Genes and signals controlling mammalian hematopoiesis.

控制哺乳动物造血的基因和信号。

• 批准号: 10685190
• 负责人: PAUL E LOVE
• 金额: $ 236.8万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/10685190
• 关键词: Acute T Cell Leukemia; Affinity; Area; Autoimmune Diseases; Binding; Biochemical; Blood Cells; Blood Platelets; CD3 Antigens; CD3E gene; Catalytic Domain; Cell Cycle; Cell Differentiation process; Cell Maintenance; Cells; Childhood Leukemia; Complex; DNA Binding; Data; Development; Diagnosis; Discrimination; Ensure; Enterobacteria phage P1 Cre recombinase; Erythrocytes; Erythroid; Erythropoiesis; Feedback; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Transcription; Hematopoiesis; Hematopoietic System; Hematopoietic stem cells; Human; ITAM; Immune checkpoint inhibitor; Immunotherapy; Individual; Integral Membrane Protein; Investigation; Knock-in; Knockout Mice; Knowledge; LIM Domain; Laboratories; Libraries; Life; Ligands; Literature; Lymphocyte; Maintenance; Malignant Neoplasms; Mature T-Lymphocyte; Mediating; Modeling; Mus; Myeloid Cells; PTPN6 gene; Pathway interactions; Peptides; Phenotype; Protein Binding Domain; Protein Family; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Receptor Signaling; Research; Role; SKP Cullin F-Box Protein Ligases; Signal Transduction; Signaling Molecule; Surface; System; T cell regulation; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Testing; Thymocyte Development; Thymocyte Selection; Thymus Gland; Transgenes; Transgenic Organisms; Tumor stage; Tyrosine; Work; cancer therapy; conditional knockout; experimental study; hematopoietic stem cell self-renewal; inhibitor; interest; mouse model; mutant; neoplastic cell; notch protein; novel; progenitor; programs; prototype; receptor; reconstitution; recruit; response; screening; self-renewal; stem-like cell; therapeutic target; thymocyte; transcriptome sequencing; tumor; tumorigenesis; virtual

Our current research is focused in four main areas: 1) characterization of the role of T cell antigen receptor (TCR) signals in T cell development. 2) identification and analysis of signal 'tuning' molecules that function downstream of the TCR that augment or inhibit TCR signaling. 3) Identification and characterization of previously undefined proteins that have important roles in T cell development. 4) Studies of the genes controlling the maintenance and self-renewal of Hematopoietic Stem Cells (HSCs) that may also have roles in stem cell like T cell tumors (T-ALL). Examining the role of T cell antigen receptor (TCR) signaling in thymocyte development. Signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs; ITAMs) are contained within four different subunits of the multimeric TCR complex (CD3-gamma, -delta, -epsilon, -zeta). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously generated CD3-zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR signaling. The results of these studies indicated that TCR-ITAMs are functionally analogous but appear to act in concert to amplify TCR signals. In current studies, we are using conditional gene inactivation systems to more precisely analyze the importance of ITAM signaling at specific stages of development. We generated a "knock-in" model where mice express wild-type CD3-zeta but can be induced to express signaling incompetent (ITAM mutant) CD3-zeta by Cre-recombinase. Using this model, we made the surprising discovery that the CD3-zeta ITAMs can perform both activating/amplifying and inhibitory roles in TCR signaling depending upon the affinity of the TCR/ligand interaction. We found that CD3-zeta ITAMs regulate ligand discrimination by inhibiting signaling by low affinity TCR/ligand interactions and by contributing to activation by high affinity TCR/ligand interactions. In current studies, we are seeking to leverage this discovery to enhance the activity of T cells that express low affinity tumor