CELL INTERACTIONS IN THREE DIMENSIONAL TISSUE CULTURE

三维组织培养中的细胞相互作用

• 批准号: 6432567
• 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/6432567
• 关键词: B lymphocyte; HIV infections; T lymphocyte; antibody formation; cell cell interaction; dendritic cells; human immunodeficiency virus 1; human tissue; macrophage; pathologic process; tetanus toxoid; tissue /cell culture; virus replication

Complex cell-cell interactions in tissues are the basis for their normal and pathological behavior. HIV infection disrupts multiple cell interactions in the lymphoid tissue where critical events of HIV disease occur. In vivo HIV-1 infection is transmitted by viruses that in vitro recognize chemokine receptor CCR5 (R5 variants), while viruses dominating later stages of HIV disease often recognize either CXCR4 alone (X4 variants) or in addition to CCCR5 (R5X4 variants). This switch of viral tropism often coincides with dramatic loss of CD4+ T cells. In ex vivo tissues R5 infection leads to severe depletion of CD4+ T cells, whereas R5 infection depletes CD4+ T cells only mildly. It is not known why R5 HIV-1 infection ex vivo does not lead to a massive CD4+ T cell depletion, as it occurs in X4 HIV-infection. To answer this question we determined whether viral coreceptor preferences dictate the selective depletion of T- cells expressing particular coreceptors. We measured depletion of coreceptor-specific CD4+ T-cell subsets in histocultures inoculated with sets of X4 and R5 HIV-1 variants. Flow cytometry of cells isolated from cultured uninfected control tissues revealed that the CD4+ T lymphocyte population comprised subsets of cells differentially expressing CCR5 and CXCR4 HIV-1 coreceptors on their surface. CD4+ T lymphocytes were unequally distributed between CCR5+ and CXCR4+ cell subtypes; about 80% of CD4+ T lymphocytes were exclusively CXCR4+ whereas 4% of CD4+ T lymphocytes were exclusively CCR5+. 6% of CD4+ T cells expressed both CXCR4 and CCR5, and in the remaining CD4+ T cells neither CCR5 nor CXCR4 was detected on the cell surface. Infection with the X4 variants LAV.04 and NL4-3 or with a primary isolate 92US076 caused severe depletion of total CD4+ T cells, only 10 to 20% of CD4+ T cells remained in the infected lymphoid tissues on days 12 to 13 post-infection. In contrast, infection with all the tested R5 variants (laboratory strains BaL, SF162 and with a primary isolate 91US056) caused only mild depletion: about 90% of CD4+ T cells remained in the tissues compared to matched uninfected controls. However, when individual subsets of CD4+ T cells were examined, we found that R5 variants selectively depleted CCR5+/CD4+ T cells. No CCR5-/CD4+ T lymphocytes were significantly depleted by either of R5 HIV-1 variants tested. The depletion of CCR5+/CD4+ T lymphocytes accounts for the entire loss of CD4+ T cells in R5 HIV-1-infected lymphoid tissues. In contrast to R5 HIV-1, all three tested X4 variants severely depleted all subsets of CD4+ T cells, although these viruses depleted CXCR4+/CD4+ T cells slightly more efficiently than lymphocytes from other subsets. These data indicate that both X4 and R5 HIV-1 variants are highly cytopathic for their cognate CD4+ T cell targets in human lymphoid tissue. To test whether HIV variants that are classified as dual-tropic based on their usage of co- receptors in transfected cell lines, behave as dual-tropic in ex vivo lymphoid tissues, we used cloned R5X4 isolate 89.6 and its two R5X4 chimeras 89-v345.SF and 89-v345.FL which are isogenic except for specific env sequences. We treated tissues with specific coreceptor ligands, RANTES to inhibit viral entry through CCR5, and AMD3100 to block viral entry through CXCR4. R5X4 HIV-1 isolate 89.6 was almost completely inhibited by AMD3100, but was minimally affected by RANTES. Infection of 89-v345.SF was almost completely blocked by RANTES and was slightly inhibited by AMD3100. Infection with 89-v345.FL was almost equally sensitive to AMD3100 and RANTES. Thus, in the complex microenvironment of human lymphoid tissue some R5X4 variants behave as more X4, while others are more R5. Preferential usage of CXCR4 by these variants correlated with their cytopathicity: 20% of CD4+ T cells remained in 89.6-, 85% in 89- v345.SF-, and 50% in 89-v345.FL-infected tissues compared with uninfected controls. Similar to the pattern of cytopathicity of monotropic HIV variants, 89-v345. SF depleted only CCR5+/CD4+ T cells whereas, 89.6 and 89-v345. FL depleted all CD4+ T cell subsets. In summary, dual-tropic viruses may use only one co- receptor in lymphoid tissue, and for both mono- and dual-tropic HIV- 1 variants their cytopathicity toward the general CD4+ T cell population in lymphoid tissue is closely associated with use of CXCR 4. Apparent low cytopathicity of variantspreferentially using CC R5 in human lymphoid tissue is explained by the low frequency of CCR5 expressing cells.

