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

CD 8+细胞数量的增加是HIV的一贯特征， FIV感染。 在艾滋病病毒的情况下，这种异常的稳态是显着的， 通过L-选择素（LS）阳性初始CD 8+细胞的进行性损失， LS阴性CD 8+效应细胞的伴随扩增，使得 CD 8 + LSneg细胞可能占总数的80-90 在晚期无症状感染阶段循环CD 8+细胞。 FIV 感染诱导CD 8+亚群的进行性扩增 其特征在于β链的显著减少。 这种CD 8 + betalo 表型可包括多达90%的总血液CD 8+细胞 晚期无症状感染者。 我们利用了beta 链下调至FACStar分选高度富集的CD 8 + betalo， CD 8 + betahi表型，并已表明CD 8 + betalo表型 合成高水平的IL 10和IFN γ mRNA，并具有有效的抗 FIV活动 此外，我们已经通过2色和3色FACS显示， CD 8 + betalo细胞是效应表型（CD 8 + betaloLSneg CD 44 hi）和CD 8 + betahi是幼稚表型（CD 8 + betahiLSneg CD44hi）。 这项提议将检验FIV诱导一种 CD 8 +betaloLSneg CD 44 hi效应子表型的慢性扩增， 不仅具有有效的抗病毒活性， 贩运和对继发感染的免疫抑制反应。 将设计实验以进一步表征以下表型： 这种CD 8 +betaloLSneg亚群及其在淋巴和 非淋巴组织在不同阶段的FIV感染。 荧光 将进行染料标记以评估CD 8+的运输潜力 在血液和LN之间以及血液和肺之间的betaloLSneg细胞 响应T.弓形虫感染 将设计RT-qcPCR研究 以确定CD 8 +betaloLSneg表型是否在以下水平受到调节： 以及是否与病毒载量有关。 纯化 CD 8 +betaloLSneg（效应子）和CD 8 +betahiLSpos（初始）的亚群将 通过RT-qcPCR测定细胞因子和趋化因子mRNA，以解决 假设CD 8 + betaloLSneg具有细胞因子/趋化因子 CTL和/或T抑制细胞的特征。 体外实验将 确定CD 8 + 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 PBMC的。 研究还将阐明CD 8 +betaloLSneg 细胞对肺巨噬细胞发挥免疫抑制作用， 降低的细胞因子应答和T.弓形虫 速殖子 这些实验将共同验证假设 FIV诱导新的CD 8 +betaloLSneg T- 抑制子样激活表型，不仅介导对 FIV，但由于其改变的组织运输（L-选择素的损失） 并且细胞因子表达谱（例如IL 10）被选择性地募集 进入炎症部位并抑制免疫反应， 病原体

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.