T Cells in the Pathogenesis of Systemic Sclerosis

描述（由申请人提供）：系统性硬化症（SSc）是一种慢性 以广泛纤维化、微血管纤维内膜 增殖和自身抗体产生。T细胞和单核细胞浸润 SSc患者的早期皮肤病变。我们已经证明 5/5例SSc患者的皮肤活检中含有寡克隆群体 的T细胞。在患有SSc的女性中已经发现了造血胎儿细胞， 她以前怀孕过。母亲体内这些胎儿T细胞的激活 导致被称为母体SSc的临床疾病，其类似于GVHD。 母体细胞的微嵌合体已经在那些没有被发现的女性中被发现。 以前怀孕或男性。这些母体T细胞的激活导致 后代SSc.在本RO 1应用程序中要测试的假设是： 是否T细胞浸润皮肤活检患者与母亲SSc是 以及它们是否识别母体来源的同种异体抗原， 直接或间接抗原识别途径;（B）T细胞是否 来自患有后代SSc的患者的浸润性皮肤活检是母体的 来源和它们是否识别后代来源的同种异体抗原， 直接或间接的抗原识别途径。我们的具体目标是：1. 为了确定是否有新鲜的（未在培养中扩增的）T细胞浸润， 来自患有母体SSc或后代SSc的患者的皮肤活组织检查包含 相当大比例的单克隆T细胞以确定是否这些 克隆扩增的T细胞是母体SSc中的胎儿来源的或母体SSc中的胎儿来源的。 起源于后代SSc。2.识别T细胞识别的抗原 使用克隆扩增的α和β链TCR转录物， 母体SSc或后代SSc皮肤活检。a.以克隆的方式表达 在SSc皮肤活组织检查中扩增TCR转录物至ap TCR阴性J.RT3T3.5 Jurkat细胞，或通过用逆转录病毒感染进入正常CTL 向量。B.为了确定这些转导或转染的T细胞是否具有 克隆扩增的TCR，通过直接或间接识别同种异体抗原， 识别途径3.通过有限稀释法开发特异性T细胞克隆 同种异体抗原或推定的SSc抗原（CMV和DNA拓扑异构酶I） 来自特定目标#1中研究的相同SSc患者。a.以确定是否 这些T细胞克隆在母体SSc中是胎儿来源的，或者在母体SSc中是母体来源的。 分支SSc。以确定它们是否能识别同种异体抗原。B.到 表征这些T细胞克隆的表型和功能， 确定它们是否识别：（i）同种异体抗原;通过直接或 间接识别途径;（ii）上述推定的SSc抗原。到 确定这些T细胞克隆是否表现出抑制活性， 同种异体特异性反应或CTL活性。C.为了比较TCR序列 利用这些抗原特异性T细胞克隆的那些新鲜的（不 在培养中扩增）来自相同患者的SSc浸润T细胞， 以确定是否克隆。这些新鲜的浸润性T细胞群 具有与抗原特异性T细胞相同的抗原特异性 克隆D.为了鉴定Thl、Th 2、抑制性、无能性和CTL T细胞克隆， 以上开发的那些，基于其基因的表达模式， 使用DNA阵列技术。

DESCRIPTION (provided by applicant): Systemic Sclerosis (SSc) is a chronic disease characterized by extensive fibrosis, microvascular fibrointimal proliferation and autoantibody production. T cells and monocytes infiltrate early skin lesions of patients with SSc. We have demonstrated that skin-biopsies in five of five patients with SSc contain oligoclonal populations of T cells. Hemopoeitic fetal cells have been identified in women with SSc who had previously been pregnant. Activation of these fetal T cells in the mother results in clinical disease designated as maternal SSc, which resembles GVHD. Microchimerism of maternal cells has been identified in women who have not been previously pregnant or in men. Activation of these maternal T cells results in offspring SSc. The hypothesis to be tested in this RO1 application is: (a) whether T-cells infiltrating skin-biopsies from patients with maternal SSc are of fetal origin and whether their recognize alloantigens of maternal origin, by the direct or the indirect antigen recognition pathways; (b) whether T-cells infiltrating skin-biopsies from patients with offspring SSc are of maternal origin and whether their recognize alloantigens of offspring origin, by the direct or the indirect antigen recognition pathways. Our specific aims are: 1. To determine whether fresh (not expanded in culture) T cells infiltrating skin-biopsies from patients with maternal SSc or offspring SSc contain substantial proportions of monoclonal T cells. To determine whether these clonally expanded T cells are of fetal origin in maternal SSc or of maternal origin in offspring SSc. 2. To identify the antigens recognized by T cells employing the clonally expanded alpha- and beta-chain TCR transcripts in maternal SSc or offspring SSc skin-biopsies. a. To express the clonally expanded in SSc skin-biopsies TCR transcripts into ap TCR-negative J.RT3T3.5 Jurkat cells by transfection, or into normal CTL by infection with retroviral vectors. b. To determine whether these transduced or transfected T cells with the clonally expanded TCR, recognize alloantigen by the direct or the indirect recognition pathway. 3. To develop by limiting dilution T-cell clones specific for alloantigens or for putative SSc antigens (CMV and DNA topoisomerase I) from the same SSc patients studied in specific aim #1. a. To determine whether these T-cell clones are of fetal origin in maternal SSc or maternal origin in offsping SSc. To determine whether they recognize alloantigen. b. To characterize these T-cell clones phenotypically and functionally and to determine whether they recognize: (i) alloantigens; by the direct or the indirect recognition pathway; (ii) the putative SSc antigen(s) listed above. To determine whether these T-cell clones exhibit suppressor activity to allospecific responses or to CTL activity. c. To compare the TCR sequences utilized by these antigen-specific T-cell clones to those of fresh (not expanded in culture) SSc infiltrating T cells from the same patients, in order to determine whether clonal. populations of these fresh infiltrating T cells have the same antigenic specificities to those of antigen-specific T-cell clones. d. To identify Thl, Th2, suppressor, anergic and CTL T-cell clones from those developed above, on the basis of expression patterns of their genes, using DNA array technology.