Virus Specific CTL following T cell depleted SCT

描述（由申请人提供）：同种异体干细胞移植（SCT）的T细胞消耗（TCD）显著降低移植物抗宿主病（GVHD）的风险，但导致感染性并发症的风险增加，其中最严重的两种是eb病毒（EBV）诱导的淋巴细胞增生性疾病和巨细胞病毒（CMV）感染。先前的研究表明，细胞毒性T淋巴细胞可以培养并输注到SCT受体中，导致抗原特异性免疫重建，但由于需要用单独的抗原呈递细胞扩增CTL，这可能是不切实际的。初步研究表明，B淋巴母细胞样细胞系（BLCL）通过逆转录病毒转导实现CMVpp65表达的可行性，表达pp65的BLCL刺激可扩增识别CMVpp65和EBV抗原的CTL。在本研究中，我们将比较同种异体TCD SCT受体与随机不接受CTL治疗的对照组TCD SCT患者预防性CMV/EBV特异性CTL的临床、免疫学和病毒学效果。CD4和CD8抗原特异性免疫重构（CTL前体频率）以及CMV和EBV DNA水平（通过实时PCR）将在移植后的间隔时间内进行比较。我们将使用编码pp65缺失突变的重组痘苗确定哪些CMVpp65表位在干细胞供体中具有免疫优势，然后使用脉冲到APC上的合成肽确定确切的表位。将CTL供体的显性表位与CTL受体输注后的显性表位进行比较，以确定供体识别的免疫显性表位是否保留在受体中，如果没有，哪些表位与移植后最相关。同样令人感兴趣的是效应细胞的类型和CD4和CD8 CMV特异性T细胞在CTL受体和对照组中CMV再激活期间的动态变化。这些研究将确定预防性抗原特异性CTL对病毒再激活和免疫重建的影响，并将为将来将抗原呈递和T细胞扩增系统扩展到其他病原体和肿瘤抗原奠定基础。

DESCRIPTION (provided by applicant): T cell depletion (TCD) of allogeneic stem cell transplants (SCT) dramatically reduces the risk of graft vs host disease (GVHD) but results in a heightened risk for infectious complications, two of the most serious being Epstein Barr virus (EBV) induced lymphoproliferative disease and cytomegalovirus (CMV) infection. Previous studies have shown that cytotoxic T lymphocytes can be cultured and infused into SCT recipients resulting in antigen specific immune reconstitution, but this can be impractical due to the need of expanding CTL with separate antigen presenting cells. Preliminary studies have demonstrated the feasibility of retroviral transduction of B lymphoblastoid cell lines (BLCL) to achieve expression of CMV pp65, and stimulation with BLCL expressing pp65 results in expansion of CTL recognizing both CMVpp65 and EBV antigens. In this proposal, we will compare the clinical, immunologic, and virologic effects of prophylactic CMV/EBV specific CTL in recipients of allogeneic, TCD SCT with a control group of TCD SCT patients randomized to not receive CTL. CD4 and CD8 antigen specific immune reconstitution (CTL precursor frequencies) and CMV and EBV DNA levels (by real time PCR) will be compared in these groups at intervals post-transplant. We will determine which CMVpp65 epitopes are immunodominant in stem cell donors using recombinant vaccinia encoding pp65 deletion mutations, and then the exact epitopes will be determined using synthetic peptides pulsed onto APC. Dominant epitopes of the CTL donors will be compared with those found in CTL recipients post-infusion, to determine whether immunodominant epitopes recognized by the donor are retained in the recipient, and if not, which epitopes are most relevant post-transplant. Also of interest are the types of effector cells and the dynamic changes in CD4 and CD8 CMV specific T cells during CMV reactivation in recipients of CTL and the control group. These studies will determine the impact on viral reactivation and immune reconstitution of prophylactic antigen-specific CTL and will serve as a groundwork for extending this system of antigen presentation and T cell expansion to other pathogens as well as tumor antigens in the future.