The Molecular Biology Of Experimental and Clinical Immun

实验和临床免疫的分子生物学

• 批准号: 7322321
• 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/7322321
• 关键词: Molecular; Biology; Experimental; Clinical; Immun

The mission of the Clinical and Molecular Immunology Group within the Clinical Immunology Section of the Laboratory of Immunology is to conduct clinical trials and laboratory based research in pursuit of achieving clinical immune tolerance. Cellular, molecular and clinically based laboratory studies are aimed at understanding the basis of immune tolerance with the long-term goal of developing new therapies for allograft transplantation and the treatment of autoimmune and inflammatory diseases. I. Induction of Clinical Immunotolerance. To date, autoimmune disease and transplant graft rejection have 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. We are currently completing one trial to induce immune tolerance and a third trial will start this year. Both are Phase I/II Clinical Studies to Evaluate the Induction of Immune Tolerance in Patients with Autoimmune disease. Study 04-EI-0115 has now been underway for 2 years. In this study we utilize two pharmacological agents; daclizumab, a monoclonal antibody against the interluekin-2 receptor that can control autoimmune uveitis but does not appear to block the induction of tolerance, and sirolimus (rapamycin), a drug that can induce experimental immune tolerance. Study participants have been successfully tapered off daclizumab, and are now being tapered off sirolimus. They will be followed off of all medications for 6 months before it is determined whether they have been cured of their uveitis. The new studies will attempt to achieve tolerance through two entirely different mechanisms. One will be through the deletion of peripheral autoreactive T cells. A characteristic of autoreactive cells is their expression of CD25, the alpha chain of the high affinity IL-2 receptor. We propose to eliminate these cells by administering denileukin diftitox, a recombinant protein composed of the amino acid sequences for diphtheria toxin fragments A and B fused to the sequences for interleukin-2. By targeting the cytocidal activity of diphtheria toxin to autoreactive T cells via CD25, we hope to induce immune tolerance in these patients and thus in essence ?cure? them of their disease. The other new study seeks to induce immune tolerance by expanding and enhancing the function of regulatory immune cells called CD56 bright NK cells. One important challenge in these clinical studies is to develop tools that will allow us to detect and monitor the fate of autoreactive T cells that have become tolerized. Studies of host protein incorporation into HIV might lend insight into the development of such tools. 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 demonstrated 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 on 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?ve found that naive and memory CD4 T cells exhibit biphasic CD40L expression and in both, the late phase is CD28-dependent and inhibited by daclizumab independently of cell division. In contrast to mouse, human late phase CD40L is a consequence of CD28 signaling and IL-2, not the principal Th1/Th2 polarizing cytokines. This fundamental difference between man and mouse in the regulation of CD40L has profound implications for mouse models of B cell maturation, transplant tolerance, allergy and autoimmune disease. Collectively, our results indicate that IL-2 has a broader immunologic role than the expansion and maintenance of CD25+Tregs. IL-2R blockade could represent one component of an alternative strategy to anti-CD40L immunotherapy for the induction of immune tolerance. In contrast to late CD40L expression, early expression is completely independent of cytokines. However, we?ve discovered that early CD40L expression, like late expression, is dependent on the presence of antigen presenting cells (APC), but through different and unknown cell surface receptors. The CD40L costimulatory activity of APC is dependent upon cell-cell contact with monocytes and does not require monocyte activation. Our current efforts are focused on identifying the unknown surface ligand on APC that augments the induction of early CD40L expression and the molecular mechanisms underlying its biphasic expression. III. 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. Biochemical studies carried out to identify proteins that bind the IL-2 mRNA have focused on the 3'UTR of the mRNA, which contains a cis-dominant instability element. We have identified a half-dozen RNA binding proteins that specifically interact with distinct sequence elements within the 3?UTR. One such protein is 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 CD28-mediated stabilization. The controversial nature of this observation prompted us to generate an IL-2 deletion mutant expected not to bind HuR. Characterization of this mutant reveals a partial defect in CD28-mediated stabilization, indicating that HuR, if not sufficient, may be necessary for IL-2 mRNA stabilization. However, we subsequently identified additional HuR binding sites downstream of the one we initially characterized and deleted. This discovery both complicates our interpretation of the mutant phenotype and raises the possibility that HuR binding to the IL-2 mRNA is in part regulated by differential polyadenylation as these newly identified HuR binding sites that are located downstream of the 5? most polyadenylation signal (the IL-2 transcript has 3 potential polyadenylation signals). We are currently assessing the role of these additional HuR binding sites and working to characterize the binding and function of the other IL-2 mRNA binding proteins we?ve identified.

