A novel approach to tracking antigen-experienced CD4 T cells into functional compartments via tandem deep and shallow TCR clonotyping

• Published in: The Journal of immunology (1950) Vol. 191; no. 11; pp. 5430 - 5440
• Main Authors: Estorninho, Megan, Gibson, Vivienne B, Kronenberg-Versteeg, Deborah, Liu, Yuk-Fun, Ni, Chester, Cerosaletti, Karen, Peakman, Mark
• Format: Journal Article
• Language: English
• Published: United States 01.12.2013
• Subjects: Autoantigens - immunology; Autoantigens - metabolism; CD4-Positive T-Lymphocytes - immunology; Cell Tracking - methods; Clonal Selection, Antigen-Mediated - genetics; Clone Cells; Diabetes Mellitus, Type 1 - immunology; Diabetes Mellitus, Type 1 - therapy; Epitopes, T-Lymphocyte - metabolism; Genetic Variation - immunology; High-Throughput Nucleotide Sequencing; HLA-DR4 Antigen - metabolism; Humans; Immunologic Memory; Immunotherapy, Adoptive; Insulin-Secreting Cells - metabolism; Interferon-gamma - secretion; Protein Binding - immunology; Receptors, Antigen, T-Cell, alpha-beta - genetics; Receptors, Antigen, T-Cell, alpha-beta - immunology; T-Cell Antigen Receptor Specificity - genetics; T-Lymphocyte Subsets - immunology
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.1300622

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.