DataSheet_1_In-Depth Analysis of the Impact of Different Serum-Free Media on the Production of Clinical Grade Dendritic Cells for Cancer Immunotherapy.docx

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.

以树突状细胞（Dendritic cell, DC）为基础的抗肿瘤疫苗已被证实为安全的免疫治疗策略，但在临床试验中往往难以获得稳定且显著的疗效。其临床转化受阻的部分原因在于疫苗生产缺乏标准操作规程，具体而言，即体外DC分化过程中最佳培养条件的定义尚未明确。本研究旨在对比三种临床级无血清培养基——DendriMACS、AIM-V及X-VIVO 15，以及添加胎牛血清的罗斯威尔帕克纪念研究所培养基（RPMI），在支持单核细胞来源树突状细胞（Mo-DCs）体外分化方面的效能。在上述不同培养条件下，本研究对未成熟Mo-DCs的表型特征、细胞代谢组学谱、成熟刺激应答反应、细胞因子分泌能力、同种异体T细胞刺激活性，以及抗原特异性CD8+ T细胞致敏能力与自体自然杀伤（NK）细胞活化情况进行了系统性分析。相较于DendriMACS培养基培养获得的DC，经AIM-V或X-VIVO 15培养基分化得到的未成熟Mo-DCs，其CD1c、CD1a的表达水平更低，而CD11c的表达水平更高。经成熟刺激后，仅AIM-V或X-VIVO 15培养基培养的DC可呈现完全成熟的表型，这印证了其具备更强的辅助T细胞1（Th1）亚群极化能力、抗原特异性CD8+ T细胞致敏能力以及NK细胞活化能力。与AIM-V或X-VIVO 15培养基来源的DC共培养得到的CD8+ T细胞与NK细胞，同样展现出更优异的细胞溶解活性。基于1H核磁共振的代谢组学分析显示，DC更强的免疫刺激能力与氨基酸和葡萄糖的分解代谢增强呈显著正相关。总体而言，本研究结果凸显了精准定义癌症免疫治疗临床应用中DC生产所用培养基的重要性。此外，在DC分化过程中调控代谢状态，可作为一种优化其理想细胞特性的潜在策略。