CMV/IMMUNE INTERACTIONS IN TRANSPLANT ARTERIOSCLEROSIS

巨细胞病毒(CMV)被认为是一种在 移植相关动脉硬化(TxAA) 同种异体心脏移植物长期存活的主要限制 目前还没有令人满意的医疗干预措施。为了努力 定义了解释这种联系的机制，我们提出CMV- 感染的移植物内皮细胞可以启动宿主免疫激活级联反应， 随之而来的细胞因子的局部释放可以使近端 未感染的内皮细胞达到增强同种异体免疫原性的状态，理想情况下 准备好通过循环宿主细胞免疫成分进行免疫攻击。 因此，本提案中描述的实验旨在测试 假设1)巨细胞病毒感染的内皮细胞(EC)可以刺激 异体和自体溶细胞T细胞(CTL)的产生 对未感染的EC表现出混杂的裂解活性，2)这些 相互作用是通过非传统机制发生的，这些机制高度 依赖于共刺激信号，3)未感染的内皮细胞 由CMV反应的T细胞精心设计的细胞因子激活可以作为 T细胞迁移和/或细胞溶解的底物，以及4)这些 在临床上，这些事件可以在很大程度上发生 相应浓度的免疫抑制剂。CTL前驱体 将确定体外刺激T细胞群体的频率 ~(51)Cr标记感染细胞放射性标记释放的测定 或未感染的自体或异体EC靶标。要确定 人类白细胞抗原和黏附分子在这些反应中的作用 和/或目标人群将丧失人类白细胞抗原I类和/或人类白细胞抗原 免疫磁性或荧光激活的II类阳性细胞 细胞分选，或在存在阻断的情况下进行细胞溶解检测 抗体。免疫荧光流式细胞术将用于识别 反应性T细胞亚群表现为幼稚或记忆、阿尔法/贝塔或伽马/三角洲， 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，内皮细胞 单分子层将通过跨井插入件下的培养来激活 含有T细胞和CMV感染的EC，然后检测 它们促进跨内皮T细胞迁移的能力和它们的 在缺乏或存在的情况下对CTL介导的裂解的敏感性 临床相关免疫抑制剂的不同浓度。 这些研究的结果将阐明保护性和/或 对这种病毒的致病免疫反应，可能会识别 迄今未被描述的免疫激活途径，并有助于阐明 巨细胞病毒在移植相关疾病发生发展中的作用 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.