Cellular Mechanisms of PTLD in Transplant Resipients

描述（由申请人提供）： 移植后淋巴增生性疾病（PTLD）是一种形态复杂的移植并发症，其范围从良性B细胞增生到恶性淋巴瘤。EB病毒（EBV）B细胞淋巴瘤是PTLD的标志，与显著的发病率和死亡率相关。在之前的资助期间，我们确定了特定的信号通路，尽管在移植受体的循环中存在显著频率的EBV特异性CD 8 + T细胞，但这些信号通路赋予EBV感染的B细胞生存优势。我们还确定了免疫抑制剂西罗莫司（RAPA）可以直接抑制EBV感染的B细胞的生长。本研究的目的是确定有助于EBV相关B细胞淋巴瘤自主生长的免疫改变。为了实现这一目标，我们提出了三个具体目标。具体目标1将检查EBV感染的B细胞对死亡受体诱导的细胞凋亡具有抗性的机制。通过二维凝胶电泳分析和DNA微阵列分析来表征Fas-Fas配体和TRAIL-DR 4/DR 5细胞死亡途径中的近端事件，包括死亡诱导信号复合物（DISC）的组装。将检查诱饵受体DcR 1和DcR 2在抗TRAIL细胞毒性中的作用。还将确定EBV潜伏基因LMP 1和EBNA 2对细胞凋亡抗性的贡献。在特定目标2中，将使用含有EBV抗原的免疫显性表位的MHC/肽四聚体来鉴定移植受体中的EBV特异性CD 8 + T细胞，并使用细胞内染色和细胞因子和细胞溶解功能的体外测定来评估这些T细胞的功能特性。将在我们的SCID/EBV B细胞淋巴瘤模型中确定免疫抑制对功能性EBV特异性CD 8 + T细胞产生的影响。特异性目标3将确定RAPA抑制EBV感染的B细胞生长的机制，特别关注细胞凋亡、细胞周期蛋白和Jak/STAT途径。还将确定IL-10途径的体内阻断对肿瘤生长的影响。阐明PTLD中涉及的免疫机制将为合理设计器官移植严重并发症的新治疗方法提供新的机会。

DESCRIPTION (provided by applicant): Post-transplant lymphoproliferative disorder (PTLD) is a morphologically complex complication of transplantation that ranges from benign B cell hyperplasia to malignant lymphoma. Epstein-Barr virus (EBV) B cell lymphomas are the hallmark of PTLD and are associated with significant morbidity and mortality. During the previous funding period we identified specific signaling pathways that confer a survival advantage to EBV-infected B cells despite the presence of significant frequencies of EBV-specific CD8+ T cells in the circulation of transplant recipients. We also established that the immunosuppressive sirolimus (RAPA) can directly inhibit the growth of EBV-infected B cells. The objective of this research is to define the immune alterations that contribute to the autonomous growth of EBV-associated B cell lymphomas. To accomplish this objective we propose three Specific Aims. Specific Aim 1 will examine the mechanism by which EBV-infected B cells are resistant to death receptor-induced apoptosis. The proximal events, including assembly of the death inducing signaling complex (DISC), in the Fas-Fas ligand and TRAIL-DR4/DR5 cell death pathways will be characterized by two-dimensional gel electrophoresis analysis and DNA microarray. The role of the decoy receptors DcR1 and DcR2 in resistance to TRAIL cytotoxicity will be examined. The contribution of the EBV latent genes LMP1 and EBNA2 to resistance to apoptosis will also be determined. In Specific Aim 2 MHC/peptide tetramers containing immunodominant epitopes of EBV antigens will be used to identify EBV-specific CD8+ T cells in transplant recipients and the functional properties of these T cells will be assessed using intracellular staining and in vitro assays for cytokine and cytolytic function. The effects of immunosuppression on the generation of functional EBV-specific CD8+ T cells will be determined in our SCID/EBV B cell lymphoma model. Specific Aim 3 will determine the mechanism by which RAPA inhibits growth of EBV-infected B cells with specific focus on apoptosis, cell cycle proteins and the Jak/STAT pathway. The effect of in vivo blockade of the IL-10 pathway on tumor growth will also be determined. Elucidation of the immune mechanisms involved in PTLD will provide novel opportunities for the rational design of new treatments for this serious complication of organ transplantation.