T CELL RESPONSE TO A PERIODONTAL PATHOGEN

我们主要关注牙周病原体放线菌伴生放线杆菌(AA)。感染这种细菌的个体会产生特定的体液免疫反应；一些被血清抗体识别的抗原已经被识别出来。另一方面，AA上的T细胞表位尚未确定。为此，我们采用了一种创新的、基于T细胞杂交瘤的方法来分析T细胞对AA的反应。最初，用活细菌口服接种小鼠，产生了一组T细胞杂交瘤。令我们惊讶的是，大约50%与AA反应的T细胞对这种口腔病原体产生的一种毒力因子--白毒素具有特异性。为了进一步研究AA的免疫应答，我们建议：1.通过直接筛选AA基因组文库，克隆编码其他T细胞表位的基因。将确定T细胞杂交瘤识别的AA蛋白的特性，并产生用于AIMS 2和AIMS 3的重组多肽。目的2：表征体内对单个AA抗原的免疫反应的性质。小鼠将：a)用纯化的重组肽免疫，b)用细菌免疫，或c)口服接种活的AA。然后将通过抗体产生、T细胞激活和细胞因子产生(Th1与Th2)以及在小鼠炎症模型中的保护作用来评估对单个T细胞表位的免疫激活。目的3.确定小鼠中的主要T细胞抗原在AA感染患者中是否具有类似的刺激作用。具体地说，EOP患者的外周血淋巴细胞将与单独的重组AA多肽一起体外培养，并通过细胞因子产生和光谱分析评估T细胞刺激。这些目标将：i)提供关于这种病原体上T细胞抗原表位的第一证据，以及ii)评估在小鼠中占主导地位的T细胞表位与在人类中发现的T细胞表位之间的关系。长期目标是开发和验证评估宿主-寄生虫相互作用、疫苗效力和牙周病免疫保护的模型。

We have focused on the periodontal pathogen, Actinobacillus actinomycetemcomitans (Aa). Individuals infected with this bacterium generate a specific humoral immune responses; some of the antigens recognized by serum antibodies have already been identified. On the other hand, the T cell epitopes on Aa have not yet been defined. Towards this end, we have undertaken an innovative, T cell hybridoma-based approach in order to dissect the T cell responses to Aa. In preliminary mice were orally inoculated with live bacteria and a panel of T cell hybridomas was generated. To our surprise, approximately 50% of the T cells reactive with Aa were specific for leukotoxin, a virulence factor produced by this oral pathogen. In order to characterize the immune response to Aa further, we now propose to: Aim 1. Clone genes that encode other T cell epitopes by direct screening of an Aa genomic library. The identities of the Aa proteins recognized by the T cell hybridomas will be determined and recombinant peptides generated for use in Aims 2 and 3. Aim 2: Characterize the nature of the immune response in vivo to individual Aa antigens. Mice will be: a) immunized with purified recombinant peptides, b) immunized with bacteria, or c) orally inoculated with viable Aa. Immune activation to individual T cell epitopes will then be assessed by studies of antibody production, T cell activation and cytokine production (Th1 versus Th2), and protection in a murine inflammation model. Aim 3. Determine whether the predominant T cell antigens in mice are similarly stimulatory in Aa-infected patients. Specifically, peripheral blood lymphocytes from EOP patients will be cultured in vitro with individual recombinant Aa peptides and T cell stimulation assessed by cytokine production and by spectrotyping. These aims will: i) provide the first evidence regarding T cell antigenic epitopes on this pathogen and ii) assess the relationship between T cell epitopes that are immunodominant in mice and those seen in humans. The long term goal is to develop and validate a model for evaluating host-parasite interactions, vaccine potency, and immune protection for periodontal diseases.