The Molecular Biology Of Immune Tolerance

免疫耐受的分子生物学

• 批准号: 6826757
• 负责人: Jack Ragheb
• 金额: --
• 依托单位: NATIONAL EYE INSTITUTE
• 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/6826757
• 关键词: CD28 molecule; CD40 molecule; T cell receptor; T lymphocyte; autoimmunity; flow cytometry; helper T lymphocyte; human tissue; immune tolerance /unresponsiveness; immunogenetics; interferon gamma; interleukin 12; interleukin 2; leukocyte activation /transformation; molecular biology

The mission of the Clinical and Molecular Immunology Group within the Clinical Immunology Section is to perform cellular, molecular and clinical studies aimed at understanding the basis of immune tolerance. A long-term objective is to develop new therapies for allograft transplantation and the treatment of autoimmune and inflammatory diseases. I. CD28 Signaling Signaling through the CD28 receptor during T cell activation exerts a profound influence on the outcome of T cell receptor (TCR) engagement. Failure to receive a costimulatory signal through CD28 results in an unresponsive state termed anergy or in T cell death; both of which contribute to the induction and maintenance of immune tolerance. Thus CD28 signaling is a critical determinant of T cell activation and is an important factor in the development of both auto- and allo- immunity. While signal transduction through the T cell receptor has been extensively characterized, the CD28 signaling pathway is poorly understood. To decipher this pathway, we've utilized a mouse model system in which CD28 signaling is responsible for greater than 99% of T cell IL-2 production. Earlier work had shown that this CD28 dependent regulation of IL-2 expression is not at the transcriptional or translational levels but rather a consequence of increased IL-2 mRNA stability. Our earlier work using sequence tagged genomic IL-2 reporter constructs demonstrated that sequences within the 3' untranslated region of the mouse IL-2 mRNA are responsible for mRNA instability but cannot confer CD28 responsiveness upon a heterologous reporter mRNA. In addition, we discovered the presence of an additional mRNA instability element located within exon 3 and that sequences within exon 2 and the coding region of exon 4 are required for CD28 mediated IL-2 mRNA stabilization. Within these same regions of the IL-2 mRNA we have identified a sequence motif that may be critical for CD28 responsiveness. To test the role of this putative CD28 response element (RE) nearly a dozen mutants have been generated and stable cell lines carrying these mutations have been established. Unexpectedly, analyses of these constructs indicate that CD28 mediated stabilization of the IL-2 mRNA in the cytoplasm appears to be coupled to splicing of the pre-mRNA in the nucleus. While this finding has complicated our studies on the role of the exonic CD28REs, we are actively pursuing this novel finding to determine how and why these two processes are coupled. The results of these studies will assist us in the identification of RNA binding proteins that interact with cytokine mRNAs in a CD28 specific fashion, thus providing us with a protein probe to help elucidate the CD28 signal transduction pathway. