Molecular Immunology Of Tolerance

耐受性的分子免疫学

• 批准号: 6534940
• 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/6534940
• 关键词: CD28 molecule; T lymphocyte; autoimmunity; chemical stability; gene induction /repression; homologous transplantation; human tissue; immune tolerance /unresponsiveness; immunogenetics; inflammation; interleukin 2; leukocyte activation /transformation; messenger RNA; nervous system transplantation; posttranscriptional RNA processing

The principal 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 likely to be 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 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. We have shown that the AUUUA sequences within the 3' untranslated region of the IL-2 mRNA responsible for mRNA instability do not confer CD28 responsiveness. In addition, we discovered the presence of an additional mRNA instability element located within exon 3. The instability conferred by this element appears to be enhanced, rather than retarded by CD28 costimulation. Recent evidence indicates that CD28 may also negatively regulate other genes during human T cell activation, particularly the HIV coreceptor CCR5. This system that was developed to study CD28 regulation of IL-2 is now also being used to address whether post-transcriptional mechanisms play a role in the negative regulation of CCR5 in T cells. We have determined that sequences upstream of the 3'UTR, localized to 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. The results of these studies should be submitted for publication in the next six months. Information from these studies will assist us in the identification of RNA binding proteins that interact with cytokine mRNAs in a CD28 specific fashion. Studies carried out to identify proteins that bind the IL-2 mRNA initially focused on the 3'UTR of the mRNA but are now being extended to examine binding to the putative CD28RE. To date, constitutively expressed and TCR regulated sequence specific proteins that bind the 3'UTR have been found. One of these appears to be HuR, the mammalian homolog of a protein (ELAV) involved in the embryonic development of the neural system in Drosophila. In nonlymphoid cells HuR is associated with mRNA stabilization, however, we do not observe a change in HuR binding to the IL-2 mRNA in association with CD28 costimulation. Studies of the IL-2 mRNA 5' UTR have identified an alternative transcriptional start site for the IL-2 gene. The biological significance of this finding is being pursued. 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 results of these studies should be submitted for publication early next year. II. Molecular Consequences of IL-2 Receptor Blockade Laboratory investigations to understand the mechanism(s) by which an antibody (HAT) against the alpha chain of the high affinity IL-2 receptor, which is used in our clinical trials, acts to ameliorate autoimmune uveitis have just completed their first phase. We found that HAT blocks the expression of multiple cytokines (both Th1 and Th2) implicated in the pathogenesis of autoimmune disease. Interestingly, HAT blocks IFN-gamma expression as effectively as an antibody against IL-12, the cytokine principally associated with the induction of IFN-gamma. We have gone on to define the mechanisms by which HAT inhibits IFN-gamma production. HAT inhibits IL-12 dependent IFN-gamma production by blocking the expression of CD40L, which itself is critical for the induction of IL-12 by antigen presenting cells. In addition, HAT can directly inhibit the production of IL-12 independent IFN-gamma from T cells by mechanisms that have not been fully elucidated. These findings have important implications for the choice of immunosuppressive regimen (e.g. HAT vs. anti- IL-12) employed in the setting of transplantation or autoimmune disease. III. Oral Tolerance Evidence from a number of labs suggests that there exists within the lymphatic tissue of the gastrointestinal tract regulatory cells that function to maintain tolerance to foreign (e.g. plant) proteins that traverse the GI tract. This has led to clinical attempts to treat autoimmune disease by feeding the putative antigen to patients. Out of necessity, these studies either utilized an animal homolog of the human antigen or limited quantities of impure recombinant human antigen; factors that possibly contributed to the inconclusive results that were obtained. Attempts to induce/reestablish systemic tolerance via the oral route might be enhanced if sufficient quantities of pure human antigen could be administered to patients. Once again, the use of DNA encoding the relevant antigen (i.e. a DNA "vaccine") may represent a potential solution to this problem as it can be produced in very large quantities to a very high purity. Recent work by our collaborator at John Hopkins has demonstrated that DNA can be effectively delivered to the GI tract via the oral route. We have found that oral administration of a human IRBP DNA vaccine appears to slightly reduce the experimental disease induced in mice by the human protein antigen administered with a very powerful adjuvant. Approaches that might better reflect the clinical situation and methods to enhance the protective effect are being investigated.