specific TCRs for cancer treatment in humans. Identifying molecules that 'fine-tune' the TCR signal. Our results with TCR-ITAM mutant mice suggested that other signaling molecules can compensate for the reduction in TCR ITAMs. An initial search for candidate compensatory molecules led us to CD5, a TCR associated trans-membrane protein that inhibits TCR signaling. Importantly, we found that CD5 surface expression is regulated by and parallels TCR signal intensity. Thus, rather than simply functioning as a static inhibitory co-receptor, CD5 regulation by TCR signaling provides a feedback mechanism to 'fine-tune' the overall TCR signaling response during thymocyte selection since the expression of CD5 depends upon the intensity of TCR signaling. An obvious benefit of such fine-tuning of the TCR signaling response would be to enable the generation of a T cell repertoire with the maximum possible diversity since it would allow a broader range of TCRs to pass through the signaling threshold 'window' of positive selection. Since little is known about how CD5 regulates TCR signaling, we initiated a project to characterize CD5 function, both genetically and biochemically. We have also begun a search for additional tuning molecules using microarray and RNA-Seq screens and literature searches. We believe that the identification of such molecules will be relevant to the diagnosis and treatment of human autoimmune diseases and cancer (similar to checkpoint inhibitors) since they function to set the activation threshold for naive T cells. In current studies we are testing if conditional deletion of CD5 in T cells enhances their tumor reactivity in the mouse model. Identification of new genes important for T cell development. Themis: We identified a novel T-lineage restricted protein designated Themis and generated germline and conditional Themis deficient mice. Themis is the prototype of a new family of proteins in metazoans that are defined by the presence of a novel domain called the CABIT module. We discovered that CABIT modules function by binding to tyrosine phosphatase catalytic domains and inhibit catalytic activity. By inhibiting the tyrosine phosphatase SHP-1 Themis enhances TCR signaling in developing thymocytes enabling them to successfully undergo positive selection mediated by low affinity self-peptides. FBXL12: We recently identified and characterized a new T cell protein called Fbxl12. We found that the Fbxl12 functions as a subunit of an SCF ubiquitin ligase complex that degrades the cell cycle inhibitor Cdkn1b resulting in proliferation. Previous data had shown that a related protein, Fbxl1 also targets Cdkn1b for degradation in thymocytes. We investigated the function of Fbxl12 by generating conditional knockout mice, comparing the phenotype to Fbxl1 knockout mice, and examining the phenotype of Fbxl1/Fbxl12 double knockout mice. We found that both Fbxl1 and Fbxl12 are required for proliferation of thymocytes at a stage called beta-selection which is controlled by concurrent signals transduced by the pre-TCR and Notch. Our results demonstrated that Fbxl1 expression is induced by Notch whereas Fbxl12 expression is induced by the pre-TCR and that Fbxl1 and Fbxl12 function additively to degrade Cdkn1b. This mechanism explains the requirement for simultaneous Notch and pre-TCR signaling for beta-selection associated proliferation. Role of Ldb1 in hematopoiesis. The hematopoietic system is composed of a functionally diverse group of cells that originate from a common hematopoietic stem cell (HSC) capable of long-term self-renewal and multi-lineage differentiation. Self-renewal ensures that a pool of HSCs persists throughout life, whereas differentiation leads to loss of self-renewal capability and the continuous generation of all circulating blood cells including lymphocytes, myeloid cells, erythrocytes and platelets. Several