Fields R C, Osterholzer J J, Fuller J A, Thomas E K, Geraghty P J, Mulé J J
Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109-0666, USA.
J Immunother. 1998 Sep;21(5):323-39. doi: 10.1097/00002371-199809000-00001.
In order to improve upon preclinical tumor vaccine strategies that employ dendritic cells (DC), we now have compared short-term cultures of spleen- and GM-CSF/IL-4-stimulated bone marrow (BM) to determine if differences exist in phenotype and function of murine DC derived from primary and secondary hematolymphoid organs. Although cultures of BM contained a lower percentage of DC compared to spleen, their capacity to stimulate a primary allogeneic mixed leukocyte reaction (MLR) and to uptake fluorescent dextran was substantially greater. In addition, the overall yields of DC per animal was at least twofold greater from BM compared to spleen. Cultures of BM harvested at day 3, 6, or 9 stimulated comparable levels of primary allo-MLR on a per-cell basis. However, there was a consistent loss (at least twofold) of all cells occurring beyond day 6 as compared with cell yields from earlier time points. Importantly, we also improved on methods to rapidly obtain highly enriched DC (> 90%) from BM, which has obviated the reported prior need for complex antibody and complement treatments to remove contaminating mature T and B lymphocytes, Ia-bearing cells, and granulocytes before DC generation. In contrast, although similar purity of DC with similar phenotype and function could be obtained from the spleen, substantial loss in yield occurred, suggesting a further difference in DC between the two tissue sources. The overall yield of DC derived from spleen and BM cultures could be substantially increased by in vivo pretreatment of the donor animals with recombinant Flt3-L. Collectively, these studies demonstrate that notable differences exist in DC preparations derived from spleen vs. BM and that BM provides the preferred source of DC that can be rapidly enriched to high purity for use in further vaccine development.
为了改进采用树突状细胞（DC）的临床前肿瘤疫苗策略，我们现在比较了脾脏以及经GM-CSF/IL-4刺激的骨髓（BM）的短期培养物，以确定源自原发性和继发性血液淋巴器官的小鼠DC在表型和功能上是否存在差异。尽管与脾脏相比，BM培养物中DC的百分比更低，但其刺激原发性同种异体混合淋巴细胞反应（MLR）和摄取荧光葡聚糖的能力要强得多。此外，每只动物的DC总产量从BM获得的至少是脾脏的两倍。在第3、6或9天收获的BM培养物在每个细胞的基础上刺激了相当水平的原发性同种异体MLR。然而，与早期时间点的细胞产量相比，第6天之后所有细胞持续损失（至少两倍）。重要地，我们还改进了从BM快速获得高度富集的DC（>90%）的方法，这消除了之前报道的在DC产生之前需要复杂的抗体和补体处理以去除污染的成熟T和B淋巴细胞、Ia阳性细胞和粒细胞的需求。相比之下，尽管可以从脾脏中获得具有相似表型和功能的类似纯度的DC，但产量有大量损失，这表明两种组织来源的DC存在进一步差异。通过用重组Flt3-L对供体动物进行体内预处理，源自脾脏和BM培养物的DC的总产量可以大幅增加。总体而言，这些研究表明，源自脾脏与BM的DC制剂存在显著差异，并且BM提供了首选的DC来源，可以快速富集至高纯度以用于进一步的疫苗开发。
