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to measure gene expression in t_h1/17, t_h17, t_h1, and dn cells sorted from frozen pbmc with qpcr, total rna was isolated and digested with dnase i with the rnaqeous micro total rna isolation kit. cdna was synthesized with the superscript vilo master mix and pre-amplified for 14 cycles with the taqman preamp master mix following the manufactures instruction. qpcr analysis was run and analyzed with the viia 7 real-time pcr system (life technologies) using the taqman fast universal pcr master mix (2x) and qpcr primers purchased from thermofisher scientific. the comparative threshold cycle method and an internal control (2m) were used for normalization of the target genes. relative expression was calculated as: c _t=c _{tgene of interest}c _t2m; c _t=c _{t cell subset of interest}mean of c _{t dn of healthy control}; the relative change of gene expression between the expression level of sample of interest and the mean expression level of all dn samples in healthy controls was given by this formula: (2 ^ct)10. all qpcr reactions were performed in duplicate.

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