Genes and signals controlling mammalian hematopoiesis.

控制哺乳动物造血的基因和信号。

• 批准号: 10007492
• 负责人: PAUL E LOVE
• 金额: $ 219.78万
• 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/10007492
• 关键词: Acute T Cell Leukemia; Affinity; Area; Autoimmune Diseases; B-Lymphocytes; Binding; Biochemical; Blood Cells; Blood Platelets; CD3 Antigens; CD3E gene; Cell Cycle; Cell Differentiation process; Cell Maintenance; Cells; Childhood Leukemia; Complement; Complex; DNA Binding; Data; Development; Diagnosis; Ensure; Enterobacteria phage P1 Cre recombinase; Erythrocytes; Feedback; Gene Expression; Gene Targeting; Generations; Genes; Genetic Transcription; Hematopoiesis; Hematopoietic; 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; Lymphocyte; Maintenance; Mature T-Lymphocyte; Mediating; Modeling; Mus; Myeloid Cells; PTPN6 gene; Pathway interactions; Peptides; Phenotype; Process; Protein Binding Domain; Protein Family; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Receptor Signaling; Research; Role; SKP Cullin F-Box Protein Ligases; Signal Pathway; Signal Transduction; Signaling Molecule; Surface; System; T cell regulation; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Thymocyte Development; Thymocyte Selection; Thymus Gland; Transgenes; Transgenic Organisms; Tumor stage; Tyrosine; Work; autoreactivity; base; experimental study; hematopoietic stem cell self-renewal; inhibitor/antagonist; interest; mouse model; mutant; neoplastic cell; notch protein; novel; progenitor; programs; prototype; receptor; receptor function; reconstitution; recruit; response; screening; self-renewal; stem-like cell; therapeutic target; thymocyte; transcriptome sequencing; tumor; tumorigenesis

Our 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 and that may represent targets for immunotherapy in humans. 3) Identification and characterization of new molecules 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 (zeta, CD3-gamma, -delta, -epsilon). 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 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 demonstrated that TCR-ITAMs are functionally analogous but appear to act in concert to amplify TCR signals. The full complement of TCR ITAMs was found to be critical for thymocyte selection, the process by which potentially useful immature T cells are instructed to survive and differentiate further-(positive selection), and potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). In current studies, we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of development and after T cell development and thymocyte selection is finished. We generated a "knock-in" model where mice express wild-type zeta but can be induced to express signaling incompetent (ITAM mutant) zeta by Cre-recombinase. Using this model we can test the effect of reduced TCR signaling potential on TCR function at various stages of development or in particular T cell subsets. 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 FACS-based 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. The identification of such molecules may be relevant to the diagnosis and treatment of human autoimmune diseases and tumors (similar to checkpoint inhibitors) since these molecules function to set the activation threshold for naive T cells. Identification of new genes important for T cell development. Themis: We identified a novel T-lineage restricted protein designated Themis. Biochemical studies indicate that Themis functions in the TCR signaling pathway and may have an important role is helping to sustain TCR signaling in thymocytes undergoing positive selection. Germline and and conditional Themis deficient mice have been generated and their phenotype reveals an important role for this protein in late thymocyte development and in thymocyte selection. 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 the tyrosine phosphatase SHP-1 and inhibiting SHP-1 catalytic activity. By inhibiting 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 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 is induced by Notch whereas Fbxl12 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 Hematopoietic Stem (HSC) cell maintenance and self-renewal. 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 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 suggest that Ldb1-nucleated multi-subunit transcription complexes may control HSC maintenance. Deletion of Ldb1 in HSCs resulted in loss of HSCs revealing that Ldb1 complexes function as 'master regulators' of the transcriptional program regulating HSC maintenance/self-renewal. Role of Ldb1 complexes in T-ALL. 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 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 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 targets for treatment of human T-ALL.

我们的研究集中在四个主要领域：1）表征T细胞抗原受体（TCR）信号在T细胞发育中的作用。 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，并且可能代表人类免疫疗法的靶标。 3）在T细胞发育中具有重要作用的新分子的鉴定和表征。 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的活化基序； ITAMS）包含在多聚体TCR复合物的四个不同亚基中（Zeta，CD3 -Gamma，-delta，-epsilon）。 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 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信号传导。这些研究的结果表明，TCR-ITAMS在功能上是类似的，但似乎协同起作用用于扩大TCR信号。发现TCR ITAM的完整补体对于胸腺细胞的选择至关重要，该过程指示潜在有用的未成熟T细胞生存和分化进一步的（阳性选择），并且可能导致可能导致自身免疫性疾病的自身反应性细胞在胸腺中被删除（负选择）。 在当前的研究中，我们正在使用条件基因表达系统来分析在发育的特定阶段以及T细胞发育和胸腺细胞选择后完成TCR信号的重要性。我们产生了一个“敲门”模型，其中小鼠表达野生型Zeta，但可以通过Cre-Recombinase诱导Zeta表达信号传导不符（ITAM突变体）。使用此模型，我们可以测试在开发的各个阶段或尤其是T细胞子集的TCR信号电位降低对TCR功能的影响。 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。对候选补偿性分子的最初基于FACS的搜索使我们进入了CD5，CD5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屏幕来搜索其他调整分子。这种分子的鉴定可能与人类自身免疫性疾病和肿瘤的诊断和治疗有关（类似于检查点抑制剂），因为这些分子的功能可以设置天真T细胞的激活阈值。 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受限蛋白指定为主题。生化研究表明，TRES在TCR信号通路中起作用，并且可能发挥重要作用是有助于维持在接受阳性选择的胸腺细胞中TCR信号传导。生殖线和有条件的主体缺乏小鼠，它们的表型在胸腺细胞晚期发育和胸腺细胞选择中揭示了该蛋白质的重要​​作用。 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SHP-1结合并抑制SHP-1催化活性来发挥作用。通过抑制SHP-1主题，可以增强胸腺细胞中TCR信号传导，从而使它们能够成功地经历低亲和力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泛素连接酶复合物的亚基，该子单位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是由Notch诱导的，而FBXL12是由TRE-TCR诱导的，FBXL1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在造血茎（HSC）细胞维持和自我更新中的作用。 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）。自我更新确保HSC库持续一生，而分化导致所有循环血细胞的连续产生，包括淋巴细胞，髓样细胞，红细胞和血小板。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在调节造血干细胞中自我更新/分化细胞命运决策方面的关键功能，并表明LDB1-核核的多积分转录复合物可以控制HSC维持。 HSC中LDB1的删除导致HSC的损失，表明LDB1复合物是调节HSC维护/自我更新的转录程序的“主调节器”。 LDB1复合物在T-ALL中的作用。 当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，这些细胞的异常更新易患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在肿瘤发生之前是自我更新胸腺细胞所必需的，并且是小鼠诱导ETP T-ALL所必需的。这些结果将LDB1复合物确定为治疗人类T-ALL的靶标。