临床免疫学部分临床和分子免疫学组的主要任务是进行细胞，分子和临床研究，以理解免疫耐受性的基础。一个长期目标是开发新的同种异体移植疗法，并治疗自身免疫性疾病和炎症性疾病。 I.在T细胞激活过程中通过CD28受体信号传导信号传导对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，并且可能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。我们已经表明，负责mRNA不稳定性的IL-2 mRNA的3'未翻译区域内的AUUUA序列不会赋予CD28响应能力。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。该元素所赋予的不稳定性似乎是在增强的，而不是受CD28 costaulation的阻碍。最近的证据表明，在人T细胞激活期间，CD28也可能对其他基因，尤其是HIV共受体CCR5进行负调节。该系统用于研究IL-2调节的CD28调节的系统现在也用于解决转录后机制是否在T细胞中CCR5的负调控中起作用。我们已经确定，CD28介导的IL-2 mRNA稳定化需要3'UTR上游，位于外显子2和外显子4的编码区域的序列。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。这些研究的结果应在接下来的六个月内提交出版。这些研究的信息将帮助我们鉴定以CD28特定方式与细胞因子mRNA相互作用的RNA结合蛋白。进行的研究是为了鉴定结合最初集中在mRNA的3'UTR的IL-2 mRNA的蛋白质，但现在正在扩展以检查与假定的CD28RE的结合。迄今为止，已经发现了结合3'UTR的组成型和TCR调节的序列特异性蛋白。其中之一似乎是HUR，是果蝇神经系统胚胎发育的蛋白质（ELAV）的哺乳动物同源物。在非淋巴细胞中，HUR与mRNA稳定有关，但是，我们没有观察到与CD28共刺激相关的HUR与IL-2 mRNA结合的变化。 IL-2 mRNA 5'UTR的研究已确定了IL-2基因的替代转录起始位点。这一发现的生物学意义正在追求。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。这些研究的结果应在明年年初提交出版。 ii。 IL-2受体阻断实验室研究的分子后果，以了解对高亲和力IL-2受体的α链的抗体（HAT）的机制，该抗体（在我们的临床试验中使用）可以改善自身免疫性渗出性炎炎，刚刚完成了第一阶段。我们发现，HAT阻止了与自身免疫性疾病发病机理有关的多种细胞因子（TH1和TH2）的表达。有趣的是，HAT像对IL-12的抗体一样有效地阻止了IFN-GAMMA的表达，IL-12（主要与IFN-GAMMA诱导的细胞因子）表达。我们继续定义HAT抑制IFN-GAMMA产生的机制。 HAT通过阻止CD40L的表达来抑制IL-12依赖性IFN-gamma的产生，这本身对于通过抗原呈递细胞诱导IL-12至关重要。此外，HAT可以通过尚未完全阐明的机制直接抑制从T细胞中IL-12独立的IFN-gamma产生。这些发现对在移植或自身免疫性疾病的情况下采用免疫抑制方案（例如帽子与抗IL-12）具有重要意义。 iii。来自许多实验室的口服耐受性证据表明，在胃肠道调节细胞的淋巴组织中存在，这些细胞在遍及胃肠道的易耐受性（例如植物）蛋白质上作用。这导致了通过向患者喂养假定的抗原来治疗自身免疫性疾病的临床尝试。出于必要，这些研究要么利用人类抗原的动物同源物，要么利用有限的不纯净重组人类抗原。可能导致获得的不确定结果的因素。如果可以对患者施用足够数量的纯人类抗原，则可以通过口腔途径诱导/重新建立全身性耐受性。再一次，使用编码相关抗原的DNA（即DNA“疫苗”）可能代表了该问题的潜在解决方案，因为它可以大量生产至非常高的纯度。我们在约翰·霍普金斯（John Hopkins）的合作者最近的工作表明，DNA可以通过口头途径有效地传递到胃肠道。我们发现，通过用非常强大的辅助剂施用的人蛋白抗原诱导的人IRBP DNA疫苗的口服略微减少了小鼠诱导的实验疾病。可以更好地反映临床状况和增强保护作用的方法的方法。