T Cell Receptor Repertoire Analysis

T cell receptor (TCR) diversity was evaluated using complementarity-determining region 3 (CDR3) spectratyping. RNA was extracted from PBMCs using the RNeasy® Mini Kit (Qiagen), quantified using a NanoDrop® ND-1000 spectrophotometer (LabTech), and converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The TCRβ variable region was amplified using twenty-three Vβ primers (Fig. S6) followed by a run-off reaction with FAM-labeled Cβ primer and CDR3 fragment length analysis by capillary electrophoresis using the 3130 Genetic Analyzer (ThermoFisher). Data were analyzed using Peak Scanner (Applied Biosystems). Using healthy control samples, a scoring system was developed to define electropherograms as normal, abnormal, or highly abnormal (Fig. S7). Gaussian distributions were a subjective description of each Vβ family to demonstrate the visual change in the TCR repertoire, rather than a statistical description.