TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer.

Tumor antigen-driven selection may expand T cells having T cell receptors (TCRs) of shared antigen specificity but different amino acid or nucleotide sequence in a process known as TCR convergence. Substitution sequencing errors introduced by TCRβ (TCRB) repertoire sequencing may create artifacts resembling TCR convergence. Given the anticipated differences in substitution error rates across different next-generation sequencing platforms, the choice of platform could be consequential. To test this, we performed TCRB sequencing on the same peripheral blood mononuclear cells (PBMC) from individuals with cancer receiving anti-CTLA-4 or anti-PD-1 using an Illumina-based approach (Sequenta) and an Ion Torrent-based approach (Oncomine TCRB-LR). While both approaches found similar TCR diversity, clonality, and clonal overlap, we found that Illumina-based sequencing resulted in higher TCR convergence than with the Ion Torrent approach. To build upon this initial observation we conducted a systematic comparison of Illumina-based TCRB sequencing assays, including those employing molecular barcodes, with the Oncomine assay, revealing differences in the frequency of convergent events, purportedly artifactual rearrangements, and sensitivity of detection. Finally, we applied the Ion Torrent-based approach to evaluate clonality and convergence in a cohort of individuals receiving anti-CTLA-4 blockade for cancer. We found that clonality and convergence independently predicted response and could be combined to improve the accuracy of a logistic regression classifier. These results demonstrate the importance of the sequencing platform in assessing TCRB convergence

Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing

Background: Polymorphism within the human T-cell receptor beta variable (TRBV) gene has been proposed as a risk factor for autoimmune disease and immune-related adverse events (IRAEs) during immunotherapy. Previous efforts to evaluate TRBV polymorphism by whole genome sequencing have been hampered by the repetitive nature of the T-cell receptor beta (TCRB) locus. We present a novel long-amplicon TCRB repertoire sequencing approach to enable TRBV haplotype analysis from peripheral blood. Methods: Peripheral blood leukocyte total RNA from 81 Caucasians was used for sequencing of TCRB chains via the Oncomine TCRB-LR assay (amplicon spanning CDR1, 2 and 3) and the Ion Gene Studio S5. VDJ rearrangements were annotated by comparison to the IMGT database, then mined to construct TRBV allele profiles for each individual including, where detected, novel alleles not present in the ImMunoGeneTics (IMGT) database. Finally, TRBV allele profiles were subjected to principal component analysis and k-means clustering to identify TRBV allele haplotypes. Results: Clustering analysis revealed the presence of six major sets of coincident TRBV alleles, which we term haplotype groups. Allelic diversity varied markedly across haplotype groups, with approximately one third of the cohort showing limited TRBV allelic diversity and few uncommon alleles compared to members of other groups. Analysis revealed 37 putatively novel TRBV alleles that are absent from the IMGT database. Conclusion: We demonstrate a straightforward and cost-efficient method for TRBV haplotype analysis from long-amplicon TCRB sequencing data