Use of Adjuvants to Improve Skin Genetic Immunizations

描述（申请人提供）：黑色素瘤是最具侵袭性的肿瘤之一，发病率和死亡率都在增加。虽然黑色素瘤是免疫原性肿瘤，可以通过免疫介导的自发消退来潜在地消除，但这种现象极为罕见。更常见的是，由于缺乏肿瘤特异性抗原（Ags）或肿瘤特异性耐受状态的发展，黑素瘤不能被免疫系统识别。基于诱导肿瘤特异性CD4+ T辅助1 （Th1）淋巴细胞和CD8+细胞毒性T细胞（ctl），通过DNA疫苗对黑色素瘤的免疫策略是很有前途的治疗工具。皮肤由于其易接近性和自身的免疫系统，是基因免疫的最佳部位，而基因枪（GO）是诱导转基因Ags在皮肤中高表达的一种极好的方法。皮肤的专业银加工和提呈细胞[皮肤树突状细胞（dc）：表皮朗格汉斯细胞和真皮dc]可以通过gg免疫原位转染，或者它们可以内化来自邻近皮肤细胞的转基因蛋白。因此，通过GG直接或间接转染皮肤dc，可以在体内设计皮肤免疫系统。在外周组织（即皮肤）中dc激活的早期阶段，适当的免疫刺激信号的存在“指示”dc完全成熟并诱导有效的抗肿瘤th1偏倚免疫反应。然而，虽然GG诱导抗肿瘤CD8+ ctl，但GG是否能够产生有效的CD4+ th1偏向免疫应答（维持有效的细胞抗肿瘤免疫所必需的），而是诱导Th2细胞，仍然存在争议。该提议的假设是，在th1驱动佐剂P神经激肽1受体激动剂（SP-NK1a）和咪喹莫特存在的情况下，使用GG在皮肤中表达转基因黑色素瘤Ags（在GG免疫部位成熟并使th1极化的皮肤dc），将产生完全激活的皮肤dc，具有有效刺激肿瘤ag特异性T细胞的能力。在这些条件下产生的皮肤源性dc将偏向于初始肿瘤特异性T细胞向Th1细胞的分化，并促进ctl的发展，产生有效和持久的抗肿瘤免疫反应。为了验证这一假设，我们提出了以下具体目的：目的1：研究在th1驱动佐剂咪喹莫特和SP-NK1a存在的情况下，从GG转染皮肤迁移的dc的功能。目的2：分析体内th1驱动佐剂在皮肤GG基因免疫诱导的免疫应答结果中的作用；目的3：分析th1驱动佐剂极化转染了GG的人皮肤中不同皮肤迁移dc亚群的能力。

DESCRIPTION (provided by applicant): Melanomas are one the most aggressive tumors with increasing incidence and mortality. Although melanomas are immunogenic tumors that could be potentially eliminated by immune-mediated spontaneous regression, this phenomenon is extremely rare. More frequently, melanomas are not recognized by the immune system due to the absence of tumor-specific antigens (Ags) or development of a state of tumor-specific tolerance. Immunization strategies against melanoma by means of DNA vaccines are promising therapeutic tools based on the induction of tumor-specific CD4+ T helper 1 (Th1) lymphocytes and CD8+ cytotoxic T cells (CTLs). Due to its easy accessibility and intrinsic immune system, the skin is an optimal site for genetic immunization and the gene gun (GO is an excellent approach to induce high expression of transgenic Ags in the skin. Professional Ag processing and presenting cells of the skin [cutaneous dendritic cells (DCs): epidermal Langerhans cells and dermal DCs] can be transfected in situ by GG-immunization, or alternatively, they may internalize transgenic proteins from neighboring skin cells. Therefore, through direct of indirect transfection of skin DCs by the GG, it is possible to engineer in vivo the skin immune system. The presence of appropriate immuno-stimulatory signals during the early stages of DCs activation in peripheral tissues (i.e. skin) "instructs" DCs to fully mature and to induce an effective anti-tumor Th1-biased immune response. However, although the GG induces anti-tumor CD8+ CTLs, it is still controversial whether the GG is able to generate an effective CD4+ Th1-biased immune response (required to maintain efficient cellular anti-tumor immunity) and it rather induces Th2 cells. The hypothesis of this proposal is that the use of the GG to express transgenic melanoma Ags in the skin in the presence of the Th1-driving adjuvants substance P neurokinin 1 receptor agonist (SP-NK1a) and Imiquimod (to mature and to Th1-polarize skin DCs at the site of GG-immunization) will generate fully activated skin DCs with ability to stimulate efficiently tumor Ag-specific T cells. Skin-derived DCs generated under these conditions will bias the differentiation of naive tumor-specific T cells into Th1 cells and promote development of CTLs, generating an effective and long-lasting anti-tumor immune response. To test the hypothesis, we propose the following specific aims: Aim 1: To investigate the function of DCs migrated from skin transfected with GG in the presence of Th1-driving adjuvants Imiquimod and SP-NK1a. Aim 2: To analyze in vivo the role of Th1-driving adjuvants in the outcome of the immune responses induced by GG genetic immunization of skin, and Aim 3: To analyze the capacity of the Th1-driving adjuvants to polarize different subpopulations of skin migratory DCs from human skin transfected with the GG.