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An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences.

ABSTRACT: T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.

SUBMITTER: Gurun B

PROVIDER: S-EPMC10291816 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences.

Gurun Burcu B Horton Wesley W Murugan Dhaarini D Zhu Biqing B Leyshock Patrick P Kumar Sushil S Byrne Katelyn T KT Vonderheide Robert H RH Margolin Adam A AA Mori Motomi M Spellman Paul T PT Coussens Lisa M LM Speed Terence P TP

BMC genomics 20230626 1

T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification b ...[more]

PMID: 37365517

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Project description:To maintain a persistent infection viruses such as hepatitis C virus (HCV) employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilize either antigen stimulation (e.g., ELISpot, proliferation assays, cytokine production) or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently, high-throughput sequencing (HTS) technologies have been developed for the analysis of T cell repertoires. In the present study, we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR)3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing antiviral therapy, we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study; however, the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high junction region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted (1395)HSKKKCDEL(1403) and HLA-A*0101-restricted (1435)ATDALMTGY(1443) epitopes. The pipeline developed in this proof of concept study provides a platform for the design of future experiments to accurately address the question of whether T cell responses contribute to SVR upon antiviral therapy. This pipeline represents a novel technique to analyze T cell dynamics in situations where conventional antigen-dependent methods are limited due to suppression of T cell functions and highly diverse antigenic sequences.

Dataset Information

An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences.

Publications

An open protocol for modeling T Cell Clonotype repertoires using TCRβ CDR3 sequences.

Similar Datasets

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