Inactivation of CTL by HSV-infected cells

描述（由申请人提供）：疱疹病毒很好地适应宿主，并采用多种机制与宿主免疫反应共存。最近，我们描述了单纯疱疹病毒（HSV）感染的细胞发送负面信号持久抑制正常CTL功能的能力，我们将这种现象称为“失活”。缺乏Us3激酶的HSV突变株不诱导失活。失活需要感染单纯疱疹病毒的细胞与CTL直接接触，并且似乎是通过病毒编码配体产生的。失活的CTL不执行颗粒介导的细胞毒性，也不分泌细胞毒性细胞因子TNF和ifn - γ，以响应通过T细胞受体的刺激，但在PMA/离子霉素刺激后保留合成细胞因子的能力。失活的CTL没有凋亡的迹象，并且至少保持失活12-48小时。综上所述，这些数据表明HSV正在利用一种未知的、极其强大的信号通路，可以导致对CTL活性的深刻而持久的抑制。为了研究失活过程中涉及的分子事件，我们提出以下建议。具体目标确定单纯表达HSV蛋白Us3是否足以达到灭活效果，还是需要与其他病毒蛋白相互作用。我们将在成纤维细胞中分离其他病毒基因表达Us3，并确定表达Us3的细胞是否能灭活CTL。具体目标2。鉴定向CTL传递失活信号的配体，以及Us3在其表达中的作用。我们的初步数据表明，失活配体是病毒而不是细胞起源。我们将通过表达单个HSV候选蛋白和使用单个基因缺失的突变HSV菌株来识别发送失活信号的配体。具体目标3。确定与CTL失活相关的细胞内信号事件，以及失活对通过T细胞受体的信号传导的影响。我们将研究一个90 kda蛋白磷酸化在失活但不受控制的CTL中的作用。我们还将评估失活对T细胞受体连接引起的细胞内信号事件的影响。阐明失活途径的分子细节将为CTL的调控提供基础的见解。对这一途径的理解也可能为药理操纵CTL功能提供合理的靶点。

DESCRIPTION (provided by applicant): The herpesviruses are well adapted to their hosts, and employ a variety of mechanisms to co-exist with the host immune response. Recently, we have described the ability of herpes simplex virus (HSV)-infected cells to send a negative signal that durably inhibits normal CTL function, a phenomenon we have called "inactivation". Mutant HSV strains lacking the Us3 kinase do not induce inactivation. Inactivation requires direct contact between HSV-infected cells and CTL, and appears to result via a virally encoded ligand. Inactivated CTL do not perform granule-mediated cytotoxicity or secrete the cytotoxic cytokines TNF and IFN-gamma in response to stimulation via the T cell receptor, yet retain the ability to synthesize cytokines after stimulation by PMA/ionomycin. Inactivated CTL show no signs of apoptosis, and remain inactivated for at least 12-48 hours. Taken together, these data suggest that HSV is exploiting an unidentified and extremely powerful signaling pathway that can lead to a profound and durable inhibition of CTL activity. To investigate the molecular events involved in the inactivation process, we propose the following. Specific Aim 1. Determine whether expression of the HSV protein Us3 alone is sufficient for the inactivation effect, or requires interaction with other viral proteins. We will express Us3 in isolation from other viral genes in fibroblasts, and determine whether Us3-expressing cells can inactivate CTL. Specific Aim 2. Identify the ligand transmitting the inactivating signal to CTL, and the role of Us3 in its expression. Our preliminary data suggest that the inactivating ligand is of viral rather than cellular origin. We will identify the ligand sending the inactivating signal by expressing individual HSV candidate proteins, and using mutant HSV strains with deletions of individual genes. Specific Aim 3. Determine the intracellular signaling events associated with inactivation of CTL, and the effects of inactivation on signaling via the T cell receptor. We will investigate the role of a 90-kDa protein phosphorylated in inactivated but not control CTL. We will also evaluate the effect of inactivation on intracellular signaling events resulting from T cell receptor ligation. Elucidation of the molecular details of the inactivation pathway will provide fundamental insight into the regulation of CTL. The understanding of this pathway may also suggest rational targets for pharmacological manipulation of CTL function.