A comparative analysis of human and canine iNKT cells for ACT

项目总结(见说明)： 采用细胞疗法(ACT)的圣杯是从 可以安全有效地跨越异基因障碍进行移植的健康捐赠者，以改善 汽车疗法的可得性和有效性。不变自然杀伤T细胞(INKT)是一个异质性群体 表达不变的TcRα的“非常规”T细胞识别非多态的糖脂- 提出了MHC分子CD1d。因此，未经编辑的同种异体iNKT细胞(allo-iNKT)可以被采用 在没有引起GVHD的情况下转移。此外，iNKT细胞抑制传统T细胞引起的GVHD，并 抑制同种异体细胞的排斥反应，促进实体器官移植的终身耐受性。此外，iNKT细胞具有 天然的肿瘤趋向性，杀伤CD1d+肿瘤相关巨噬细胞，激活肿瘤中的效应性免疫细胞。 鉴于这些特性，我们假设allo-iNKT可以安全有效地用于推广allo-cart 坚持不懈，增强抗肿瘤免疫力。 INKT功能的复杂性可能归因于不同的iNKT亚群表现出细胞毒性与 监管属性。了解子集的异质性将使最“理想的”iNKT子集能够 被选为联合ACT的评估对象。与小鼠不同，犬iNKT细胞具有相似的表型和 人类iNKT细胞的生物学特征，表明患有自发性癌症的宠物狗可能代表着 使用iNKT细胞研究联合异基因移植的理想临床前模型。在这里，为了与目标保持一致 支持犬类免疫治疗临床试验和产生免疫试剂的分局 IOTN有兴趣生成现成的购物车单元平台，我们将执行比较方法来 通过集成的单细胞RNAseq/CITEseq方法研究可行的iNKT细胞异质性 人iNKT和转移学习用于从scRNA中识别犬iNKT亚群的表型标记 序列号数据。此外，为了扩展犬NK研究的免疫反应工具箱，一个全面的单链抗体 噬菌体展示文库将用于简单的淘洗实验，以产生和验证急需的狗- 针对NK激活和抑制受体的特异性抗体将对了解 犬NK激活和抑制的机制。总之，这些研究将奠定必要的基础 为下一代和体内测试结合异体基因的最佳iNKT细胞产品奠定基础 免疫功能良好的犬癌患者的iNKT/CART治疗，目标是加速新的allo-ACT 将这些策略引入人类诊所。 相关性(请参阅说明)： INKT细胞的自然特性有助于安全的同种异体过继转移和抑制 同种异体反应的反应，否则将消除异种车产品。在这里，我们将提供 对人和狗的iNKT细胞进行必要的比较评估，使未来能够进行体内测试 用于联合应用allo-iNKT/CART疗法的最佳翻译相关iNKT产品 犬癌患者，旨在加速将新的allo-ACT策略应用于人类临床

PROJECT SUMMARY (See instructions): The holy grail of adoptive cell therapies (ACT) is the generation of fit, ready-to-use, CART products from healthy donors that could be safely and effectively transferred across allogeneic barriers to improve the availability and efficacy of CAR-therapies. Invariant Natural Killer T cells (iNKT) are a heterogenous population of `non-conventional' T cells expressing an invariant TCRα which recognizes the non-polymorphic, glycolipid- presenting MHC molecule CD1d. As such, unedited allogeneic iNKT cells (allo-iNKT) can be adoptively transferred without causing GVHD. Further, iNKT cells suppress GVHD caused by conventional T cells and inhibit rejection of allogeneic cells promoting lifelong tolerance of solid organ grafts. In addition, iNKT cells have natural tumor tropism, kill CD1d+ tumor associated macrophages and activate effector immune cells in the tumor. Given these properties, we hypothesize that allo-iNKT could be safely and effectively used to promote allo-CART persistence and enhance anti-tumor immunity. The complexity of iNKT function may be attributed to distinct iNKT subsets that exhibit cytotoxic versus regulatory properties. Understanding subset heterogeneity would enable the most “desirable” iNKT subsets to be selected for evaluation of combination ACT. Unlike mice, canine iNKT cells share similar phenotypic and biological features to human iNKT cells, suggesting that pet dogs with spontaneous cancer may represent the ideal pre-clinical model to investigate combination allo-ACT using iNKT cells. Here, in alignment with the aims of PRECINCT to support canine immunotherapy clinical trials and generate immunological reagents and with the interest of IOTN to generate an off-the-shelf CART cell platform, we will perform a comparative approach to investigate actionable iNKT cell heterogeneity through an integrated, single cell RNAseq/CITEseq approach in human iNKT and employ transfer learning to impute phenotypic markers of canine iNKT subsets from scRNA seq data. Furthermore, to expand the immunoreagent toolbox for canine NK studies, a comprehensive scFv phage display library will be used in simple panning experiments to generate and validate much needed canine- specific antibodies against NK activation and inhibitory receptors that will be of value in understanding the mechanisms governing canine NK activation and inhibition. Together, these studies will lay the necessary groundwork for future generation and in vivo testing of optimal iNKT cell products for combination allo- iNKT/CART therapies in immune competent canine cancer patients, with the goal to accelerate novel allo-ACT strategies into the human clinic. RELEVANCE (See instructions): The natural properties of iNKT cells lend themselves to safe allogeneic adoptive transfer and suppression of alloreactive responses that would otherwise eliminate allo-CART products. Here, we will provide the necessary comparative evaluation of human and canine iNKT cells that will enable future in vivo testing of optimal, translationally relevant iNKT products for combination allo-iNKT/CART therapies in immune competent canine cancer patients, aiming to accelerate novel allo-ACT strategies into the human clinic