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

描述（由申请方提供）：低剂量口服耐受性定义为通过应答重复摄入特定抗原而产生的调节性T细胞（TcR）主动抑制针对该抗原的全身免疫应答。虽然广泛的研究，涉及的具体机制，在响应口服抗原的调节细胞的产生仍然知之甚少。虽然先前的研究已经证明口服抗原可以诱导CD 4+和CD 8 + T细胞，但CD 8 + T细胞的作用仍然存在争议。我们的初步数据证明，用OVA的MHC I类免疫显性肽（SIINFEKL）喂养C57 BL/6小鼠产生对随后用全OVA蛋白免疫的耐受性，并且这种应答依赖于CD 8 + T细胞的存在。我们的假设是，CD 8 + T细胞是在正常免疫环境中对口服抗原的致耐受性应答的主要成分。SIINFEKL直接与MHC I类分子结合，因此仅刺激CD 8 + T细胞。我们建议利用这一点来评估CD 8 + T细胞在口服耐受性中的作用，独立于CD 4 + T细胞，使用完整的免疫系统。在具体目标1中，我们将证实SIINFEKL喂养通过将耐受性转移至具有从抗原喂养小鼠分离的CD 8 + T细胞的初始小鼠来诱导CD 8 + T细胞的产生，然后通过喂养荧光标记的肽或蛋白质来定位抗原摄取和呈递的位点。在具体目标2中，我们将首先使用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，从而确定CD 8 + T细胞如何诱导抑制的机制。然后，我们将使用流式细胞术通过用负载抗原的五聚体染色SIINFEKL特异性细胞，并用针对CD 8 + T细胞功能或调节功能的表型标志物的不同组合的抗体共染色来鉴定所涉及的介质。最后，在具体目标3中，我们将确定TCR和肽-MHC复合物之间的高亲和力相互作用在通过喂食SIINFEKL的肽变体诱导CD 8 + TCR中的重要性。这是特别重要的，因为这是第一个研究TCR-抗原亲和力在口服耐受性中产生TCR的重要性的研究。虽然许多研究已经证明了低剂量口服耐受性在治疗各种自身免疫性疾病动物模型中的治疗潜力，但几项临床试验发现其在人类疾病的改善中仅略微成功。这些试验的结果已经证明，虽然口服给予抗原可以影响全身免疫应答，但是需要更全面地理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.