years ago we initiated experiments aimed at identifying genes important for HSC generation and maintenance. Our initial studies focused on the role of LIM domain binding protein-1 (Ldb1) in hematopoiesis as prior work had suggested a function for Ldb1 in the hematopoietic system. These experiments revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells and suggested that Ldb1-nucleated multi-subunit transcription complexes control HSC maintenance. We also found that another Ldb1 complex with different subunits controls the expression of erythroid genes demonstrating that Ldb1 functions as a key factor in modular transcription complexes that regulate multiple gene programs. Role of Ldb1 complexes in T-ALL. Our current studies are focused on exploring the role of Ldb1 complexes in the maintenance/self-renewal of immature thymocytes that resemble HSCs. Our results suggest that abnormal self-renewal of these cells predisposes to T-Cell Acute Lymphoblastic Leukemia (T-ALL). Using a model of T-ALL that resembles an aggressive form of Early T Progenitor human T-ALL (ETP T-ALL) we found that Ldb1 is required for abnormal self-renewal of thymocytes prior to oncogenesis and is required for the induction of ETP T-ALL in mice. These results identify Ldb1 complexes as potential targets for treatment of human T-ALL.

我们目前的研究集中在四个主要领域：1）T细胞抗原受体（TCR）信号在T细胞发育中的作用的表征。 2）鉴定和分析TCR下游功能的信号“调谐”分子增强或抑制TCR信号传导。 3）在T细胞发育中具有重要作用的先前未定义的蛋白质的鉴定和表征。 4）研究控制造血干细胞（HSC）的维持和自我更新的基因，这些基因也可能在干细胞（如T细胞肿瘤）（T-ALL）等干细胞中起作用。 检查T细胞抗原受体（TCR）信号在胸腺细胞发育中的作用。 信号转导序列（称为免疫受体酪氨酸基于激活基序； ITAMS）包含在多聚体TCR复合物（CD3 -GAMMA，-DELTA，-DELTA，-EPSILON，-ZETA）的四个不同亚基中。 ITAM中的Di-tyrosine残基在TCR参与和功能下被磷酸化，以募集TCR复合物等信号分子，例如蛋白酪氨酸激酶，从而启动T细胞激活级联反应。 To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously generated CD3-zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these TCR信号传导的改变。这些研究的结果表明，TCR-ITAMS在功能上是类似的，但似乎共同起作用用于扩增TCR信号。在当前的研究中，我们正在使用条件基因失活系统来更精确地分析ITAM信号在特定发展阶段的重要性。我们产生了一个“敲门”模型，其中小鼠表达野生型CD3-Zeta，但可以诱导通过Cre-Recombinase表达信号传导不符（ITAM突变体）CD3-Zeta。使用该模型，我们提出了一个令人惊讶的发现，即CD3-Zeta ITAM可以根据TCR/配体相互作用的亲和力在TCR信号传导中执行激活/扩增和抑制作用。我们发现，CD3-Zeta Itams通过抑制低亲和力TCR/配体相互作用的信号传导以及高亲和力TCR/配体相互作用来调节配体歧视。在当前的研究中，我们试图利用这一发现来增强T细胞的活性，该T细胞表达低亲和力肿瘤特异性TCR对人类的癌症治疗。 识别“微调” TCR信号的分子。 我们使用TCR-ITAM突变小鼠的结果表明，其他信号分子可以补偿TCR ITAM的减少。对候选代码分子的初步搜索使我们进入了CD5，这是一种抑制TCR信号传导的TCR相关的跨膜蛋白。重要的是，我们发现CD5表面表达受到TCR信号强度的调节。因此，TCR信号通过CD5调节而不是简单地充当静态抑制性共受体，而是为“微调”胸腺细胞选择过程中总体TCR信号反应的反馈机制，因为CD5的表达取决于TCR信号的强度。 TCR信号响应的这种微调的一个明显好处是，可以使具有最大可能多样性的T细胞库生成，因为它将允许更广泛的TCR通过正选择的信号传导阈值“窗口”。 由于CD5如何调节TCR信号传导知之甚少，因此我们启动了一个项目来表征CD5功能，无论是在遗传和生化上。我们还开始使用微阵列和RNA-Seq屏幕和文献搜索来搜索其他调谐分子。我们认为，这种分子的鉴定将与人类自身免疫性疾病和癌症的诊断和治疗有关（类似于检查点抑制剂），因为它们起作用以设定幼稚T细胞的激活阈值。在当前的研究中，我们正在测试T细胞中CD5的条件缺失是否会在小鼠模型中增强其肿瘤反应性。 鉴定新基因对T细胞发育很重要。 Themis：我们确定了一种新型的T-line限制蛋白质指定为主题，并产生了种系和有条件的主体缺乏小鼠。 Themis是后生动物中新的蛋白质家族的原型，该蛋白质是由一个名为Cabit模块的新型域名定义的。我们发现Cabit模块通过与酪氨酸磷酸酶催化结构域结合并抑制催化活性来发挥作用。通过抑制酪氨酸磷酸酶SHP-1主题增强了开发胸腺细胞的TCR信号传导，从而使它们能够成功地接受由低亲和自肽介导的阳性选择。 FBXL12：我们最近确定并表征了一种称为FBXL12的新T细胞蛋白。我们发现FBXL12充当SCF泛素连接酶复合酶复合物的亚基，从而降解了细胞周期抑制剂CDKN1B，从而导致增殖。先前的数据表明，相关蛋白FBXL1还靶向CDKN1B以降解胸腺细胞。我们通过生成条件敲除小鼠，将表型与FBXL1基因敲除小鼠进行比较，研究了FBXL12的功能，并检查了FBXL1/FBXL12双基因敲除小鼠的表型。我们发现，在称为β选择的阶段，FBXL1和FBXL12都是胸腺细胞增殖所必需的，该阶段是由Pre-TCR和Notch传递的并发信号控制的。我们的结果表明，FBXL1的表达是由Notch诱导的，而FBXL12表达是由TRE-TCR诱导的，FBXL1和FBXL12和FBXL12函数可将CDKN1B添加函数。该机制解释了同时缺口和TCR前信号传导对β选择相关增殖的要求。 LDB1在造血中的作用。 造血系统由功能多样的细胞组成，该细胞源自能够长期自我更新和多条形分化的常见造血干细胞（HSC）。自我更新可确保HSC库持续一生，而分化会导致自我更新能力的丧失，并连续产生所有循环的血细胞，包括淋巴细胞，髓样细胞，红细胞和血浆。几年前，我们启动了旨在识别对HSC生成和维护重要的基因的实验。我们的初步研究集中于LiM结构域结合蛋白-1（LDB1）在造血中的作用，因为先前的工作提出了造血系统中LDB1的功能。这些实验揭示了LDB1在造血干细胞中调节自我更新/分化细胞命运决策方面的关键功能，并提出LDB1-核核的多积分转录复合物控制HSC维护。我们还发现，另一种带有不同亚基的LDB1复合物控制红细胞基因的表达，表明LDB1是调节多个基因程序的模块化转录复合物的关键因素。 LDB1复合物在T-ALL中的作用。 我们目前的研究集中在探索LDB1复合物在类似于HSC的未成熟胸腺细胞的维持/自我更新中的作用。我们的结果表明，这些细胞异常的自我更新易于T细胞急性淋巴细胞白血病（T-All）。使用T-All模型，类似于早期T祖细胞人类T-ALL（ETP T-ALL）的侵略性形式，我们发现LDB1在肿瘤发生之前需要异常的胸腺细胞自我更新，并且在小鼠中诱导ETP T-ALL是必需的。这些结果将LDB1复合物确定为治疗人类T-ALL的潜在靶标。