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

在HIV感染中，大量HIV特异性CD8+和CD4+T淋巴细胞功能失调， 巧合显示T细胞活化的负性调节因子PD-1的表达增加，PD1结扎 由PD-L1和PD-L2导致的大多数T细胞功能的抑制包括增殖、细胞因子 生产和生存。然而，PD-1下游的分子机制仍不清楚。这个 该项目的主要目标是确定导致T细胞功能障碍的分子机制 在PD-1结扎后，并确定将允许拯救艾滋病毒特异性的这种功能障碍的策略 CD8细胞。我们将按顺序检验PD1干扰上游TCR信号通路的假设 影响所有T细胞的功能。我们已经证明，PD-1连接导致SHP-1的磷酸化，并且 SHP-2磷酸酶。此外，PD1的交联会通过抑制p56Lck的激活而抑制其活性。 抑制激酶CSK对酪氨酸Y505的磷酸化。在具体目标1中，我们将确定 HIV感染者PD-1hi耗竭细胞的近端信号缺陷及CSK是否 通过PD1连接被招募到免疫突触，或者是否由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激酶CSK和In 磷酸酶SHP-1和SHP-2被招募到TCR复合体中。荧光(福斯特)共振 将进行能量转移(FRET)实验，以确定与负面影响有关的伙伴 P56Lck活性的调节。这些负调控因子的招募将在下列细胞中进行评估 不同程度的功能障碍。我们将使用针对CSK、SHP-1、SHP-2和Fyn-T的siRNA，并 激活形式的LCK在这种功能障碍的极端情况下挽救T细胞的T细胞功能。在……里面 目标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Core C合作)PD-1上免疫突触形成的动力学 HIV特异性CD8+T细胞表现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，如 在有效激活的T细胞中观察到。该项目产生的结果可能会导致确定 新的治疗靶点，可以允许在艾滋病毒感染期间进行免疫重建。

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.