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首页> 外文期刊>Journal of Translational Medicine >Monocyte-derived dendritic cells loaded with a mixture of apoptoticecrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8+ T lymphocytes
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Monocyte-derived dendritic cells loaded with a mixture of apoptoticecrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8+ T lymphocytes

机译：单核细胞衍生的树突状细胞载有凋亡/坏死性黑色素瘤细胞的混合物有效地将gp100和MART-1抗原交叉呈递给特定的CD8 + T淋巴细胞

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Background In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptoticecrotic by γ irradiation (Apo-Nec cells). Methods We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100. Results Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 ± 10.5%) at 12 hs of coculture. By 12–24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC – Dextran uptake (iDC: 81 ± 5%; DC/Apo-Nec 33 ± 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3β. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-γ secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change. Conclusion We conclude that the use of a mixture of four apoptoticecrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3β and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.
机译：背景技术在本研究中，我们以严格的方式证明了，当与由γ导致凋亡/坏死的人类黑素瘤细胞混合物共培养时，人类单核细胞衍生的未成熟树突状细胞（DC）可以有效地交叉呈现肿瘤相关抗原。辐射（Apo-Nec细胞）。方法我们评估了在共培养的不同时间对DC和Apo-Nec细胞进行PKH26和PKH67染色后，通过FACS对Apo-Nec细胞的吞噬作用。该过程的动力学还通过电子显微镜观察。还研究了DC的成熟，监测特定标志物的表达，向特定趋化因子的迁移以及体外交叉呈现天然黑素瘤相关的Ags MelanA / MART-1和gp100的能力。结果共培养12 h，未成熟DC（iDC）（55±10.5％）有效吞噬Apo-Nec细胞。到12–24小时，我们观察到DC内部消化的Apo-Nec细胞被消化，并且空泡成为细胞加工的一部分。载有Apo-Nec细胞可诱导DCs成熟，达到LPS处理所达到的水平，方法如下：i）FITC-右旋糖酐摄取减少（iDC：81±5％; DC / Apo-Nec 33±12％）; ii）CD80，CD86，CD83，CCR7，CD40，HLA-1和HLA-II的细胞表面上调，以及iii）体外向MIP-3β迁移的增加。从HLA-A * 0201供体分离出的DC / Apo-Nec在共培养6小时和最多48小时后，能够诱导对MelanA / MART-1和gp100 Ags特异的CTL克隆分泌> 600 pg / mlIFN​​-γ，展示了本地Ag的高效交叉展示。共培养24小时后在DC / Apo-Nec中检测到细胞内IL-12，而IL-10没有改变。结论我们得出结论，使用四种凋亡/坏死性黑色素瘤细胞系的混合物是天然黑色素瘤Ags的合适来源，可为DC提供成熟信号，增加向MIP-3β的迁移并允许Ag交叉展示。该策略可用于黑色素瘤患者的疫苗接种。

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