Genetic, Cellular And Molecular Mechanisms In Autoimmune

自身免疫的遗传、细胞和分子机制

• 批准号: 6826498
• 负责人: Rachel R. Caspi
• 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/6826498
• 关键词: CD antigens; T lymphocyte; arrestins; autoantigens; autoimmune disorder; cellular immunity; cellular pathology; chemokine; chemotaxis; cholera toxin; cytokine receptors; disease /disorder model; gene therapy; genetically modified animals; helper T lymphocyte; immune tolerance /unresponsiveness; immunogenetics; laboratory mouse; laboratory rat; pathologic process; pertussis toxin; retina; toll like receptor; transforming growth factors; uveitis; vector vaccine

Cellular and molecular mechanisms involved in T cell-mediated autoimmunity against immunologically privileged retinal antigens are being studied. The questions are aimed at elucidating the natural development and maintenance of self-tolerance to retinal antigens, and at defining the processes that lead to their pathological breakdown. The goal is to use this knowledge for designing novel and rational strategies for immunotherapy. The experimental approaches utilize the model of experimental autoimmune uveoretinitis (EAU), which resembles immune-mediated uveitic diseases in humans that can lead to blindness. EAU is induced in mice and rats by immunization with retinal antigens such as IRBP, Arrestin (S-Ag), or their component peptide epitopes, or by infusion of cultured lymphocytes that recognize these antigens. The mechanisms controlling disease susceptibility and pathogenesis are being defined at the genetic, developmental, and immunological levels. Novel approaches to disease regulation are devised based on these findings. Natural tolerance to IRBP is being investigated by using mice that differ in IRBP expression. 1) IRBP-transgenic (TG) mice that express the N-terminal half of IRBP extraocularly under control of the MHC class II promoter. 2) IRBP knockout (KO) mice that have no detectable expression of IRBP and 3) wild type (WT) mice expressing IRBP in the eye and pineal. Using these mice, we found that thymic selection plays a major role in tuning the threshold of susceptibility to ocular autoimmunity. The mechanism involves elimination of highly IRBP reactive T cells and release of CD4+CD25+ regulatory T cells by the thymus. Extraocular expression of IRBP can enhance this tolerance and abolishes the ability to develop EAU in a transgene-specific fashion. These results suggested that thymic tolerance has a greater role than hitherto realized in setting the threshold of immune and autoimmune responsiveness to sequestered retinal antigens. In contrast, peripheral tolerance appears to be an underutilized mechanism that may be exploited therapeutically. Based on this conclusion, we are studying therapeutic induction of tolerance in adult mice by two distinct genetic approaches. 