PCR and Sequencing

75–100 ng of DNA was amplified through multiplex PCR of TRBV–TRBD–TRBJ gene rearrangements following the BIOMED-2 protocol [10]. Purification of PCR products and library preparation were performed as described previously [11, 12]. Paired-end sequencing was performed using the MiSeq Reagent Kit v2 (2 × 250 bp) on the MiSeq Benchtop Sequencer (Illumina, San Diego, CA, USA). To increase library diversity, PhiX was spiked-in at a 20% concentration. Raw FASTQ files were uploaded to the interactive ARResT/Interrogate immunoprofiler[13] for annotation of the reads and data exploration. QC metrics for the sequencing data are included in Table S3. A mean of 80,607 high-quality sequences was retrieved per sample. Unproductive sequences were removed from further analyses. Additional analyses were performed in R (version 3.5.0, R Foundation for Statistical Computing, Vienna, Austria). For determination of overlap of clonotypes among samples, clonotypes below 50 reads (potential sequencing artifacts) were removed. Shannon’s diversity was calculated using the package Abdiv [14]. The VDJdb database[15] was used, to study the presence of sequences with known antigen specificity. A matching clonotype was defined based on V, CDR3, J, and HLA-type restriction. Although the HLA type of the age-matched healthy control was unknown, the clonotypes of the healthy control that were matching with HLA-A-restricted clonotypes specific for EBV and CMV in the VDJdb database were all restricted to HLA-A*02:01.