Oxygen as a master immunologic switch

描述（由申请人提供）：恶性脑肿瘤是35岁以下人群癌症相关死亡的主要原因。个体化疫苗形式的免疫治疗在选择性神经胶质瘤患者中显示出免疫活性和临床反应，毒性最小。有许多正在进行的临床试验利用培养的肿瘤细胞作为疫苗抗原的来源，用于治疗多种肿瘤。然而，关于细胞培养条件如何影响疫苗接种后的免疫反应和最终临床反应，我们知之甚少。一致使用的条件是肿瘤细胞在常压氧气（20% O2）中扩增。我们已经确定组织培养中使用的氧浓度是肿瘤细胞疫苗免疫原性的主要决定因素。我们的数据导致了一个有吸引力的中心假设，即肿瘤细胞培养中的氧张力作为一个主要的免疫开关，决定了疫苗接种诱导的免疫反应的类型和强度。我们已经重复地证明，在5% O2中培养的胶质瘤细胞裂解物（ctl）在人和小鼠系统中具有优于在20% O2中培养的胶质瘤细胞裂解物的效应功能。这种差异深刻地影响了免疫治疗的疗效，胶质瘤和乳腺癌小鼠模型的存活率显著提高。我们证明，与20% O2裂解液疫苗相比，5% O2裂解液疫苗可引起更优越的CTL增殖、细胞因子精化、肿瘤杀伤功能和向肿瘤部位的运输。相反，20%氧裂解物疫苗可增强抗体反应。尽管肿瘤反应性抗体反应降低，但5% O2疫苗需要B细胞才能达到治疗效果，这揭示了B细胞在CTL启动中的假定作用。此外，我们有证据表明，在5% O2环境中生长的胶质瘤细胞上调toll样受体（TLR） 2配体，因为在几种系统中需要TLR2来区分5%和20% O2裂解物的免疫原性。本课题旨在阐明“氧开关效应”的分子基础，以便合理提高肿瘤细胞疫苗的疗效。在特定目标1中，我们将确定和优化调节免疫原性的肿瘤细胞内在变化。增加肿瘤细胞缺氧诱导因子（HIF）的药理学策略将与细胞培养中的实际缺氧进行比较，随后将测定免疫原性。此外，我们将验证HIF2a是诱导TLR2配体表达的分子开关的假设。在特定目标2中，我们将验证我们的假设，即B细胞是必需的，因为igg -肿瘤裂解物复合物触发Fc受体介导的抗原交叉呈递以增强CTL反应。在特定目标3中，我们将确定5% O2裂解物的辅助作用是否由于膜联蛋白A2的表达，这是一种新的TLR2配体，我们最近发现在缺氧时上调。总的来说，这些知识将促进突破性的方法来提高免疫治疗胶质瘤和其他非中枢神经系统肿瘤的疗效。

DESCRIPTION (provided by applicant): Malignant brain tumors are the leading cause of cancer-related death in people under the age of 35. Immunotherapy in the form of personalized vaccines has demonstrated immunologic activity and clinical responses in select glioma patients with minimal toxicity. There are numerous ongoing clinical trials that utilize cultured tumor cells as the source of vaccine antigen for the treatment of a wide array of tumors. However, very little is known about how cell culture conditions affect the immune response and ultimate clinical response following vaccination. The consistent condition used is expansion of tumor cells in atmospheric oxygen (20% O2). We have identified the oxygen concentration used in tissue culture as a primary determinant of the immunogenicity of tumor cell vaccines. Our data have led to the attractive central hypothesis that the oxygen tension in a tumor cell culture acts as a master immunologic switch, dictating the type and strength of immune response induced by vaccination. We have reproducibly shown that lysates from glioma cells cultured in 5% O2 prime cytotoxic T lymphocytes (CTLs) with superior effector functions relative to lysate from glioma cells cultured in 20% O2 in human and murine systems. This difference profoundly affects the efficacy of immunotherapy, as shown by significant improvements in survival in murine models of glioma and breast carcinoma. We demonstrated that administration of 5% O2 lysate vaccination caused superior CTL proliferation, cytokine elaboration, tumoricidal function, and trafficking to tumor sites relative to 20% O2 lysate vaccines. Conversely, 20% O2 lysate vaccines enhance antibody responses. Despite reduced tumor-reactive antibody responses, the 5% O2 vaccines require B cells for therapeutic efficacy, revealing a putative role of B cells in CTL priming. Additionally, we have evidence that glioma cells grown in 5% O2 upregulate toll-like receptor (TLR) 2 ligands because TLR2 is required in several systems to distinguish the immunogenicity of 5% and 20% O2 lysates. The goal of this proposal is to elucidate the molecular basis of the "oxygen switch effect" in order to rationally improve the efficacy of tumor cell vaccines. In specific aim 1 we will determine and optimize tumor cell intrinsic changes that modulate immunogenicity. Pharmacologic strategies to increase hypoxia inducible factors (HIF) in tumor cells will be compared to actual hypoxia in cell cultures that will subsequently be assayed for immunogenicity. Additionally, we will test the hypothesis that HIF2a is the molecular switch that induces expression of TLR2 ligands. In specific aim 2, we will test our hypothesis that B cells are required because IgG-tumor lysate complexes trigger Fc receptor-mediated cross presentation of antigens to enhance CTL responses. In specific aim 3, we will determine if the adjuvant effect of 5% O2 lysates is due to expression of Annexin A2, a novel putative TLR2 ligand we recently found to be upregulated in hypoxia. Collectively, this knowledge will facilitate groundbreaking approaches to enhance the efficacy of immunotherapy for glioma and other non-central nervous system tumors.