Expression and selection of productively rearranged TCRβ VDJ genes are sequentially regulated by CD3 signaling in the development of NK1.1+ αβ T cells

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Abstract
The generation of thymic NK1.1+αβT (NKT) cells involves positive selection of cells enriched for Vα14/Vβ8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent β selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3ζ/η–/– and/or p56lck–/–) and TCRα-deficient mice. In contrast to wild-type mice, NK1.1+ thymocytes in CD3 signaling-deficient mice are ~10-fold reduced in number, do not exhibit Vα14–Jα281 rearrangements and fail to express αβTCR at the cell surface. However, they exhibit TCRβ VDJ rearrangements and pre-Tα mRNA, suggesting that they contain pre-NKT cells. Strikingly, pre-NKT cells of CD3ζ/Lck double-deficient mice fail to express TCRβ mRNA and protein. Whereas in wild-type NKT cells TCRβ VDJ junctions are selected for productive Vβ8 and against productive Vβ5 rearrangements, Vβ8 and Vβ5 rearrangements are non-selected in pre-NKT cells of CD3 signaling-deficient mice. Thus, pre-NKT cell development in CD3 signaling-deficient mice is blocked after rearrangement of TCRβ VDJ genes but before expression of TCRβ proteins. Most NKT cells of TCRα-deficient mice exhibit cell surface γδTCR. In contrast to pre-NKT cells of CD3 signaling-deficient mice, ~25% of NKT cells of TCRα-deficient mice exhibit intracellular TCRβ polypeptide chains. Moreover, both Vβ8 and Vβ5 families are selected for in-frame VDJ joints in the TCRβ+ NKT cell subset of TCRα-deficient mice. The data suggest that CD3 signals regulate initial TCRβ VDJ gene expression prior to β selection in developing pre-NKT cells.