Profiling T Cell Receptor Repertoire

Freshly biopsied specimens were immediately frozen at −80 °C. Thirty samples obtained at baseline and on-treatment from 15 patients were analyzed. Total RNA was extracted using RNAlater-ICE Frozen Tissue Transition Solution (Thermo Fisher Scientific) according to the instructions provided by the manufacturer. After the PCR step was performed using a primer set that enriched the V, J, and C regions of hTRA (human TCR alpha), the PCR products were measured using a Qubit Fluorometer (Thermo Fisher Scientific) and checked by electrophoresis for quality. Eleven of 30 samples were below the required level for analysis and excluded from further analysis. Purified products were sequenced by Mi-Seq (Illumina, San Diego, US), and sequence data were analyzed by Repertoire Genesis version 20180912 software (Repertoire Genesis, Osaka, Japan). According to the number of sequenced unique reads for complementarity-determining region three (CDR3) of TRA, the diversity (Shannon-Weaver index H, Inverse Simpson’s index 1/gamma, Pielou’s evenness) or clonality (diversity evenness score [DE50]) of the TCR repertoire in each sample were calculated^{18 (link)}.