CD4+ T CELLS RESPONDING TO SALMONELLA INFECTION

CD4 T 细胞对沙门氏菌感染的反应

• 批准号: 6626383
• 负责人: BRAD T COOKSON
• 金额: $ 33.81万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626383
• 关键词: SDS polyacrylamide gel electrophoresis; Salmonella infections; bacteria infection mechanism; bacterial antigens; bacterial genetics; bactericidal immunity; biomarker; cellular immunity; enzyme linked immunosorbent assay; flow cytometry; genetic mapping; genetic strain; helper T lymphocyte; laboratory mouse; phagocytosis; vesicle /vacuole; virulence

DESCRIPTION (Adapted from the Applicant's Abstract): Salmonella, Mycobacterium and Histoplasma are facultative intracellular pathogens that live inside phagosomes of host macrophages. They all cause AIDS-defining illnesses, and the investigator's long-term goal is to understand the development of immunity against such pathogens. CD4+ T cells are also required for immune mice to resist virulent Salmonella, providing a model of protective host functions which can successfully combat a macrophage-tropic infection. However, the specific bacterial antigens (Ags) recognized by Salmonella-immune hosts are largely unknown. Two proteins expressed in the surface-exposed "compartment" of Salmonella are recognized by CD4+ T cells from immune mice. One is a flagellar protein also recognized by T cells from humans immunized with Salmonella. The other is an unidentified protein expressed by most Enterobacteriaceae, including E. coli, Yersinia, Shigella, and Enterobacter. Both proteins are regulated in a fashion suggesting part of the Salmonella intracellular survival strategy is to down-regulate expression of bacterial surface Ags recognized by CD4+ T cells. In AIM I, the diversity of Salmonella Ags recognized by CD4+ T cells from immune mice will be determined using SDS-PAGE fractionated bacteria as Ag, and bacterial expression of these Ags will be characterized with respect to compartmentalization and regulation using T cell clones. The studies will provide insight into the nature of Ags recognized by CD4+ T cells, the environmental signals affecting bacterial processing Salmonella for T cell responses. In AIM 2, genes encoding Ags recognized by T cell clones will be identified by expression cloning or sequencing analysis of biochemically purified Ags. This work may reveal gene products useful as markers of cellular immunity to Salmonella in humans. In AIM 3, murine infection with Salmonella strains expressing a model Ag in various compartments of the bacterial cell will be used to directly test if compartmentalization of bacterial Ag alters its significance for surveillance by T cells. Primary and secondary CD4+ T cell responses generated by these stains will be quantified using ELISPOT and flow cytometry, and the effectiveness of an Ag-specific immune response against these strains will be tested in vivo. These studies will provide insight into the nature of Ags recognized by CD4+ T cells responding to pathogens similarly adapted for life in phagosomes. In AIM 4, the functional importance of Ags identified in AIMS 1 & 2 will be determined by testing purified Ags for their ability to stimulate protective immunity against challenge by virulent Salmonella. The protective Ags identified will be excellent candidates for components of subunit vaccines and markers of cellular immunity in humans.

描述（改编自申请人的摘要）：沙门氏菌、分枝杆菌 和组织胞浆菌是生活在体内的兼性细胞内病原体 宿主巨噬细胞的吞噬体。它们都会导致艾滋病定义的疾病，并且 研究人员的长期目标是了解免疫力的发展 针对此类病原体。免疫小鼠也需要 CD4+ T 细胞 抵抗剧毒沙门氏菌，提供保护宿主功能的模型 它可以成功对抗巨噬细胞嗜性感染。然而， 沙门氏菌免疫宿主识别的特异性细菌抗原 (Ag) 是 很大程度上不为人知。两种蛋白质在表面暴露的“区室”中表达 沙门氏菌可被免疫小鼠的 CD4+ T 细胞识别。一种是鞭毛 蛋白质也被沙门氏菌免疫人类的 T 细胞识别。这 另一种是大多数肠杆菌科细菌表达的未知蛋白质， 包括大肠杆菌、耶尔森氏菌、志贺氏菌和肠杆菌。两种蛋白质都是 以表明沙门氏菌细胞内存活的部分方式进行调节 策略是下调细菌表面Ags的表达 CD4+ T 细胞。在 AIM I 中，CD4+ T 识别的沙门氏菌 Ag 多样性 来自免疫小鼠的细胞将使用 SDS-PAGE 分级细菌进行测定 作为 Ag，这些 Ag 的细菌表达将根据以下方面进行表征 使用 T 细胞克隆进行区室化和调节。这些研究将 深入了解 CD4+ T 细胞识别的 Ag 的性质， 影响 T 细胞细菌加工的环境信号 沙门氏菌 回应。在 AIM 2 中，T 细胞克隆识别的编码 Ag 的基因将被 通过表达克隆或生化测序分析鉴定 纯化的Ag。这项工作可能揭示可用作细胞标记的基因产物 人类对沙门氏菌的免疫力。在 AIM 3 中，小鼠沙门氏菌感染 在细菌细胞的各个区室中表达模型Ag的菌株 将用于直接测试细菌 Ag 的区室化是否改变 其对于 T 细胞监视的意义。初级和次级 CD4+ T 细胞 这些染色剂产生的反应将使用 ELISPOT 和流式细胞仪进行量化 细胞计数，以及 Ag 特异性免疫反应的有效性 这些菌株将在体内进行测试。这些研究将提供深入了解 CD4+ T 细胞识别的 Ag 的性质对病原体的反应类似 适应吞噬体的生活。在 AIM 4 中，Ag 的功能重要性 AIMS 1 和 2 中确定的抗原将通过测试纯化的 Ag 的活性来确定 刺激保护性免疫力抵抗有毒物质攻击的能力 沙门氏菌。已确定的保护性 Ag 将是优秀的候选者 亚单位疫苗的成分和人类细胞免疫的标志物。