title: Visualization and quantification of monoallelic TCRα gene rearrangement in αβ T cells.
abstract: T-cell receptor α (TCRα) chain rearrangement is not constrained by allelic exclusion and thus αβ T cells frequently have rearranged both alleles of this locus. Thereby, stepwise secondary rearrangements of both TCRα loci further increase the odds for generation of an α-chain that can be positively selected in combination with a pre-existing TCRβ chain. Previous studies estimated that approximately 2-12% of murine and human αβ T cells still carry one TCRα locus in germline configuration, which must comprise a partially or even fully rearranged TCRδ locus. However, these estimates are based on a relatively small amount of individual αβ T-cell clones and αβ T-cell hybridomas analyzed to date. To address this issue more accurately, we made use of a mouse model, in which a fluorescent reporter protein is introduced into the constant region of the TCRδ locus. In this TcrdH2BeGFP system, fluorescence emanating from retained TCRδ loci enabled us to quantify monoallelically rearranged αβ T cells on a single-cell basis. Via fluorescence-activated cell sorting analysis, we determined the frequency of monoallelic TCRα rearrangements to be 1.7% in both peripheral CD4(+) and CD8(+) αβ T cells. Furthermore, we found a skewed 5' Jα gene utilization of the rearranged TCRα allele in T cells with monoallelic TCRα rearrangements. This is in line with previous descriptions of a tight interallelic positional coincidence of Jα gene segments used on both TCRα alleles. Finally, analysis of T cells from transgenic mice harboring only one functional TCRα locus implied the existence of very rare unusual translocation or episomal reintegration events of formerly excised TCRδ loci.
authors.affiliation: Institute of Immunology, Hannover Medical School, Hannover, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Winter
authors.affiliation: Institute of Immunology, Hannover Medical School, Hannover, Germany.
authors.forename: Elham
authors.initials: E
authors.lastname: Kashani
authors.affiliation: Institute of Immunology, Hannover Medical School, Hannover, Germany.
authors.forename: Vijaykumar
authors.initials: V
authors.lastname: Chennupati
authors.affiliation: Institute of Immunology, Hannover Medical School, Hannover, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Föhse
authors.affiliation: Institute of Immunology, Hannover Medical School, Hannover, Germany.
authors.forename: Immo
authors.initials: I
authors.lastname: Prinz
mesh_terms: D000483:Alleles; D000818:Animals; D015870:Gene Expression; D005787:Gene Frequency; D015329:Gene Rearrangement, T-Lymphocyte; D017930:Genes, Reporter; D056426:Genetic Loci; D051379:Mice; D008822:Mice, Transgenic; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000378:metabolism*
chemical_list: D016693:Receptors, Antigen, T-Cell, alpha-beta