Genetic and Biochemical Approaches to Tyrosine Kinase and Lymphocyte Signaling

2015 在过去一年，我们的工作涵盖了几个主要领域： I. Tec激酶：影响Tec激酶Btk的突变引起遗传性疾病X连锁无丙种球蛋白血症，其特征在于异常的B细胞发育和功能。在过去的15年中，我们发现T淋巴细胞中表达的Tec激酶Itk和Rlk是T细胞信号传导的重要调节剂：Itk和Rlk的突变不会阻止T细胞发育和功能，但通过激活PLC-g和Ca++动员影响T细胞受体信号传导强度来改变结果。证实了它的重要性，Itk的突变已被描述在一个深刻的EBV诱导的致命免疫缺陷。 最近，我们集中在突变对CD 4 + T辅助细胞产生细胞因子模式的影响。在过去的一年里，我们专注于Itk在Th 9细胞发育中的作用，Th 9细胞是哮喘和气道超敏反应的重要贡献者。先前的工作已经证明Itk是导致哮喘的II型免疫反应所必需的。我们现在已经证明Itk是IL-9表达的严格要求，并将这种缺陷与TCR信号转导缺陷联系起来。我们进一步表明，IL-2可以挽救Itk缺陷型T细胞中的多种缺陷，包括诱导IRF 4，一种与TCR信号强度相关的转录因子。我们的工作提供了IL-2促进次优活化T细胞功能的机制，并表明Itk是IL-9介导疾病的治疗靶点（Gomez-Rodriguez et al，2016）。此外，作为T辅助细胞分化和基因靶向方面的专家，Gomez-Rodriguez博士为其他几个实验室的工作做出了贡献。 二.磷酸肌醇3激酶（PI 3 K）δ：作为一项合作研究的一部分，我们之前帮助描述和表征了影响PI 3 K δ的激活突变，PI 3 K δ是一种造血特异性的PI 3 K催化亚基成员，在患有鼻窦-肺感染、粘膜淋巴小结、循环淋巴细胞减少、淋巴细胞增殖和EBV病毒血症的患者中。我们的工作集中于表征这些患者中的CD 8+细胞缺陷，其显示下游PI 3 K靶标的活化升高，包括增加的pAKT和mTOR下游靶标（Lucas等人，Nature Immunol.2014）。为了进一步了解这些缺陷，我们已经生成了一个小鼠模型，并使用这些小鼠来提供对PI 3 K在免疫稳态和功能中的需求的新见解。 三. SAP和Tfh细胞和体液免疫反应的调节：我们工作的另一个主要焦点是SAP，其突变导致遗传性疾病X连锁增殖综合征（XLP 1），其特征是致命的EBV感染，淋巴瘤和抗体缺陷。SAP是一种小的含有SH 2的衔接子，其结合SLAM家族共刺激受体的细胞内尾中的磷酸化酪氨酸残基。我们先前产生了SAP缺陷小鼠，其概括了XLP的特征，包括感染后T细胞活化增加和抗体产生减少（Czar等人PNAS）。值得注意的是，SAP-/- T细胞未能为B细胞提供产生生发中心和长期抗体应答的必要信号，这是成功接种疫苗的标志。这项工作有助于定义T细胞亚群Tfh细胞的重要性，Tfh细胞是为B细胞提供信号以形成生发中心所需的（Qi et al，Nature，2008; Cannons et al，Immunity，2010）。我们的工作提供了对Tfh细胞的发育和功能中T：B细胞相互作用的需求的深入了解，Tfh细胞是向B细胞提供信号以形成生发中心和长期体液免疫所需的关键辅助T细胞群体，这是对大多数免疫的保护性应答的关键特征。在过去的一年中，我们使用RNAseq来评估Tfh特异性基因表达特征。我们发现，转录因子TCF 1，Wnt信号通路的一个组成部分，在Tfh细胞中选择性表达，以响应病毒感染。使用条件性敲除小鼠和shRNA敲除，我们和其他人最近提供了TCF 1是Tfh对病毒感染的应答所必需的证据（Wu et al，Cell Reports，2015）。我们的工作有助于深入了解这一重要的T辅助细胞群体的调节，这使得生物体能够对不同的感染性生物体和疫苗做出适当的反应（Cannons et al Trends Immunol.2013）。在最近的工作中，我们已经发现类似的信号传导和转录网络是慢性感染的长期CD 8细胞应答所必需的（Wu等人，提交）。 四.为了提高我们探测免疫系统的能力，我们已经开发了新的CRISPR介导的工具，以在小鼠和原代T细胞中检测多个基因（Huang et al PLOS One 2016）。我们正在使用这些工具来探测SLAM家族成员和参与Tfh细胞分化的其他基因的功能。

2015 In the last year, our work has covered several major areas: I. Tec Kinases: Mutations affecting the Tec kinase, Btk, cause the genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. Over the last 15 years, we showed that the Tec kinases expressed in T lymphocytes, Itk and Rlk, are important modulators of T cell signaling: mutations of Itk and Rlk do not prevent T cell development and function, but alter outcomes by affecting T cell receptor signaling strength via activation of PLC-g and Ca++ mobilization. Confirming its importance, mutations of Itk have been described in a profound EBV-induced lethal immunodeficiency. Recently, we have focused on the effects of mutations on patterns of cytokine production by CD4+ T helper cells. In the last year, we focused on the role of Itk in the development of Th9 cells, important contributors to asthma and airway hypersensitivity. Previous work had demonstrated that Itk was required for Type II immune responses that lead to asthma. We have now shown that Itk is strictly required for the expression of IL-9 and link this defect to TCR signaling defects. We further show that IL-2 can rescue multiple defects in Itk-deficient T cells, including the induction of IRF4, a transcription factor linked to TCR signaling strength. Our work provides insight into mechanisms by which IL-2 promotes the function of sub-optimally activated T cells and suggests Itk as a therapeutic target for IL-9-mediated diseases (Gomez-Rodriguez et al, 2016). Furthermore, as an expert in T helper differentiation and in gene-targeting, Dr. Gomez-Rodriguez has contributed to the work of several other laboratories. II. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we previously helped describe and characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific member of the PI3K catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets (Lucas et al, Nature Immunol. 2014). To further understand these defects, we have generated a mouse model and are using these mice to provide new insight into the requirements for PI3K in immune homeostasis and function. III. SAP and the regulation of Tfh cells and humoral immune responses: Another major focus of our work is SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1),characterized by fatal EBV-infection, lymphomas, and antibody defects. SAP is a small SH2 containing adaptor that binds phosphorylated tyrosine residues in the intracellular tails of SLAM family co-stimulatory receptors. We previously generated SAP-deficient mice, which recapitulate features of XLP, including increased T cell activation and decreased antibody production upon infection (Czar et al PNAS). Notably, SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination. This work has helped define the importance of a subset of T cells, Tfh cells, that are required to provide signals for B cells to form germinal centers (Qi et al, Nature, 2008; Cannons et al, Immunity, 2010). Our work has provided insight into the requirement for T:B cell interactions in the development and function of Tfh cells, the critical helper T cell population required for providing signals to B cells for germinal center formation and long-term humoral immunity, a key feature of protective responses to most immunizations. In the last year, we have used RNAseq to evaluation Tfh-specific gene-expression signatures. We found that the transcription factor TCF1, a component of the Wnt signaling pathway, is selectively expressed in Tfh cells in response to viral infection. Using conditional knockout mice and shRNA knockouts, we and others recently provided evidence that TCF1 is required for Tfh responses to viral infection (Wu et al, Cell Reports, 2015). Our work helps provide insight into the regulation of this important T helper cell population, which permits an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013). In recent work, we have found that similar signaling and transcriptional networks are required for longterm CD8 cell responses to chronic infection (Wu et al, submitted). IV. To increase our ability to probe the immune system, we have developed new CRISPR mediated tools to inactivate multiple genes in mice and in primary T cells (Huang et al PLOS One 2016). We are using these tools to probe function of the SLAM family members and of other genes involved in Tfh cell differentiation.