IL-2 Family Cytokines and Receptors-- Mechanisms of Regulation & Action

IL-2 家族细胞因子和受体——调节机制

• 批准号: 8557958
• 负责人: Warren J Leonard
• 金额: $ 134.67万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557958
• 关键词: Adoptive Immunotherapy; Adult; Allergic; Animals; Area; Autoimmune Process; B-Lymphocytes; Binding; Binding Sites; Biological; Biological Process; Boxing; CD27 Antigens; CD4 Positive T Lymphocytes; Cell Differentiation process; Cell physiology; Cells; ChIP-seq; Clinical; Collaborations; Complex; Consensus; Coupled; Cytokine Receptors; Cytokine Signaling; DNA Sequence Analysis; Data; Defect; Dendritic Cells; Development; Disease; Disease model; Electrophoretic Mobility Shift Assay; Elements; Event; Family; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Helminths; Helper-Inducer T-Lymphocyte; Host Defense; Human; Human T-lymphotropic virus 1; IRF4 gene; Immune response; Immune system; Indium; Inflammatory; Inflammatory Bowel Diseases; Interleukin 2 Receptor; Interleukin 2 Receptor Gamma; Interleukin-10; Interleukin-15; Interleukin-17; Interleukin-2; Interleukin-4; Interleukin-7; Interleukin-9; Leucine Zippers; MAPK Signaling Pathway Pathway; Malignant - descriptor; Malignant Neoplasms; Mediating; Molecular Profiling; Mus; Mutagenesis; Mutate; N Domain; N-terminal; Normal Cell; PRDM1 gene; Phenotype; Population; Process; Property; Proteins; Proto-Oncogene Proteins c-jun; Psoriasis; RNA; Regulation; Regulatory T-Lymphocyte; Reporting; Response Elements; Role; STAT protein; STAT3 gene; STAT5A gene; STAT5B gene; Signal Transduction; Spinal Cord Diseases; Structure; System; T-Cell Activation; T-Cell Leukemia; T-Cell Receptor; T-Lymphocyte; T-bet protein; Th2 Cells; Time; Transcription Factor AP-1; Transgenic Mice; Transgenic Model; Tropical Spastic Paraparesis; Variant; Virus; X-Linked Severe Combined Immunodeficiency; base; cancer therapy; chromatin immunoprecipitation; cytokine; dimer; genome-wide; human disease; in vivo; insight; interleukin-12 receptor; neoplastic; neoplastic cell; novel; pathogen; protein expression; receptor; response; three dimensional structure; transcription factor; tumor

The interleukin-2 receptor and related cytokine/cytokine receptor systems are being studied to understand critical components of the T cell immune response in normal and neoplastic cells. Following T-cell activation, IL-2 and IL-2 receptors are induced; the magnitude and duration of the T-cell immune response is controlled by the amount of IL-2 produced, the levels of receptors expressed, and the time course of these events. Expression of IL-2Ra is interestingly high in cells infected with HTLV-I, the cause of adult T cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). Three chains of the IL-2 receptor exist, IL-2Ra, IL-2Rb, and gc, with IL-2Ra and IL-2Rb being significantly regulated at the level of transcription. gc is a shared chain also used by the receptors for IL-4, IL-7, IL-9, IL-15, and IL-21, and is the protein that is mutated in XSCID. We have focused primarily on the types of signals induced by some of these cytokines, particularly the activation of STAT proteins (signal transducers and activators of transcription), and the mechanism by which they regulate cytokine/STAT target genes. Given our prior observations that STAT5A or STAT5B transgenic mice develop tumors, which was consistent with STAT5 being implicated in malignant transformation and elevated in a range of human tumors, this is an important area for both normal and pathological states. Moreover, humans and mice with defective STAT protein expression have a range of immunological defects. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation being important for host defense to viruses and other intracelllular pathogens, Th2 differentiation being vital in allergic disorders and related to helminths, and Th17 differentiation being vital in a range of inflammatory disorders, including psoriasis and inflammatory bowel disease. We previously showed that IL-2 is important for Th2 differentiation and reported that IL-2 regulates expression of the IL-4 receptor in a STAT5-dependent manner and critically controls priming of cells for Th2 differentiation. Moreover, using genome-wide Ilumina-based ChIP-Seq (chromatin immunoprecipitation coupled to DNA sequencing) analysis, we previously discovered broad regulation of Th2 differentiation via STAT5A and STAT5B, substantially extending earlier studies focused on STAT5A. Moreover, we had discovered that IL-2-mediated IL-4Ra induction was critical in priming cells for Th2 differentiation. In the prior year, we substantially extended these findings by showing that IL-2 via STAT5 