Immunoregulation by TLR-activated TIM-1+ ProB Cells in Transplantation

TLR 激活的 TIM-1 ProB 细胞在移植中的免疫调节

• 批准号: 10455069
• 负责人: DAVID M ROTHSTEIN
• 金额: $ 43.75万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455069
• 关键词: Address; Adult; Allograft Tolerance; Allografting; Antigens; B cell differentiation; B-Lymphocytes; Bone Marrow; Cell Count; Cell Lineage; Cells; Cues; Dependence; Engraftment; Environment; Genetic Transcription; Immune response; Impairment; Inflammatory; Interleukin-10; Knockout Mice; Lead; Ligation; MS4A1 gene; Maintenance; Mediating; Methods; Modeling; Monoclonal Antibodies; Nature; Pathway interactions; Peripheral; Phenotype; Population; Proliferating; Property; Protocols documentation; Receptor Signaling; Regulation; Regulator Genes; Regulatory T-Lymphocyte; Rest; Role; Signal Pathway; Site; Therapeutic; Time; Translating; Transplantation; Transplantation Tolerance; base; heart allograft; immunoregulation; islet allograft; knockout gene; member; mouse model; nano-string; single-cell RNA sequencing; transcriptome sequencing

Abstract (Project 2) Rather than use passive transfer of well differentiated adult immunoregulatory cells as a means to create transplant tolerance, we have discovered that a population CpG activated pro-B cells are extraordinarily potent, far more potent on a per cell basis than Tregs and far easier to prepare. Transfer of only 40,000, but not 90,000, induces permanent engraftment and probably tolerance in MHC incompatible islet and cardiac allograft mouse models. We have discovered that CpG induces expression TIM-1 a potential master switch for expression of an immunoregulatory module in mature Bregs (see Project 1/ Kuchroo). We also learned that ligation of TIM-1 by anti-TIM-1 mAb induces expression of IL-10, a member of the regulatory model discovered by Kuchroo. The stepwise contributions of CpG and activation of the TIM-1 pathway are examined through RNAseq and NanoString based analysis. To more precisely characterize the fate and phenotype, proliferative and differentiation of CpG activated pro-B cells, lineage-tracking methods are utilized throughout. We know that CpG activated pro-B cells proliferate and differentiate after transfer when introduced in optimal, not excessive, cell number. Depending upon environmental cues CpG activated pro-B cells can differentiate into a variety Bregs but CpG activated pro-B cells from RAG KO mice do not generate Bregs. In this project we will analyze the cellular basis of tolerance believing that CpG activated B regs and their progeny create an environment conducive to activation, expansion and retention of donor specific Tregs. The specific but multifaceted requirements for B cell differentiation, including the nature of the niche impairing expansion and differentiation of CpG-ProBs, in the acquisition of tolerance will be analyzed through use of a panel of carefully selected gene knockout mice. The role of T and B regs in the induction and maintenance of tolerance will be determined through time course protocols that selectively destroy Tregs or the mature progeny of CpG activate pro cells. The diversity of CpG-ProB progeny and their expression of a regulatory module that is IL-10 inclusive will be examined by single cell RNAseq of CpG-ProBs and their progeny at the transplant.

摘要（项目2） 而不是使用被动转移分化良好的成体免疫调节细胞作为创造的手段 移植耐受性，我们发现 CpG 激活的亲 B 细胞群非常有效， 从每个细胞的角度来看，它比 Tregs 更有效，而且更容易准备。只转账4万，但不 90,000，在 MHC 不相容的胰岛和心脏同种异体移植物中诱导永久植入并可能耐受 鼠标模型。我们发现 CpG 诱导表达 TIM-1，这是一个潜在的主开关 成熟 Breg 中免疫调节模块的表达（参见项目 1/Kuchroo）。我们还了解到 抗 TIM-1 mAb 连接 TIM-1 可诱导 IL-10 的表达，IL-10 是发现的调节模型的成员 库克鲁. CpG 的逐步贡献和 TIM-1 通路的激活通过 基于 RNAseq 和 NanoString 的分析。为了更准确地表征命运和表型，增殖 以及 CpG 激活的 pro-B 细胞的分化，整个过程中都采用了谱系追踪方法。我们知道 当以最佳状态引入时，CpG 激活的 pro-B 细胞在转移后会增殖和分化，而不是 细胞数量过多。根据环境提示，CpG 激活的 pro-B 细胞可以分化为 多种 Bregs，但来自 RAG KO 小鼠的 CpG 激活的 pro-B 细胞不会产生 Bregs。在这个项目中我们将 分析耐受性的细胞基础，相信 CpG 激活的 B regs 及其后代创造了 有利于供体特异性Tregs的激活、扩增和保留的环境。具体但是 B 细胞分化的多方面要求，包括损害扩张的生态位的性质和 CpG-ProB 的分化，在耐受性的获得中，将通过使用一组仔细的分析来分析 选择基因敲除小鼠。 T 和 B 调节器在诱导和维持耐受性中的作用将是 通过选择性破坏 Tregs 或 CpG 激活的成熟后代的时间过程协议来确定 亲细胞。 CpG-ProB 后代的多样性及其包含 IL-10 的调节模块的表达 将在移植时通过 CpG-ProB 及其后代的单细胞 RNAseq 进行检查。