CELL INTERACTIONS IN THREE DIMENSIONAL TISSUE CULTURE

组织中复杂的细胞-细胞相互作用是其 正常和病态的行为。艾滋病毒感染扰乱了多个 淋巴组织中的细胞相互作用 艾滋病就会发生。在体内，HIV-1感染通过病毒传播 在体外识别趋化因子受体CCR5(R5变体)，而 主宰HIV疾病后期的病毒通常会识别 CXCR4单独(X4变种)或与CCCR5(R5X4)一起使用 变体)。这种病毒嗜性的转变经常与 CD_4~+T细胞急剧丧失。在体外组织中，R5感染导致 导致CD4+T细胞严重耗尽，而R5感染则耗尽 仅有少量的CD4+T细胞。目前尚不清楚为什么R5 HIV-1感染会 体内并不会导致大量的CD4+T细胞耗尽 在X4艾滋病毒感染中。为了回答这个问题，我们确定了 病毒共受体偏好决定T-T细胞的选择性耗竭 表达特定辅受体的细胞。我们测量了耗尽的 组织培养物中共受体特异性的CD4+T细胞亚群 以及一组X4和R5 HIV-1变种。细胞的流式细胞术 从培养的未感染的对照组织中分离出 CD4+T淋巴细胞群由多个细胞亚群组成 CCR5和CXCR4 HIV-1辅助受体在其上的差异表达 浮出水面。CD_4~+T细胞在CCR5~+之间分布不均 和CXCR4+细胞亚群；约80%的CD4+T淋巴细胞是 只有CXCR4+，而4%的CD4+T淋巴细胞是 CCR5+。6%的CD4+T细胞同时表达CXCR4和CCR5，在 其余的CD4+T细胞既没有CCR5也没有CXCR4在 细胞表面。感染X4变种LAV.04和NL4-3或 与初级分离物92US076一起导致总CD4+严重耗尽 T细胞，只有10%~20%的CD4+T细胞残留在感染者体内 感染后12~13天的淋巴组织。相比之下， 感染所有测试的R5变种(实验室菌株Bal， SF162和初级分离物91US056)仅引起轻微 耗竭：约90%的CD4+T细胞残留在组织中 与匹配的未感染的对照组相比。然而，当个人 检查了CD4+T细胞亚群，我们发现R5变异体 选择性耗尽CCR5+/CD4+T细胞。无CCR5-/CD4+T细胞 R5 HIV-1的任何一种都会显著耗尽淋巴细胞 经过测试的变种。CCR5+/CD4+T淋巴细胞的耗竭 R5 HIV-1感染淋巴中CD4+T细胞的全部丧失 纸巾。与R5 HIV-1不同，这三种病毒都测试了X4变种 严重耗尽了所有的CD4+T细胞亚群，尽管这些 病毒耗尽CXCR4+/CD4+T细胞的效率略高于 其他亚群的淋巴细胞。这些数据表明，X4和 R5 HIV-1变异株对其同源CD4+T细胞具有高度的细胞病变作用 人类淋巴组织中的细胞靶点。来测试HIV变种是否 根据它们对co-的使用而被归类为双向性的 转基因细胞中的受体在EX中表现为双向性 体内淋巴组织，我们用克隆的R5X4分离物89.6和它的两个 R5X4嵌合体-v345.SF和-v345.FL，除 用于特定的环境序列。我们用特定的药物处理组织 共受体配体，RANTES，以抑制病毒通过CCR5进入，以及 AMD3100通过CXCR4阻止病毒入侵。R5X4 HIV-1分离株89.6 几乎完全被AMD3100抑制，但最低限度 受RANTES影响。89-v345.SF的感染几乎完全 被RANTES阻断，被AMD3100轻微抑制。感染 对于89-v345，FL对AMD3100和RANTES的敏感性几乎相同。 因此，在人类淋巴组织的复杂微环境中，一些 R5X4变种的行为更像X4，而其他变种更像R5。 这些变异体优先使用CXCR4与它们的 细胞病变：20%的CD4+T细胞保留在89.6-，85%保留在89- V345.SF-，与89-v345.FL感染组织相比，50% 未感染的对照组。类似于细胞致病的模式 单性艾滋病毒变异株，89-v345。SF仅耗尽CCR5+/CD4+T细胞 细胞，而-v345和89.6。FL耗尽所有的CD4+T细胞 子集。总而言之，双嗜性病毒可能只使用一个共同的- 淋巴组织中的受体以及单嗜性和双嗜性艾滋病毒- 1个变异体对一般的CD4+T细胞具有细胞病变作用 淋巴组织中的群体与CXCR的使用密切相关 4.参考使用CC R5的变异体的明显低细胞致病性 CCR5在人类淋巴组织中的表达频率较低 表达细胞。

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.