Transcriptome Analysis of T Cell Subsets

The CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ T cells were prepared as described above. The T cells were lysed with TRIzol reagent (Thermo Fisher Scientific) and stored at − 80 °C. The lysates were sent to Genewiz Japan Corp for RNA-seq and related analyses. In brief, RNA was extracted with chloroform/isopropanol and recovered from the supernatants using RNA Clean and Concentrator-5 columns (ZymoResearch) following the manufacturer’s instructions. The RNA purity was assessed with an Agilent 2100 Bioanalyzer. The RNA was subjected to library preparation with the TaKaRa SmartSeq Stranded Kit (Takara Bio) and sequenced with Illumina Hiseq (Illumina). Sequences were mapped to grch38 with HISAT2 (version 2.0.1). Differentially expressed genes were counted using the DESeq2 package in R (version 3.6.3). Up- and downregulated genes were defined as those (i) differentially expressed in peripheral and lesional blood cells with a P-value < 0.05, and (ii) having a greater than twofold change in the average normalized number of peripheral and lesional blood cells. Gene ontology analysis and enrichment analysis using the Jensen DISEASES dataset of differentially expressed genes was performed using the Enrichr webtool (https://maayanlab.cloud/Enrichr/) and Metascape (https://metascape.org/)^{48 (link)–50 (link)}.