imgt deep sequencing of immune repertoire

Our workflow begins with the cDNA derived from PBL total RNA, which is used as input for multiplex polymerase chain reaction (PCR) through framework 1 (FR1) and constant gene primers to generate ~330 bp TCRB amplicons spanning the three beta chain CDR regions. To the best of our knowledge, this represents the first NGS-based method to permit haplotype-level resolution of the TRB locus. To circumvent the challenge in measuring TRBV polymorphism by WGS, we developed a method for the detection of TRBV polymorphism by next-generation sequencing (NGS) of rearranged TCR beta (TCRB) chains from peripheral blood leukocytes (PBL). Of note, germline-encoded polymorphism in the cytotoxic T-lymphocyte-associated antigen 4 and programmed death-1 pathways has already been linked to autoimmune disease, highlighting the potential mechanistic link between CPI-mediated IRAEs and chronic autoimmune disease. These ranges from common and typically manageable cutaneous IRAEs such as eczema, pruritus, and vitiligo, where the evidence supports a role for autoreactive resident memory T cells, to rare and severe IRAEs such as fulminant type 1 diabetes, where comparatively less is understood with respect to the potentially causative T-cell populations. First, the T-cell receptor (TCR) locus is repetitive and structurally complex, impeding the measurement of variation by traditional short-read WGS or microarray-based methods second, single amino acid substitutions within the framework or CDR 1 and 2 regions of the rearranged TCRB chain are known to significantly alter TCR affinity for HLA and third, adverse events during immunotherapy may manifest as acute versions of chronic autoimmune diseases that have been separately linked to TRBV and HLA polymorphism. Despite previous efforts, three lines of reasoning support the notion that germline-encoded T-cell receptor beta variable (TRBV) polymorphism could be a key determinant of adverse events during CPI.

Identifying such biomarkers could allow for personalized drug selection and dosing to enable safer and more effective immunotherapy, particularly in light of the increasing use of combination CPI regimens having a significant incidence of severe adverse events. Efforts to identify germline variants associated with adverse events using whole-genome sequencing (WGS) or microarrays have yet to reveal markers predictive of adverse events following immunotherapy. Conclusion: We demonstrate a straightforward and cost-efficient method for TRBV haplotype analysis from long-amplicon TCRB sequencing data.Ĭheckpoint blockade immunotherapy (CPI) can elicit anticancer T-cell responses mediating durable progression-free survival but may also promote T-cell destruction of healthy tissue to elicit immune-related adverse events (IRAEs). Analysis revealed 37 putatively novel TRBV alleles that are absent from the IMGT database. 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. Results: Clustering analysis revealed the presence of six major sets of coincident TRBV alleles, which we term haplotype groups. Finally, TRBV allele profiles were subjected to principal component analysis and k-means clustering to identify TRBV allele haplotypes. 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. 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.

We present a novel long-amplicon TCRB repertoire sequencing approach to enable TRBV haplotype analysis from peripheral blood. 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. 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.

Imgt deep sequencing of immune repertoire