Cell Interactions in Three Dimensional Tissue Culture

三维组织培养中的细胞相互作用

• 批准号: 8149276
• 负责人: Leonid B. Margolis
• 金额: $ 154.86万
• 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/8149276
• 关键词: Cell Communication; tissue culture

Selective transmission of R5 HIV-1 variants, gatekeeping X4 HIV-1. To enter target cells, HIV-1 uses CD4 and a coreceptor that in vivo is either CCR5 (for R5 HIV-variants) or CXCR4 (for X4 HIV-1 variants). Although both R5 and X4 HIV-1 variants are present in body fluids (semen, blood, cervico-vaginal and rectal secretions), almost exclusively, R5 HIV-1 appears to transmit infection and dominates the early stages of HIV disease. Moreover, recent sequence analysis showed that a single virus initiates the infection in the majority of cases. Therefore, there is a gatekeeper that selects R5 over X4 HIV-1 and may operate among R5 HIV-1 variants to select a single variant. Careful analysis of such potential gatekeeping mechanisms associated with each of the transmission routes shows that they are too imperfect to explain the almost perfect selection of R5 over X4 in HIV transmission. We suggest that instead of one strong gatekeeper there are many weak ones, whose superimposition is sufficient to protect against X4 HIV-1 infection and potentially select transmittable variants from a pool of R5 HIV-1 variants. In conclusion, we think that the principle of multiple barriers is more general and not restricted to the protection against X4 HIV-1, but can be applied to other phenomena when one factor has a selective advantage over the other(s). In the case of gatekeepers for HIV-1 transmission, the task is to identify these factors and to decipher their molecular mechanisms. The knowledge of the gatekeepers localization and function may enable us to facilitate existing barriers against R5 transmission and to erect new ones against all HIV-1 variants. In general, understanding the gatekeeping mechanisms of HIV-1 transmission is critically important for the development of effective HIV-1-preventive measures (microbicides, virustats, and vaccines). Experimental study of the early barriers in HIV-1 transmission to cervico-vaginal tissue ex vivo As the cervico-vaginal tissue is the first gateway for HIV-1 during heterosexual transmission, the female lower genital tract may serve as the first gatekeeper, conferring protection against at least some HIV-1 variants. Using surgical samples of cervico-vaginal tissue (available commercially), we developed an ex vivo system to study HIV male-to-female vaginal transmission, and investigated the gatekeeping mechanisms in this system. Understanding these mechanisms requires a robust knowledge of the phenotypes, and of the functional features of the cells in the female lower genital mucosa, which are implicated in the early events of HIV-1 transmission. Such knowledge was lacking in the published literature. Using an original flow-cytometry protocol, we found that in the cervico-vaginal tissue, CD4 and CD8 T cells are predominantly of the highly differentiated effector memory phenotype with a variable pattern of expression of activation markers. The high prevalence of effector memory cells among cervico-vaginal T cells may be critical for the highly efficient defense against various incoming pathogens. We identified the phenotype, as well as the activation and differentiation status, of CD4 T cells that are preferentially infected by HIV-1 that is transmitted to cervico-vaginal tissue ex vivo. Preferential infection of activated CD38+ CD4 T cells was followed by a similar activation of HIV-1-uninfected bystander cells, creating a vicious circle between R5 HIV-1 replication and the number of cell targets that support this infection. These results provide new insights into the dependence of HIV-1 infection and the dissemination of the activation of cervico-vaginal lymphocytes. Remarkably, isolated blocks of cervico-vaginal tissue retain an in vivo gatekeeping mechanism that selects between R5 and X4 HIV-1 infection in spite of the broad expression of both CXCR4 and CCR5, the co receptors for X4 and R5 HV-1. We attributed the higher susceptibility of cervico-vaginal tissue to R5 HIV-1 infection over X4 HIV-1 infection, to the low abundance of the CD27+ CD28+ effector memory CD4 T cells. In the system of cervico-vaginal tissue ex vivo, we are now investigating potential barriers that select for the R5 HIV-1 virions that transmit infection through the mucosal barriers. Determinants of protective barriers among high-risk seronegative persons Theoretical and experimental laboratory evidences of the existence of multiple gatekeeping mechanisms need to be translated into clinical research. Growing epidemiological literature indicates that there are persons who have been exposed to HIV infection but who have remained uninfected. We suggest launching a complex study to investigate the natural barriers of resistance to HIV infection by using a cohort of uninfected persons where the risks for infection are thought to be high. In addition to the 32 base pair deletion in the CCR5 gene which, at the date of this report, is the only mechanism associated with a high protection level from HIV infection, these cohorts will allow the investigation of alternate mechanisms of protection. According to our concept a number of imperfect gatekeepers contribute to the protection from infection. Moreover, this protection may not be consistent over time, and may be affected by various factors such as hormonal environment, innate and adaptive immunity as well as the individuals microbiome so that an individual apparently protected for one period of high-risk exposure, may lose this relative protection at other times. A consortium of laboratories including our Section has been established to identify natural gatekeepers in vivo.

