Immunization with Genetically Modified HSCs.

描述(申请人提供)：用体外产生的树突状细胞(DC)免疫多肽抗原(Ag)或转导表达Ag的重组病毒是一种很有前途的免疫治疗和预防艾滋病毒/艾滋病、其他传染病和癌症的方法。然而，基于DC的免疫受到一些关键限制的限制，包括被转移的DC向次级淋巴器官的迁移有限，以及它们在受者体内的快速消除。目前尚不清楚基于DC的疫苗是否能够在不需要频繁重新免疫的情况下提供长期保护。 在这一应用中，提出了一种新的免疫治疗和长期免疫的方法，该方法基于可激活诱导的抗原在转基因DC中的表达。我们假设：在启动子的控制下，将表达抗原的慢病毒载体转导的自体造血干细胞(HSCs)移植到成熟激活的DC上，会导致一种“受控旁观者激活”状态，在这种状态下，抗原重复表达并呈现给有限数量的HSC来源的DC，这些DC目前被持续的感染或炎症激活。这导致长期维持高水平的抗原特异性记忆T细胞。 为了验证这一假设，本文提出了两个具体目标。在特定的目的1，CCL17启动子在小鼠体内的表达模式将在转导的HSCs移植后表现出来。将研究转基因表达的组织分布，不同DC刺激的诱导性，以及不同程度的去髓术对嵌合体水平的影响。在特定目标2中，将评估在CCL17启动子控制下用编码OVA抗原的慢病毒转导HSCs的受者对抗原特异性T细胞反应的诱导和维持。OVA抗原呈递的程度将通过四聚体染色、细胞内细胞因子染色以及体内过继转移的OT-I和OT-II转基因T细胞的增殖来监测。最后，保护性免疫反应的存在将通过对表达OVA抗原的牛痘病毒的攻击来评估。 拟议的研究将测试一种通过移植转基因HSCs来诱导持久免疫反应的新方法。与体外转导的树突状细胞免疫相比，这种策略有几个关键的优点，如果被证明可行，可能会在艾滋病毒/艾滋病、癌症和其他慢性疾病的免疫治疗中发挥很大作用。

DESCRIPTION (provided by applicant): Immunization with ex vivo-generated dendritic cells (DCs) pulsed with peptide antigen (Ag) or transduced with Ag-expressing recombinant virus represents a promising approach to both the immunotherapy and prevention of HIV/AIDS, other infectious diseases, and cancer. However, DC-based immunization is constrained by some critical limitations, including the limited migration of transferred DCs to secondary lymphoid organs and their rapid elimination in recipients. It is presently unclear whether DC-based vaccines can provide long-lasting protection without the need for frequent re-immunization. In this application, a novel approach to immunotherapy and long-term immunization is proposed based on activation-inducible Ag expression in genetically modified DCs. We hypothesize that: Transplantation with autologous hematopoietic stem cells (HSCs) transduced with lentiviral vector expressing the Ag under the control of a promoter restricting its expression to mature activated DCs results in a state of "controlled by-stander activation" in which the antigen is repeatedly expressed and presented by a limited population of HSC-derived DCs that are currently activated by an ongoing infection or inflammation. This results in a long-term maintenance of high levels of Ag-specific memory T cells. To test this hypothesis, two specific aims are submitted. In Specific Aim 1, the expression pattern of CCL17 promoter will be characterized in mice following the transplantation of transduced HSCs. The tissue distribution of transgene expression, inducibility with different DC stimuli, and the effect of various degrees of myeloablation on the level of chimerism will be studied. In specific Aim 2, the induction and maintenance of Ag-specific T cell responses will be assessed in the recipients of HSCs transduced with lentivirus encoding the OVA Ag under the control of CCL17 promoter. The extent of OVA Ag presentation will be monitored by tetramer staining, intracellular cytokine staining, and by proliferation of adoptively transferred OT-I and OT-II transgenic T cells in vivo. Finally, the presence of protective immune responses will be assessed by a challenge with vaccinia virus expressing OVA Ag. The proposed studies will test a novel approach to the induction of durable immune responses by transplantation of genetically modified HSCs. This strategy offers several critical advantages over the immunization with ex vivo-transduced DCs and, if proven feasible, may be highly instrumental in the immunotherapy of HIV/AIDS, cancer, and other chronic diseases.