1) an immunodominant IRBP fragment is expressed in autologous B cells by ex vivo transduction with a retroviral construct. Infusion of these cells into genetically compatible recipients results in a profound and long-term tolerance that can both prevent and reverse EAU. 2) IV or IM vaccination with a DNA plasmid encoding an IRBP fragment. This method is efficient in preventing disease, but less so in reversing disease elicited by already primed T cells. The mechanism of protection in both DNA vaccination and B cell therapy appears to involve inactivation of high-affinity Ag specific T cells. This conclusion is based on data showing that although immunological hyporesponsiveness is induced, protection is not transferable and does not involve the two major known apoptosis pathways involving Fas/FasL (death receptor driven) or Bcl-2 regulated (mitochondrial). Transforming growth factor beta (TGF-b) is an immunoinhibitory factor found in aqueous humor of the eye and known to suppress T cell responses. We found that although it can inhibit the activation and function of immature (primary culture) uveitogenic effector T cells, polarized uveitogenic T cells (long term line) are insensitive to TGF-b and may even be enhanced. The mechanism involves direct effects on the T cell as well as indirect effects through the antigen-presenting cell. In chronic uveitis polarized effector T cells may help drive the disease. This may explain persistence of uveitis in the face of the high TGF-b levels found in the eye. Innate immune responses to microbes, that create a pro-inflammatory environment, are thought to play a role as environmental triggers of autoimmunity. For this reason we have made efforts to delineate the role of adjuvant effects in EAU. Pertussis toxin (PT) enhances autoimmune disease in experimental models. PT administered at the time of immunization enhances the Th1 response, which underlies EAU pathology, but can also inhibit EAU if given later, by preventing effector cell migration through blockade of chemokine signaling. We have now resolved these opposing effects of PT. Whereas the disease-inhibitory ability resides in the A subunit, the disease-enhancing activity resides primarily in the B subunit. In contrast to PT, cholera toxin, which shares structural, but not functional, homology with PT, appears to inhibit EAU through induction of immune deviation. Using gene knockout mice developed at Osaka University in Japan, we are studying the TOLL receptor dependence of adjuvant effects in EAU. Preliminary data using TLR-deficient mice indicate that different TLRs can have redundant functions in promoting EAU induction. In collaboration with the Oppenheim group at NCI we demonstrated that lymphocytes and immature dendritic cells exhibit chemotactic responses to the retinal antigens S-Ag and IRBP. The chemokine receptors CXCR5 and CXCR3 mediated the chemotactic effect of IRBP, while only CXCR3 was required for the S-Ag signal. We hypothesize that these responses may underlie attraction of immunocytes to sites of tissue damage and may thus facilitate processes of tissue repair and regeneration. In conjunction with adjuvant effects such as a concomitant infection, autoimmune-prone individuals having inadequate immunological control mechanisms, such responses may lead to development of an adaptive response of the Th1 phenotype, leading to autoimmune tissue pathology. These studies will help to understand disease modulation by adjuvant effects. EAU, similarly to uveitis in humans, is