Chen B, Shi Y, Smith J D, Choi D, Geiger J D, Mulé J J
Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109-0666, USA.
Blood. 1998 Jun 15;91(12):4652-61.
Because dendritic cells (DC) are critically involved in both initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor antigen-pulsed DC in vitro. The identification of in vitro conditions that would generate large numbers of DC with more potent antigen-presenting cell (APC) capacity would be an important step in the further development of clinical cancer vaccine approaches in humans. We have focused attention on identifying certain exogenous cytokines added to DC cultures that would lead to augmented human DC number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days with added granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). At day 7, cultures contained cells that displayed the typical phenotypic and morphologic characteristics of DC. Importantly, we have found that the further addition of tumor necrosis factor alpha (TNFalpha) at day 7 resulted in a twofold higher yield of DC compared with non-TNFalpha-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFalpha (when added at day 7) demonstrated marked enhancement in their capacity to stimulate a primary allogeneic mixed leukocyte reaction (8-fold increase in stimulation index [SI]) as well as to present soluble tetanus toxoid and candida albicans (10- to 100-fold increases in SI) to purified CD4+ T cells. These defined conditions allowed for significantly fewer DC and lower concentrations of soluble antigen to be used for the pulsing of DC to efficiently trigger specific T-cell proliferative responses in vitro. When compared with non-TNFalpha-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFalpha from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFalpha over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFalpha added to cultures of cytokine-driven human DC under serum-free conditions, which resulted in an enhanced number and function of these APC. On the basis of these results, we plan to initiate clinical vaccine trials in patients that use tumor-pulsed DC generated under these defined conditions.
由于树突状细胞（DC）在启动原发性和增强继发性宿主免疫反应中都起着关键作用，因此人们将注意力集中在利用DC进行疫苗策略，以增强对肿瘤相关抗原的反应性。我们之前曾报道，在体外用肿瘤抗原脉冲DC刺激后，可诱导主要组织相容性复合体II类特异性T细胞反应。确定能够产生大量具有更强抗原呈递细胞（APC）能力的DC的体外条件，将是人类临床癌症疫苗方法进一步发展的重要一步。我们将注意力集中在确定添加到DC培养物中的某些外源性细胞因子上，这些因子会导致人类DC数量和功能的增加。通过贴壁于塑料培养皿富集外周血单核细胞（PBMC）中的DC祖细胞，然后将贴壁细胞在无血清XVIVO-15培养基（SFM）中培养7天，并添加粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）。在第7天，培养物中含有表现出DC典型表型和形态特征的细胞。重要的是，我们发现，在第7天进一步添加肿瘤坏死因子α（TNFα），与不含TNFα的DC培养物在第14天时相比，DC产量提高了两倍。此外，在TNFα存在下（第7天添加）培养14天的DC，在刺激原发性同种异体混合淋巴细胞反应的能力方面有显著增强（刺激指数[SI]增加8倍），以及向纯化的CD4+T细胞呈递可溶性破伤风类毒素和白色念珠菌的能力增强（SI增加10至100倍）。这些确定的条件允许使用显著更少的DC和更低浓度的可溶性抗原对DC进行脉冲处理，以在体外有效触发特异性T细胞增殖反应。与未添加TNFα的培养物相比，这些DC还表现出CD83以及共刺激分子CD80和CD86的表面表达增加。在2天或4天后从DC培养物中去除TNFα会降低其对DC产量、表型和功能的增强作用。因此，TNFα在7天内持续存在是实现观察到的最大增强作用所必需的。总体而言，我们的研究结果指出了在无血清条件下添加到细胞因子驱动的人类DC培养物中的外源性TNFα的重要性，这导致了这些APC数量和功能的增强。基于这些结果，我们计划在患者中启动临床疫苗试验，使用在这些确定条件下产生的肿瘤脉冲DC。
