Ncounter human immunology v2 gene expression code set

Frozen PBMCs were thawed, stained as above, and sorted on a BD FACS Aria II (BD Biosciences) into CD4⁺CD25^hiCD127^lo Tregs, CD4⁺CD25^lo-intCD127^hi conventional T cells (Tcons), and CD14⁺CD16⁻ classical monocytes. Sorted cells were suspended in RLT buffer (Qiagen, Hilden, Germany). Expression of immune-related genes was quantified using the nCounter Human Immunology v2 gene expression codeset (Nanostring, Seattle, WA) according to the manufacturer’s instructions. This codeset determines expression of 579 immune-related transcripts, controlling for technical variation with 14 spiked-in positive and negative controls, and for RNA content with 15 internal reference genes. Gene expression above background was taken to be ≥30 transcripts per lane. Gene ontology and pathway analysis were performed using Ingenuity pathway analysis (Qiagen, Redwood City, CA). The nCounter Human Immunology v2 gene list was set as the background gene list for Ingenuity pathway analysis.