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA initially focused on the 3'UTR of the mRNA. We identified one such protein to be HuR, the mammalian homolog of the Drosophila ELAV (embryonic lethal abnormal vision) gene. It is widely held that HuR binding stabilizes labile mRNAs such as c-myc and IL-3, however, we found no association between HuR binding to the IL-2 mRNA and its CD28-mediated stabilization. The controversial nature of our observation has prompted us to determine the biological significance of this finding. One hypothesis that is being pursued is that such binding is related to transport of the mRNA from the nucleus to the cytoplasm. This proposal is supported by our observation that HuR appears to translocate from the nucleus to the cytoplasm upon T cell activation. Along these lines we have generated several IL-2 deletions that would be postulated to abolish HuR binding and the phenotype of these mutants is being characterized. In addition to furthering our elucidation of the CD28 costimulatory pathway, these observations may have important consequences for our understanding of how IL-2 gene expression is regulated at both the transcriptional and post-transcriptional levels. The initial results of these studies have been submitted for publication. II. Molecular Consequences of IL-2 Receptor Blockade Laboratory investigations to understand the mechanism(s) by which blockade of CD25, the high affinity IL-2 receptor (a therapeutic modality in transplantation, allergic, and autoimmune disease) inhibits immune activation have completed their initial phase of study. We found that receptor blockade inhibits the expression of multiple cytokines (both Th1 and Th2) implicated in the pathogenesis of autoimmune disease and graft rejection. We have gone on to show that inhibition of IFN-gamma production occurs through both IL-12 dependent and IL-12 independent pathways. Furthermore, inhibition via the IL-12 dependent pathway is a consequence of blocking CD40L expression, which itself is critical for the induction of IL-12 from monocytes. Furthermore, our results reveal for the first time that both IFN-gamma production and CD40L expression are biphasic and that the latter, but not the initial phase of expression, is highly dependent of IL-2R signaling. These findings have important implications for the choice of immunosuppressive regimen (e.g. anti- IL-2R vs. anti- IL-12) employed in the setting of transplantation or autoimmune disease. These observations are being extended to understand at the molecular level the activation pathways involved in CD40L expression. We expected early expression to occur on memory CD4 cells and late expression to be on naive cells. Though CD40L expression on resting cells is restricted to the CD4 memory population, early expression on activated cells occurred proportionately on naive and memory T cells while late expression was predominately on naive cells. Early expression, like late expression, is dependent on cell-cell contact with monocytes, but through different cell surface receptors. In contrast, early expression is independent of cytokines while late expression is wholly dependent on IL-2 and partially dependent on INF-gamma. Furthermore, the late phase can be completely restored in cells that have not been CD28 costimulated by exogenous IL-2. Since others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model, it will be of critical importance to understand the pathways controlling the expression of this important determinant of immune tolerance. Recently, a great deal of scientific interest has focused on a