Analysis of the T Cell Repertoire

T 细胞库分析

• 批准号: 9153507
• 负责人: RICHARD J. HODES
• 金额: $ 42.52万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/9153507
• 关键词: Alleles; Antibody Response; Antigen Receptors; Antigens; Architecture; Ataxia Telangiectasia Patients; B-Lymphocytes; CD28 gene; CD3 Antigens; CD40 Ligand; CD80 gene; CD8B1 gene; Cell Communication; Cell Lineage; Cell Surface Receptors; Cell surface; Cells; Chromatin; Chromosomes; Clinical; Code; Critical Pathways; DNA; DNA Damage; DNA Double Strand Break; Defect; Dendritic Cells; Development; Dissection; Epithelial Cells; Epithelium; Event; Exclusion; Exhibits; Failure; Frequencies; Generations; Genes; Genetic Recombination; Genomics; Hybrid Cells; Hybrids; Imagery; Immune response; Immune system; Immunoglobulin Class Switching; Immunoglobulin Constant Region; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Individual; Ionizing radiation; Knock-out; Knockout Mice; Lymphocyte; Lymphoid Cell; Mature T-Lymphocyte; Mature Thymocyte; Measurement; Measures; Mediating; Mediator of activation protein; Memory; Molecular Conformation; Mus; Mutation; Nature; Organogenesis; Pathway interactions; Physiological; Play; Population; Process; Property; Proteins; Receptors, Antigen, B-Cell; Regulation; Resolution; Role; Self Tolerance; Signal Transduction; Specificity; Staging; T-Cell Development; T-Cell Receptor; T-Cell Receptor Genes; T-Cell Receptor gamma-Chain; T-Lymphocyte; TNF receptor-associated factor 3; TNFRSF5 gene; TNFSF5 gene; TRA@ gene cluster; TRB@ gene cluster; Testing; Thymic epithelial cell; Thymocyte Development; Thymus Gland; Time; Transgenes; Transgenic Mice; V(D)J Recombination; alpha-beta T-Cell Receptor; ataxia telangiectasia mutated protein; beta Chain Antigen T Cell Receptor; deep sequencing; functional outcomes; inhibitor/antagonist; insight; joint formation; mouse model; mutant; novel; receptor; receptor expression; research study; response; sensor; thymocyte

Mice with genetically altered expression of costimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40, or of the costimulatory receptors CD28 and CD154 (CD40 ligand) have been analyzed for thymic development and expressed T cell repertoire. Disruption of the CD28-B7 pathway or of the CD40-CD154 pathway had minimal effect on development of normal thymic cortico-medullary architecture. In contrast, disruption of both pathways resulted in a profound failure of thymic development. These findings identify a previously unappreciated role of redundant costimulatory pathways mediating an essential function in thymic development. The effect of these costimulatory pathways on selection of the T cell repertoire was studied in parallel. Two pathways of negative selection have been identified. One pathway is CD40L-dependent and acts at a relatively early stage in intra-thymic development and is mediated by a non-cell-autonomous mechanism. The second is CD40L-independent and occurs later in intra-thymic or post-thymic development. Interestingly, we have observed that when negative selection is abrogated by inactivation of the CD40L pathway, functional self tolerance is maintained by a non-deletional mechanism. This non-deletional tolerance is mediated by a CD28-dependent pathway. Thus, redundant pathways exist to protect against self reactivity in the thymus, and disruption of both C40L (deletional) and CD28 (non-deletional) mechanisms of self tolerance results in a population of highly self-reactive thymic T cells. These findings have elucidated the roles of costimulatory pathways in thymic T cell repertoire selection and self tolerance. Most recently, we have analyzed the signals that mediate T cell-epithelial cell cross-talk during thymic development. We have observed that CD28 costimulation is essential for optimal induction of TNFab in single-positive (SP) thymocytes, and that combined defects in CD40-CD40L and TNFab pathways result in defects in medullary thymic eipthelial cell (mTEC) development that are similar to those in combined CD40-CD40L and CD28-B7 disruption, and are in fact as profound as those seen in complete absence of mature SP TCRab thymocytes. We found that selective inactivation in TEC of Traf3, an inhibitor of alternative NFkB signaling, restores mTEC development in the complete absence of SP thymocytes and does so through a RelB-dependent pathway. These findings indicate a novel role for CD28-B7 in addition to other mediators of T cell-TEC cross-talk and demonstrate that any and all signals provided by mature thymocytes for mTEC development are provided by activation of RelB-dependent alternative NFkB signaling. The ataxia telangiectasia mutated (ATM) protein plays a central role in sensing and responding to chromatin changes including DNA double strand breaks (dsb) generated by insults such as ionizing radiation. Lymphoid cells are unique in undergoing physiologic dsb in the processes of Ig class switch recombination (CSR) and T or B cell receptor V(D)J recombination, and a role for ATM in these processes has been suggested by clinical observations in ataxia telangiectasia (AT) patients with mutations in the Atm gene as well as by studies of mice deficient in ATM. We have characterize a defect in T cell development in ATM-deficient mice that is associated with decreased efficiency in V-J rearrangement of the endogenous T cell receptor (TCR)-alpha locus, with consequent reduction in the number of mature TCR-expressing CD4+ and CD8+ thymic T cells. CD4+CD8+ thymocytes from ATM-deficient mice exhibit a reduction in TCRalpha V-J rearrangement as measured by real-time genomic PCR, while containing an increased frequency of unresolved TCR Jbeta coding ends (CE), indicating a delay in TCRalpha V-J coding joint formation. The absence of ATM thus results in reduced frequency of successfully completed TCRalpha recombination, leading to decreased TCRalpha/beta expression and decreased positive selection of mature CD4+ and CD8+ T cells, defining a pivotal role for ATM in T cell development. We have subsequently analyzed the role of ATM in earlier stages of T cell development, including TCR beta rearrangement. In the absence of ATM, VDJ rearrangement of TCRb is defective during the early DN2/3 stage of thymocyte development. This defect has been characterized by direct immuno-FISH visualization of increased DSB foci at the TCRb locus, by decreased productive VDJ recombination, and by decreased expression of TCRb protein. This defect in VDJ recombination reults in functional outcomes that include defective DN3-DN4 and DN-DP developmental transitions, as well as in altered TCRb repertoire as analyzed by deep sequencing of TCRb CD3 regions. These findings thus provide novel insights into the mechanism and functional consequences of ATM during T cell development. Most recently, we have characterized the expression of novel hybrid TCR chains generated by trans-recombination of two distinct TCR loci. Trans recombined genes are expressed at the level of hybrid cell surface receptor chains. Hybrid chains resulting from trans-recombination of TCR gamma variable regions and TCR beta constant regions were identified, and allowed testing of the effect of variable regions on lineage choice (TCR alpha-beta or gamma-delta lineages) and MHC restriction (proposed to be determined by evolutionarily conserved TCR beta V regions). We found that cells expressing these hybrid receptors develop into CD4 and CD8 SP T cells (resembling alpha-beta T cells), undergo MHC-restricted thymic selection despite absence of TCR beta variable region, and support naive and memory mature T cell development. Transgenic mice expressing hybrid receptors are being generated to further define the function of these novel receptors. Costimulatory pathways play critical roles during lymphocyte development as well as during differentiation of mature immune responses. The dissection of specific cell interaction pathways mediating these effects is, however, incomplete. To dissect in detail the nature of these interactions, we have initiated the generation of conditional knock-out and mutant transgenic mouse models, which have not previously existed. To date, we have generated the first (to our knowledge) conditional CD80 (B7-1) knockout mice and have begun the characterization of cell-specific B7 expression during T cell development, and in functional properties of T cells, B cells, dendritic cells, and thymic epithelium. Early results have identified previously undescribed distinct pathways for T helper-dependent antibody responses as elucidated by their costimulatory requirements. We have very recently used a similar strategy to generate conditional CD40 knockouts, with founders now being analyzed. A central property of the adaptive antigen-specific immune system is the expression by individual T and B lymphocytes of a single antigen receptor, mediating their unique antigen specificity. The expression of only one of the two alleles of a TCR or BCR, known as allelic exclusion, is critical to this process, but the mechanisms mediating allelic exclusion are not fully characterized. In initial experiments, we have found that ATM, best understood as a mediator of DNA damage response, plays a substantial role in TCR beta allelic exclusion. Mature T cells in ATM-deficient mice have an increased proportion of cells expressing two cell surface TCR Vbeta products. Using TCR deep sequencing, measurement of DNA breaks, locus conformation, and the effect of rearranged TCR transgenes, we have identified a novel mechanism of locus regulation occurring at a later stage of T cell development than initial TCR beta rearrangement.

