FIV/HIV IMMUNOPATHOGENESIS--A CASE FOR THE CD8+ CELL

FIV/HIV 免疫发病机制——CD8 细胞的一个案例

基本信息

批准号：
6532755
负责人：
Wayne A Tompkins
金额：
$ 29.73万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2004-07-31
项目状态：
已结题

项目摘要

Elevation in the number of CD8+ cells is a consistent feature of HIV and FIV infections. In the case of HIV, this abnormal homeostasis is marked by a progressive loss of L-selectin (LS) positive naive CD8+ cells and a concomitant expansion of LS negative CD8+ effector cells, such that the CD8+ LSneg cells may represent 80-90 percent of the total circulating CD8+ cells in the late asymptomatic stage infection. FIV infection induces the progressive expansion of a CD8+ subset characterized by a marked reduction of the beta chain. This CD8+ betalo phenotype may comprise as much as 90 percent of total blood CD8+ cells at late-stage asymptomatic infection. We have taken advantage of beta chain down-regulation to FACStar sort highly enriched CD8+ betalo and CD8+ betahi phenotypes, and have shown that the CD8+ betalo phenotype synthesizes high levels of IL10 and IFNgamma mRNA, and has potent anti- FIV activity. In addition, we have shown by 2- and 3-color FACS that the CD8+ betalo cells is an effector phenotype (CD8+ betaloLSneg CD44hi) and the CD8+ betahi is a naive phenotype (CD8+ betahiLSneg CD44hi). This proposal will test the hypothesis that FIV induces a chronic expansion of a CD8+betaloLSneg CD44hi effector phenotype that not only has potent antiviral activity but exhibits abnormal tissue trafficking and immunosuppressive responses to secondary infections. Experiments will be designed to further characterize the phenotype of this CD8+betaloLSneg subset and its distribution in lymphoid and nonlymphoid tissues at different stages of FIV infection. Fluorescent dye labeling will be done to assess the trafficking potential of CD8+ betaloLSneg cells between blood and LN, and between blood and the lung in response to T. gondii infection. RT-qcPCR studies will be designed to determine if the CD8+betaloLSneg phenotype is regulated at the level of gene transcription and if it is related to virus load. Purified subsets of CD8+betaloLSneg (effector) and CD8+betahiLSpos (naive) will be assayed by RT-qcPCR assays for cytokine and chemokine mRNA to address the hypothesis that the CD8+ betaloLSneg has the cytokine/chemokine profile of a CTL and/or T suppressor cell. In vitro experiments will determine if CD8+ betaloSneg cells are CTL's or virus suppressor cells and whether they suppress mitogen and antigen-specific recall responses by PBMC. Studies will also address the hypothesis that CD8+betaloLSneg cells exert immunosuppressive effects on lung macrophages resulting in decreased cytokine responses and uncontrolled replication of T. gondii tachyzoites. These experiments will collectively test the hypothesis that FIV induces the chronic expansion of a novel CD8+betaloLSneg T- suppressor-like activation phenotype that not only mediates immunity to FIV, but because of its altered tissue trafficking (loss of L-selectin) and cytokine expression profile (e.g. IL10) is selectively recruited into inflammatory sites and suppresses immune responses to secondary pathogens.

CD8+ 细胞数量升高是 HIV 的一致特征， FIV 感染。就艾滋病毒而言，这种异常的体内平衡是明显的 L-选择素 (LS) 阳性幼稚 CD8+ 细胞逐渐丧失， LS阴性CD8+效应细胞的伴随扩增，使得 CD8+ LSneg 细胞可能占总数的 80-90% 感染晚期无症状阶段的循环CD8+细胞。国际静脉注射感染诱导 CD8+ 亚群逐渐扩增其特点是β链显着减少。这个 CD8+ 贝塔洛表型可能占全血 CD8+ 细胞的 90% 处于无症状感染者的晚期。我们已经利用了测试版链下调至 FACStar 排序高度富集的 CD8+ betalo 和 CD8+ betahi 表型，并且已表明 CD8+ betalo 表型合成高水平的 IL10 和 IFNgamma mRNA，并具有有效的抗- 国际排联活动。此外，我们通过 2 色和 3 色 FACS 表明 CD8+ betalo 细胞是一种效应表型 (CD8+ betaloLSneg CD44hi) 和 CD8+ betahi 是幼稚表型 (CD8+ betahiLSneg CD44hi）。该提案将检验 FIV 诱发的假设 CD8+betaloLSneg CD44hi 效应子表型的慢性扩张不仅具有有效的抗病毒活性，而且表现出异常组织贩运和对继发感染的免疫抑制反应。实验将被设计为进一步表征表型这个 CD8+betaloLSneg 子集及其在淋巴和淋巴中的分布 FIV 感染不同阶段的非淋巴组织。荧光将进行染料标记以评估 CD8+ 的贩运潜力血液和 LN 之间以及血液和肺之间的 betaloLSneg 细胞响应弓形虫感染。将设计 RT-qcPCR 研究确定 CD8+betaloLSneg 表型是否在水平上受到调节基因转录的情况以及是否与病毒载量有关。纯化的 CD8+betaloLSneg（效应器）和 CD8+betahiLSpos（原始）的子集将通过 RT-qcPCR 检测细胞因子和趋化因子 mRNA 来解决 CD8+ betaloLSneg 具有细胞因子/趋化因子的假设 CTL 和/或 T 抑制细胞的概况。体外实验将确定 CD8+ betaloSneg 细胞是 CTL 还是病毒抑制细胞以及它们是否抑制有丝分裂原和抗原特异性回忆反应通过 PBMC。研究还将解决 CD8+betaloLSneg 的假设细胞对肺巨噬细胞产生免疫抑制作用，导致细胞因子反应减少和弓形虫复制失控速殖子。这些实验将共同检验假设 FIV 诱导新型 CD8+betaloLSneg T- 的慢性扩增抑制样激活表型不仅介导免疫 FIV，但由于其组织运输发生改变（L-选择素丢失）并选择性地招募细胞因子表达谱（例如 IL10）进入炎症部位并抑制继发性免疫反应病原体。