免疫学实验室临床免疫学部分临床和分子免疫学组的使命是进行临床试验和基于实验室的研究，以追求实现临床免疫耐受性。细胞，分子和基于临床的实验室研究旨在理解免疫耐受性的基础，其长期目标是开发新的同种异体移植疗法以及自身免疫性和炎症性疾病的治疗。 I.诱导临床免疫力耐受性。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，这是在没有持续免疫抑制或自身免疫性疾病治疗的情况下长期接受接枝的先决条件。我们目前正在完成一项试验以诱发免疫耐受性，第三次试验将于今年开始。两者都是I/II期临床研究，以评估自身免疫性疾病患者的免疫耐受性的诱导。研究04-EI-0115现在已经进行了2年。在这项研究中，我们利用两种药理学剂。 Daclizumab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，但似乎并不能阻止耐受性的诱导，Sirolimus（Rapamycin）（一种可以诱导实验性免疫耐受性的药物）。研究参与者已成功地逐渐减少了daclizumab，现在正在逐渐减少西罗莫司。在确定是否已治愈葡萄膜炎之前，将遵循所有药物的6个月。新的研究将尝试通过两种完全不同的机制实现容忍度。一种将通过删除外周自动反应性T细胞。自动反应性细胞的一个特征是它们的表达CD25是高亲和力IL-2受体的α链。我们建议通过管理Denileukin diftitox来消除这些细胞，这是一种由白喉毒素片段A和B融合到interleukin-2序列的氨基酸序列的重组蛋白。通过靶向白喉毒素通过CD25靶向自动反应性T细胞，我们希望在这些患者中诱导免疫耐受性，从而本质上可以治愈？他们的疾病。另一项新研究试图通过扩展和增强称为CD56明亮NK细胞的调节性免疫细胞的功能来诱导免疫耐受性。 在这些临床研究中，一个重要的挑战是开发工具，使我们能够检测和监测已耐受性的自动反应性T细胞的命运。将宿主蛋白纳入HIV的研究可能会洞悉此类工具的开发。 ii。 IL-2受体阻滞的分子后果。实验室研究以了解阻断CD25的机制，高亲和力IL-2受体（一种移植，过敏性和自身免疫性疾病的治疗方式）首次证明了IFN-GAMMA产生和CD40L表达的首次表达，但最初的表达是最初的，但最初的表达是最初的，但最初的表达是最初的，这是第一次抑制免疫激活。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。我们发现，幼稚和记忆CD4 T细胞表现出双相CD40L表达，在这两种情况下，晚期均依赖于CD28依赖性，并且由Daclizumab抑制了与细胞分裂无关。与小鼠相反，人类晚期CD40L是CD28信号传导和IL-2的结果，而不是主要TH1/TH2极化细胞因子。在CD40L调节中，人与小鼠之间的这种基本差异对B细胞成熟，移植耐受性，过敏和自身免疫性疾病的小鼠模型具有深远的影响。总的来说，我们的结果表明，IL-2比CD25+Tregs的扩展和维持具有更广泛的免疫学作用。 IL-2R阻滞可以代表抗CD40L免疫疗法的替代策略的一个组成部分，以诱导免疫耐受性。与晚期CD40L表达相反，早期表达完全独立于细胞因子。但是，我们发现，早期的CD40L表达（如晚期表达）取决于抗原呈递细胞（APC），但通过不同和未知的细胞表面受体。 APC的CD40L共刺激活性取决于细胞 - 细胞与单核细胞的接触，并且不需要单核细胞激活。我们目前的努力集中在识别APC上未知的表面配体，该配体增强了早期CD40L表达的诱导和其双相表达的分子机制。 iii。 CD28信号传导。 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。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，其中包含顺式优势不稳定性元件。我们已经确定了六个RNA结合蛋白，该蛋白与3？utr中的不同序列元素特别相互作用。一种这样的蛋白质是Hur，Hur，果蝇的哺乳动物同源物（胚胎致死异常）基因。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，与IL-2 mRNA的HUR结合不足以用于CD28介导的稳定性。这一观察结果的有争议的性质促使我们产生一个IL-2缺失突变体，预计不会绑定HUR。该突变体的表征表明，CD28介导的稳定性中有部分缺陷，表明HUR（即使不够）对于IL-2 mRNA稳定可能是必需的。但是，随后，我们确定了我们最初表征和删除下游下游的其他HUR绑定位点。这一发现既使我们对突变表型的解释变得复杂，又提高了HUR与IL-2 mRNA结合的可能性部分受差分聚腺苷酸化的调节，因为这些新鉴定出的位于5的新鉴定的HUR结合位点？大多数聚腺苷酸化信号（IL-2转录本具有3个潜在的聚腺苷酸化信号）。我们目前正在评估这些额外的HUR结合位点的作用，并致力于表征我们确定的其他IL-2 mRNA结合蛋白的结合和功能。