具有遗传刺激分子CD80（B7-1），CD86（B7-2）和CD40或共刺激受体CD28和CD154（CD40配体）表达的小鼠已被分析，以进行胸膜发育并表达了T细胞依存量。 CD28-B7途径的破坏或CD40-CD154途径的破坏对正常胸腺皮质 - 糖体系结构的发展影响最小。相反，两种途径的破坏都导致胸腺发育的严重失败。这些发现确定了先前未欣赏的冗余共刺激途径的作用，该途径介导了胸腺发育中的重要功能。这些共刺激途径对选择T细胞库的选择的影响并联。已经确定了两种负面选择途径。一个途径是CD40L依赖性的，在胸腔内发育中起作用相对较早，并由非细胞自主机制介导。第二个是与CD40L无关的，发生在胸腔内或胸腔后发育中。有趣的是，我们观察到，当通过CD40L途径失活消除阴性选择时，功能性自耐力将通过非缺血机制维持。这种非残留耐受性是由CD28依赖性途径介导的。因此，存在冗余途径，以防止胸腺中的自我反应性，并破坏自耐力的C40L（缺失）和CD28（非缺血）机制，导致高度自我反应性胸腺T细胞的种群。这些发现阐明了共刺激途径在胸腺T细胞库的选择和自耐力中的作用。最近，我们分析了在胸腺发育过程中介导T细胞上皮细胞串扰的信号。 We have observed that CD28 costimulation is essential for optimal induction of TNFab in single-positive (SP) thymocytes, and that combined defects in CD40-CD40L and TNFab pathways result in defects in medullary thymic eipthelial cell (mTEC) development that are similar to those in combined CD40-CD40L and CD28-B7 disruption, and are in fact as profound as在完全没有成熟的SP tcrab胸腺细胞的情况下看到的。我们发现，替代NFKB信号传导的抑制剂TEC的选择性失活在完全不存在SP胸腺细胞的情况下恢复MTEC的发育，并通过RELB依赖性途径进行。这些发现表明，除了T细胞-TEC串扰的其他介体外，CD28-B7的新作用是一种新的作用，并证明，成熟胸腺细胞提供的任何信号用于MTEC发育，通过激活RELB依赖性替代性NFKB信号传导提供。毛细血管炎突变（ATM）蛋白在感应和响应染色质变化中起着核心作用，包括由电离辐射等侮辱产生的DNA双链断裂（DSB）。淋巴样细胞在IG类转换重组（CSR）和T或B细胞受体V（D）J重组的过程中经历生理DSB中是独一无二的，并且已经通过ATAXIA TELANGICTASIA（AT）在ATM Gene和ATM Gene的突变患者的ATM和ATM aTT ATM的临床观察表明，ATM在这些过程中的作用。我们表征了ATM缺陷小鼠T细胞发育中的缺陷，这与内源性T细胞受体（TCR） - α基因座的V-J重排效率降低有关，从而减少了表达TCR CDCR CD4+和CD8+胸腺T细胞的成熟数量。通过实时基因组PCR测量的TCRALPHA V-J重排的CD4+ CD8+胸腺细胞表现出降低的Tcralpha V-J重排的降低，同时含有增加未解决的TCR JBETA编码的频率（CE），表明TCRALPHA V-J编码关节延迟。因此，ATM的不存在导致成功完成TCRALPHA重组的频率降低，导致tcralpha/beta表达降低，并降低了成熟的CD4+和CD8+ T细胞的阳性选择，从而定义了ATM在T细胞发育中的关键作用。随后，我们分析了ATM在T细胞开发的早期阶段的作用，包括TCRβ重排。在没有ATM的情况下，在胸腺细胞发育的DN2/3早期，TCRB的VDJ重排有缺陷。该缺陷的特征是TCRB基因座在TCRB基因座，生产性VDJ重组减少以及TCRB蛋白表达降低的直接免疫鱼可视化增加了DSB焦点。 VDJ重组中的这种缺陷在功能结果中的缺陷，包括有缺陷的DN3-DN4和DN-DP发育过渡，以及通过TCRB CD3区域进行深入测序分析的变化TCRB曲目。因此，这些发现为T细胞开发过程中ATM的机制和功能后果提供了新的见解。最近，我们表征了通过两个不同的TCR基因座的反式重组产生的新型混合TCR链的表达。反式重组基因在杂化细胞表面受体链的水平上表达。鉴定了TCR伽马可变区域和TCRβ常数区域引起的混合链，并允许测试可变区域对谱系选择（TCR Alpha-Beta或Gamma-Delta Leneages或MHC限制）的影响（建议通过进化保存的TCR Beta vecions确定）。我们发现，表达这些杂种受体的细胞发展为CD4和CD8 SP T细胞（类似于α-beta T细胞），尽管没有TCRβ变量区域，但仍接受MHC限制的胸腺限制性选择，并支持天真和记忆成熟的T细胞发育。正在生成表达杂种受体的转基因小鼠，以进一步定义这些新型受体的功能。共刺激途径在淋巴细胞发育以及成熟免疫反应的分化过程中起着关键作用。但是，介导这些作用的特定细胞相互作用途径的解剖是不完整的。为了详细阐述这些相互作用的性质，我们启动了以前尚未存在的条件敲除和突变的转基因小鼠模型的产生。迄今为止，我们已经生成了第一个（据我们所知）条件CD80（B7-1）敲除小鼠，并开始在T细胞发育过程中表征细胞特异性B7表达，以及在T细胞，B细胞，树突状细胞和胸皮上皮的功能特性中。早期结果已经确定了先前未描述的T舵依赖性抗体反应的不同途径，以阐明其共刺激要求。我们最近已经使用了类似的策略来产生条件CD40敲除，现在对创始人进行了分析。自适应抗原特异性免疫系统的中心特性是单个抗原受体的单个T和B淋巴细胞的表达，从而介导了其独特的抗原特异性。 TCR或BCR的两个等位基因之一（称为等位基因排除）的表达对于此过程至关重要，但是介导等位基因排除的机制尚未完全表征。在最初的实验中，我们发现，最好理解为DNA损伤反应的介体，在TCRβ等位基因排除中起着重要作用。 ATM缺陷小鼠中成熟的T细胞的表达两种细胞表面TCR VBETA产物的细胞比例增加。使用TCR深度测序，DNA断裂的测量，基因座构象以及重排的TCR转基因的效果，我们已经确定了在T细胞发育后期发生的新型基因座调节机制，而不是初始TCR Beta重排。