induces expression of IL-12Rb1 and IL-12Rb2 and that the induction of IL-12Rb2 is critical for Th1 differentiation and we defined the mechanism of regulation of IL-12Rb2. Additionally, we showed that IL-2 via STAT5 also regulates the T box protein, T-bet. Interestingly, in contrast to the induction of IL-12R proteins, IL-2 inhibits expression of IL-6Ra and gp130, helping to explain the inhibition of Th17 differentiation. Consistent with the ability of Tbx21 to inhibit Th17 differentiation, expression of Tbx21 in Th17 cells resulted in increased IFNg but decreased expression of IL-17A. These results indicated a very broad effect of IL-2 via STAT5 on T helper cell differentation. In the current review year, we have continued to study the role of IL-2 in Th differentiation. We also reported major discoveries related to the role of STAT5 tetramerization in vivo. In addition to forming dimers, a number of STAT proteins can form tetramers via N-terminal region (N-domain)-mediated oligomerization of STAT dimers. Using the previously defined structure of the N-domain for a different STAT, we predicted the key residues in STAT5A and STAT5B for N-domain oligomerization and confirmed them by mutagenesis and electrophoretic mobility shift assays. We then made single and double knockin mice for STAT5A and STAT5B to generate animals that formed STAT5 dimers but not tetramers. Using Affymetrix arrays and RNA-Seq, we defined the role of STAT5 tetramerization for gene expression. Using ChIP-Seq, we also defined the consensus motifs that were required for STAT5 dimer versus tetramer formation. We also coupled the ChIP-Seq data to RNA-Seq data to define tetramer-regulated genes and to defined key binding sites. We found that a key set of genes required STAT5 tetramers for normal expression. We also found that STAT5 tetramers were essential for normal T cell expansion/proliferation as well as survival. We also demonstrated in an inflammatory bowel disease model that STAT5 tetramers are needed for normal development of regulatory T cells. During this year, we also collaborated with Dr. K. Christopher Garcia at Stanford, studying the actions of wild type IL-2 versus novel IL-2 variants, a project with potential clinical ramifications. These studies in part use the pmel-1 T cell receptor transgenic model of adoptive immunotherapy for cancer in collaboration with Dr. Nicholas Restifo, NCI. We have also collaborated with Dr. Garcia on a project in which they compared the three dimensional structure of IL-2 complexed to its receptors to that of IL-15 bound to its receptor. These studies have provided key mechanistic and structural insights into the functional differences between IL-2 and IL-15, which are highly related and share IL-2Rbeta and gc as receptor components but nevertheless possess distinctive biological functions. Although IL-2 primarily signals via cis-signaling and IL-15 via trans-signaling, these cytokines have essentially identical activation of STAT, PI3K/Akt, and Ras/MAPK signaling pathways. Moreover, gene expression profiles are very similar, although not identical. Thus, these cytokines have almost indistinguishable signaling properties despite different biological responses. This study has substantially elucidated structural and mechanistic aspects of IL-2 and IL-15 signaling. Previously, we demonstrated that IL-21 regulated expression of the Prdm1 gene that encodes BLIMP1 via a response element that depends on STAT3 and IRF4. This led to our discovering in the past year that in contrast to its known ability to cooperate with PU.1 in B cells to act via Ets-IRF composite elements (EICEs), IRF4 cooperates with BATF/JUN family proteins to act via AP1-IRF composite elements (AICEs) in T cells, as well as in B cells. We demonstrated critical cooperative regulation of important genes via these AICEs and demonstrated cooperative binding of IRF4, BATF, and JUN family proteins, with markedly diminished IRF4 binding in Batf-deficient cells and markedly diminished BATF binding in Irf4-deficient cells. We demonstrated critical regulation of key genes, including for example those encoding IL-10 and IL-17 via AICEs. In collaborative studies with Ken Murphy, it was demonstrated that there were important compensatory roles for BATF factors in dendritic cell development mediated by BATF-IRF interactions involving the leucine zipper domain of BATF. Overall, the above findings enhance our understanding of mechanisms by which the gc family cytokines regulate gene expression and biologically important processes. In addition, these findings have implications related to the treatment of cancer, autoimmune, and other diseases.

