Immunization with Genetically Modified HSCs.

描述（由申请人提供）：用肽抗原（Ag）脉冲或用表达Ag的重组病毒转导的离体产生的树突状细胞（DC）进行免疫接种代表了免疫治疗和预防HIV/AIDS、其他传染病和癌症的有希望的方法。然而，基于DC的免疫受到一些关键限制的限制，包括转移的DC向次级淋巴器官的有限迁移以及它们在接受者中的快速消除。目前尚不清楚基于DC的疫苗是否可以提供持久的保护，而无需频繁重新免疫。 在本申请中，基于基因修饰的DC中激活诱导的Ag表达，提出了一种免疫治疗和长期免疫的新方法。我们假设：用慢病毒载体转导的自体造血干细胞（HSC）移植导致“受控的旁观者活化”状态，其中抗原由目前由正在进行的感染或炎症活化的有限的HSC衍生的DC群体重复表达和呈递，所述慢病毒载体在限制其表达至成熟活化DC的启动子的控制下表达Ag。这导致长期维持高水平的Ag特异性记忆T细胞。 为了检验这一假设，提出了两个具体目标。在特异性目的1中，将在移植转导的HSC后在小鼠中表征CCL 17启动子的表达模式。将研究用不同DC刺激诱导的转基因表达的组织分布，以及不同程度的骨髓消融对嵌合体水平的影响。在具体目标2中，将在用在CCL 17启动子控制下编码OVA Ag的慢病毒转导的HSC的受体中评估Ag特异性T细胞应答的诱导和维持。将通过四聚体染色、细胞内细胞因子染色以及通过体内过继转移的OT-I和OT-II转基因T细胞的增殖来监测OVA Ag呈递的程度。 最后，通过用表达OVA Ag的牛痘病毒攻击来评估保护性免疫应答的存在。 拟议的研究将测试一种通过移植转基因HSC诱导持久免疫应答的新方法。这种策略提供了几个关键的优势，免疫与离体转导的DC，如果被证明是可行的，可能是非常有用的免疫治疗艾滋病毒/艾滋病，癌症和其他慢性疾病。

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.