The slan antigen identifies the prototypical non-classical CD16+-monocytes in human blood [Bulk RNA-seq]

Peripheral monocytes in humans are conventionally divided into classical (CL, CD14++CD16−), intermediate (INT, CD14++CD16+) and non-classical (NC, CD14dim/−CD16++) cells, based on their expression levels of CD14 and CD16. A major fraction of the NC-monocytes has been shown to express the 6-sulfo LacNAc (slan) antigen, but whether these slan+/NC-monocytes represent the prototypical non-classical monocytes or whether they are simply a sub-fraction with identical features as the remainder of NC monocytes is still unclear. Therefore, we analysed transcriptome, proteome, cell surface markers and production of discrete cytokines by peripheral slan+/NC- and slan−/NC-monocytes, in comparison to total NC-, CL- and INT- monocytes. By bulk RNA-seq and proteomic analysis, we found that slan+/NC-monocytes express higher levels of genes and proteins specific of NC-monocytes than slan−/NC-monocytes do. Unsupervised clustering of scRNA-seq data generated one cluster of NC- and one of INT-monocytes, where all slan+/NC-monocytes were allocated to the NC-monocyte cluster, while slan−/NC-monocytes were found, in part (13.38 %), within the INT-monocyte cluster. In addition, total NC- and slan−/NC-monocytes, but not slan+/NC-monocytes, were found by both bulk RNA-seq and scRNA-seq to contain a small percentage of natural killer cells. Altogether, in addition to comparatively characterize total NC-, slan−/NC- and slan+/NC-monocyte transcriptomes and proteomes, our data prove that slan+/NC-, but not slan−/NC-, monocytes are more representative of prototypical NC-monocytes. Monocytes subsets were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of CD14, CD16 and slan membrane markers and analyzed using Bulk RNA-seq. slan−/NC-monocytes were isolated concurrently with other monocyte subsets (including slan+/NC-, total NC-, INT-, and CL-monocytes), which were previously investigated in our previous study by bulk RNA-seqs (GSE136107: GSM4041235, GSM4041236, GSM4041237, GSM4041243, GSM4041244, GSM4041245, GSM4041246, GSM4041247, GSM4041248, GSM4041249, GSM4041250, and GSM4041251), and compared with them. Re-analysed Samples PRJNA561329/GSE136107: GEO Sample SRA Runs BioSample Title GSM4041235 SRX6747882 SAMN12614816 CL_mono_1 GSM4041236 SRX6747883 SAMN12614814 CL_mono_2 GSM4041237 SRX6747884 SAMN12614812 CL_mono_3 GSM4041243 SRX6747890 SAMN12614850 INT_mono_1 GSM4041244 SRX6747891 SAMN12614849 INT_mono_2 GSM4041245 SRX6747892 SAMN12614848 INT_mono_3 GSM4041246 SRX6747893 SAMN12614847 NC_mono_1 GSM4041247 SRX6747894 SAMN12614846 NC_mono_2 GSM4041248 SRX6747895 SAMN12614845 NC_mono_3 GSM4041249 SRX6747896 SAMN12614844 slan_plus_mono_1 GSM4041250 SRX6747897 SAMN12614843 slan_plus_mono_2 GSM4041251 SRX6747898 SAMN12614842 slan_plus_mono_3