HIV-1 Nef对人造血干细胞分化发育为T细胞的影响及其转录调控

Human immunodeficiency virus type 1(HIV-1) infection causes CD4+T cell depletion in patients. In our previous work we found that one of the mechanisms of CD4+ T cell depletion in HIV-1 infection is through reduction of thymic naïve T cell outputs, in which HIV-1 encoded protein Nef was found to play a key role. It has been shown that HIV-1 can infect hematopoietic stem cells (HSCs) and inhibit their capacity of hematopoiesis. However, it still remains elusive whether reduction of thymic output of naïve T cells is due to HIV’s inhibitory effect on HSC through Nef. In this project, we propose to investigate the effect of Nef on the differentiation and development from HSC to T cells and to determine the underlying mechanisms. OP9-DL1 cell system and NSG humanized mice will be employed to study the effect of Nef on the differentiation and development from HSC to T cells in vitro and in vivo, respectively. To understand the underlying mechanisms, we will use RNA-seq technique to explore Nef-induced changes of intracellular factors and signaling pathways in the early-stage of thymocytes. The findings from the proposed study will add to our understanding HIV pathogenesis and ultimately developing mechanism-based novel strategies from root of T cell development to restore the immune functions of HIV-infected patients.

人类免疫缺陷病毒-1(HIV-1) 感染导致患者体内CD4+ T细胞耗竭。我们在前期工作中发现: “胸腺新生T细胞产出减少”是HIV-1感染中CD4+ T细胞耗竭的机制之一，HIV-1编码的蛋白Nef在其中起关键作用。既往研究表明，HIV-1能感染并抑制造血干细胞（HSC）的功能，但目前还不清楚Nef是否通过参与HIV-1对HSC功能的抑制而减少胸腺新生T细胞的产出。本研究旨在探讨Nef对HSC分化发育为T细胞的影响及机制。我们将通过OP9-DL1细胞系统和NSG人源化小鼠分别在体外和体内研究Nef对HSC分化发育为T细胞的影响，并使用RNA-seq技术探索Nef导致早期发育阶段的胸腺细胞内细胞因子和信号通路的变化。本研究将为进一步解析HIV的发病机制和从T细胞发育源头寻找恢复HIV感染病人免疫功能的新方法提供依据。

尽管抗病毒治疗很成功，CD4+T细胞的水平在某些HIV-1感染者中并不能完全恢复。在前期工作中，我们在感染了嗜CXCR4的HIV-1LAI (LAI)的人缘化小鼠中，发现病毒蛋白Nef是导致CD4+T细胞和CD4+CD8+ 胸腺细胞快速丢失的主要因子。但Nef对早期T细胞发育的影响却还不清楚。本项目拟研究LAI nef对造血干/前体细胞（hematopoietic stem/progenitor cells, HSPCs) 发育为T淋巴细胞的影响。通过HSPC-OP9-DL1 细胞共培养和人缘化小鼠，我们在体外和体内研究了以上内容，采用RNA-seq研究了nef对HSPCS中基因表达指纹的影响。HSPCs从人脐血中分离。我们发现nef在HSPCs中的表达阻碍了HSPCS向T淋巴细胞的发育。这一阻滞作用在体外和移植了表达nef的HSPCs人缘化小鼠中都存在。令人惊奇的是，在人缘化小鼠中，nef的表达更倾向于抑制CD4+T细胞的生成。这种发育缺陷并不是CD34+细胞丢失的结果。RNA-seq分析揭示Nef影响了HSPCs中176个基因的表达，这些基因包括那些对造血细胞发育非常重要的TNF、TLR和 NOD样受体信号通路中的基因。我们的结果表明Nef抑制HSPCs向T细胞的发育，尤其是CD4+T细胞。这一结果提示针对Nef的治疗方法有可能纠正HIV-1相关血液系统异常，尤其是T细胞的发育缺陷。