subpopulation of CD4 T cells that are characterized in part by expressing CD25 on their surface. These resting cells, in contradistinction to activated CD25+ CD4 T cells, appear in the mouse to play an important role in the homeostasis of the immune system and in establishing tolerance. An analogous population has been defined in humans but their in vivo significance is unknown. We have a patient population that has been treated with a monoclonal antibody against CD25 for over 4 years, but contrary to what might have been predicted from the mouse experiments, these patients are healthy; showing no evidence of immune dysregulation. This patient population presents a unique opportunity to study the functional significance of these CD4/CD25+ regulatory cells in humans. We have begun to do so by further characterizing these cells in normal blood donors in an attempt to better define this population phenotypically. By using 4-color multiparameter flow cytometry, we?ve been able to enrich this population 20-fold. We are currently assessing whether this phenotypic population is also enriched for regulatory cell function.

临床免疫学部分中临床和分子免疫学组的使命是进行细胞，分子和临床研究，以了解免疫耐受性的基础。一个长期目标是开发新疗法，用于同种异体移植以及自身免疫性和炎症性疾病的治疗。 I. CD28信号 T细胞活化过程中通过CD28受体的信号传导对T细胞受体（TCR）参与的结果产生了深远的影响。未能通过CD28接收共刺激信号会导致无反应状态被称为Anergy或T细胞死亡；两者都有助于免疫耐受性的诱导和维持。因此，CD28信号传导是T细胞激活的关键决定因素，并且是自动免疫和同种免疫力发展的重要因素。尽管通过T细胞受体的信号转导已广泛表征，但CD28信号通路的理解很少。为了破译这一途径，我们使用了一个小鼠模型系统，其中CD28信号传导造成了T细胞IL-2产生的99％以上。较早的工作表明，IL-2表达的CD28依赖性调节不是在转录或翻译水平上，而是IL-2 mRNA稳定性增加的结果。我们使用序列标记的基因组IL-2报告基因构建体的较早工作表明，小鼠IL-2 mRNA的3'未翻译区域内的序列负责mRNA的不稳定性，但不能在异源记者mRNA上赋予CD28响应性。此外，我们发现了位于外显子3中的附加mRNA不稳定性元件的存在，并且外显子2中的序列和外显子4的编码区域是CD28介导的IL-2 mRNA稳定所必需的。在IL-2 mRNA的这些相同区域中，我们确定了一个序列基序，这对于CD28响应性至关重要。为了测试该推定的CD28响应元件（RE）的作用，已经产生了几乎十二个突变体，并建立了携带这些突变的稳定细胞系。出乎意料的是，对这些构建体的分析表明，CD28介导的IL-2 mRNA在细胞质中的稳定似乎与核中MRNA的剪接耦合。尽管这一发现使我们对外显子CD28RES的作用的研究变得复杂，但我们正在积极追求这一新发现，以确定这两个过程的耦合方式以及为什么。这些研究的结果将有助于我们以CD28特异性方式鉴定与细胞因子mRNA相互作用的RNA结合蛋白，从而为我们提供蛋白质探针，以帮助阐明CD28信号转导途径。进行的生化研究是为了鉴定结合最初集中在mRNA 3'UTR的IL-2 mRNA的蛋白质。我们确定了一种这样的蛋白质是HUR，是果蝇Elav（胚胎致死异常视力）基因的哺乳动物同源物。人们普遍认为，HUR结合可以稳定不稳定的mRNA，例如C-MYC和IL-3，但是，我们发现HUR与IL-2 mRNA与IL-2 mRNA的结合与其CD28介导的稳定性之间没有关联。我们观察的有争议的性质促使我们确定了这一发现的生物学意义。一种被提出的假设是，这种结合与mRNA从细胞核到细胞质的转运有关。我们的观察结果支持了这一建议，即在T细胞激活时，HUR似乎从细胞核转移到细胞质。沿着这些线条，我们产生了几种IL-2缺失，这些缺失将被假设以废除HUR结合，并且这些突变体的表型正在表征。除了进一步阐明CD28共刺激途径外，这些观察结果可能对我们对如何在转录和转录后水平调节IL-2基因表达的理解产生重要的影响。这些研究的最初结果已提交出版。 ii。 IL-2受体阻滞的分子后果 实验室研究以了解阻断CD25，高亲和力IL-2受体（移植，过敏和自身免疫性疾病的治疗方式）的机制的机制。我们发现受体阻滞抑制了与自身免疫性疾病和移植排斥的发病机理有关的多种细胞因子（TH1和TH2）的表达。我们继续表明，通过IL-12依赖性和IL-12独立途径，对IFN-gamma产生的抑制作用发生。此外，通过IL-12依赖途径抑制是阻断CD40L表达的结果，这本身对于从单核细胞诱导IL-12至关重要。此外，我们的结果首次揭示了IFN-GAMMA的产生和CD40L表达都是双相的，而后者（但不是表达的初始阶段）高度依赖于IL-2R信号传导。这些发现对于在移植或自身免疫性疾病的情况下采用的免疫抑制方案（例如抗IL-2R与抗IL-12）具有重要意义。这些观察结果正在扩展到在分子水平上理解CD40L表达所涉及的激活途径。我们预计早期表达会在记忆CD4细胞上发生，并且晚期表达将在天真的细胞上发生。尽管在静息细胞上的CD40L表达仅限于CD4记忆群，但活化细胞的早期表达是在天真和记忆T细胞上成比例地发生的，而晚期表达则主要在幼稚的细胞上。早期表达，例如晚表达，取决于细胞 - 细胞与单核细胞的接触，但通过不同的细胞表面受体。相比之下，早期表达与细胞因子无关，而晚期表达完全取决于IL-2，部分依赖于INF-GAMMA。此外，晚期可以完全恢复在未被外源性IL-2加工CD28的细胞中。由于其他人已经表明，仅阻止CD40L可以在灵长类动物移植模型中诱导长期耐受性，因此了解控制这种免疫耐受性的重要决定因素表达的途径至关重要。最近，很大一部分科学的兴趣集中在CD4 T细胞的亚群上，这些细胞的一部分是通过表面表达CD25来表征的。这些静止的细胞与活化的CD25+ CD4 T细胞相反，在小鼠中出现，在免疫系统的体内稳态中起着重要作用并在建立耐受性中起着重要作用。人类已经定义了类似的人群，但它们的体内意义尚不清楚。我们有一个患者人群已接受针对CD25的单克隆抗体治疗已有4年以上，但与从小鼠实验中预测的情况相反，这些患者是健康的。没有显示免疫失调的证据。该患者人群为研究人类这些CD4/CD25+调节细胞的功能意义提供了独特的机会。我们已经开始通过进一步表征正常献血者中的这些细胞来做到这一点，以便更好地在表型上定义这一人群。通过使用4彩色多参数流式细胞仪，我们能够丰富该人群20倍。我们目前正在评估该表型种群是否也用于调节细胞功能。