The balance between tolerance and immunity is important for the outcome of an infection or cancer, and dendritic cells (DCs) are key regulators of this balance. DC-specific transcript (DC-SCRIPT) is a protein expressed by DCs and has been demonstrated to suppress both TLR-mediated expression of IL-10 and glucocorticoid receptor–mediated transcription of glucocorticoid-induced leucine zipper (GILZ). Because GILZ is known to promote IL-10 production, we investigated whether these two processes are linked. Dual-knockdown and inhibition experiments demonstrated that neither GILZ nor glucocorticoid receptor play a role in TLR-induced IL-10 production after DC-SCRIPT knockdown. The NF-κB pathway is another route involved in IL-10 production after DC activation. Strikingly, inhibition of NF-κB led to a decreased TLR-mediated IL-10 production in DC-SCRIPT knockdown DCs. Moreover, DC-SCRIPT knockdown DCs showed enhanced phosphorylation, acetylation, and IL10 enhancer binding of the NF-κB subunit p65. These data demonstrate that besides nuclear receptor regulation, DC-SCRIPT also modulates activation of NF-κBp65 after TLR activation in human DCs.

The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel–tumor cell coculture assay.