CMV/IMMUNE INTERACTIONS IN TRANSPLANT ARTERIOSCLEROSIS

巨细胞病毒（CMV）已被认为是一种加重剂， 移植相关动脉硬化（TxAA）， 心脏移植物长期存活的主要限制，没有 目前仍有令人满意的医疗干预。为了努力 定义解释这种关联的机制，我们建议CMV- 受感染的移植物内皮细胞可以启动宿主免疫激活级联反应， 并且随之而来的细胞因子的局部释放可以提高近端的 将未感染的内皮细胞转化为增强的同种异体免疫原性状态， 通过循环宿主细胞免疫成分准备免疫攻击。 因此，本提案中描述的实验旨在测试 假设1）CMV感染的内皮细胞（EC）可以刺激 产生同种异体和自体溶细胞性T细胞（CTL）， 对未感染的EC表现出混杂的裂解活性，2）这些 非传统机制的相互作用， 依赖于共刺激信号，3）未感染的内皮细胞 由CMV应答性T细胞精心制作的细胞因子激活的细胞因子可以充当 用于T细胞迁移和/或细胞溶解的底物，和4）这些 在临床上， 相关浓度的免疫抑制剂。CTL前体 将确定体外刺激T细胞群体的频率 通过测量从51 Cr标记的感染细胞释放的放射性标记， 或未感染的自体或同种异体EC靶。确定 HLA和粘附分子在这些反应中的作用，刺激因子 和/或靶群体将耗尽HLA I类和/或HLA II类阳性细胞免疫磁性或通过荧光激活 细胞分选，或在存在阻断的情况下测定细胞溶解 抗体的免疫荧光流式细胞术将用于鉴定 应答性T细胞亚群如幼稚或记忆，α/β或γ/δ， 以及Vbeta类型。最后，为了模拟CMV触发的细胞因子- 可能发生在移植物/宿主界面的介导事件， 单层细胞将通过跨孔插入物下的培养物活化 含有T细胞与CMV感染的EC的组合，然后测试 它们促进跨内皮T细胞迁移的能力和它们的 在不存在或存在以下物质的情况下对CTL介导的裂解的易感性 不同浓度的临床相关免疫抑制剂。 这些研究的结果将阐明保护和/或 对该病毒的致病性免疫反应，可能识别 迄今为止未描述的免疫激活途径，并帮助阐明 CMV在移植相关性肿瘤发生中的作用 动脉硬化

Cytomegalovirus (CMV) has been implicated as an exacerbating agent in the development of transplantation-associated arteriosclerosis (TxAA), a major limitation in long-term cardiac allograft survival for which no satisfactory medical intervention is yet available. In an effort to define mechanisms explaining this association, we propose that CMV- infected graft endothelia can initiate a host immune activation cascade, and that the consequent localized release of cytokines can raise proximal uninfected endothelium to a state of enhanced alloimmunogenicity, ideally poised for immune attack by circulating host cellular immune components. Thus the experiments described in this proposal are designed to test the hypotheses that 1) CMV-infected endothelial cells (EC) can stimulate the generation of allogeneic and autologous cytolytic T cells (CTL) which exhibit promiscuous lytic activity against uninfected EC, 2) that these interactions occur by non-traditional mechanisms which are highly dependent upon costimulatory signals, 3) that uninfected endothelia activated by cytokines elaborated by CMV-responsive T cells can serve as a substrate for T cell transmigration and/or cytolysis, and 4) that these events can occur to a significant extent in the presence of clinically relevant concentrations of immunosuppressive agents. CTL precursor frequencies will be determined for populations of in vitro-stimulated T cells by measurement of radiolabel release from 51Cr-labelled infected or uninfected autologous or allogeneic EC targets. To determine the contribution of HLA and adhesion molecules in these responses, stimulator and/or target populations will be depleted of HLA class I and/or HLA class II-positive cells immunomagnetically or by fluorescence-activated cell sorting, or assayed for cytolysis in the presence of blocking antibodies. immunofluorescence flow cytometry will be used to identify responsive T cell subsets as naive or memory, alpha/Beta or gamma/delta, as well as by Vbeta type. Finally, to simulate CMV-triggered cytokine- mediated events which may occur at the graft/host interface, endothelial monolayers will be activated by culture beneath trans-well inserts containing T cells in combination with CMV-infected EC, then tested for their ability to promote trans-endothelial T cell migration and their susceptibility to CTL-mediated lysis in the absence or presence of various concentrations of clinically relevant immunosuppressive agents. Results of these studies will elucidate aspects of protective and/or pathogenic immune responses to this virus, potentially identify heretofore undescribed pathways of immune activation, and help elucidate the role of CMV in the development of transplantation-associated arteriosclerosis.