title: A new method for quantitative analysis of the T cell receptor V region repertoires in healthy common marmosets by microplate hybridization assay.
abstract: The common marmoset, Callithrix jacchus, is one of the smallest primates and is increasingly used for an experimental nonhuman primate model in many research fields. Analysis of T cell receptor (TCR) repertoires is a powerful tool to investigate T cell immunity in terms of antigen specificity and variability of TCR expression. However, monoclonal antibodies specific for many TCR Vα or Vβ chains have not been created. We have recently identified a large number of TCRα chain variable (TRAV) and TCRβ chain variable (TRBV) sequences from a cDNA library of common marmosets. The purpose of this study is to develop a new method for analysis of TCR repertoires in the common marmoset using this sequence information. This method is based on a microplate hybridization technique using 32 TRAV-specific and 32 TRBV-specific oligoprobes following an adaptor-ligation PCR. This enables the easy quantitation of the respective TRAV and TRBV expression levels. No cross-hybridization among specific-oligoprobes and very low variances in repeated measures of the same samples was found, demonstrating high specificity and reproducibility. Furthermore, this method was validated by an antihuman Vβ23 antibody which specifically bound to marmoset Vβ23. Using this method, we analyzed TCR repertoires from various tissue samples (PBMCs, spleen, lymph node and thymus) and isolated T cell subpopulations (CD4+CD8+, CD4+CD8− and CD4−CD8+) from the thymus of 10 common marmosets. Neither tissue-specific nor T cell subpopulation-specific differences was found in TRAV and TRBV repertoires. These results suggest that, unlike mice, TCR repertoires in the common marmoset are not affected by endogenous superantigens and are conserved among individuals, among tissues, and among T cell subpopulations. Thus, TCR repertoire analysis with high specificity and reproducibility is a very useful technique, with the potential to replace flow cytometric analysis using a panel of TRV-specific antibodies, many of which remain unavailable.
authors: Department of Rheumatology and Clinical Immunology, Clinical Research Center for Allergy and Rheumatology, Sagamihra National Hospital, National Hospital Organization, Kanagawa, Japan.
authors: Kazutaka
authors: K
authors: Kitaura
authors: Yoshiki
authors: Y
authors: Fujii
authors: Takaji
authors: T
authors: Matsutani
authors: Kenji
authors: K
authors: Shirai
authors: Satsuki
authors: S
authors: Suzuki
authors: Tomohiko
authors: T
authors: Takasaki
authors: Shin
authors: S
authors: Shimada
authors: Yoshie
authors: Y
authors: Kametani
authors: Takashi
authors: T
authors: Shiina
authors: Shuji
authors: S
authors: Takabayashi
authors: Hideki
authors: H
authors: Katoh
authors: Kouetsu
authors: K
authors: Ogasawara
authors: Ichiro
authors: I
authors: Kurane
authors: Ryuji
authors: R
authors: Suzuki
mesh_terms: D000818:Animals; D002144:Callithrix / Q000235:genetics* / Q000276:immunology; D005260:Female; D015723:Gene Library; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008198:Lymph Nodes / Q000166:cytology / Q000378:metabolism; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D015203:Reproducibility of Results; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism
chemical_list: D015345:Oligonucleotide Probes; D016693:Receptors, Antigen, T-Cell, alpha-beta