune system and in the setting of shared environmental exposures with humans. Humans and dogs also share a paired evolutionary history which has led to greater similarities between canine and human genomes and microbiomes than between mouse and human. Together, these traits make dogs an advantageous translational model to study cancer immunology and cancer immunotherapy. Dog clinical trials allow for the study of complex immune interactions during therapy while also addressing long-term efficacy and toxicity of cancer immunotherapies. However, immune dissection requires the development of robust and validated assays and reagents, and high impact studies using dog immunotherapy are premature until a deeper understanding of dog immunology and immune biomarkers is realized, especially for natural killer (NK) cells. This proposal will build on our exciting preliminary data characterizing dog NK cells, including first-in-dog clinical trials of adoptive transfer of dog NK cells and novel cytokine delivery with inhaled IL-15. In collaboration with the School of Medicine, the School of Veterinary Medicine, and the Data Intensive Biology Lab which performs cross-species genomic, transcriptomic, and metagenomic sequence analyses, we will examine dog NK differential gene expression across 1) multiple stimulatory and steady-state conditions; 2) from dog blood and tumor bio-specimens; and 3) from responding and non-responding dogs treated with inhaled IL-15 on an investigational dog clinical trial. We will utilize multiple applications of high-throughput sequencing technologies (RNASeq and single cell RNA sequencing) to establish more robust and validated gene signatures of resting and activated dog NK cells from both ex vivo and in vivo conditions with further analysis of dog sarcoma-infiltrating NK cells. We will then compare these gene signatures to data from high throughput sequencing of murine and human NK cells to bridge species differences and facilitate clinical translation. Ultimately, knowledge of the role of canine NK cells in immune defense and cancer therapy is limited, and it remains unclear whether this is due to biological differences across species or limited reagents for investigation. Results from this proposal will extend the important link that canine studies provide between murine pre-clinical studies and human clinical trials, for cancer immunotherapy research in general but especially for NK studies where high impact clinical translation has remained elusive.