肿瘤发生中髓系祖细胞的功能与发育

Myeloid-derived suppressor cells (MDSCs）are critically involved in the development of cancers and other diseases. So far, studies surrounding MDSCs have been centered on its two major subsets, monocyte- and granulocyte-like MDSC. However, the precise nature regarding the precursors or progenitors of the two MDSC subpopulations remains unknown. Our recent work clearly shows that both mouse embryonic stem cells and bone marrow hematopoietic stem cells are able to generate a cell population closely comparable to the granulocyte/macrophage progenitors (GMPs) in the context of differentiation potentials. Somewhat unexpectedly, these in vitro-derived cells display strong suppressive activity against polyclonal- and allo-antigen-stimulated T cells, suggesting a putative MDSC subset unappreciated previously. Furthermore, our preliminary data also indicate that GMPs freshly isolated from tumor-bearing mice possess similarly functional capability. Based on these findings, we hypothesize that immune-inhibitory function is likely a shared inherent property for all myeloid-restricted progenitors at different developmental stages. To test this hypothesis, the present proposal, by utilizing a mouse tumor model, will focus on addressing three crucial issues: 1) The earliest progenitor population with suppressive activity emerged along the pathway of bone marrow hematopoietic stem cell differentiation toward myeloid cells; 2) The molecular basis of myeloid progenitors acquiring suppressive function; 3) The stage-specific features and underlying mechanisms of suppressive myeloid progenitor development. These studies represent the first attempt to explore systematically the immune-regulatory function of myeloid progenitors and dissect their developmental patterns during tumor growth. At the end of this project, we will have i)identified novel members of the MDSC family,which encompass primarily the myeloid-resitiricted progenitor compartment; ii)characterized their behavior changes over the course of tumor progression,and iii)uncovered the molecular mechanisms responsible for their action and development. The results from this application will not only substantially broaden the scope of MDSC biology as well as offer a unique entry into solution of the long-standing questions relating to MDSC origin and their abnormal accumulation under pathological conditions, but also have tremendous potential in helping develop MDSC-based therapeutic strategies for a variety of clinical settings.

髓源性抑制细胞（Myeloid-derived suppressor cells，MDSCs）与肿瘤等疾病的发生密切相关。迄今，MDSC研究主要囿于单核细胞样和粒细胞样MDSC两个亚群，但对其前体细胞的确切性质尚不清楚。我们的工作表明，胚胎和造血干细胞在体外可产生一种在分化潜能上相当于粒细胞/巨噬细胞祖细胞(GMP)但具T细胞抑制活性的细胞群。初步试验也显示，体内来源的GMP具有类似的能力。据此，我们推测免疫抑制可能是髓系祖细胞的共同功能特性。为验证此假说，本项目将利用小鼠肿瘤模型探讨三个问题：1）髓系发生通路中最早出现的抑制性细胞群；2）髓系祖细胞获得抑制功能的分子基础；3）髓系祖细胞发育的特征与调控机理。这些研究将让我们鉴定出新的MDSC亚群并阐明其功能和发育特点与调节机制。所得结果不仅将拓展MDSC的认识和有助于MDSC起源及异常扩增等问题的解决，还将推动基于MDSC治疗策的发展。

髓系细胞发生加剧及其所导致的免疫抑制是肿瘤免疫异常或肿瘤免疫逃逸的关键事件之一。本项目致力于探讨肿瘤进程中髓系发生通路早期细胞群的免疫调节功能及发育。我们首先通过非特异性抑制（用抗CD3+抗CD28抗体刺激T细胞）实验和抗原特异性实验（混合淋巴细胞反应，mixed leukocyte reaction ，MLR)）, 证明荷瘤小鼠骨髓髓系祖细胞群CMPs （Common myeloid progenitors,共同髓系祖细）和GMPs （Granulocyte/Macrophage progenitors，粒细胞/巨噬细胞祖细胞）具有极强的T细胞增殖抑制能力，其活性甚至强过经典的髓源性抑制细胞（MDSCs）。源自正常小鼠的CMPs和GMPs也呈弱抑制活性。髓系祖细胞（GMPs）表达诱导型一氧化氮合酶（iNOS）并分泌高水平NO。利用iNOS特异性抑制剂的试验进一步表明，髓系祖细胞的免疫抑制活性在很大程度上依赖于NO的产生。GMPs还能够有效地诱导调节性T细胞（Tregs），这可能构成髓系祖细胞实现其免疫抑制的间接途径。在肿瘤状态下，骨髓和外周血中髓系祖细胞（GMPs，但不包括CMPs）以及MPPs（多能祖细胞）和HSCs (造血干细胞）的相对含量和绝对数量显著增加。细胞周期分析和体内标记试验显示，荷瘤鼠中这些细胞群处于更为活跃的增殖状态。在肿瘤条件培养基下，荷瘤小鼠来源的GMPs和CMPs以及MPPs和HSCs均比正常小鼠的相应细胞群具有更强的集落形成能力，所形成的集落中含有更多的粒细胞和较少的巨噬细胞。髓系发育相关转录因子检测显示， IRF8 、PU1和 RUNX1在肿瘤小鼠造血干/祖细胞群中的表达发生不同程度的下调。通过慢病毒转染敲低正常小鼠GMPs和CMPs中IRF8的表达，导致类似荷瘤小鼠相应细胞群分化潜能的变化（产生较多的粒细胞和较少的巨噬细胞）。概括起来，该项目让我们鉴定出了髓源性抑制细胞家族的两个新成员，证明髓系祖细胞除扮演免疫细胞发育中间体的角色外，还可以作为效应细胞直接参与免疫调节。此外，我们还初步理清了肿瘤状态下髓系祖细胞发育的特征及调控机制。髓系祖细胞处于经典髓源性抑制细胞的上游，对其进行适当干预将更有助于肿瘤免疫抑制的解决，因而有望发展成为肿瘤等疾病的新治疗靶点。