警报素HMGN1诱导抗肿瘤免疫的作用及其机制研究

Alarmins are endogenous mediators that rapidly become available in peripheral tissues in response to danger signals and are capable of enhancing the induction of innate and adaptive immune responses by promoting the recruitment and maturation of antigen presenting cells (APCs), especially dendritic cells (DCs). We have previously shown that high-mobility group nucleosome-binding protein 1 (HMGN1) is a potent alarmin that induces antigen-specific Th1 immune response by interacting with Toll-like receptor 4 (TLR4). In the previous study, we investigated whether HMGN1 can generate antitumor immunity. Inoculation of EG7, a mouse thymoma transfected to overexpress OVA, into Hmgn1-/- and littermate-matched Hmgn1+/+ mice revealed that the tumor grew much faster in Hmgn1-/- mice than in Hmgn1+/+ mice. In addition, EG7-bearing Hmgn1-/- mice had fewer splenic OVA-specific CD8+ cells, suggesting that endogenous HMGN1 contributes to the development of antitumor immune responses. To determine whether exogenous HMGN1 could also enhance antitumor defense, we inoculated EG7-N1, an HMGN1-expressing EG7 tumor cell line, and parental EG7 into C57BL/6 mice. EG7-N1 tumors grew slower than EG7 tumors in mice, while both cell lines proliferated equally in vitro, suggesting that the murine immune system was more resistant to HMGN1-expressing tumors. To verify the capability of alarmin HMGN1 to enhance antitumor immune responses, we constructed a series of eukaryotic expressing plasmids encoding the genes of HMGN1, gp100 (a murine melanoma-associated antigen), or HMGN1-gp100 fusion gene, and used these plasmids as DNA vaccines. C57BL/6 mice vaccinated by gene gun with various plasmids were subcutaneously implanted with B16F1 melanoma and monitored for tumor growth for 4 weeks. Mice vaccinated with HMGN1-gp100 plasmid inhibited tumor growth, whereas control mice developed tumors. Further analysis revealed that T cells from mice immunized with HMGN1-gp100 plasmid generated gp100-specific cytotoxic activity. These data illustrated that HMGN1 contributes to the generation of antitumor immunity and suggested that the alarmin HMGN1 may be used as an effective tumor vaccine adjuvant. In this study, we will investigate the receptors related to the DC-recruiting function of HMGN1, the core sequences of the alarmin functions of HMGN1, the cytological features and molecular mechanisms of HMGN1-induced antitumor immunity, and the potential of HMGN1 to counter the immunosuppressive properties of cancers. This project will provide deeper understanding of the critical roles of alarmin HMGN1 in antitumor immunity.

HMGN1是新发现的警报素分子，能够募集和活化树突状细胞（DC），刺激Th1型免疫反应。HMGN1主要通过TLR4活化DC，而募集DC的受体未知，其在抗肿瘤免疫中的作用远未阐明。前期的青年科学基金项目中，我们发现EG7淋巴瘤在Hmgn1-/-小鼠中较在野生型小鼠中生长更为迅速，而在野生型小鼠中，分泌表达HMGN1的EG7肿瘤较对照组生长缓慢。我们通过构建以HMGN1为佐剂的DNA疫苗，在小鼠恶性黑色素瘤模型中证实HMGN1作为佐剂能够诱导肿瘤特异性Th1型免疫反应，有效抑制肿瘤生长。这些结果提示HMGN1在抗肿瘤免疫的诱导中起重要作用。本项目将进一步探索HMGN1诱导抗肿瘤免疫的机制，包括其募集DC所涉及的受体、其警报素功能的核心序列，并在恶性黑色素瘤模型中探讨其诱导抗肿瘤免疫的细胞学特征及分子机制，其结果将深化我们对肿瘤免疫的认识，为肿瘤生物治疗提供新的思路和理论依据。

HMGN1是新发现的警报素分子，能够募集和活化树突状细胞（DC），刺激Th1型免疫反应。本项目探索HMGN1诱导抗肿瘤免疫的机制，包括其募集DC所涉及的受体、其警报素功能的核心序列，并在动物模型中探讨其诱导抗肿瘤免疫的细胞学特征及分子机制。.经过本课题的研究，我们确认全长HMGN1可以活化DC，而不含核小体结合结构域（nucleosomal binding domain, NBD）的HMGN1突变体则不能有效活化DC。我们进而单独合成了NBD蛋白，发现单独的NBD蛋白即可有效活化DC。由此我们证明，NBD区是HMGN1活化DC的关键区域。本课题组同样检验了NBD区募集DC的能力，发现全长HMGN1可以募集DC，不含NBD区的HMGN1突变体则不能募集DC；相反，合成的NBD蛋白则可以募集DC。综上，这一部分的结果证明，HMGN1的NBD区是其警报素功能的核心区。我们还通过一系列GST-Pull-Down实验和细胞迁移实验鉴定出HMGN1可以通过Integrin-β1依赖的方式募集DC，从而初步鉴定了HMGN1募集DC所涉及的受体。在一系列动物实验中，本课题组进一步证明不论是表达HMGN1的肿瘤细胞、携带HMGN1编码序列的腺病毒，还是HMGN1蛋白直接瘤内注射，均可以诱导Th1极化的肿瘤特异性免疫反应，从而阐明了HMGN1诱导抗肿瘤免疫的细胞学特征。由此，本课题组成功地完成了计划书的预定目标。.本研究深化了我们对警报素HMGN1在抗肿瘤免疫中关键作用的认识，为肿瘤生物治疗提供了新的思路和理论依据。