RNA-seq analysis for characterization of the TCR repertoire

Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonistic anti-CD40 antibody 2C10R4, rapamycin, soluble tumor necrosis factor receptor and anti-interleukin 6 receptor antibody induce long-term (=1 year) tolerance to islet allografts in 5 of 5 nonsensitized, MHC class I-disparate, and 1 MHC class II DRB allele-matched rhesus macaques. Tolerance in our preclinical model is associated with a regulatory network, involving antigen-specific Tr1 cells exhibiting a distinct transcriptome and indirect specificity for matched MHC class II and mismatched class I peptides. Apoptotic donor leukocyte infusions warrant continued investigation as a cellular, nonchimeric and translatable method for inducing antigen- specific tolerance in transplantation. Overall design: Animals (Amazo, Rocket and Nebula) were infused with Apoptotic Donor Leucocytes on day -7 and day +1. PBMCS were isolated from serial blood samples collected post-ADL infusion and used for TCR sequencing. Total RNA was extracted from frozen PBLs using Rneasy Plus Universal (Qiagen) and first strand cDNA was created from the poly A tailed fraction of total RNA, SMART™ Approach was used for Full-Length cDNA Library Construction. Enriched VDJ amplicons from each sample were uniquely dual-indexed by PCR for multiplexing compatibility during sequencing. Indexed amplicon libraries were pooled equimolarly and cleaned with SPRI beads (Ampure XP, Beckman Coulter). The final pool was sequenced on a MiSeq (Illumina) 300 bp paired end run (v3 kit). Raw, QC-ed short reads were cleaned via trimmomatic81 using following parameters (ILLUMINACLIP:all_illumina_adapters.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:70). The preprocessed reads were directly input into MiXCR82 for TCR profiling with default setting (https://mixcr.readthedocs.io/en/latest/rnaseq.html). KnownTRB clonetypes in rhesus monkeys was used as reference. The resulted TRB clonetypes were further filtered using customized threshold with a clone fraction of = 0.5%. Data from Rocket is presented in the manuscript.