Reads were trimmed on the basis of quality with Trimmomatic, version 0.36 (Bolger et al., 2014). Bases with quality scores below Q10 (inferred base cell accuracy below 90%) were trimmed, and reads shorter than 50 base pair were dropped. The remaining reads were aligned in the STAR mapping algorithm, version 2.4.0 (Dobin and Gingeras, 2015) to the human reference genome, version GRCh38.p7 (GENCODE release 25). The files were converted from SAM format to the more compressed BAM format with SAMtools, version 1.3 (Li et al., 2009). The count tables were obtained using HTSeq, version 0.6.1 (Anders et al., 2015). ENSEMBL IDs were converted to HGNC symbols using biomaRt, version 2.30.0 (Durinck et al., 2005). Further analysis of the data was undertaken in R, version 3.6.0, and Rstudio, version 1.0.143. Protein coding genes with over 10 reads in total were retained for the analysis. The DESeq2 package, version 1.24.0 (Love et al., 2014) was used for normalization and differential gene expression analysis.

Differentially expressed genes (adjusted P-value < 0.05 and fold change over 1.5) for monocyte versus monocyte-derived LC (moLC) and skin LC versus moLC comparisons were split into genes with higher expression in monocytes (2625/4301) and higher expression in moLCs (1676/4301) and genes with higher expression in LC (1073/1801) and higher expression in moLCs (728/1801), respectively. The lists of differentially expressed genes were converted from HGNC symbols to Entrez Gene IDs with biomaRt, version 2.30.0 (Durinck et al., 2005) and used as input for the Gene Ontology analysis performed with the clusterprofiler R package (Yu et al., 2012).

Heatmaps showing the expression of LC-related and TLR and RLR genes were constructed with gplots, version 3.0.1.1. A pseudo count of 1 was added to the DESeq2-normalized expression values, which were then log2 transformed and displayed as row z scores.

Geneset enrichment analysis was performed with the GSEA software, version 4.0.2 (Mootha et al., 2003, Subramanian et al., 2005) using the Reactome C-type lectin receptors pathway (R-HSA-5621481) and Gene Ontology terms for antigen presentation in the context of major histocompatibility complex class I (Gene Ontology:0002474) and MHC II (Gene Ontology:0002504). DESeq2-normalized expression values were used as input.

The accession number for the RNA-sequencing data is National Center for Biotechnology Information Gene Expression Omnibus GSE141048.

Characterization of moLC culture. (a) Stromal OP9-DLL4 cells were analyzed for their expression of DLL4 by flow cytometry. Isotype-control mAb is displayed in blue. (b) Cell yields from moLC cultures were determined by calculating the percentages of harvested cells from initially seeded monocytes. (c) Langerin and CD1a expression on LCs isolated from human skin (left) and moLCs (right). Isotype-control mAb is displayed in blue. (d) Gating strategy after 3 days of culture of monocytes on OP9-DLL4 plus TGF-β1 and GM-CSF. Detached FSClow OP9-DLL4 cells are mostly found in the orange part, with 80% being dead; FSChigh OP9-DLL4 cells are found together with the in vitro‒generated cells in the petrol blue part but can be excluded by their lack of CD45. FSC, forward scatter; LC, Langerhans cell; moLC, monocyte-derived LC; SSC, side scatter.

GO analysis of moLCs. Gene expression from sorted CD14+ monocytes, CD1a+Langerin+ moLCs, and CD1a+Langerin+ migratory skin LCs from two different donors were analyzed by RNA-seq. GO analysis was performed on differentially expressed genes with higher expression in monocytes versus moLCs or skin LCs versus moLCs. All differentially regulated pathways (adjusted P-value < 0.01) are listed. GO, gene ontology; LC, Langerhans cell; moLC, monocyte-derived LC; RNA-seq, RNA sequencing.

Characterization of the minor subset of CD1a+Langerin cells. (a–c) CD1a+Langerin cells were analyzed by flow cytometry after 3-day culture (0 hour) or after 24 h or 48 h in the presence (mat. cockt.) or absence (w/o) of a cytokine mat. cockt. (a) Percentages of CD1a+Langerin cells within viable CD45+ cells and the expression of (b) the maturation markers HLA-DR, CD83, CD80, and CD86 and (c) the C-type lectin receptors DEC-205 and DC-SIGN are shown. Summary graphs for four experiments are shown; mean ± SD. FMO, fluorescence minus one; h, hour; mat.cockt, maturation cocktail; w/o, without.

Functional aspects of moLCs. (a) Enrichment plots show GO terms for antigen presentation in the context of MHC I (GO:0002474) and MHC II (GO:0002504) as well as the Reactome for C-type lectin receptor pathway (R-HSA-5621481). Data are accessible through GSE141048. (b) CD1a+Langerin+ moLCs and CD1a+Langerin cells were sorted, cultured for 24 h in the presence or absence of a cytokine mat. cockt. and then cocultured in different ratios with CFSE-labeled allogeneic PBLs. After 5 days, CFSE dilution was analyzed by flow cytometry for CD4+ T cells and CD8+ T cells. Representative histograms are for CFSE dilution for 1:10 ratio. CFSE, carboxyfluorescein succinimidyl ester; GO, gene ontology; h, hour; LC, Langerhans cell; mat. cockt., maturation cocktail; MHC, major histocompatibility complex; moLC, monocyte-derived LC; PBL, peripheral blood lymphocyte.

Fluorophore-Labeled Antibodies Used for Flow Cytometry

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