Regulation of Lymphocyte Proliferation

淋巴细胞增殖的调节

• 批准号: 6559077
• 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/6559077
• 关键词: T lymphocyte; ataxia telangiectasia; biological models; cancer risk; cell differentiation; cell growth regulation; cell population study; chemical chain length; chemical structure function; enzyme activity; gene expression; genetic mapping; genetic regulation; genetically modified animals; helper T lymphocyte; immunogenetics; immunoregulation; laboratory mouse; lymphocyte proliferation; neoplastic process; northern blottings; nucleic acid structure; polymerase chain reaction; protein localization; telomerase; telomere; tissue /cell culture

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 CD4+ 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 128-fold 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 (anti-CD3/CD28). Telomerase may thus play a permissive role in T cell development and in determining the capacity of lymphoid cells for clonal expansion. In differentiating human tonsil B cells, it was demonstrated that telomerase is expressed specifically at a high level in GC B cells. Expression of telomerase in these cells may provide a mechanism for the apparent 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 by RT-PCR and Northern analaysis, 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. In studies of the mechanisms mediating telomerase regulation in lymphocytes, 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. The role of such changes in regulation of telomerase enzymatic activity is under study. 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, suggesting that TR expression may be functionally limiting 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. 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.

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CD4+ NAIVE T细胞中，端粒比在供体年龄范围内的同一供体的记忆细胞中更长。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比同一供体的记忆细胞大128倍。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。这种机制可能会扩展内存和效应B细胞的克隆膨胀能力。 端粒酶是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激活（抗CD3/CD28），端粒酶活性在周围T淋巴细胞中高度诱导。因此，端粒酶可以在T细胞发育以及确定淋巴样细胞的能力中起允许的作用。在区分人扁桃体B细胞时，证明端粒酶在GC B细胞中特别表达。端粒酶在这些细胞中的表达可能为明显的端粒延长提供了一种机制，这在从前体到GC B细胞的分化中发生。 编码端粒酶，RNA模板（TR）和逆转录酶催化成分（TERT）的两个基因的表达已通过RT-PCR和Northern Analaysis进行了分析，并且在淋巴细胞发育和激活过程中已发现两个mRNA。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。在研究介导型淋巴细胞中端粒酶调节的机制中，已经证明，包括HTERT的磷酸化以及TERT从细胞质到核的转运在内的事件与活化T细胞中端粒酶活性的诱导同时发生。这种变化在端粒酶酶活性调节中的作用正在研究。 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。对端粒长度测定的主要作用的基因座已映射到远端染色体2的5厘米区域。采用遗传上遗传缺陷的小鼠RNA模板缺乏小鼠的研究已经表明，体内观察到的端粒伸长表明，端粒（tr）依赖性且表明了小鼠的驱动率，这表明了驱动型的驱动率。表达在功能上可能限制在体内。 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。 在ATM缺陷型小鼠的小鼠模型系统中研究了telangiectia突变（ATM）基因产物突变（ATM）基因产物的功能。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。