The Molecular Biology Of Experimental and Clinical Immune Tolerance

实验和临床免疫耐受的分子生物学

• 批准号: 7594067
• 负责人: Jack Ragheb
• 金额: $ 169.62万
• 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/7594067
• 关键词: 3' Untranslated Regions; Adverse effects; Affinity; Allergic; Allografting; Antigen-Presenting Cells; Autoimmune Diseases; Autoimmune Process; B-Lymphocytes; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Models; Blocking Antibodies; CD28 gene; Cell Death; Cell Maturation; Cell Surface Receptors; Cell division; Cells; Clinic; Clinical; Clinical Immunology; Clinical Research; Clinical Trials; Conduct Clinical Trials; Daclizumab; Defect; Development; Disease; Drosophila genus; Drug usage; Elements; Embryo; End Point; Exhibits; Failure; Genes; Goals; Graft Rejection; HIV; Homologous Gene; Human; Hypersensitivity; Immune; Immune Tolerance; Immunologics; Immunology; Immunosuppression; Immunosuppressive Agents; Immunotherapy; Inflammatory; Interferon Type II; Interleukin 2 Receptor; Interleukin-12; Interleukin-2; Interleukin-3; Investigation; Laboratories; Laboratory Study; Life; Ligands; Maintenance; Mediating; Messenger RNA; Mission; Modality; Modeling; Molecular; Molecular Biology; Molecular Immunology; Monitor; Monoclonal Antibodies; Mus; Nature; Outcome; Pathway interactions; Pharmaceutical Preparations; Phase; Phenotype; Polyadenylation; Primates; Production; Protein Binding; Proteins; RNA-Binding Proteins; Regulation; Research; Role; Signal Pathway; Signal Transduction; Sirolimus; Surface; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TNFSF5 gene; Therapeutic; Therapeutic immunosuppression; Time; Transcript; Transplantation; Treatment Protocols; United States Food and Drug Administration; Up-Regulation; Vision; Work; anergy; autoimmune uveitis; autoreactive T cell; base; c-myc Genes; cytokine; innovation; insight; mRNA Stability; man; memory CD4 T lymphocyte; monocyte; mouse model; mutant; partial response; receptor; tool

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 a Phase I/II Clinical Trial to induce immune tolerance. 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. To date three subjects have completed the study. Two reached the primary study endpoint and the third had a partial response. Two additional subjects are approaching the primary study endpoint. 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 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. Weve found that nave 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. Our findings also 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. Others have shown that blocking CD40L alone can induce long-term tolerance in a primate transplant model, which has never been accomplished by any other means. Unfortunately, CD40L blocking antibodies had serious adverse effects when used in human trials. Our observations suggest IL-2R blockade could represent one component of an alternative strategy to anti-CD40L immunotherapy for the induction of immune tolerance. Collectively, our results also indicate that IL-2 has a broader immunologic role than the expansion and maintenance of CD25+Tregs. In contrast to late CD40L expression, early expression is completely independent of cytokines. However, weve 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 3UTR. 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 weve identified.

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I/II期临床试验以诱导免疫耐受性。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）。迄今为止，三个受试者已经完成了这项研究。两个达到了主要研究终点，第三个有部分响应。另外两个主题正在接近主要研究终点。 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。实验室研究以了解高亲和力IL-2受体阻断的机制（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的产生和CD40L表达是Biphasic的首次表现出来，并且不是最初的阶段，但最初是表达的，这是最初的表达，这是最初的表达，这是IFN-GAMMA的产生和CD40L的表达均高度依赖，并且是表达的最初阶段。我们发现，中殿和记忆CD4 T细胞表现出双相CD40L的表达，在这两者中，晚期均依赖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。我们的发现对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）的选择也具有重要意义。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，这从未通过任何其他方式完成。不幸的是，在人类试验中使用CD40L阻断抗体会产生严重的不利影响。我们的观察结果表明，IL-2R封锁可以代表抗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。总的来说，我们的结果还表明，IL-2比CD25+Treg的扩展和维持具有更广泛的免疫学作用。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的存在。 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3UTR内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异聚烯基化的调节，因为这些新鉴定的hur结合位点位于5个最多腺苷酸化信号的下游（IL-2转录物具有3个潜在的聚烯基化信号）。我们目前正在评估这些附加的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。