Cell Interactions in Three Dimensional Tissue Culture

选择性传播R5 HIV-1变异体，守门X4 HIV-1。 为了进入靶细胞，HIV-1使用CD 4和一个辅助受体，在体内是CCR 5（对于R5 HIV变体）或CXCR 4（对于X4 HIV-1变体）。 虽然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，但几乎完全是R5 HIV-1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。 仔细分析与每一种传播途径相关的这种潜在的守门机制表明，它们太不完善，无法解释HIV传播中R5对X4的几乎完美的选择。 我们认为，不是一个强大的看门人，而是有许多薄弱的看门人，其叠加足以防止X4 HIV-1感染，并可能从R5 HIV-1变异体库中选择可传播的变异体。 总之，我们认为多重屏障的原则是更普遍的，并不局限于对X4 HIV-1的保护，但可以应用于其他现象时，一个因素有选择性的优势。 对于HIV-1传播的守门人来说，任务是识别这些因素并破译其分子机制。 对守门人的定位和功能的了解可能使我们能够促进现有的针对R5传播的障碍，并建立针对所有HIV-1变体的新障碍。一般来说，了解HIV-1传播的守门机制对于制定有效的HIV-1预防措施（杀微生物剂、抗病毒剂和疫苗）至关重要。 HIV-1向宫颈-阴道组织传播早期屏障的体外实验研究 由于子宫颈阴道组织是HIV-1在异性间传播的第一个门户，女性下生殖道可能是第一个守门人，至少可以保护女性免受某些HIV-1变种的感染。 使用宫颈阴道组织的手术样本（可商购获得），我们开发了一种体外系统来研究HIV男性到女性的阴道传播，并研究了该系统中的守门机制。 了解这些机制需要一个强大的知识的表型，并在女性下生殖器粘膜，这是在HIV-1传播的早期事件牵连的细胞的功能特征。在已发表的文献中缺乏这种知识。 使用原始流式细胞术方案，我们发现在宫颈阴道组织中，CD 4和CD 8 T细胞主要是高度分化的效应记忆表型，具有可变的活化标志物表达模式。宫颈-阴道T细胞中效应记忆细胞的高流行率可能是高效防御各种传入病原体的关键。 我们确定了CD 4 T细胞的表型以及活化和分化状态，这些T细胞优先被HIV-1感染，HIV-1在体外传播到宫颈阴道组织。活化的CD 38 + CD 4 T细胞的优先感染之后，HIV-1未感染的旁观者细胞的类似活化，在R5 HIV-1复制和支持这种感染的细胞靶点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活化的传播提供了新的见解。 值得注意的是，宫颈阴道组织的分离块保留了在R5和X4 HIV-1感染之间进行选择的体内守门机制，尽管X4和R5 HV-1的共同受体CXCR 4和CCR 5都广泛表达。 我们将宫颈阴道组织对R5 HIV-1感染的易感性高于X4 HIV-1感染归因于CD 27 + CD 28+效应记忆CD 4 T细胞的低丰度。 在离体宫颈阴道组织系统中，我们现在正在研究潜在的屏障，以选择通过粘膜屏障传播感染的R5 HIV-1病毒粒子。 高危血清反应阴性者保护性障碍的决定因素 理论和实验室证据的存在多种守门机制需要转化为临床研究。越来越多的流行病学文献表明，有些人接触过艾滋病毒感染，但没有受到感染。我们建议开展一项复杂的研究，通过使用一组被认为感染风险很高的未感染者来调查抵抗艾滋病毒感染的天然屏障。除了CCR 5基因中的32个碱基对缺失之外，在本报告日期，这是与HIV感染的高保护水平相关的唯一机制，这些队列将允许研究替代保护机制。 根据我们的概念，一些不完美的看门人有助于保护免受感染。 此外，这种保护可能不会随着时间的推移而一致，并且可能受到各种因素的影响，例如激素环境，先天和适应性免疫以及个体微生物组，因此，在一个高风险暴露时期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建立，以确定体内的天然看门人。

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.