genetically controlled by MHC as well as non-MHC genes. We study genetic control of EAU using two approaches: 1) defining genetic markers associated with susceptibility in genetically defined rodents. We identified 3 chromosomal regions affecting EAU susceptibility, that are now being isolated in lines of consomic rats by marker-assisted selection. Genes differentially expressed by susceptible and resistant strains are being identified using DNA microarrays. These studies will permit closer identification of the genes and pathways determining susceptibility or resistance to EAU. 2) We have developed a "humanized" model of EAU using HLA-transgenic mice in which murine MHC genes have been replaced by human MHC genes. Interestingly, one of the "humanized" strains is developing strong EAU when immunized with S-Ag, which induces little or no uveitis in WT mice, but elicits responses in humans, and recognize an epitope that is recognized by uveitis patients. These results validate EAU as a model of human uveitis.

T 细胞介导的针对免疫特权视网膜抗原的自身免疫的细胞和分子机制正在研究中。这些问题旨在阐明视网膜抗原自我耐受的自然发展和维持，并确定导致其病理崩溃的过程。目标是利用这些知识来设计新颖且合理的免疫治疗策略。实验方法利用实验性自身免疫性葡萄膜视网膜炎（EAU）模型，该模型类似于人类免疫介导的葡萄膜炎疾病，可导致失明。在小鼠和大鼠中，通过使用视网膜抗原（例如 IRBP、Arrestin (S-Ag) 或其组成肽表位）进行免疫，或通过输注识别这些抗原的培养淋巴细胞来诱导 EAU。控制疾病易感性和发病机制的机制正在遗传、发育和免疫学水平上得到定义。根据这些发现设计了疾病调控的新方法。 正在通过使用 IRBP 表达不同的小鼠来研究对 IRBP 的天然耐受性。 1) IRBP 转基因 (TG) 小鼠，在 MHC II 类启动子的控制下，在眼外表达 IRBP N 末端一半。 2) IRBP 敲除 (KO) 小鼠没有可检测到的 IRBP 表达，3) 野生型 (WT) 小鼠在眼睛和松果体中表达 IRBP。使用这些小鼠，我们发现胸腺选择在调节眼自身免疫易感性阈值方面发挥着重要作用。该机制涉及胸腺消除高 IRBP 反应性 T 细胞和释放 CD4+CD25+ 调节性 T 细胞。 IRBP 的眼外表达可以增强这种耐受性，并消除以转基因特异性方式发展 EAU 的能力。这些结果表明，胸腺耐受性在设定对隔离的视网膜抗原的免疫和自身免疫反应阈值方面比迄今为止认识到的作用更大。相反，外周耐受似乎是一种未充分利用的机制，可用于治疗。 基于这一结论，我们正在通过两种不同的遗传方法研究成年小鼠耐受性的治疗诱导。 1) 通过逆转录病毒构建体离体转导，在自体 B 细胞中表达免疫显性 IRBP 片段。将这些细胞输注到遗传相容的受体中会产生深刻且长期的耐受性，从而可以预防和逆转 EAU。 2)用编码IRBP片段的DNA质粒进行IV或IM疫苗接种。这种方法可以有效预防疾病，但在逆转已启动的 T 细胞引发的疾病方面效果较差。 DNA 疫苗接种和 B 细胞治疗的保护机制似乎都涉及高亲和力 Ag 特异性 T 细胞的灭活。这一结论基于的数据显示，虽然诱导了免疫低反应，但保护作用不可转移，并且不涉及涉及 Fas/FasL（死亡受体驱动）或 Bcl-2 调节（线粒体）的两个主要已知凋亡途径。 转化生长因子 β (TGF-b) 是一种存在于眼房水中的免疫抑制因子，已知可抑制 T 细胞反应。我们发现，虽然它可以抑制未成熟（原代培养）致葡萄膜效应 T 细胞的激活和功能，但极化致葡萄膜 T 细胞（长期系）对 TGF-b 不敏感，甚至可能增强。该机制涉及对 T 细胞的直接作用以及通过抗原呈递细胞的间接作用。在慢性葡萄膜炎中，极化效应 T 细胞可能有助于推动疾病。这可以解释在眼睛中存在高 TGF-b 水平的情况下葡萄膜炎持续存在的原因。 对微生物的先天免疫反应会产生促炎环境，被认为是自身免疫的环境触发因素。为此，我们努力描述辅助效应​​在 EAU 中的作用。百日咳毒素（PT）在实验模型中增强自身免疫性疾病。免疫时给予 PT 可增强 Th1 反应，这是 EAU 病理学的基础，但如果稍后给予，也可以通过阻断趋化因子信号传导来防止效应细胞迁移，从而抑制 EAU。我们现在已经解决了 PT 的这些相反影响。疾病抑制能力存在于 A 亚基中，而疾病增强活性则主要存在于 B 亚基中。与 PT 相比，霍乱毒素与 PT 具有结构同源性，但功能不同源，似乎通过诱导免疫偏差来抑制 EAU。使用日本大阪大学培育的基因敲除小鼠，我们正在研究 EAU 中佐剂作用的 TOLL 受体依赖性。使用 TLR 缺陷小鼠的初步数据表明，不同的 TLR 在促进 EAU 诱导方面可能具有冗余功能。我们与 NCI 的奥本海姆小组合作，证明淋巴细胞和未成熟的树突状细胞对视网膜抗原 S-Ag 和 IRBP 表现出趋化反应。趋化因子受体CXCR5和CXCR3介导IRBP的趋化作用，而S-Ag信号仅需要CXCR3。我们假设这些反应可能是免疫细胞吸引组织损伤部位的基础，因此可能促进组织修复和再生过程。与伴随感染等佐剂作用相结合，免疫控制机制不足的易患自身免疫的个体，这种反应可能导致 Th1 表型的适应性反应的发展，从而导致自身免疫组织病理学。这些研究将有助于了解辅助作用对疾病的调节作用。 EAU 与人类葡萄膜炎类似，受 MHC 和非 MHC 基因的遗传控制。我们使用两种方法研究 EAU 的遗传控制：1）定义与遗传定义的啮齿动物易感性相关的遗传标记。我们鉴定了影响 EAU 易感性的 3 个染色体区域，目前正在通过标记辅助选择在健康大鼠品系中分离出这些区域。使用 DNA 微阵列可以鉴定敏感菌株和耐药菌株差异表达的基因。这些研究将有助于更仔细地鉴定决定 EAU 易感性或抗性的基因和途径。 2）我们使用HLA转基因小鼠开发了一种“人源化”EAU模型，其中小鼠MHC基因已被人类MHC基因取代。有趣的是，其中一种“人源化”菌株在用 S-Ag 免疫时会产生强烈的 EAU，在 WT 小鼠中很少或不会诱发葡萄膜炎，但会在人类中引起反应，并识别葡萄膜炎患者所识别的表位。这些结果验证了 EAU 作为人类葡萄膜炎的模型。