Novel Approach to T Cell Specificity in Sjogren's Syndrome

原发性干燥综合征(SS)是一种常见的、衰弱的疾病，其特征是外分泌组织中的淋巴细胞渗入。由于缺乏破译靶组织中抗原特异性T细胞的特异性和分化状态的策略，对SS发病机制的了解和SS有效生物疗法的开发一直受到阻碍。我们处于独特的地位来应对这一重要挑战，因为i)成功地建立了OMRF Sjogren研究诊所，ii)拥有蛋白质组学和淋巴细胞受体单细胞RT-PCR方面的专业知识，iii)最近发现了人类白细胞抗原DR3限制性T细胞 标准SS自身抗原的细胞表位和iv)招募强大的合作者，他们将与我们合作，将一种新的T细胞受体(TCR)RNA转基因技术从癌症领域转移到自身免疫领域。针对新的致病T辅助(Th)细胞类型，包括Th17和T滤泡辅助(TFH)细胞的新的生物治疗方法正在开发中，如果知道SS中主要的Th分化状态，这些新的生物治疗方法可以应用于SS。在目标1中，我们将使用系统的单细胞RT-PCR方法来 从SS患者的双唇唾液腺(SG)活检和外周血(PB)样本中分离出CD4+和CD8+T细胞的TCR谱特征。对这些数据进行评估，以寻找T细胞克隆性增殖和共同的TCR片段使用或CDRS基序的证据，将揭示CD8+T细胞参与SS的证据，并将识别在炎性病变中原位扩张的CD4+或CD8+TCR。在目标2中，我们将合成并验证DRS四聚体试剂，该试剂将检测针对主要已知SS自身抗原Ro/SS-A和La/SS-B的Th细胞。这些试剂将被用来量化这些细胞的频率，并确定它们在SG和PB中的主要记忆和Th分化状态。这些特征与血清I型干扰素活性和疾病措施的关系(S)有望导致对SS发病机制的深入了解。针对S，我们将使用蛋白质组学方法来鉴定SS患者SG中克隆扩增的T细胞识别的SG抗原，这一策略因我们的合作者强大的TCRRNA转染技术而变得可行。

Primary Sjogren's Syndrome (SS) is a common, debilitating disease characterized by lymphocytic infiltrates in exocrine tissue. Understanding of SS pathogenesis and development of effective biologic therapies for SS have been hampered by a lack of strategies for deciphering the specificities and differentiation states of antigen-specific T cells in the targeted tissue. We are in a unique position to address this important challenge due to i) establishment of the successful OMRF Sjogren's Research Clinic, ii) available expertise in proteomics and single cell RT-PCR of lymphocyte receptors, iii) recent discovery of HLA DR3-restricted T cell epitopes of the canonical SS autoantigens and iv) recruitment of strong collaborators who will work with us to transfer a novel T cell receptor (TCR) RNA transfection technology from the field of cancer to autoimmunity. Novel biologic therapies directed against new pathogenic T helper (Th) cell types including Th17 and T follicular helper (TFH) cells are being developed and could be applied to SS if predominant Th differentiation states in SS were known. In Aim 1 we will use a systematic single cell RT-PCR approach to characterize the TCR repertoire of CD4+ and CD8+ T cells isolated from paired labial salivary gland (SG) biopsy and peripheral blood (PB) samples of SS patients. Evaluation of these data for evidence of T cell clonal expansion and common TCR segment usage or CDRS motifs will disclose evidence for or against involvement of CD8+ T cells in SS and will identify CD4+ or CD8+ TCRs that are expanded in situ within the inflammatory lesion. In Aim 2, we will synthesize and validate DRS tetramer reagents that will detect Th cells specific for the primary known SS autoantigens Ro/SS-A and La/SS-B. These reagents will be exploited to quantify the frequency of these cells and to determine their predominant memory and Th differentiation states in SG and PB. The relationship(s) of these features to serum Type I interferon activity and measures of disease are expected to lead to insights into SS pathogenesis. In Aim S we will use a proteomic approach to identify SG antigens recognized by T cells that are clonally expanded in SG of SS patients, a strategy made feasible by the robust TCR RNA transfection technique of our collaborators.