T cell receptor-γδ-expressing fetal mouse thymocytes are generated without T cell receptor Vβ selection

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Abstract
We investigated whether fetal mouse T cell receptor (TCR) γδ cells have been subjected to so‐called TCRβ selection at the CD25 stage of thymus development. To this end, we carried out a comparative three‐color flow microfluorimetric analysis of TCRβδ cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day‐15 fetal TCRγδ cells were CD2+, suggesting an origin at a post‐CD25 stage. Molecular analysis of TCRβ rearrangements were also carried out. Thus, by semi‐quantitative polymerase chain reaction (PCR) amplification of Vβ6 and Vβ8 to Jβ2 rearrangements day‐15 fetal TCRγδ showed extensive TCRβ rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR‐amplified TCR VDJβ2 fragments showed that the majority (58%) were rearranged out of frame. Taken together, these phenotypic and molecular analyses suggest that fetal TCRγδ cells have not been subject to TCRβ selection.