Molecular Biology Of Experimental & Clinical Tolerance

实验分子生物学

• 批准号: 6968538
• 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/6968538
• 关键词: CD28 molecule; CD40 molecule; bioinformatics; biological signal transduction; cytokine receptors; differentiation antigens; human tissue; immune tolerance /unresponsiveness; interleukin 2; leukocyte activation /transformation; molecular biology; uveitis

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. 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 has shown that this CD28 dependent upregulation of IL-2 is a consequence of increased IL-2 mRNA stability. We?ve 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. Additional mRNA stability elements located in exon 2 and the coding region of exon 4 are required for CD28 mediated IL-2 mRNA stabilization. Reporter constructs designed to test the role of these sequences in CD28- mediated stabilization unexpectedly revealed that IL-2 mRNA stability 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 sequences, we plan to pursue this novel finding to determine how and why these two processes are coupled. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA have 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 concluded that HuR binding to the IL-2 mRNA is not sufficient for its? CD28-mediated stabilization. This work has been published in the Journal of Biological Chemistry. The controversial nature of this observation prompted us to generate IL-2 deletion mutants expected not to bind HuR. Characterization of this mutant is incomplete, but it exhibits a partial defect in CD28-mediated stabilization indicating that HuR, if not sufficient, may be necessary for IL-2 mRNA stabilization. Bioinformatic analysis of the HuR binding site in the IL-2 mRNA reveals several potential stem-loop structures in the region and we?ve identified a second, unknown 50kD protein that binds the same sequence. We are currently assessing the potential role of these stem-loop structures in HuR binding and working to identify and determine the function of the 50kD IL-2 mRNA binding protein. 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. Our results revealed 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. Others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model. As this has never been accomplished by blocking any other molecule, it will be of critical importance to understand the pathways controlling the expression of this important determinant of immune tolerance. 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. These results have been submitted for publication. Our efforts are currently focused on identifying the unknown surface ligand on monocytes that is necessary for the induction of early CD40L expression. III. Induction of Clinical Immunotolerance in Uveitis We?ve initiated an IRB approved Phase I/II Clinical Study to Evaluate the Induction of Immune Tolerance in Patients with Sight Threatening Autoimmune Uveitis (04-EI-0115). To date, autoimmune disease and transplant graft rejection has been managed using a barrage of immunosuppressive drugs. These medications often require life-long administration and have a plethora of serious side effects. With few exceptions, these same drugs block the induction of immune tolerance; a likely prerequisite for long term graft acceptance in the absence of continued immunosuppression, or for an autoimmune disease cure. The observation that immunosuppressive monotherapy with daclizumab, a monoclonal antibody against the interluekin-2 receptor, can control autoimmune uveitis but does not appear to block the induction of tolerance, presents us with a unique opportunity to clinically test the hypothesis that sirolimus therapy can result in immune tolerance. Sirolimus is a macrocyclic lactone produced by Streptomyces hygroscopicus that inhibits T lymphocyte activation and proliferation in response to both antigenic and cytokine stimulation by a mechanism that is distinct from that of other immuno-suppressants. In cells, sirolimus binds to the immunophilin, FKBP-12, to generate an immunosuppressive complex. This complex binds to and inhibits the activation of mTOR, a key regulatory kinase. This inhibition suppresses cytokine-driven T-cell proliferation, inhibiting the progression from the G 1 to the S phase of the cell cycle, which is believed to responsible for tolerance induction. The study is currently 60% enrolled.

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。 CD28信号通路知之甚少。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的CD28依赖性上调是IL-2 mRNA稳定性增加的结果。我们证明了小鼠IL-2 mRNA的3'未翻译区域内的序列是mRNA不稳定性的，但不能在异源记录器mRNA上赋予CD28响应能力。 CD28介导的IL-2 mRNA稳定化需要位于外显子2中的其他mRNA稳定性元件和外显子4的编码区域。旨在测试这些序列在CD28介导的稳定中的作用的报告构建体意外地表明，IL-2 mRNA稳定性似乎与原子核中MRNA的剪接耦合。尽管这一发现使我们对外显子序列的作用的研究变得复杂，但我们计划追求这一新颖发现，以确定这两个过程的耦合方式以及为什么。进行的生化研究是为了鉴定结合IL-2 mRNA的蛋白质，已集中在mRNA的3'UTR上。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，我们得出的结论是，HUR与IL-2 mRNA的结合不足以使其足够吗？ CD28介导的稳定。这项工作已发表在《生物化学杂志》上。这一观察结果的有争议的性质促使我们产生IL-2缺失突变体期望不绑定HUR。该突变体的表征是不完整的，但在CD28介导的稳定中表现出部分缺陷，表明IL-2 mRNA稳定可能是HUR（即使不够的话）。对IL-2 mRNA中的HUR结合位点的生物信息学分析揭示了该地区的几个潜在的干循环结构，我们识别出第二种结合相同序列的未知的50KD蛋白。我们目前正在评估这些干循环结构在HUR结合中的潜在作用，并致力于识别和确定50KD IL-2 mRNA结合蛋白的功能。 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。我们的结果首次表明，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。其他人则表明，仅阻止CD40L可以在灵长类动物移植模型中诱导长期耐受性。由于从未通过阻止任何其他分子来实现这一目标，因此了解控制这种免疫耐受性决定因素的表达的途径至关重要。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。这些结果已提交出版。目前，我们的努力集中在鉴定单核细胞上未知的表面配体，这是诱导早期CD40L表达所必需的。 iii。诱导葡萄膜炎的临床免疫耐受性 我们启动了IRB批准的I/II期临床研究，以评估视力威胁自身免疫性葡萄膜炎患者的免疫耐受性（04-EI-0115）。迄今为止，已经使用一系列免疫抑制药物来管理自身免疫性疾病和移植移植抑制。这些药物通常需要终身管理，并且具有许多严重的副作用。除少数例外，这些相同的药物阻止了免疫耐受性的诱导。在没有持续免疫抑制或自身免疫性疾病治疗的情况下，可能是长期接枝接受的可能性。 Daclizumab的免疫抑制性单药治疗是一种针对Interluekin-2受体的单克隆抗体，可以控制自身免疫性葡萄膜炎，但似乎并没有阻止耐受性的诱导，它为我们提供了一个独特的机会，可以在临床上测试苏里洛司型疗法可导致免疫耐受性的假设。 Sirolimus是由链霉菌湿生成产生的大环内酯，它通过与其他免疫抑制剂不同的机制抑制抗原和细胞因子刺激的T淋巴细胞活化和增殖。在细胞中，Sirolimus与免疫蛋白FKBP-12结合以产生免疫抑制复合物。该复合物与关键调节激酶MTOR的激活结合并抑制。这种抑制作用抑制了细胞因子驱动的T细胞增殖，从而抑制了从G 1到细胞周期的S相的进展，据信这是造成耐受性诱导的原因。该研究目前已入学60％。