Molecular Biology--Experimental/Clinical Immune Toleranc

分子生物学--实验/临床免疫耐受

• 批准号: 7139197
• 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/7139197
• 关键词: CD28 molecule; CD40 molecule; T cell receptor; bioinformatics; biological signal transduction; clinical trial phase I; clinical trial phase II; cytokine receptors; differentiation antigens; human subject; immune tolerance /unresponsiveness; immunotherapy; interleukin 2; leukocyte activation /transformation; molecular biology; monoclonal antibody; patient oriented research; sirolimus; 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 may be coupled to splicing of the pre-mRNA in the nucleus. While this finding has complicated our studies on the role of IL-2 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 CD28-mediated stabilization. This appeared in press this past year 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. In addition we?ve identified HuR binding sites downstream of the one we originally characterized. Binding to these downstream sites would by necessity be associated with a long form of the IL-2 mRNA that arises by differential polyadenylation. We are currently assessing the potential role of these additional HuR binding sites and working to identify and determine the function of the 50kD IL-2 mRNA binding protein(s). 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 nearly completed the 2nd & 3rd stages of study. Our previous results 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 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 have now defined the cytokine requirements for late CD40L expression by demonstrating that IL-2 acts directly on CD40L rather than through IL-2 dependent cytokines such as IL-4 and IFN-gamma. We have also shown, that while CD40L expression on resting cells is restricted to the CD4 memory population, unexpectedly, on activated cells early and late expression occurs proportionately on naive and memory T cells. In the 3rd stage of the project, we?ve demonstrated that early CD40L expression, like late expression, is dependent on APC-T cell contact, but through different and unknown cell surface receptors. In contrast to late CD40L expression, early expression is completely independent of cytokines. The cell contact-dependent CD40L costimulatory activity of APC is present on resting cells and is actively down regulated upon APC activation, possibly through an NF-kappa B dependent pathway. The 2nd stage results have been submitted for publication and are under review. The 3rd stage results are being prepared for submission. Our current efforts are focused on identifying the unknown surface ligand on APC that is augments the induction of early CD40L expression. III. Induction of Clinical Immunotolerance in Uveitis. 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. We are currently in the middle of one trial to induce immune tolerance in uveitis and a second trial will begin later this fall pending FDA and IRB approval. Both are Phase I/II Clinical Studies to Evaluate the Induction of Immune Tolerance in Patients with Sight Threatening Autoimmune Uveitis. Study 04-EI-0115 has now been underway for a year. 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, a drug that can induce experimental immune tolerance. Sirolimus is a macrocyclic lactone 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 G1 to S phase of the cell cycle, which is believed to responsible for tolerance induction. The study is 100% enrolled and has now reached the watershed point at which daclizumab will be tapered and discontinued. If participants can successfully be taken off daclizumab, they will remain on sirolimus alone for another 6 months before it is determined whether they have been cured of their uveitis.

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记者构建体意外地表明，IL-2 mRNA稳定性可以与原子核中MRNA的拼接耦合。尽管这一发现使我们对IL-2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的发现，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，与IL-2 mRNA的HUR结合不足以用于CD28介导的稳定性。这是在过去的一年中发表在《生物化学杂志》上的媒体上。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。此外，我们确定了我们最初表征的下游的HUR绑定位点。与这些下游位点的结合必须与差分聚腺苷酸化产生的长形式的IL-2 mRNA相关。我们目前正在评估这些额外的HUR结合位点的潜在作用，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的机制，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）几乎完成了研究的第二阶段和第三阶段。我们先前的结果首次证明了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。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。现在，我们通过证明IL-2直接作用于CD40L而不是通过IL-2依赖性细胞因子（例如IL-4和IFN-GAMMA）来定义CD40L表达晚期表达的细胞因子需求。我们还表明，尽管静息细胞上的CD40L表达仅限于CD4记忆群，但出乎意料的是，在活化细胞的早期和晚期表达上是在天真和记忆T细胞上成比例地发生的。在项目的第三阶段，我们证明了早期的CD40L表达（如晚表达）取决于APC-T细胞的接触，但通过不同的和未知的细胞表面受体。与晚期CD40L表达相反，早期表达完全独立于细胞因子。 APC的细胞接触依赖性CD40L在静息细胞上存在共刺激活性，并在APC激活后通过NF-KAPPA B依赖性途径积极调节。第二阶段的结果已提交出版，并正在审查中。第三阶段的结果正在准备提交。我们目前的努力集中在识别APC上未知的表面配体，这增强了早期CD40L表达的诱导。 iii。诱导葡萄膜炎的临床免疫耐受性。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。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。我们目前正在一项试验中，以诱导葡萄膜炎的免疫耐受性，第二次试验将于今年秋天开始，等待FDA和IRB批准。两者都是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的诱导。研究04-EI-0115现在已经进行了一年。在这项研究中，我们利用两种药理学剂。 Daclizumab，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是一种可以诱导实验性免疫耐受性的药物。 Sirolimus是一种大环内酯，可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，从而抑制了细胞周期的G1到S相的进展，这被认为是造成耐受性诱导的原因。这项研究已100％招募，现在已经到达了Daclizumab将逐渐变细和中断的流域。如果可以成功地从Daclizumab上取出参与者，他们将独自在西罗莫司（Sirolimus）上呆了6个月，然后确定是否已经治愈了葡萄膜炎。