title: The clonal composition of human CD4+CD25+Foxp3+ cells determined by a comprehensive DNA-based multiplex PCR for TCRB gene rearrangements.
abstract: The characterization of the T-cell receptor (TCR) repertoire of CD4+ regulatory T cells (T(R)) has been limited due to the RNA degradation that results following permeabilization and fixation as routinely used for intracellular staining of Foxp3. In the present study the clonal composition of human umbilical cord blood (UCB) and adult peripheral blood mononuclear cell (PBMC) CD4+ T(R) and non-T(R) was characterized by a DNA-based multiplex PCR which allowed for the consistent clonotypic characterization of cells that have undergone fixation and permeabilization. To validate this method, CD8+ T cells from two HLA A()0201 individuals were sorted and compared clonotypically based upon their ability either to secrete interferon-gamma in response to a CMV pp65 epitope or to bind to the corresponding pMHC I tetramer. Clonotypes shared between the CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- subsets were observed in all 3 UCB and in one adult PBMCs, suggesting that naïve and memory CD4+ T(R) can share the same clonotypes as CD4+ non-T(R) in humans.
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Phillip
authors.initials: P
authors.lastname: Scheinberg
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Melenhorst
authors.forename: Brenna J
authors.initials: BJ
authors.lastname: Hill
authors.forename: Keyvan
authors.initials: K
authors.lastname: Keyvanfar
authors.forename: A John
authors.initials: AJ
authors.lastname: Barrett
authors.forename: David A
authors.initials: DA
authors.lastname: Price
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Douek
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002478:Cells, Cultured; D002999:Clone Cells / Q000276:immunology; D017931:DNA Primers; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D051858:Forkhead Transcription Factors / Q000032:analysis*; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor / Q000235:genetics*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D053645:Interleukin-2 Receptor alpha Subunit / Q000032:analysis* / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000821:virology; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D020871:RNA Stability; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D050378:T-Lymphocytes, Regulatory / Q000276:immunology; D014763:Viral Matrix Proteins / Q000276:immunology
chemical_list: D017931:DNA Primers; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit; D010750:Phosphoproteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa