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. 2021 Apr 12;12:2147. doi: 10.1038/s41467-021-22375-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A Dermal macrophages, cDC2 (langerin⁻ and langerin⁺), CD14⁺CD1c⁺ cells and CD14⁺CD1c⁻ cells as well as epidermal Langerhans cells were liberated from n = 3 abdominal skin donors and FACS isolated for RNAsequencing (cDC1 were omitted from analysis due to low donor numbers). A PCA plot shows clustering of cell subsets. B Left: HIV restriction factor APOBEC3G gene expression by RNAseq for CD14⁺CD1c⁻ and CD14⁺CD1c⁺ cells (n = 3). Reads per kilobase of transcript, per million mapped reads (RPKM) is plotted with each dot representing an individual donor and lines matching donors. Rights: intracellular SAMHD1 expression on MNP subsets from abdominal skin assessed by flow cytometry, plotted as box and whisker plots, box representing the upper and lower quartile, central line representing the median, the whiskers the minimum and maximum of each tissue sample and each donor represented by an individual dot. Statistics generated using Friedman test with two-tailed Dunn’s multiple comparisons adjusted P values shown (n = 5). C Epidermal LCs and dermal MNPs were liberated from tissue using enzymatic digestions and surface receptor expression evaluated by flow cytometry and compared to in vitro monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC). Left: the geometric mean fluorescent intensity (gMFI) minus the isotype for each subset was calculated and mean plotted in a heat map (n = 2–11). Right: representative histograms of one donor for cell surface expression of three c-type lectin receptors of interest, Siglec-1, DC-SIGN, CLEC5a for all subsets and CD1a and CD141 expression on CD14⁺CD1c⁻ cells and CD14⁺CD1c⁺ compared to cDC2 and cDC1 respectively. D A heatmap was also generated from the RNAseq data to investigate the expression of the corresponding genes investigated by flow cytometry in C. E Flow cytometry analysis shows differences in cell surface expression on immature (enzymatically digested-derived cells) vs mature (spontaneous migration-derived cells) for left: cDC2 and right: CD14⁺CD1c⁺ cells. LC (black), cDC1(purple), langerin⁻ cDC2 (light blue), langerin⁺ cDC2 (dark blue), dermal macrophages (green), CD14⁺ CD1c⁻ MDM (red), CD14⁺ CD1c⁺ MDDC (orange), in vitro MDM (pink) and in vitro MDDC (crimson).