Expression and selection of productively rearranged TCR beta VDJ genes are sequentially regulated by CD3 signaling in the development of NK1.1(+) alpha beta T cells

Abstract

The generation of thymic NK1.1(+)alpha beta T (NKT) cells involves positive selection of cells enriched for V(alpha)14/V(beta)8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent beta selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3 zeta/eta(-/-) and/or p56(lck-/-)) and TCR alpha-deficient mice. In contrast to wild-type mice, NK1.1(+) thymocytes in CD3 signaling-deficient mice are approximately 10-fold reduced in number, do not exhibit V(alpha)14-J(alpha)281 rearrangements and fail to express alpha beta TCR at the cell surface. However, they exhibit TCR beta VDJ rearrangements and pre-T alpha mRNA, suggesting that they contain pre-NKT cells. Strikingly, pre-NKT cells of CD3 zeta/Lck double-deficient mice fail to express TCR beta mRNA and protein. Whereas in wild-type NKT cells TCR beta VDJ junctions are selected for productive V(beta)8 and against productive V(beta)5 rearrangements, V(beta)8 and V(beta)5 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 TCR beta VDJ genes but before expression of TCR beta proteins. Most NKT cells of TCR alpha-deficient mice exhibit cell surface gamma delta TCR. In contrast to pre-NKT cells of CD3 signaling-deficient mice, approximately 25% of NKT cells of TCR alpha-deficient mice exhibit intracellular TCR beta polypeptide chains. Moreover, both V(beta)8 and V(beta)5 families are selected for in-frame VDJ joints in the TCR beta(+) NKT cell subset of TCR alpha-deficient mice. The data suggest that CD3 signals regulate initial TCR beta VDJ gene expression prior to beta selection in developing pre-NKT cells.