Analysis of T cell receptor β chain expression by isoelectric focusing following gene amplification and in vitro translation

• Published in: Journal of immunological methods Vol. 187; no. 1; pp. 9 - 21
• Main Authors: Bouffard, Pascal, Gagnon, Christine, Cloutier, Diane, MacLean, Sheila J., Souleimani, Abdellah, Nallainathan, Dhani, Home, William A., Pilon, Nicolas, Gibson, David M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16.11.1995; Elsevier
• Subjects: Base Sequence; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Gene Amplification; Genetic Techniques; Human; Humans; In vitro transcription; In vitro translation; Isoelectric focusing; Isoelectric Focusing - methods; Molecular immunology; Molecular Sequence Data; Protein Biosynthesis; Receptors, Antigen, T-Cell, alpha-beta - analysis; Receptors, Antigen, T-Cell, alpha-beta - genetics; Repertoire analysis; T cell receptor; T-Lymphocyte Subsets - immunology; Techniques; Transcription, Genetic; β Chain; In vitro transcription; Human; Tcr; T cell receptor; In vitro translation; Repertoire analysis; IEF; Cβ; β Chain; RT-PCR; Isoelectric focusing; Vα; Vβ; CDR3; PCR; Isoelectrical focusing; Investigation method; Variable region; Beta-Peptide chain; Diversity; T-Lymphocyte; Immunologic repertory
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(95)00161-3

We describe a new approach to analysis of T cell receptor diversity based on isoelectric focusing of in vitro translation products of amplified V region genes. The method is illustrated by analysis of Vβ2 profiles in peripheral blood lymphocytes from normal donors. The primers used for Vβ2 analysis spanned the V-(D-)J junction and included the segment from amino acid residue position 53 in the variable region to residue 132 of the constant region. The isoelectric focusing patterns display approximately 13–14 bands of varying intensity. Differences in expression of Vβ2-derived peptides were detected in comparisons of the isoelectric focusing profiles from different individuals, suggesting that the method may be useful for detecting genetically determined, immune response related or disease associated differences in Tcr V region expression. The major isoelectric focusing bands have been interpreted as representing groups of Vβ2 sequences sharing Jβ region and NDN region charge similarity. Quantitative differences were detected in Vβ2 profiles of CD4 and CD8 T cell subpopulations indicating there may be selection for different charge characteristics in NDNJ sequences in the two T cell subsets. The method provides a new dimension for the detection of perturbations in the T cell repertoire.