T Cells in the Pathogenesis of Systemic Sclerosis

描述(申请人提供)：系统性硬化症(SSC)是一种慢性 以广泛的纤维化、微血管纤维内膜为特征的疾病 增殖和自身抗体的产生。T细胞和单核细胞浸润 系统性红斑狼疮患者的早期皮肤损害。我们已经证明了 5例SSc患者的皮肤活检中有5例含有寡克隆群体 T细胞的数量。在患有自发性硬化症的妇女中发现了造血细胞 之前曾怀孕过。这些胎儿T细胞在母亲体内的激活 结果在临床上被称为母体SSc，类似于GVHD。 母体细胞的微嵌合体已在未被发现的妇女中被发现 先前怀孕或在男性中。这些母体T细胞的激活导致 后代SSC。在此RO1应用程序中要测试的假设是：(A) 母系SSc患者皮肤活检组织中的T细胞是否 以及它们是否识别母体来源的同种异体抗原，通过 直接或间接抗原识别途径；(B)T细胞 子代SSc患者的浸润性皮肤活检为母体 来源及其是否识别后代来源的同种异体抗原，由 直接或间接的抗原识别途径。我们的具体目标是：1. 确定新鲜(未在培养中扩增)T细胞是否渗入 母亲或子代SSC患者的皮肤活检含有 大量的单克隆性T细胞。以确定这些是否 克隆扩增的T细胞在母体SSC或母体中起源于胎儿 起源于后代的SSC。2.鉴定T细胞识别的抗原 利用克隆扩展的α链和β链TCR转录本在 母体SSC或子代SSC皮肤活检。A.克隆表达 在SSC皮肤活检中扩增TCR转录本为AP TCR阴性J.RT3T3.5 用逆转录病毒感染Jurkat细胞或进入正常CTL 向量。B.确定这些转导或转染的T细胞是否具有 克隆化扩增的TCR，直接或间接识别同种异体抗原 识别途径。3.通过限制稀释度来开发特异性T细胞克隆 对于同种异体抗原或推定的SSC抗原(CMV和DNA拓扑异构酶I) 从相同的SSC患者中研究的特定目标#1.a.确定 这些T细胞克隆在母体SSC中来自胎儿，或在 衍生的SSC。以确定他们是否识别同种异体抗原。B.至 鉴定这些T细胞克隆的表型和功能，并 确定它们是否识别：(I)同种异体抗原；通过直接识别或 间接识别途径；(Ii)推测的SSc抗原(S)。至 确定这些T细胞克隆是否表现出抑制活性 同种异体反应或对CTL活性的反应。C.比较TCR序列 由这些抗原特异性T细胞克隆用于新鲜(不是 培养扩增)SSC渗入同一患者的T细胞，按顺序 以确定克隆是否。这些新鲜浸润性T细胞的数量 与抗原特异性T细胞具有相同的抗原特异性 克隆人。D.鉴定Th1、Th2、抑制性、无能和CTL T细胞克隆 上面发展出来的那些，基于它们基因的表达模式， 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.