Analysis of cancer-related immune suppressor mechanisms in mice

研究表明，肿瘤已经发展出许多逃避肿瘤特异性免疫反应的方法。这些机制最终不仅会促进肿瘤生长，而且会损害基于免疫的癌症治疗的效果。髓系来源的抑制细胞是最近发现的一种细胞群，它在小鼠和人类中都显示出损害肿瘤特异性免疫反应。MDSC可分为两种亚型（即单核细胞型和粒细胞型）。我们已经能够通过ifn - γ检测和定义小鼠MDSC的调控和功能，并研究了不同HCC模型中的MDSC 1。我们描述了一种新的依赖ifn - γ的MDSC抑制功能的调节机制。而ifn - γ阻断则会损害单核细胞CD11b+Gr-1dull/int的抑制功能。它对粒细胞CD11b+ gr -1高的MDSC有相反的作用。CD11b+ gr -1高粒细胞MDSC与抗原刺激的T细胞共培养，同时使用抗ifn - γ阻断抗体、ifn - γ -效应T细胞、ifn - γ -/- MDSC或STAT1 -/- MDSC阻断ifn - γ，导致CD11b+ gr -1高粒细胞MDSC中Bcl2a1表达上调，提高了MDSC-T细胞相互作用期间粒细胞亚群的存活率，增强了抑制功能。分子研究表明，抗原刺激的CD8+T细胞释放的GM-CSF诱导Bcl2a1上调，在ifn - γ存在时，磷酸化的STAT-1与Bcl2a1启动子直接相互作用抑制Bcl2a1上调。Bcl2a1过表达MDSC不仅能延长体外存活时间，增强体外抑制功能，还能改善体内抑制功能。我们的数据表明，依赖ifn - γ / STAT1的Bcl2a1调节存活，从而抑制CD11b+ gr -1高的MDSC的功能。2. 髓源性抑制细胞（MDSC）是具有免疫抑制活性的未成熟髓细胞。它们在荷瘤小鼠和患有不同类型癌症（包括肝细胞癌）的人体内积累。我们在小鼠肝细胞癌模型中检测了MDSC的生物学特性，并确定了一个模拟人类疾病的模型。在肝细胞癌小鼠中发现MDSC的积累，与所测试的模型无关。与缓慢生长的den诱导或表达myc的HCC相比，可移植肿瘤迅速诱导MDSC的全身募集，在后者中，MDSC数量仅在晚期肿瘤小鼠的肝内增加。来自皮下肿瘤小鼠的MDSC比来自den诱导的HCC小鼠的MDSC具有更强的抑制作用。皮下肿瘤小鼠中MDSC生成相关基因（GM-CSF、VEGF、IL-6、il -1 β）和迁移相关基因（MCP-1、KC、S100A8、S100A9）表达增强。相比之下，在den诱导的HCC小鼠中，只有KC水平升高。KC和GM-CSF过表达或抗KC和抗GM-CSF治疗均可控制HCC小鼠的MDSC频率。最后，在索拉非尼抗肿瘤治疗成功后，MDSC的发生频率下降。结论：我们的数据表明，MDSC积累是肝癌发生过程中的一个晚期事件，并且根据所研究的肿瘤模型有显著差异。

It has been shown that tumors have developed numerous ways to escape tumor specific immune responses. These mechanisms will ultimately not only enhance tumor growth, but also impair the effect of immune based therapies in cancer. Myeloid derived suppressor cells represent a recently identified cell population, which has been shown to impair tumor specific immune responses both in mice and human. MDSC can be divided in two subtypes (namely monocytic and granulocytic MDSC. We have been able to examine and define the regulation and function of murine MDSC by IFN-gamma and studied MDSC in different HCC models 1. We described a new IFN-gamma -dependent regulator mechanism of the suppressor function of MDSC. While IFN-gamma blockade impairs the suppressor function of monocytic CD11b+Gr-1dull/int. it has opposing effects on granulocytic CD11b+Gr-1high MDSC. Co-culture of CD11b+Gr-1high granulocytic MDSC with antigen-stimulated T cells and simultaneous blockade of IFN-gamma by the use of anti-IFN-gamma blocking antibody, IFN-gamma-/- effector T cells, IFN-gammaR-/- MDSC or STAT1 -/- MDSC led to up-regulation of Bcl2a1 in CD11b+Gr-1high granulocytic MDSC, improved survival of granulocytic subpopulation during MDSC-T cell interaction and enhanced suppressor function. Molecular studies revealed that GM-CSF released by antigen-stimulated CD8+T cells induced Bcl2a1 up-regulation, which was repressed in the presence of IFN-gamma by a direct interaction of phosphorylated STAT-1 with the Bcl2a1 promotor. Bcl2a1 overexpressing MDSC not only demonstrated prolonged survival in vitro and enhanced suppressor function in vitro but also showed improved suppressor function in vivo. Our data suggest that IFN-gamma/ STAT1 -dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of CD11b+Gr-1high MDSC. 2. Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). We examined the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease. An accumulation of MDSC was found in mice with HCC irrespectively of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL-6, IL-1beta) and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF over-expression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib. Conclusions: Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.