正在研究白细胞介素-2受体和相关的细胞因子/细胞因子受体系统，以了解正常和肿瘤细胞中T细胞免疫反应的关键成分。 T细胞激活后，诱导IL-2和IL-2受体。 T细胞免疫反应的大小和持续时间受到产生的IL-2的量，表达的受体水平以及这些事件的时间过程的控制。在感染HTLV-I的细胞中，IL-2RA的表达很高，HTLV-I是成年T细胞白血病（ATL）和热带痉挛性模拟的原因/HTLV-I相关性脊髓病（TSP/HAM）。 IL-2受体的三个链，IL-2RA，IL-2RB和GC存在，IL-2RA和IL-2RB在转录水平下受到显着调节。 GC是一个共享链也由受体用于IL-4，IL-7，IL-9，IL-15和IL-21，并且是在XSCID中突变的蛋白质。我们主要集中于这些细胞因子中的某些信号的类型，尤其是STAT蛋白的激活（信号转录器和转录的激活因子），以及它们调节细胞因子/STAT靶基因的机制。鉴于我们先前的观察到STAT5A或Stat5b转基因小鼠会发展肿瘤，这与STAT5与恶性转化有关，并且在一系列人类肿瘤中升高，这是正常状态和病理状态的重要区域。此外，具有缺陷统计蛋白表达的人类和小鼠具有一系列免疫缺陷。 T辅助细胞分化对于正常的免疫反应至关重要，Th1的分化对于宿主对病毒和其他细胞内病原体的抗体很重要，Th2分化在过敏性疾病中至关重要，并且与蠕虫有关包括牛皮癣和炎症性肠病。我们先前表明，IL-2对于TH2分化很重要，并报告IL-2以STAT5依赖性方式调节IL-4受体的表达，并严格控制细胞的TH2分化启动。此外，使用基于全基因组iLumina的芯片seq（染色质免疫沉淀与DNA测序结合）分析，我们先前通过STAT5A和STAT5B发现了对TH2分化的广泛调节，从而大大扩展了较早的研究，该研究集中在STAT5A上。此外，我们发现IL-2介导的IL-4RA诱导对于Th2分化的启动细胞至关重要。在上一年，我们通过证明IL-2通过STAT5诱导IL-12RB1和IL-12RB2的表达来实质上扩展了这些发现，并且IL-12RB2的诱导对于Th1分化至关重要，我们定义了IL调节的机制。 -12rb2。此外，我们表明IL-2通过STAT5还调节T盒蛋白T-bet。有趣的是，与诱导IL-12R蛋白的诱导相反，IL-2抑制了IL-6RA和GP130的表达，有助于解释Th17分化的抑制作用。与TBX21抑制Th17分化的能力一致，Th17细胞中TBX21的表达导致IFNG增加，但IL-17A的表达降低。这些结果表明，IL-2通过STAT5对T辅助细胞分化产生了非常广泛的影响。 在当前的审查年中，我们继续研究IL-2在分化中的作用。我们还报告了与Stat5四聚体在体内作用相关的主要发现。除了形成二聚体外，许多STAT蛋白还可以通过N末端区域（N域）介导的STAT二聚体的寡聚化形成四聚体。我们将N域域的先前定义的结构用于不同的统计数据，我们预测了N域的低聚物中STAT5A和Stat5b中的关键残基，并通过诱变和电泳迁移率转移测定法对其进行了证实。然后，我们为STAT5A和STAT5B制造了单一和双重敲击小鼠，以产生形成STAT5二聚体但不是四聚体的动物。使用Affymetrix阵列和RNA-Seq，我们定义了STAT5四聚体在基因表达中的作用。使用Chip-Seq，我们还定义了STAT5二聚体与四聚体形成所需的共识基序。我们还将CHIP-SEQ数据与RNA-Seq数据耦合，以定义四聚体调节的基因并定义了关键结合位点。我们发现，一组关键基因需要STAT5四聚体才能正常表达。我们还发现，STAT5四聚体对于正常的T细胞膨胀/增殖以及存活至关重要。我们还在炎症性肠病模型中证明了调节性T细胞的正常发育需要STAT5四聚体。 在今年，我们还与斯坦福大学的K. Christopher Garcia博士合作，研究了野生IL-2与新颖的IL-2变体的行为，这是一个潜在的临床后果的项目。这些研究部分使用了与NCI的Nicholas Restifo博士合作的PMEL-1 T细胞受体转基因模型的癌症。我们还与Garcia博士合作了一个项目，在该项目中，它们比较了IL-2的三维结构与其受体结合的IL-15的受体。这些研究为IL-2和IL-15之间的功能差异提供了关键的机械和结构见解，这些功能差异高度相关，并共享IL-2RBETA和GC作为受体成分，但仍然具有独特的生物学功能。尽管IL-2主要通过顺式信号和IL-15信号通过反式信号，但这些细胞因子基本上具有STAT，PI3K/AKT和RAS/MAPK信号通路的STAT基本相同激活。此外，基因表达谱非常相似，尽管并不相同。因此，尽管生物学反应不同，这些细胞因子几乎具有几乎无法区分的信号传导特性。这项研究已实质上阐明了IL-2和IL-15信号的结构和机械方面。 以前，我们证明IL-21通过依赖于STAT3和IRF4的响应元件编码BLIMP1的PRDM1基因的表达。这导致我们在过去的一年中发现，与已知与B细胞中PU.1合作通过ETS-IRF复合元件（EICES）作用的能力相比，IRF4与BATF/JUN家族蛋白合作通过AP1-合作T细胞以及B细胞中的IRF复合元件（AICS）。我们证明了通过这些AICS对重要基因进行关键的合作调节，并证明了IRF4，BATF和JUN家族蛋白的合作结合，并且在BATF缺陷型细胞中的IRF4结合显着降低，并且在IRF4缺乏细胞中显着减少了BATF的结合。我们证明了关键基因的批判性调节，包括例如那些通过AICE编码IL-10和IL-17的人。在与肯·墨菲（Ken Murphy）的合作研究中，证明BATF因子在涉及BATF亮氨酸拉链域的BATF-IRF相互作用介导的树突状细胞发育中存在重要的补偿作用。 总体而言，上述发现增强了我们对GC家族细胞因子调节基因表达和生物学重要过程的机制的理解。此外，这些发现具有与癌症，自身免疫性和其他疾病的治疗有关的影响。