Induction of local and systemic immune tolerance towards tumor metastasis through ectopic gene expression in antigen presenting cells of the bone marrow

在许多肿瘤实体中，抗肿瘤T细胞反应是改善预后的关键决定因素，特别是在肿瘤完全切除后防止肿瘤复发和远处转移方面。对肿瘤相关抗原（TAAs）反应的调节性T细胞（Treg细胞）抑制T细胞介导的抗肿瘤细胞毒性，其在癌症患者中的频率与生存率呈负相关。在乳腺癌患者中，骨髓驻留的肿瘤特异性Treg细胞被选择性激活，这启动了它们向外周组织的迁移，在那里它们发挥肿瘤特异性免疫抑制作用。迄今为止，骨髓中taa特异性Treg细胞激活的机制尚不清楚。我们最近使用转基因小鼠模型的研究结果表明，骨髓中表达转录因子自身免疫调节因子（Aire）的独特细胞群诱导外周组织限制性抗原的异位表达。骨髓常驻空气表达细胞（bmac）表达多种自身抗原，包括TAAs和与转移相关的基因。通过将naïve taa特异性CD4+ T细胞转化为骨髓中的Treg细胞，BMACs以抗原依赖、MHC-II受限的方式诱导骨髓和其他淋巴器官（如脾脏）中taa特异性Treg细胞的强大群体。除小鼠模型外，我们在患有各种肿瘤实体的癌症患者骨髓中检测到BMACs的频率高于健康供者。我们假设bmac在诱导肿瘤特异性免疫耐受中发挥关键作用，抵抗骨髓中弥散性癌细胞（DCCs）和外周组织中转移性肿瘤细胞。在这个项目中，我们的目标是研究(i) BMACs通过诱导肿瘤反应性Treg细胞对肿瘤生长和传播的全身免疫耐受的贡献，（ii） BMACs在骨髓中为dcc的定植创造耐受性生态位中的作用，以及（iii）肿瘤衍生炎症因子在诱导和激活BMACs的作用，这反过来又促进了对肿瘤细胞的外周耐受性。我们将利用Aire-HA和HA-TCR转基因小鼠模型以及表达ha的Balb-NeuT肿瘤细胞，以及癌症患者的活检。在小鼠模型中，我们在存在/不存在air - ha bmac的情况下监测肿瘤的发展和肿瘤细胞的传播。骨髓壁龛中BMACs、Treg细胞和DCCs相互串接将在小鼠模型和癌症患者的活组织检查中进行评估。为了确定激活BMAC中Aire表达的肿瘤源性因子，我们将确定骨髓中细胞因子的组成，并随后确定炎症因子作为阻断BMAC激活的靶标。因此，我们期望通过取消BMAC激活和Treg诱导来发现减少肿瘤发展和转移的潜在治疗效果。

Anti-tumor T cell responses are key determinants of improved prognosis in many tumor entities, particularly in regard to protection against tumor relapse and distant metastasis after complete tumor resection. Regulatory T cells (Treg cells) reactive to tumor associated antigens (TAAs) suppress T cell mediated anti-tumor cytotoxicity and their frequencies in cancer patients are negatively correlated to survival. In breast cancer patients bone marrow-resident tumor-specific Treg cells are selectively activated, which initiates their emigration into peripheral tissues where they exert tumor specific immune suppression. The mechanism underlying the activation of TAA-specific Treg cells in bone marrow is hitherto unknown. Our recent findings using transgenic mouse models demonstrate that a unique cell population in bone marrow express the transcription factor autoimmune regulator (Aire) which induces the ectopic expression of peripheral tissue-restricted antigens. Bone marrow-resident Aire-expressing cells (BMACs) express a diverse repertoire of self antigens, including TAAs and genes related to metastasis. By converting naïve TAA-specific CD4+ T cells into Treg cells in bone marrow, BMACs induce in an antigen dependent, MHC-II restricted manner robust populations of TAA-specific Treg cells both in bone marrow and in other lymphoid organs such as spleen. Besides murine models, we detected BMACs in human bone marrow at higher frequencies in cancer patients with various tumor entities than in healthy donors. We here hypothesize that BMACs play a key role in the induction of tumor specific immune tolerance against disseminated cancer cells (DCCs) in bone marrow and metastasizing tumor cells in peripheral tissues. Within this project we aim to investigate (i) the contribution of BMACs on systemic immune tolerance against tumor growth and dissemination via the induction of tumor-reactive Treg cells, (ii) the role of BMACs in creating a tolerogenic niche in bone marrow for the colonization of DCCs, and (iii) the role of tumor derived inflammatory factors for the induction and activation of BMACs, which in turn promote peripheral tolerance against tumor cells. We will exploit Aire-HA and HA-TCR transgenic mouse models along with HA-expressing Balb-NeuT tumor cells, as well as biopsies from cancer patients. With the mouse models we monitor tumor development and tumor cell dissemination in the presence/absence of Aire-HA BMACs. Bone marrow niches where BMACs, Treg cells and DCCs cross-talk with each other will be evaluated in mouse models and biopsies of cancer patients. In order to pinpoint the tumor-derived factors which activate Aire expression in BMACs, we will determine the composition of cytokines in bone marrow and subsequently identify the inflammatory factor as a target to block BMAC activation. We thereby expect to find a potential therapeutic effect of reducing tumor development and metastasis by abrogating BMAC activation and thus Treg induction.