Department of Clinical Science, University of Bergen, Bergen, Norway.
Centre for Cancer Biomarkers (CCBIO), Department of Clinical Science, University of Bergen, Bergen, Norway.
Front Immunol. 2023 Jan 12;13:1058963. doi: 10.3389/fimmu.2022.1058963. eCollection 2022.
Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function . Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.
传统的 1 型树突状细胞 (cDC1) 和传统的 2 型树突状细胞 (cDC2) 在癌症免疫治疗中作为单核细胞衍生的树突状细胞 (moDC) 的替代品受到越来越多的关注。由于外周血中 cDC 的数量较少，因此其在治疗中的应用受到限制。在本研究中，我们发现 24 小时内大量自发凋亡和 cDC 死亡是另一个挑战。研究了不同维持 cDC 活力和功能的培养基条件。从健康献血者的白细胞层中分离出 CD141+cDC1 和 CD1c+cDC2。在 24 小时和 48 小时时，发现 CellGenix DC、RPMI-1640 和 X-VIVO 15 标准培养基以及几种补充剂的 cDC1 和 cDC2 活力较低。在多种因素中，发现 GM-CSF 提高了 cDC1 和 cDC2 的活力，而 Flt3-L 和 IL-4 分别仅提高了 cDC1 和 cDC2 的活力。这三种细胞因子的组合进一步提高了两种 cDC 的活力，无论是在 24 小时还是 48 小时时间点。尽管这些细胞因子在髓样细胞分化中的作用已被广泛研究，并且临床上也在使用，但它们对成熟 cDC 的影响仍不完全清楚，特别是对促炎或耐受型 cDC 特征的影响。与 TLR 刺激 24 小时后强烈诱导相比，GM-CSF、IL-4 和 Flt3-L 细胞因子补充对 HLA-DR、CD80、CD83、CD86、PD-L1 和 PD-L2 cDC 膜表达的影响相对较小。除了一些例外，这三种细胞因子似乎对 TLR 诱导的标记表达具有许可作用。同种异体混合淋巴细胞反应表明，细胞因子促进了 T 细胞的增殖，并显示出促进 Th1 和 Th2 极化细胞因子的潜力。GM-CSF 和 Flt3-L 及其组合提高了 cDC1 摄取右旋糖酐的能力，而在 cDC2 中，GM-CSF 提高了右旋糖酐的捕获能力。数据表明，GM-CSF、IL-4 和 Flt3-L 及其组合可能有益于 DC 的活力和功能。cDC 的有限活力可能是实验和免疫治疗中的一个混杂变量。
