title: A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution.
abstract: The development of T cell effector and memory responses against foreign antigens (Ags) involves the activation, differentiation and proliferation of naive T cells expressing distinct Ag-specific TCRs. Understanding the complexity of Ag-selected TCR repertoires in individual responders in terms of the sequences selected and their relative frequencies may provide indications about how a repertoire is established and suggest ways to influence the outcome of an immune response. Most methods of repertoire analysis are unsuitable for calculating the relative in vivo frequencies of Ag-specific clones (expressing distinct TCRs) selected during an immune response, whereas sequence data obtained by single-cell PCR analysis directly reflect cell frequencies if a sufficiently large number of cells is sampled. Using a CD8 T cell response in normal mice in which Ag-selected cells are identified by cell surface phenotype and rearranged TCRBV sequences are determined by PCR amplification of genomic DNA directly from single cells, we have analyzed a large number (>200 per animal) of structurally-related Ag-specific TCRs to calculate the frequencies of distinct TCRs selected by individual mice. We found that each responder selects a unique Ag-specific TCR repertoire in which the various TCRBV sequences are present in a wide range of frequencies. However, the overall distribution of sequences is quite similar for different responder animals. Moreover, an individual's selected TCR repertoire is uniformly represented among Ag-specific CD8 cells circulating in the blood or localized in the spleen or liver. Relatively few sequences make up the bulk of the repertoire and account for the oligoclonality observed in earlier studies. We discuss various models that could account for this skewed distribution of an Ag-selected TCR repertoire.
authors.affiliation: INSERM Unit 503, Ecole Normale Supérieure de Lyon, France. janet.maryanski@ens-lyon.fr
authors.forename: J L
authors.initials: JL
authors.lastname: Maryanski
authors.forename: V
authors.initials: V
authors.lastname: Attuil
authors.forename: P
authors.initials: P
authors.lastname: Bucher
authors.forename: P R
authors.initials: PR
authors.lastname: Walker
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D005260:Female; D015329:Gene Rearrangement, T-Lymphocyte; D015236:HLA-C Antigens / Q000235:genetics / Q000276:immunology; D006801:Humans; D007114:Immunization; D008099:Liver / Q000166:cytology / Q000276:immunology; D051379:Mice; D008811:Mice, Inbred DBA; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology; D014162:Transfection
chemical_list: D015236:HLA-C Antigens; C038653:HLA-C*03 antigen; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins