Lymphocyte Proliferation and Replicative Capacity

淋巴细胞增殖和复制能力

• 批准号: 7048973
• 负责人: RICHARD J. HODES
• 金额: --
• 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/7048973
• 关键词: B lymphocyte; RNA directed DNA polymerase; T cell receptor; T lymphocyte; ataxia telangiectasia; cancer risk; cell differentiation; cell growth regulation; cell population study; enzyme activity; gene expression; genetic mapping; genetic models; genetic regulation; genetically modified animals; immunogenetics; immunoregulation; in situ hybridization; laboratory mouse; lymphocyte proliferation; neoplastic process; nucleic acid repetitive sequence; nucleic acid structure; protein localization; telomerase; telomere

The replicative history and replicative potential of human naive and memory T cells, critical parameters of lymphocyte biology, were analyzed. Telomeres are unique terminal chromosomal structures which shorten with cell division in vitro and with increased age in vivo for human somatic cells. We found that telomeres were longer in naive T cells than in memory cells from the same donors over a wide range of donor age. This suggests that the differentiation of memory cells from naive precursors occurs with substantial clonal expansion. The in vitro replicative capacity of naive cells was greater than that of memory cells from the same donors. Human CD4+ naive and memory cells thus differ in in vivo replicative history as reflected in telomeric length as well as in their residual replicative capacity. Analysis of telomere length regulation in human B cells demonstrated that germinal center(GC)B cells have significantly longer telomeres than the naive B cells that are their precursors or the memory B cells that are their progeny. These results suggest the novel possibility that normal somatic cells of the B lymphocyte lineage express a mechanism capable of extending telomere length. Such a mechanism might function to extend the capacity for clonal expansion of memory and effector B cells.Telomerase, a ribonucleoprotein enzyme that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. When telomerase activity was analyzed in normal human T lymphocytes, it was found that telomerase is expressed at a high level in thymocytes, at an intermediate level in tonsil T cells, and at a low to undetectable level in peripheral blood T cells. Moreover, telomerase activity was highly inducible in peripheral T lymphocytes by activation through CD3 and CD28. Telomerase may thus play a role in T cell development and in the capacity of lymphoid cells for clonal expansion. In human tonsil B cells, telomerase is expressed at a high level in GC B cells and may provide a mechanism for the telomere lengthening that occurs in differentiation from precursor to GC B cells.Expression of the two genes encoding the necessary and sufficient components of telomerase, RNA template (TR) and reverse transcriptase catalytic component (TERT), has been analyzed, and both mRNAs have been found to be regulated during lymphocyte development and activation. It has been further demonstrated that telomerase enzymatic activity in vivo is determined by factors in addition to steady state RNA levels of TR and TERT. It has been demonstrated that events including phosphorylation of hTERT and translocation of TERT from cytoplasm to nucleus occur concurrent with the induction of telomerase activity in activated T cells. To further study the regulation of telomerase at a transcriptional level, a series of genetically engineered mice have been constructed including: mTERT cDNA transgenics, GFP knock-in as a reporter for mTERT transcriptional activity (also resulting in mTERT knock-out inactivation), and an hTERT BAC transgenic that allows study of human TERT regulation in human versus mouse cellular environments. In addition, the role of telomere-associated proteins TIN-2 and tankyrase-2 is being analyzed through construction of both constitutive and conditional knockouts for each of the corresponding mouse genes. Initial studies indicate that constitutive inactivation of either gene results in early embryonic lethality. Conditional knockouts will be used to analyze the mechanism of these effects. A model system has been established for analysis of the genetic regulation of telomere length in mice. Inter-fertile species of mice were identified which differ significantly in telomere length. Crosses between these species have in initial experiments demonstrated that 1) a mechanism exists for substantial telomere lengthening in somatic cells in vivo, and 2) that species-specific telomere length is regulated by segregating genes that are polymorphic between these species. A locus that has predominant effect on telomere length determination has been mapped to a 5cm region of distal chromosome 2. Studies employing mice genetically deficient in telomerse RNA template have indicated that telomere elongation obsered in vivo is telomerae (TR) dependent and, unexpectedly, have shown that mice heterozygous for TR deficiency have impaired capacity for telomere elongation. Parallel studies using mTERT deficient mice demonstrated that heploinsufficiency is not seen in heterozygotes for this gene, suggesting that TR expression, not TERT expression may be functionally limiting during development in vivo. Telomerase activity has also been analyzed in mice, allowing for the first time an analysis of telomerase activity in T cells undergoing an antigen-specific response in vivo. Substantial upregulation of telomerase occurs transiently following antigen challenge, including challenge with infectious virus, demonstrating that this activity is regulated under physiologic conditions of T cell response. Studies of LCMV viral infection have demonstrated that telomerase activity is induced in responses to primary viral infection and is maintained in memory CD8 T cells as well. Telomere length is maintained without loss in the face of extensive clonal expansion, suggesting that telomerase may compensate for telomere loss under these conditions. Experiments are in progress studying the responses of telomerase-deficient mice to directly assess the role of telomerase in anti-viral immune responses.The maintenance of telomere length is being studied in subpopulations of human memory CD4 T cells, both in normal aubjects and in patients with SLE. The relationship of telomere length and telomerase activity will be evaluated as a function of replicative history and as a modulator of function in these populations. In a second clinical situation, telomere length is being analyzed in tumor-specific populations of TILs that are isolated from human tumors, expanded in vitro, and used to treat the donor patients. The hypothesis under study is that telomere length will predict in vivo persistance of T cells used in therapy as well as their clinical effectiveness.The function of the ataxia telangiectasia mutated (ATM) gene product in tumorigenesis was studied in a mouse