title: 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.
authors.affiliation: Department of Immunobiology, King's College London, London, SE1 9RT, United Kingdom.
authors.forename: Megan
authors.initials: M
authors.lastname: Estorninho
authors.forename: Vivienne B
authors.initials: VB
authors.lastname: Gibson
authors.forename: Deborah
authors.initials: D
authors.lastname: Kronenberg-Versteeg
authors.forename: Yuk-Fun
authors.initials: YF
authors.lastname: Liu
authors.forename: Chester
authors.initials: C
authors.lastname: Ni
authors.forename: Karen
authors.initials: K
authors.lastname: Cerosaletti
authors.forename: Mark
authors.initials: M
authors.lastname: Peakman
mesh_terms: D001324:Autoantigens / Q000276:immunology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D058948:Cell Tracking / Q000379:methods*; D060966:Clonal Selection, Antigen-Mediated / Q000235:genetics; D002999:Clone Cells; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology* / Q000628:therapy; D018984:Epitopes, T-Lymphocyte / Q000378:metabolism; D014644:Genetic Variation / Q000276:immunology; D015804:HLA-DR4 Antigen / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007156:Immunologic Memory; D016219:Immunotherapy, Adoptive; D050417:Insulin-Secreting Cells / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D037182:T-Cell Antigen Receptor Specificity / Q000235:genetics; D016176:T-Lymphocyte Subsets / Q000276:immunology*
chemical_list: D001324:Autoantigens; D018984:Epitopes, T-Lymphocyte; D015804:HLA-DR4 Antigen; D016693:Receptors, Antigen, T-Cell, alpha-beta; D007371:Interferon-gamma