MECHANISM FOR SELECTIVE EXPRESSION OF IFN-GAMA IN T CELL

Laurie Penix(以下简称我)完成了住院医师培训 两年半前在儿科，从那时起 密歇根大学儿科传染病研究员 华盛顿。我曾在克里斯托弗·B博士的实验室工作过。 威尔逊，寻求探索增加的易感性的基础 人类新生儿对细胞内病原体的感染。虽然 在获得必要的技能方面已经取得了相当大的进展 建立独立的实验室职业生涯，额外的一段时间 由拟议的中央情报局提供支持的培训至关重要。我相信，Dr。 威尔逊的实验室，以及它与 免疫学系的其他调查人员和总体 华盛顿大学的科学界，将培养我的 在此期间发展成为一名独立调查员 临床研究人员奖。 我们实验室和其他实验室之前的数据表明，一个深刻的 T细胞产生干扰素-γ的缺陷 新生儿，是导致这种易感性增加的一个因素。 这种缺陷是相对选择性的，因为IL-2的产生 成人T细胞和新生儿T细胞之间没有差别。这反映了缺乏 新生儿的记忆T细胞，因为推测是幼稚的和记忆的T细胞 产生同等数量的IL-2，而初始T细胞(包括50- 65%的成人和95%的新生儿T细胞产生的干扰素减少了10倍。 伽马。记忆性T细胞和幼稚T细胞之间的差异是由于 干扰素-γ基因转录启动减弱。我加入了 并开始研究转录调控 干扰素-伽马基因，长期目标是识别顺式- 与其绑定的元素和因素，它们解释了显著的 该基因在幼稚T细胞中的差异表达。我的初步数据 强烈建议元素，这是必要和充分的 LacZ报告基因构建物的T细胞和激活特异性表达 存在于干扰素-γ转录起始点的111个碱基以内； 其中包括结合转录因子GATA-3的元件。 我将检验这样一种假设，即表达/激活的差异 结合在-111到+61关键调控元件上的蛋白质 干扰素-γ基因的区域，与差异表达有关 这种基因在幼稚T细胞和记忆T细胞中的表达。这件事将由两个人完成 相互补充的办法(目标1和目标2)，并行进行。要定义 干扰素-γ启动子中提供T细胞的最小顺式元件 激活后的特定表达式，我将使用瞬变 Jurkat T细胞的转染实验。识别变性人 与关键顺式元素相互作用的因素，我将使用电泳法 迁移率变化分析、UV交联、甲基化干扰和 部分亲和纯化。一旦这些要素和因素 如上所述，在目标3中，我将探讨它们在差异中的作用 干扰素-γ的表达。这将通过比较交易行为 成人记忆T细胞提取液中存在的因子 从新生儿和成人的幼稚T细胞中提取。这 信息将增强我们对T的分子基础的理解 细胞发育，并可能为潜在的治疗提供洞察力 提高新生儿细胞免疫功能的途径。

Laurie Penix (hereafter referred to as I) completed residency training in Pediatrics two and one-half years ago, and since that time has been a fellow in Pediatric Infectious Diseases at the University of Washington. I have worked in the laboratory of Dr. Christopher B. Wilson, seeking to explore the basis for the increased susceptibility of the human neonate to infections with intracellular pathogens. Although considerable progress has been made in acquiring the skills necessary to establish an independent laboratory career, an additional period of training supported by the proposed CIA is critical. I believe that Dr. Wilson's laboratory, and the environment provided by its relationship to other investigators in the Department of Immunology and the overall scientific community at the University of Washington, will foster my development into an independent investigator during the tenure of this Clinical Investigator Award. Previous data from our laboratory and others, indicated that a profound deficit in the production of interferon-gamma (IFN-gamma) by T cells from neonates, was a contributing factor to this increased susceptibility. This defect was relatively selective in that the production of IL-2 did not differ between adult and neonatal T cells. this reflected the lack of memory T cells in neonates, since putative naive and memory T cells produce comparable amounts of IL-2, whereas naive T cells (comprising 50- 65% of adult but > 95% of neonatal T cells) produce 10-fold less IFN- gamma. The differences between memory and naive T cells were due to diminished initiation of transcription of the IFN-gamma gene. I joined the laboratory and began to investigate the transcriptional regulation of the IFN-gamma gene, with the long term goal of identifying cis- elements and factors binding thereto, which account for the markedly different expression of this gene in naive T cells. My preliminary data strongly suggest that elements, which are necessary and sufficient for T cell- and activation-specific expression of lacZ reporter constructs are present within 111 bp of the IFN-gamma transcription start site; included therein are elements which bind the transcription factor GATA-3. I will examine the hypothesis that differences in expression/activation of proteins binding to the key regulatory elements in the -111 to +61 region of the IFN-gamma gene, contribute to the differential expression of this gene in naive and memory T cells. This will be done by two complementary approaches (Aims 1 and 2), pursued in parallel. to define the minimal cis-elements in the IFN-gamma promoter that confer T cell specific expression following activation, I will use transient transfection assays in Jurkat T cells. To identify the trans-acting factors interacting with the key cis-elements, I will use electrophoretic mobility shift assays, UV cross-linking, methylation interference and partial affinity purification. Once these elements and factors are delineated, in Aim 3 I will explore their role in the differential expression of IFN-gamma. This will be done by comparing trans-acting factors present in extracts from adult memory T cells to those present in extracts from naive T cells from neonates and adults. This information will enhance our understanding of the molecular basis of T cell development and may provide insight into potential therapeutic approaches to enhance cell-mediated immunity in neonates.