Cell Interactions In Three Dimensional Tissue Culture

三维组织培养中的细胞相互作用

• 批准号: 6541163
• 负责人: Leonid B. Margolis
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541163
• 关键词: B lymphocyte; HIV infections; T lymphocyte; antibody formation; cell cell interaction; chemokine; dendritic cells; human immunodeficiency virus 1; human tissue; pathologic process; tissue /cell culture; virus replication

Critical events in HIV disease occur in lymphoid tissue where HIV kills infected CD4+ cells as well as many non-infected cells, disrupting a complex system of cell-cell interactions that mediate normal immune response. The general aim of the project is to determine the underlying mechanism(s) of HIV-1-mediated immunopathogenesis using our system of ex vivo-infected human tonsils. In vivo the ability of HIV-1 to use CXCR4 coreceptor exclusively or in addition to CCR5 coreceptor is often associated with massive loss of CD4+ T cells and rapid disease progression. The working hypothesis we are currently testing is that the HIV-1 env gene that determines the usage of CXCR4, exclusively or in addition to CCR5, is sufficient to induce T cell depletion and immunosuppression. Also, we investigate the contribution of HIV accessory genes, in particular that of nef gene to viral tissue cytopathicity. Earlier we showed that coreceptor specificity determines cytopathicity of mono-tropic HIV-1 variants of B subtype. This year we investigated (i) whether co-receptor specificity determinines cytopathicity of HIV-1 variants of subtypes other than B; (ii) whether the cytopathicity of dual-tropic R5X4 HIV-1 variants of B subtype is determined by their preferential usage of a particular co-receptor, and (iii) whether the contribution of an HIV accessory gene nef to HIV pathogenesis is associated with modulation of cell surface receptors. To investigate the ex vivo cytopathicity of R5 and X4 HIV-1 variants of non-B subtypes, we used primary isolates of subtypes A, E, and C and prototypic subtype B variants. We observed no relationship between the maximal level of viral replication and either coreceptor usage or viral subtype. However, we did observe a strong correlation between viral coreceptor usage and CD4+ T lymphocyte depletion across all subtypes tested. All R5 HIV-1 infections resulted in mild depletion of CD4+ T cells, whereas all X4 HIV-1 infections caused severe depletion of CD4+ T cells irrespective of their subtype. Thus, non-B HIV-1 subtypes are similar to HIV-1 subtype B in that coreceptor specificity determines cytopathicity in human lymphoid tissue infected ex vivo. To find out whether differential cytopathicity of R5X4 viruses is also related to their preferential use of a particular coreceptor, we infected CCR5 delta32 tissue with three R5X4 HIV-1 variants: 89.6, 89-v345.FL, and 89-v345.SF; the latter two are isogenic to 89.6 except for the V3-V5 region of gp120. Variant 89.6 severely depletes tissue of CD4+ T cells, whereas 89-v345.SF depletes only mildly. Variant 89-v345.FL was an intermediate depletor. Both in CCR5 delta 32 and in normal lymphoid tissue, 89.6 and 89-v345.FL replicated with similar kinetics. In contrast, 89-v345.SF did not replicate in lymphoid tissue lacking CCR5. In agreement with these results, replication of 89.6 was completely inhibited by an CXCR4 ligand AMD3100 but was insensitive to a CCR5 ligand RANTES, whereas 89-v345.SF replication was almost completely inhibited by RANTES but not by AMD3100. Replication of 89-v345.FL was reduced both by AMD3100 and by RANTES. Among two studied primary isolates, which were dual-tropic in transfected cell lines, one was not inhibited by RANTES but was almost completely blocked by AMD3100. Thus, in lymphoid tissues some R5X4 isolates appear to utilize CXCR4 exclusively, whereas others use CCR5 exclusively. R5X4 HIV variants that use CXCR4 preferentially are highly cytopathic, whereas those preferentially using CCR5 deplete CD4+ T cells mildly. Hence, preferential coreceptor usage determines cytopathicity of R5X4 variants. Cell surface receptors may mediate the effects of an accessory gene nef on HIV infection. Tissues were infected with NL4-3 clones bearing nef alleles from sequential patient's isolates, as well as with clones bearing recombinant alleles. The HIV-1 variants containing the nef alleles isolated at later time points showed two- to-three fold higher levels of replication than those containing the nef alleles initially isolated at the early time point. Higher levels of replication correlated with efficient depletion of CD4+ T cells. There was a significant correlation between Nef activity in CD4 down-regulation and the efficiency of virus replication in ex vivo-infected tissue, suggesting a link between Nef-mediated CD4 downregulation, viral replication, and disease progression. In summary, coreceptor preference of HIV-1 variants is a sufficient determinant of viral cytopathicity for monotropic viruses of B and non-B subtypes as well as for dual-tropic viruses; and down-modulation of cell surface receptors may mediate nef-effects on HIV-1 infection in lymphoid tissue.

