A Novel Approach to Tracking Antigen-Experienced CD4 T Cells into Functional Compartments via Tandem Deep and Shallow TCR Clonotyping

Abstract

Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCRβ-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier–based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II–restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; “ultraprivate”). Other AST clonotypes share CDR3β amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.

High-throughput T cell receptor sequencing identifies clonally expanded CD8+ T cell populations in alopecia areata

“…Tracking of individual T cell clones using high-throughput sequencing has proven to be a sensitive method to monitor minimal residual disease after treatment for acute or chronic lymphoblastic leukemia and CTCL and for monitoring T cell clones among tumor infiltrating lymphocytes in solid tumors (30)(31)(32)(33)(34)(35)(36)(37). Further, high-throughput TCR sequencing has increased our understanding of T cell repertoire diversity (38)(39)(40)(41)(42), T cell memory formation (43), and response to infection and aging (44)(45)(46). In studies of autoimmune diseases, several recent studies show the potential utility of this technique in ulcerative colitis, multiple sclerosis, psoriasis, and type 1 diabetes (47-52).…”

High-Throughput Immunogenetics for Clinical and Research Applications in Immunohematology: Potential and Challenges

“…Errors introduced during the amplification and sequencing phases complicate data interpretation, particularly in the case of IG gene repertoire analysis, where discriminating between SHM versus error may prove challenging. Among the error correction algorithms that have been proposed (75)(76)(77), the most promising approach relies on molecular barcoding of each individual template molecule to improve data quality and sequencing accuracy (78)(79)(80). Barcoding strategies have also been developed on an mRNA template but suffer from limited sequencing depth (79).…”

Identifying and Tracking Low-Frequency Virus-Specific TCR Clonotypes Using High-Throughput Sequencing

“…Once the pathogen has been cleared, a subset of T cells with TCRs specific to the pathogen remain as long-lived memory cells. The unique DNA rearrangements have the potential to serve as a stable biomarker, cataloging an individual's functional T cell memory and immunological history (Emerson and DeWitt, 2017;Estorninho et al, 2013).…”