Targeting the PD-1 signaling pathway to rescue HIV T cell dysfunction

靶向 PD-1 信号通路挽救 HIV T 细胞功能障碍

• 批准号: 8318856
• 负责人: Rafick Pierre Sekaly
• 金额: $ 26.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8318856
• 关键词: Acute; Affect; Agonist; Antibodies; Antigen-Presenting Cells; Biochemical; Biological Assay; CD28 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Count; Cell Proliferation; Cell physiology; Cells; Chronic; Chronic Phase; Collaborations; Complex; Cytoplasmic Tail; Data; Defect; Disease; Dominant-Negative Mutation; Energy Transfer; Epitopes; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Frequencies; Functional disorder; Gene Expression; Generations; HIV; HIV Infections; HIV-1; Human; Image; Immune; Immune System Diseases; Immune response; Immune system; Immunoprecipitation; Individual; Infection; Lead; Life; Ligands; Ligation; Lipid Bilayers; Lymphocytic choriomeningitis virus; Memory; Molecular; Monitor; Mus; Mutate; Mutation; Natural History; PTPN11 gene; PTPN6 gene; Pathway interactions; Patients; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Population; Production; Protein Tyrosine Kinase; RNA Interference; Recruitment Activity; Regulation; Reporting; Signal Pathway; Signal Transduction; Signaling Molecule; Staging; Synapses; System; T memory cell; T-Cell Activation; T-Cell Activation Pathway; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Tyrosine Phosphorylation; Up-Regulation; Validation; Viral; Virus; Virus Diseases; Western Blotting; cell type; cellular imaging; crosslink; cytokine; exhaust; exhaustion; immunological synapse; immunological synapse formation; induced pluripotent stem cell; new therapeutic target; overexpression; prevent; reconstitution; research study; response

In HIV infection a large frequency of HIV specific CD8+ and CD4+ T lymphocytes are dysfunctional and coincidentally show an increased expression of the negative regulator of T cell activation PD-1, PD1 ligation by PD-L1 and PD-L2 leads to the inhibition of most T cell functions including proliferation, cytokine production and survival. However, the molecular mechanisms downstream of PD-1 remain unknown. The major objective of this project is to define the molecular mechanisms that render T cells dysfunctional following PD-1 ligation and to identify strategies that will allow the rescue of such dysfunction in HIV specific CD8 cells. We will test the hypothesis that PD1 interferes with upstream TCR signaling pathways in order to affect all T cell functions. We have shown that PD-1 ligation leads to the phosphorylation of SHP-1 and SHP-2 phosphatases. Moreover PD1 crosslinking leads to the inhibition of p56Lck activation through the phosphorylation of the tyrosine Y505 by the inhibitory kinase Csk. In Specific Aim 1 we will determine the proximal signaling defect in PD-1 hi exhausted cells from HIV infected patients and assess whether Csk is recruited to the immunological synapse by PD1 ligation or whether it is released from Cbp/PAG by the SHP- 2 phosphatase. We will determine by quantitative immunoprecipitation if the inhibitory kinase Csk and the in phosphatases SHP-1 and SHP-2 are recruited to the TCR complex. Fluorescence (Forster) resonance energy transfer (FRET) experiments will be carried out to identify the partners involved in the negative regulation of p56Lck activity. The recruitment of these negative regulators will be assessed in cells showing varying degrees of dysfunction . We will use siRNAs specific for Csk, SHP-1, SHP-2 and Fyn-T and activated forms of Lck to rescue T cell function in T cells at the extremes of this gradient of dysfunction. In Aim 2, live cell imaging using total internal reflection fluorescence microscopy (TIRFM) will be used to visualize, (in collaboration with Core C) the dynamics of the immunological synapse formation upon PD-1 crosslinking in HIV specific CD8+ T cells showing varying degrees of dysfunction. . We will assess whether inhibition of the kinase Csk and the phosphatases SHP-112 will restore the integrity of the synapse as observed in efficiently activated T cells. Results emanating from this project may lead to the identification of novel therapeutic targets that can allow immune reconstitution during HIV infection.

在HIV感染中，大量HIV特异性CD 8+和CD 4 + T淋巴细胞功能失调， 巧合地显示T细胞活化的负调节因子PD-1、PD 1连接的表达增加 PD-L1和PD-L2的免疫抑制导致大多数T细胞功能的抑制，包括增殖、细胞因子 生产与生存。然而，PD-1下游的分子机制仍然未知。的 这个项目的主要目标是确定使T细胞功能失调的分子机制 在PD-1连接后，并确定将允许在HIV特异性免疫缺陷病毒中挽救这种功能障碍的策略。 CD 8细胞。我们将检验PD 1干扰上游TCR信号通路的假设， 影响所有T细胞功能我们已经证明PD-1连接导致SHP-1的磷酸化， SHP-2磷酸酶。此外，PD 1交联导致p56 Lck激活的抑制，通过 酪氨酸Y505被抑制性激酶Csk磷酸化。在具体目标1中，我们将确定 在来自HIV感染患者的PD-1 hi耗竭细胞中的近端信号传导缺陷，并评估Csk是否是 通过PD 1连接招募到免疫突触，或者是否通过SHP从Cbp/PAG释放， 2磷酸酶。我们将通过定量免疫沉淀来确定抑制性激酶Csk和抑制性蛋白激酶Csk是否在细胞内表达。 磷酸酶SHP-1和SHP-2被募集至TCR复合物。荧光（福斯特）共振 将进行能量转移（FRET）实验，以确定参与负离子的伙伴。 p56 Lck活性的调节。将在细胞中评估这些负调节剂的募集， 不同程度的功能障碍我们将使用对Csk、SHP-1、SHP-2和Fyn-T特异的siRNA， 激活形式的Lck，以挽救处于这种功能障碍梯度极端的T细胞中的T细胞功能。在 目的2，使用全内反射荧光显微镜（TIRFM）的活细胞成像将用于 可视化，（与核心C合作）PD-1免疫突触形成的动力学 在HIV特异性CD 8 + T细胞中的交联显示出不同程度的功能障碍。.我们将评估是否 抑制激酶Csk和磷酸酶SHP-112将恢复突触的完整性， 在有效激活的T细胞中观察到。本项目的结果可能会导致识别 新的治疗靶点可以在HIV感染期间进行免疫重建。