HIV疾病的关键事件发生在淋巴组织中，其中HIV杀死受感染的CD 4+细胞以及许多未感染的细胞，破坏介导正常免疫应答的细胞-细胞相互作用的复杂系统。该项目的总体目标是使用我们的离体感染人扁桃体系统确定HIV-1介导的免疫发病机制的潜在机制。在体内，HIV-1仅使用CXCR 4辅助受体或除了CCR 5辅助受体之外还使用CXCR 4辅助受体的能力通常与CD 4 + T细胞的大量损失和快速疾病进展相关。我们目前正在测试的工作假设是，决定CXCR 4的使用的HIV-1 env基因，完全或除了CCR 5，足以诱导T细胞耗竭和免疫抑制。此外，我们还研究了HIV辅助基因，特别是nef基因对病毒组织细胞病变的贡献。早先我们发现辅助受体特异性决定了B亚型单嗜性HIV-1变异体的细胞病变性。今年，我们研究了（i）辅助受体特异性是否决定了HIV-1亚型（B亚型除外）变异体的细胞病变性;（ii）双重嗜性R5 X4 HIV-1亚型（B亚型）变异体的细胞病变性是否由它们优先使用特定辅助受体决定;（iii）HIV辅助基因nef对HIV发病机制的贡献是否与细胞表面受体的调节有关。为了研究非B亚型的R5和X4 HIV-1变体的离体细胞病变性，我们使用了A、E和C亚型的原代分离株以及原型亚型B变体。我们没有观察到病毒复制的最大水平和辅助受体的使用或病毒亚型之间的关系。然而，我们确实观察到病毒辅助受体的使用与所有检测亚型的CD 4 + T淋巴细胞耗竭之间存在很强的相关性。所有R5 HIV-1感染均导致CD 4 + T细胞轻度耗竭，而所有X4 HIV-1感染均导致CD 4 + T细胞严重耗竭，无论其亚型如何。因此，非B HIV-1亚型与HIV-1亚型B相似之处在于共受体特异性决定了离体感染的人淋巴组织中的细胞病变性。为了发现R5 X4病毒的不同细胞病变性是否也与它们优先使用特定的辅助受体有关，我们用三种R5 X4 HIV-1变体：89.6、89-v345.FL和89-v345.SF感染CCR 5 delta 32组织;后两种与89.6是等基因的，除了gp 120的V3-V5区域。变体89.6严重消耗组织的CD 4 + T细胞，而89-v345.SF仅轻度消耗。变体89-v345.FL是一种中间耗竭剂。在CCR 5 δ 32和正常淋巴组织中，89.6和89-v345.FL以相似的动力学复制。相反，89-v345.SF在缺乏CCR 5的淋巴组织中不复制。与这些结果一致，89.6的复制被CXCR 4配体AMD 3100完全抑制，但对CCR 5配体RANTES不敏感，而89-v345.SF复制几乎被RANTES完全抑制，但不被AMD 3100抑制。AMD 3100和RANTES均降低了89-v345.FL的复制。在两个研究的主要分离株，这是双嗜性的转染细胞系中，一个是不抑制RANTES，但几乎完全阻断AMD 3100。因此，在淋巴组织中，一些R5 X4分离株似乎只利用CXCR 4，而其他分离株只利用CCR 5。优先使用CXCR 4的R5 X4 HIV变体是高度致细胞病变的，而优先使用CCR 5的那些HIV变体轻度消耗CD 4 + T细胞。因此，优先辅助受体的使用决定了R5 X4变体的致细胞病变性。细胞表面受体可能介导辅助基因nef对HIV感染的影响。用携带来自连续患者分离物的nef等位基因的NL 4 -3克隆以及携带重组等位基因的克隆感染组织。在较晚时间点分离的含有nef等位基因的HIV-1变异体显示出比在较早时间点最初分离的含有nef等位基因的HIV-1变异体高2 - 3倍的复制水平。较高水平的复制与CD 4 + T细胞的有效耗竭相关。Nef在CD 4下调中的活性与离体感染组织中病毒复制的效率之间存在显著相关性，表明Nef介导的CD 4下调、病毒复制和疾病进展之间存在联系。总之，HIV-1变异体的共受体偏好性是B和非B亚型单嗜性病毒以及双嗜性病毒的病毒致细胞病变性的充分决定因素;细胞表面受体的下调可能介导淋巴组织中HIV-1感染的nef效应。