Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood
Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood
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DOI：
10.1016/0022-1759(96)00079-8
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发表时间：
1996-09-27
影响因子：
2.2
通讯作者：
Bhardwaj, N
中科院分区：
文献类型：
--
作者：
Bender, A;Sapp, M;Bhardwaj, N
我们研究了一种从人血中的非增殖祖细胞中产生相当数量的成熟树突状细胞的改进方法。该方法使用1%人血浆代替10%胎牛血清，包括两个步骤。第一步或“引发”阶段是在补充有GM-CSF和IL-4的培养基中培养去除T细胞的单核细胞6-7天。第二步或“分化”阶段需要暴露于巨噬细胞条件培养基。该培养基不能被几种已知的细胞因子如TNF-α、IL-1、IL-6、IL-12和IL-15替代，并且不能被单独或组合的IL-1、TNF-α、IL-6或IL-12的中和抗体抑制。使用这种两步法，我们获得了可观的产量。从40 ml血液中产生约1-3 × 10（6）成熟树突状细胞，而从非细胞因子处理的血液中产生< 0.1 × 10（6）成熟树突状细胞。树突状细胞来源于主要在CD 14（+）和粘附的血液单核细胞的放射抗性群体中发现的祖细胞，并且具有成熟细胞的所有特征。它们包括星状细胞形状、不粘附塑料和非常强的T细胞刺激活性。强APC功能对于MLR中同种异体T细胞的增殖和同基因T细胞产生I类限制性CTL应答流感病毒都是明显的。一组树突状细胞限制性标志物也表达，包括CD 83、p55和核周CD 68。当细胞因子被去除时，所有这些树突状细胞特性都保留至少3天，表明这些群体是稳定的和终末分化的。我们认为，这些细胞将是有效的，在体内作为佐剂的主动免疫治疗。
We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood, The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or 'priming' phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-alpha, IL-1, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-alpha, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1-3 x 10(6) mature dendritic cells are generated from 40 mi of blood vs. < 0.1 x 10(6) from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CDl4(+) and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity, Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, p55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy.