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

抗肿瘤T细胞应答是许多肿瘤实体中改善预后的关键决定因素，特别是在完全肿瘤切除后防止肿瘤复发和远处转移方面。对肿瘤相关抗原（TAA）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，并且它们在癌症患者中的频率与存活率负相关。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中的迁移，在外周组织中它们发挥肿瘤特异性免疫抑制。骨髓中TAA特异性Treg细胞活化的潜在机制迄今未知。我们最近使用转基因小鼠模型的研究结果表明，一个独特的细胞群体在骨髓中表达的转录因子自身免疫调节因子（Aire），诱导外周组织限制性抗原的异位表达。骨髓驻留Aire表达细胞（BMAC）表达多种自身抗原，包括TAA和转移相关基因。通过将原始TAA特异性CD 4 + T细胞转化为骨髓中的Treg细胞，BMAC以抗原依赖性、MHC-II限制性方式在骨髓和其他淋巴器官如脾中诱导稳健的TAA特异性Treg细胞群。除了小鼠模型外，我们在患有各种肿瘤实体的癌症患者中检测到比健康供体更高频率的人骨髓中的BMAC。我们假设BMAC在诱导肿瘤特异性免疫耐受中起关键作用，该免疫耐受针对骨髓中的播散性癌细胞（DCC）和外周组织中的转移性肿瘤细胞。在该项目中，我们的目标是研究（i）BMAC通过诱导肿瘤反应性Treg细胞对针对肿瘤生长和扩散的全身免疫耐受的贡献，（ii）BMAC在骨髓中为DCC的定殖创造致耐受性小生境中的作用，以及（iii）肿瘤源性炎症因子对BMAC的诱导和激活的作用，这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转基因小鼠模型与表达HA的Balb-NeuT肿瘤细胞以及癌症患者的活组织检查沿着。使用小鼠模型，我们在存在/不存在Aire-HA BMAC的情况下监测肿瘤发展和肿瘤细胞播散。将在小鼠模型和癌症患者的活检中评价BMAC、Treg细胞和DCC相互串扰的骨髓小生境。为了查明激活BMAC中Aire表达的肿瘤衍生因子，我们将确定骨髓中细胞因子的组成，随后将炎症因子鉴定为阻断BMAC激活的靶点。因此，我们期望发现通过消除BMAC活化并因此消除Treg诱导来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.