Regulation of Lymphocyte Proliferation and Replicative C

淋巴细胞增殖和复制的调节

• 批准号: 7291736
• 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/7291736
• 关键词: Regulation; Lymphocyte; Proliferation; Replicative

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,suggesting that 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. We further demonstrated that telomerase enzymatic activity in vivo is determined by factors in addition to steady state RNA levels of TR and TERT. Specifically, 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 functions of telomere-associated proteins TIN-2, tankyrase-2, and tankyrase-1 are being analyzed through construction of both constitutive and conditional knockouts for each of the corresponding mouse genes. Initial studies demonstrated that constitutive inactivation of TIN-2 results in early embryonic lethality. Conditional knockouts will be used to analyze the mechanism of these effects. Tankyrase-2 knockout mice are viable and are currently being phenotyped. In addition, tankyrase-1 deficient mice have been constructed, and are being bred to homozygosity and in crosses to tankyrase-2 to screen for redundant and non-redundant functions between these two relate

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的同一供体中的记忆细胞中的端粒更长，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。我们进一步证明，除了TR和TERT的稳态RNA水平外，体内的端粒酶酶活性还取决于因素。具体而言，包括HTERT的磷酸化以及TERT从细胞质到核的易位的事件与激活的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，通过构建每个相应的小鼠基因的构成型和条件敲除，正在分析与端粒相关蛋白TIN-2，Tins-2和Tankyrase-1的功能。最初的研究表明，TIN-2的组成型失活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。 Tankyrase-2基因敲除小鼠可行，目前正在表型。此外，已经构建了坦克酶-1不足的小鼠，并且正在繁殖到纯合性，并在十字架到坦克酶-2中以筛选这两种关系之间的冗余和非冗余功能