R5 HIV-1 变体的选择性传播，X4 HIV-1 的把关。 为了进入靶细胞，HIV-1 使用 CD4 和体内辅助受体，该辅助受体是 CCR5（对于 R5 HIV 变异体）或 CXCR4（对于 X4 HIV-1 变异体）。 尽管 R5 和 X4 HIV-1 变体都存在于体液（精液、血液、宫颈阴道和直肠分泌物）中，但几乎全部是 R5 HIV-1 似乎可以传播感染并在 HIV 疾病的早期阶段占主导地位。 此外，最近的序列分析表明，在大多数情况下，单一病毒引发感染。 因此，有一个看门人选择 R5 而不是 X4 HIV-1，并且可以在 R5 HIV-1 变体中操作以选择单个变体。 对与每种传播途径相关的此类潜在把关机制的仔细分析表明，它们太不完善，无法解释在 HIV 传播中 R5 相对于 X4 的近乎完美选择。 我们建议，不是一个强大的守门人，而是许多弱守门人，它们的叠加足以防止 X4 HIV-1 感染，并有可能从 R5 HIV-1 变体库中选择可传播的变体。 总之，我们认为多重屏障的原理更为普遍，并不限于针对 X4 HIV-1 的保护，而是可以应用于当一个因素比其他因素具有选择性优势时的其他现象。 对于 HIV-1 传播的守门人来说，任务是识别这些因素并破译它们的分子机制。 了解守门人的定位和功能可能使我们能够消除现有的 R5 传播障碍，并针对所有 HIV-1 变种建立新的障碍。一般来说，了解 HIV-1 传播的把关机制对于制定有效的 HIV-1 预防措施（杀菌剂、病毒剂和疫苗）至关重要。 HIV-1离体宫颈阴道组织传播早期障碍的实验研究 由于宫颈阴道组织是异性传播过程中 HIV-1 的第一个门户，因此女性下生殖道可能充当第一个把关人，提供针对至少某些 HIV-1 变种的保护。 使用宫颈阴道组织的手术样本（市售），我们开发了一种离体系统来研究艾滋病毒男性到女性的阴道传播，并研究了该系统中的把关机制。 了解这些机制需要对女性下生殖器粘膜细胞的表型和功能特征有深入的了解，这些细胞与 HIV-1 传播的早期事件有关。已发表的文献中缺乏此类知识。 使用原始的流式细胞术方案，我们发现在宫颈阴道组织中，CD4 和 CD8 T 细胞主要是高度分化的效应记忆表型，具有可变的激活标记表达模式。宫颈阴道T细胞中效应记忆细胞的高流行可能对于高效防御各种传入病原体至关重要。 我们鉴定了 CD4 T 细胞的表型以及激活和分化状态，这些细胞优先被 HIV-1 感染，并在体外传播到宫颈阴道组织。优先感染激活的 CD38+ CD4 T 细胞后，未感染 HIV-1 的旁观者细胞也会被类似激活，从而在 R5 HIV-1 复制和支持这种感染的细胞靶标数量之间形成恶性循环。 这些结果为 HIV-1 感染的依赖性和宫颈阴道淋巴细胞激活的传播提供了新的见解。 值得注意的是，尽管 X4 和 R5 HV-1 的共同受体 CXCR4 和 CCR5 广泛表达，但分离的宫颈阴道组织块保留了在 R5 和 X4 HIV-1 感染之间进行选择的体内看门机制。 我们将宫颈阴道组织对 R5 HIV-1 感染的敏感性高于 X4 HIV-1 感染的原因归因于 CD27+ CD28+ 效应记忆 CD4 T 细胞的丰度较低。 在离体宫颈阴道组织系统中，我们现在正在研究选择通过粘膜屏障传播感染的 R5 HIV-1 病毒粒子的潜在屏障。 高危血清阴性人群保护屏障的决定因素 存在多种把关机制的理论和实验实验室证据需要转化为临床研究。越来越多的流行病学文献表明，有些人曾接触过艾滋病毒感染者，但并未受到感染。我们建议开展一项复杂的研究，通过使用一群被认为感染风险较高的未感染者来调查抵抗艾滋病毒感染的自然屏障。截至本报告发布之日，CCR5 基因中的 32 个碱基对缺失是与 HIV 感染高保护水平相关的唯一机制，除此之外，这些队列还将允许研究替代保护机制。 根据我们的概念，许多不完美的守门人有助于防止感染。 此外，这种保护可能随着时间的推移而不一致，并且可能受到多种因素的影响，例如荷尔蒙环境、先天性和适应性免疫以及个体微生物组，因此在一段高风险暴露期间明显受到保护的个体可能在其他时间失去这种相对保护。包括我们部门在内的一个实验室联盟已经成立，以识别体内天然的守门人。