The Role of CD8+ cells in the Development of Oral Tolerance

描述(申请人提供)：低剂量口服耐受被定义为通过调节T细胞(Treg)对特定抗原的系统免疫反应的主动抑制，该调节性T细胞(Treg)是在重复摄入该抗原时产生的。尽管进行了广泛的研究，但对口腔抗原产生调节细胞的具体机制仍知之甚少。虽然先前的研究表明，口服抗原可以同时诱导CD4+和CD8+Tregs，但CD8+T细胞的作用仍然存在争议。我们的初步数据表明，用C57BL/6小鼠饲喂MHC‘I类OVA免疫优势肽(SIINFEKL)可产生对OVA全蛋白后续免疫的耐受性，这种反应依赖于CD8+T细胞的存在。我们的假设是，在正常的免疫环境中，CD8+T细胞是对口服抗原的耐受反应的主要组成部分。SIINFEKL直接与MHC I类分子结合，因此只刺激CD8+T细胞。我们建议利用这一点来评估CD8+Tregs在口服耐受中的作用，独立于CD4+T细胞，使用完整的免疫系统。在特定的目的1中，我们将证实SIINFEKL喂养通过将耐受转移到从抗原喂养的小鼠中分离的CDS“T细胞来诱导CD8+Tregs的产生，然后通过喂养荧光标记的多肽或蛋白来定位抗原摄取和提呈的位置。在具体目标2中，我们将首先通过体外抑制试验确定抑制机制是否需要同源或非同源相互作用，从而建立CD8+Tregs如何诱导抑制的机制。然后，我们将使用流式细胞术，通过用抗原负载的五聚体对SIINFEKL特异性细胞进行染色，并与针对CD8+T细胞功能或调节功能的不同表型标记组合的抗体共标记，来确定参与其中的介质。最后，在特定的目标3中，我们将确定TCR与多肽-MHC复合体之间的高亲和力相互作用在喂养SIINFEKL的多肽变体诱导CD8+Tregs过程中的重要性。这一点特别重要，因为这是第一次研究TCR抗原亲和力在口服耐受树突状细胞产生中的重要性。虽然许多研究已经证明了低剂量口服耐受在治疗各种自身免疫性疾病动物模型中的治疗潜力，但一些临床试验发现，它在改善人类疾病方面只取得了轻微的成功。这些试验的结果表明，虽然口服抗原可以影响全身免疫反应，但需要更全面地了解诱导口服耐受的机制，以便优化治疗方案。

DESCRIPTION (provided by applicant): Low dose oral tolerance is defined as the active suppression of a systemic immune response to a specific antigen by regulatory T cells (Tregs) generated in response to repeated ingestion of that antigen. Though ex- tensively studied, the specific mechanisms involved in the generation of regulatory cells in response to oral antigen remain poorly understood. Although previous studies have demonstrated that oral antigen can in- duce both CD4+ and CD8+ Tregs, the role of CD8+ T cells remains controversial. Our preliminary data dem- onstrate that feeding C57BL/6 mice with the MHC Class' I immunodominant peptide of OVA (SIINFEKL) generates tolerance to subsequent immunization with the whole OVA protein, and that this response is de- pendent on the presence of CD8+ T cells. Our hypothesis is that CD8+ T cells are a major component of the tolerogenic response to oral antigen in a normal immune setting. SIINFEKL binds directly to MHC Class I molecules and therefore stimulates only CD8+ T cells. We propose to harness this to assess the role of CD8+ Tregs in oral tolerance, independently of CD4+ T cells, using an intact immune system. In specific aim 1, we will confirm that SIINFEKL feeding induces the generation of CD8+ Tregs by transfer of tolerance to na- ive mice with CDS" T cells isolated from antigen fed mice, and then localize the sites of antigen uptake and presentation by feeding fluorescent-labeled peptide or protein. In specific aim 2, we will establish the mechanism of how CD8+ Tregs induce suppression, by first establishing whether the mechanism of suppres- sion requires cognate or noncognate interactions using in-vitro suppression assays. We will then identify the mediators involved using flow cytometry by staining SIINFEKL-specific cells with antigen-loaded pentamer, and costaining with antibody against different combinations of phenotypic markers for CD8+ T cell function or regulatory function. Finally, in specific aim 3, we will establish the importance of high affinity interactions be- tween TCR and peptide-MHC complexes in the induction of CD8+ Tregs by feeding peptide variants of SIINFEKL. This is of particular importance as this is the first study to investigate the importance of TCR- antigen affinity in the generation of Tregs in oral tolerance. While numerous studies have demonstrated the therapeutic potential of low dose oral tolerance in the treatment of various animal models of autoimmune disease, several clinical trials have found it to be only marginally successful in the amelioration of disease in humans. The results of these trials have demonstrated that while oral administration of antigen can influ- ence systemic immune responses, there is a need for a more complete understanding of the mechanisms underlying the induction of oral tolerance in order to optimize treatment protocols.