组织中复杂的细胞间相互作用是其发挥作用的基础 正常和病理行为。 HIV感染扰乱了多重 淋巴组织中的细胞相互作用，其中关键事件 发生艾滋病毒疾病。体内HIV-1感染是通过病毒传播的 体外识别趋化因子受体 CCR5（R5 变体），而 主导艾滋病毒疾病后期的病毒通常会识别 单独的 CXCR4（X4 变体）或 CCCR5 的补充（R5X4 变体）。这种病毒向性的转变通常与 CD4+ T 细胞急剧减少。离体组织中 R5 感染导致 CD4+ T 细胞严重耗竭，而 R5 感染则耗竭 CD4+ T 细胞仅轻度。目前尚不清楚为什么 R5 HIV-1 感染前 体内不会导致大量 CD4+ T 细胞耗竭，因为它发生 X4 HIV 感染。为了回答这个问题，我们确定是否 病毒辅助受体的偏好决定了 T-的选择性消耗 表达特定辅助受体的细胞。我们测量了消耗 接种的组织培养物中的辅助受体特异性 CD4+ T 细胞亚群 具有 X4 和 R5 HIV-1 变体组。细胞的流式细胞术 从培养的未感染对照组织中分离发现 CD4+ T 淋巴细胞群由细胞亚群组成 差异表达 CCR5 和 CXCR4 HIV-1 辅助受体 表面。 CD4+ T 淋巴细胞在 CCR5+ 之间分布不均 和 CXCR4+ 细胞亚型；大约80%的CD4+T淋巴细胞 仅 CXCR4+，而 4% 的 CD4+ T 淋巴细胞仅 CCR5+。 6% 的 CD4+ T 细胞同时表达 CXCR4 和 CCR5，并且在 剩余的 CD4+ T 细胞上既没有检测到 CCR5 也没有检测到 CXCR4 细胞表面。 X4 变种 LAV.04 和 NL4-3 感染或 初级分离株 92US076 导致 CD4+ 总量严重耗尽 T细胞，感染者体内只剩下10%到20%的CD4+T细胞 感染后第 12 至 13 天的淋巴组织。相比之下， 感染所有测试的 R5 变体（实验室菌株 BaL、 SF162 和初级分离株 91US056）仅引起轻微的 耗竭：约 90% 的 CD4+ T 细胞保留在组织中 与匹配的未感染对照相比。然而，当个体 检查了 CD4+ T 细胞亚群，我们发现 R5 变体 选择性耗尽 CCR5+/CD4+ T 细胞。无 CCR5-/CD4+ T 淋巴细胞被 R5 HIV-1 显着耗尽 已测试变体。 CCR5+/CD4+ T 淋巴细胞的消耗 R5 HIV-1 感染的淋巴组织中 CD4+ T 细胞全部丧失 组织。与 R5 HIV-1 相比，所有三个测试的 X4 变体 严重耗尽所有 CD4+ T 细胞亚群，尽管这些 病毒消耗 CXCR4+/CD4+ T 细胞的效率略高于 来自其他亚群的淋巴细胞。这些数据表明 X4 和 R5 HIV-1 变体对其同源 CD4+ T 具有高度细胞病变性 人体淋巴组织中的细胞靶标。检测HIV是否有变异 根据其共热带的使用被归类为双热带 转染细胞系中的受体，在前表现出双向性 体内淋巴组织，我们使用克隆的R5X4分离株89.6及其两个 R5X4 嵌合体 89-v345.SF 和 89-v345.FL 是同基因的，除了 对于特定的 env 序列。我们用特定的方法处理组织 辅助受体配体、RANTES 通过 CCR5 抑制病毒进入，以及 AMD3100 可阻止病毒通过 CXCR4 进入。 R5X4 HIV-1 分离物 89.6 AMD3100 几乎完全抑制，但影响最小 受 RANTES 影响。 89-v345.SF 的感染几乎完全 被 RANTES 阻断，并被 AMD3100 轻微抑制。感染 89-v345.FL 对 AMD3100 和 RANTES 几乎同样敏感。 因此，在人体淋巴组织复杂的微环境中，一些 R5X4 变体的行为更像 X4，而其他变体则更像 R5。 这些变体对 CXCR4 的优先使用与其 细胞病变：20% 的 CD4+ T 细胞保留在 89.6- 中，85% 保留在 89- 中 与 89-v345.FL 感染组织相比，v345.SF- 和 50% 未感染的对照。类似于细胞病变的模式 单向性 HIV 变体，89-v345。仅 SF 耗尽 CCR5+/CD4+ T 细胞，而89.6和89-v345。 FL 耗尽所有 CD4+ T 细胞 子集。总之，双向病毒可能只使用一种共向病毒 淋巴组织中的受体，以及单向和双向 HIV- 1 个变体对一般 CD4+ T 细胞具有细胞病变性 淋巴组织中的人群与 CXCR 的使用密切相关 4.优先使用CC R5的变体明显低细胞病变性 人类淋巴组织中 CCR5 的频率较低 表达细胞。