B7-H6分子在淋巴瘤中的表达及其抗肿瘤作用研究

B7 family molecules play important roles in antitumor immunity mediated by cellular immunity and our research team is engaged in the study on the effects of B7 family in hematological malignances.Our previous studies have shown that B7-1 and CD40L played an immunotherapeutic effect on lymphoma synergistically, knockdown of PD-L1 and B7-H3can inhibit proliferation, induce apoptosisin vitro and in vivo, and it is related to chemotherapy sensitivity. B7-H6 is another important molecule in B7 family which is found in recent years. B7-H6 is specially expressed in tumor cell surface, and NK cell surface. NK cells are activated when B7-H6 binds to natural toxicity receptor NKp30 on the surface of NK cell to kill tumor cells. Cytotoxicity of NK cells plays an important role in cellular immune therapy. This research will explore the relationship betweenexpression of B7-H6 and the function of NK cellsin hematologic malignancies and theirclinical significance. The role of B7-H6 in the proliferation, apoptosis, invasion and metastasis in B cell non-Hodgkin’s lymphomas (B-NHL) and related mechanisms will be studied by knockdown of B7-H6 and preparation of anti-B7-H6 monoclonal antibody in vitro and in vivo. This study is expected to elucidate the role of B7-H6 in tumorgenesis and progression in B-NHL and its downstream molecular mechanisms, provide new tumor biomarkers and potential therapeutic targets for B-NHL.

共刺激分子B7家族在细胞免疫介导的抗肿瘤免疫中发挥着重要作用，本课题组一直致力于B7家族分子的研究，前期研究结果显示B7-1/CD40L联合可以增强肿瘤免疫，体内外敲减PD-L1、B7-H3均可抑制淋巴瘤细胞株的增殖、诱导凋亡，还可以增加化疗敏感性。B7-H6是近年发现的B7家族另一重要分子，特异表达于肿瘤细胞表面，与NK细胞表面受体NKp30结合后可激活NK细胞的肿瘤免疫，NK细胞的杀伤功能在细胞免疫治疗中也起了重要作用。本课题拟通过检测B7-H6在恶性淋巴瘤中的表达及患者NK细胞的数量和功能，分析其与临床诊断、分期、疗效和预后的关系；通过基因敲减及制备单克隆抗体在体内外研究B7-H6对B细胞淋巴瘤增殖、凋亡、侵袭的作用及相关蛋白和信号通路。结果可揭示B7-H6及NK细胞在B细胞淋巴瘤发生发展中的作用及其下游分子机制，为B细胞淋巴瘤提供新的肿瘤标记物和潜在治疗靶点。

共刺激分子B7家族在细胞免疫介导的抗肿瘤免疫中发挥着重要作用，本课题组一直致力于B7家族分子的研究。B7-H6是近年发现的B7家族另一重要分子，特异表达于肿瘤细胞表面，与NK细胞表面受体NKp30结合后可激活NK细胞的肿瘤免疫。本项目主要研究内容包括QT-PCR、Western blot及免疫组化检测示B7-H6在NHL细胞株及患者病理组织中较正常对照表达均有明显升高。B7-H6与T-LBL的不良预后因素相关，但T-LBL中B7-H6不同表达组的生存时间无差异。成功构建敲减B7-H6分子的Jurkat、Raji细胞株，实验组细胞增殖明显受抑，凋亡增加，细胞周期阻滞于G0/G1期，迁移及侵袭能力发生明显下降，小鼠体内皮下成瘤能力明显下降。对定量磷酸化修饰蛋白组学结果进行差异蛋白富集及差异位点聚类分析显示：Ras、HIF-1信号通路受B7-H6分子影响最为明显。通过蛋白验证B7-H6敲减后Ras/MEK/ERK通路的激活明显受抑制。构建B7-H6过表达Jurkat、Raji细胞株，证实过表达B7-H6分子可以促进细胞增殖，抑制凋亡，解除G0/G1期细胞阻滞，促进细胞迁移及侵袭。对各组加入MEK抑制剂后由B7-H6引起的Ras/MEK/ERK通路的激活、上述肿瘤的生物学特性发生部分或完全逆转。使用转录组RNA测序技术对敲减与否的Jurkat细胞进行检测，结果显示差异表达基因为RAG-1。蛋白质互作质谱数据库和保守序列的生信分析预测B7-H6存在结合XPO-5蛋白的结构序列。选取B7-H6的受体NKp30作为靶标，自主设计及制备了NKp30 CART细胞，体外杀伤实验显示双价NKp30-CART对T-LBL细胞系具有良好的杀伤效果。研究结果揭示了B7-H6在NHL发生发展中的作用及其下游分子机制，为非霍奇金淋巴瘤患者提供了新的肿瘤标记物和潜在的细胞免疫治疗靶点。