T CELL RESPONSE TO A PERIODONTAL PATHOGEN

我们已经集中在牙周病原体，放线菌放线菌（Aa）。感染这种细菌的个体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鉴定出来。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解剖T细胞对Aa的反应。在初步实验中，小鼠口服接种活菌，产生一组T细胞杂交瘤。令我们惊讶的是，大约50%的与Aa反应的T细胞对白毒素是特异性的，白毒素是这种口腔病原体产生的一种毒力因子。为了进一步表征对Aa的免疫反应，我们现在提出：目的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蛋白的身份，并生成用于Aims 2和Aims 3的重组肽。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)用活Aa口服接种。对单个T细胞表位的免疫激活将通过抗体产生、T细胞激活和细胞因子产生（Th1 vs Th2）以及小鼠炎症模型中的保护研究来评估。目标3。确定小鼠的主要T细胞抗原是否在aa感染患者中具有类似的刺激作用。具体来说，EOP患者的外周血淋巴细胞将在体外培养，使用单个重组Aa肽和T细胞刺激，通过细胞因子产生和光谱分型评估。这些目标将：i)提供关于该病原体上T细胞抗原表位的第一个证据，ii)评估小鼠免疫优势T细胞表位与人类免疫优势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.