model system of ATM-deficient mice. These mice develop a high incidence of thymic lymphomas with characteristic chromosomal translocations within the T cell receptor (TCR) locus. Studies of mice deficient in recombinase associated gene (RAG) activity as well at ATM demonstrated that susceptibility to tumorigenesis in ATM-deficient mice is generalized, and is not limited to RAG-dependent rearrangements in the TCR locus. The mechanism of this tumorigenesis and the identification of genes involved in translocation are under study. In collaboration with the lab of Thomas Ried (NCI), in situ hybridization with BAC contigs has identified the regions of recombination reproducibly occurring in ATM-deficient mice, and sequencing strategies will next be applied to defining the recombination and the involved genes. These studies may lead to identification of novel and previously undescribed oncogenes.

分析了人类天真和记忆T细胞的复制史和复制潜力，淋巴细胞生物学的关键参数。端粒是独特的末端染色体结构，随着人体体细胞细胞的体外细胞分裂而缩短体内年龄。我们发现，在幼稚的T细胞中，端粒比在广泛的供体年龄范围内的记忆细胞中的记忆细胞更长。这表明记忆细胞与幼稚前体的分化发生了大量克隆膨胀。幼稚细胞的体外复制能力大于同一供体的记忆细胞的体外复制能力。因此，人类CD4+天真和记忆细胞在体内复制史上有所不同，如端粒长度以及其残留复制能力所反映的。对人B细胞中端粒长度调节的分析表明，生发中心（GC）B细胞的端粒明显更长，而端粒比其前代的前体或记忆B细胞的天真B细胞更长。这些结果表明，B淋巴细胞谱系的正常体细胞表达了一种能够延长端粒长度的机制。这种机制可能会扩大记忆和效应B细胞克隆膨胀的能力。Telomerase是一种能够合成端粒重复序列的核糖核蛋白酶，在种系和恶性细胞中表达，在大多数正常的人类体细胞中不存在。因此，端粒酶的选择性表达已被认为是生殖细胞和恶性细胞永生的基础。当在正常人T淋巴细胞中分析端粒酶活性时，发现端粒酶在胸腺细胞中的高水平表达，扁桃体T细胞中的中间水平，在外周血T细胞中以低至不可检测的水平表达。此外，通过通过CD3和CD28的激活，端粒酶活性在周围T淋巴细胞中高度诱导。因此，端粒酶可能在T细胞发育和淋巴样细胞的能力中起作用。在人类扁桃体B细胞中，端粒酶在GC B细胞中以高水平表达，并可能为端粒延长提供一种机制，这是从前体到GC B细胞的分化中发生的。在淋巴细胞发育和激活期间。进一步证明，体内端粒酶酶活性除了TR和TERT的稳态RNA水平以外，还取决于因素。已经证明，包括HTERT的磷酸化以及TERT从细胞质到核的转运的事件与激活的T细胞中端粒酶活性的诱导同时发生。 To further study the regulation of telomerase at a transcriptional level, a series of genetically engineered mice have been constructed including: mTERT cDNA transgenics, GFP knock-in as a reporter for mTERT transcriptional activity (also resulting in mTERT knock-out inactivation), and an hTERT BAC transgenic that allows study of human TERT regulation in human versus mouse cellular environments.此外，正在通过为每个相应的小鼠基因的构造型和条件敲除构建端粒相关蛋白TIN-2和Tankyrase-2的作用。最初的研究表明，任何两个基因的构成失活会导致早期胚胎致死性。条件敲除将用于分析这些作用的机制。已经建立了模型系统，以分析小鼠端粒长度的遗传调节。鉴定出小鼠的系间种类，这些小鼠在端粒长度上有显着差异。这些物种之间的杂交在初始实验中表明1）存在一种机制，可以在体内大量端粒延长端粒，而2）该物种特异性的端粒长度受这些物种之间多态性的基因进行分离来调节。 A locus that has predominant effect on telomere length determination has been mapped to a 5cm region of distal chromosome 2. Studies employing mice genetically deficient in telomerse RNA template have indicated that telomere elongation obsered in vivo is telomerae (TR) dependent and, unexpectedly, have shown that mice heterozygous for TR deficiency have impaired capacity for telomere elongation.使用MTERT缺乏小鼠的平行研究表明，该基因的杂合子中没有看到HeploInsussible，这表明TR表达而不是TERT表达在体内发育过程中可能会在功能上限制。端粒酶活性也已经在小鼠中进行了分析，这首先允许在体内经历抗原特异性反应的T细胞中端粒酶活性分析。端粒酶的实质性上调是在抗原挑战之后瞬时发生的，包括传染病的挑战，表明该活性在T细胞反应的生理条件下受到调节。 LCMV病毒感染的研究表明，端粒酶活性在对原发性病毒感染的反应中诱导，并保持在记忆CD8 T细胞中。面对广泛的克隆扩张，端粒长度无需损失而保持不变，这表明端粒酶可以在这些条件下弥补端粒损失。实验正在进行中，研究了端粒酶缺陷小鼠的反应，直接评估端粒酶在抗病毒免疫反应中的作用。在正常Aubjects和SLE患者中，在人体记忆CD4 T细胞的亚群中都研究了远程念珠菌长度的维持。端粒长度和端粒酶活性的关系将作为复制历史的函数以及这些人群的功能调节剂的函数进行评估。在第二个临床情况下，正在分析从人类肿瘤分离的TIL的肿瘤特异性种群中，在体外扩张并用于治疗供体患者。研究的假设是，端粒长度将预测治疗中使用的T细胞的体内持久性以及它们的临床有效性。在肿瘤发生中，在肿瘤发生中，链移texia telangictia突变（ATM）基因产物的功能在ATM缺乏小鼠的小鼠模型系统中进行了研究。这些小鼠在T细胞受体（TCR）基因座中具有特征性染色体易位的胸腺淋巴瘤的高发生率。对重组酶相关基因（RAG）活性缺乏小鼠的研究以及ATM的研究表明，ATM缺乏小鼠中对肿瘤发生的易感性已概括，并且不限于TCR基因座中的RAG依赖性重排。该肿瘤发生的机理和易位涉及的基因的鉴定正在研究中。与Thomas Ried（NCI）的实验室合作，与BAC重叠群的原位杂交已经确定了在ATM缺乏的小鼠中可重复发生的重组区域，接下来将应用测序策略来定义重组和所涉及的基因。这些研究可能导致鉴定出新的和以前未描述的癌基因。