Myeloid-derived suppressor cells (MDSCs) are a significant obstacle for immunotherapy of cancer. It is of great clinical relevance to study the mechanism of MDSCs accumulation in mouse spleens and establish a stable method to obtain high-purity MDSCs in vitro for further research. Here, we established a new method for amplifying a large number of highly pure MDSCs in vitro. To mimic the microenvironment of MDSCs development in vivo, mouse splenic stroma feeder cells and serum-free medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) were used to induce myeloid precursors in mouse bone marrow cells, which differentiate into MDSCs. Development and immunological functions of the cells were monitored both in vivo and in vitro. A total of 4 × 10⁸ MDSCs could be obtained from the bone marrow from one mouse, the ratio of CD11b⁺Gr-1⁺ MDSCs could reach 93.8% ± 3.3% after nine days of culture in vitro. Cultured MDSCs maintained a similar immunophenotype with MDSCs found in tumor-bearing mice. Colony forming assay in vitro and in vivo demonstrated that these were myeloid precursor cells. These cells generated high levels of reactive oxygen species and arginase 1 to prevent proliferation of CD8⁺ T cells in vitro. These also increased regulatory T (Treg) cells in blood while promoting the growth of lymphoma in vivo. In addition, cultured MDSCs effectively inhibited acute graft-versus-host disease (aGVHD). Our findings suggest that mouse splenic stroma plays an important role in the generation of MDSCs and represent a preliminary mechanism for the accumulation of MDSCs in spleens, and thereby lay the foundation for basic research and the clinical application of MDSCs.

骨髓来源的抑制细胞（MDSCs）是癌症免疫疗法的重要障碍。研究MDSCs在小鼠脾脏中的蓄积机理，建立稳定的体外高纯度MDSCs制备方法，具有重要的临床意义。在这里，我们建立了一种在体外扩增大量高纯度MDSC的新方法。为了模拟MDSCs体内发育的微环境，小鼠脾脏基质饲养细胞和含有粒细胞-巨噬细胞集落刺激因子（GM-CSF）的无血清培养基被用于诱导小鼠骨髓细胞中的髓样前体，后者分化为MDSCs。体内监测细胞的发育和免疫功能和体外。从一只小鼠的骨髓中总共可以得到4×10 ^8个MDSC ，体外培养9天后CD11b ⁺ Gr-1 ⁺ MDSC的比例可以达到93.8％±3.3％。培养的MDSC保持与荷瘤小鼠中发现的MDSC相似的免疫表型。在体外和体内的集落形成试验表明，它们是髓样前体细胞。这些细胞产生高水平的活性氧和精氨酸酶1，以防止CD8 ⁺ T细胞在体外增殖。这些还增加了血液中的调节性T（Treg）细胞，同时促进淋巴瘤的生长体内。此外，培养的MDSC可有效抑制急性移植物抗宿主病（aGVHD）。我们的研究结果表明，小鼠脾基质在MDSCs的产生中起着重要作用，并代表了脾脏中MDSCs积累的初步机制，从而为MDSCs的基础研究和临床应用奠定了基础。