IL-35诱导急性髓细胞白血病细胞免疫逃逸的作用及机制研究

Acute myeloid leukemia (AML) is a common hematological malignancy-related with immune defection. In our previous studies, we have found that tumor immune escape mechanism governed by regulatory T cells (Treg) was a key factor contributing to the pathogenesis of AML. The classical immunosuppressive cytokines TGF-β and IL-10 secreted by Tregs have also been considered to be involved in the pathophysiological process of AML. However, recent studies have reported that blockade of TGF-β and IL-10 does not abrogate the suppressive activity of Treg, suggesting other mechanisms may be responsible for Treg-mediating suppression. ..IL-35, as a novel cytokine, belongs to IL-12 cytokines family and plays an important role in the development of immune tolerance. Interestingly, we have examined the expression of IL-35 in 28 newly diagnosed, 27 complete remission and 7 relapse adult AML patients, and found that IL-35 were much higher in newly diagnosed and relapse groups than that in control and remission groups. Therefore, we proposed that IL-35 is correlated positively to adult AML occurrence and development, and plays a suppressive role in antitumor immune response. However, the exact mechanism of IL-35 mediated suppressive role in AML is yet to be elucidated. ..Recent studies have demonstrated that: (1) IL-35 is mainly produced by Treg and significantly suppresses the effector T cells (Teff) responses. Moreover, the predominant mechanism of IL-35 suppression is related to its ability to improve Treg differentiation, proliferation and function. Especially, IL-35 mediates a novel Treg subpopulation named iTr35, which constitutes a key mediator of infectious tolerance and tumor immune escape; (2) As an important and newly identified T cells transcript factor, basic leucine zipper transcription factor 2 (BACH2) plays a key role in the differentiation, proliferation and function of Treg. BACH2 might promote the expression and function of Treg/iTr35 by up-regulating IL-35...In this study, we will explore the following three issues: Firstly, the correlation among IL-35, BACH2 and Treg/iTr35 in AML; Secondly, the role of IL-35 in AML immune escape; Lastly, the effect of BACH2 and Treg/iTr35 on IL-35 expression and function in AML. This study will reveal IL-35 biological characteristics and understand deeply the mechanisms of AML cells immune escape and provide a potential target or new approach for the treatment of AML.

AML是一种发病机制与免疫因素相关的常见血液肿瘤。本课题组在前期研究中已证实Treg诱导了AML细胞的免疫逃逸，并发现在这一病理过程中新型细胞因子IL-35可能发挥了重要作用，但机制不明。最新研究表明IL-35主要来源于Treg并为其增殖和功能所必需，而且还诱导产生一种Treg新亚群（iTr35）而级联放大Treg的作用；BACH2是T细胞发育的重要转录因子，可以明显增强Treg的分化、增殖和功能。我们认为BACH2可能通过靶向上调IL-35而促进Treg/iTr35的表达和功能。本项目拟分析AML中IL-35与BACH2和Treg/iTr35的相关性，并利用体内外实验探讨IL-35诱导AML细胞免疫逃逸的作用，再通过干预BACH2和Treg/iTr35探讨IL-35在诱导AML细胞免疫逃逸过程中的上游调控和下游效应机制，以期进一步揭示AML发病机理，为今后治疗AML提供实验依据和新思路

急性髓细胞白血病（AML）是最常见的成人血液肿瘤，但临床疗效不佳，有待于进一步研究其发病机制和开发新的治疗方法。Tregs是诱导AML细胞逃逸机体抗肿瘤免疫应答的关键因素，然而去除Tregs只能带来短暂性疗效，而中和IL-10或TGF-β的活性也并不能完全阻断Tregs的免疫抑制作用，这提示Tregs在AML中的表达调控网络非常复杂，而且除了经典的抑制性细胞因子IL-10和TGF-β，或许还有其他细胞因子介导了它的免疫调节作用。.最新研究表明IL-35主要来源于Tregs并为其增殖和功能所必需，而且还诱导产生一种Treg新亚群（iTr35）而级联放大Tregs的作用；BACH2是T细胞发育的重要转录因子，可以明显增强Tregs的分化、增殖和功能。我们认为BACH2可能通过靶向上调IL-35而促进Tregs/iTr35的表达和功能，本项目即以此为出发点来在AML中展开研究。.结果如下：（1）通过对免疫细胞及细胞因子的检测，发现AML患者处于明显的免疫紊乱状态，有利于AML细胞免疫逃逸。（2）通过多种实验方法对IL-35蛋白和基因的分析，发现AML患者体内存在IL-35的高表达且与疾病发生发展密切相关。（3）通过细胞分选和功能研究，发现AML中Tregs在静息状态下不表达IL-35但经活化后高表达IL-35，iTr35在静息和活化状态下均高表达IL-35，还发现部分AML细胞也表达IL-35。（4）通过体外刺激和细胞共培养实验，发现IL-35不仅直接抑制Teffs的免疫应答，还促进Tregs和iTr35的表达和功能。（5）通过细胞增殖和凋亡实验，发现IL-35通过结合其受体直接作用于AML细胞，显著促进AML细胞的增殖和抑制AML细胞的凋亡。（6）通过免疫表型和细胞因子分析，发现自体CIK细胞中含有大量的Tregs和IL-35，而通过调整培养方案或加入DCs干预Tregs和IL-35的表达，能显著提高CIK细胞杀伤AML细胞能力。（7）通过表达和功能实验，发现AML中高表达BACH2且与Treg及IL-35表达正相关，干预Treg中BACH2后能够下调其表达IL-35的水平。.总之，本项目通过以IL-35为中心切入点的系列实验研究，初步明确了IL-35在AML中的表达、来源、功能以及可能的作用机制及表达调整机制，揭示了IL-35在AML细胞免疫逃逸过程中的重要作用。