The Vα14i-TCR knock-in mouse produces large numbers of correctly selected, bona fide NKT cells.

<p>(A) Schematic representation of the knock-in transgene. The <i>Vα14</i> promoter, <i>loxP</i> (triangle)-flanked STOP cassette, and pre-rearranged <i>Vα14i</i> (Vα14-Jα18, red square) sequences were inserted 3′ of <i>Jα1</i> and 5′ of the first <i>Cα</i> exon (coding exons are highlighted in blue); 4pA = 4 SV40 polyadenylation sites. AH, arms of homology. <i>Eα</i>, enhancer (black oval). (B) Representative proportions of NKT cells and conventional T cells of total lymphocytes in thymus and spleen. Numbers indicate mean percentages ± SD of at least seven age-matched mice per genotype. (C) Representative proportions of splenic CD4+, CD8+, and DN (CD4− CD8−) NKT cells. Numbers indicate mean percentages ± SD of seven mice per genotype. (D) The Vβ repertoires of splenic NKT cells of the indicated genotypes. Bars indicate means and error bars SD of three independent experiments. (E) Representative flow cytometric analysis of the indicated cell-surface proteins on conventional CD4+ T cells and NKT cells. (F) Intracellular flow cytometric staining of PLZF, GATA-3, ROR-γt, and Th-POK in the depicted NKT cells. Numbers indicate means of the median fluorescence intensities (MFIs), normalized to CD4+ tetramer− T cells of CTR animals, or percentage of ROR-γt+ cells among DN NKT cells; calculated from three animals per genotype. Histograms are representative of three independent experiments with eight mice in total. Throughout the figure, NKT cells were gated as tetramer+ TCRβ+, conventional (conv) T cells as tetramer− TCRβ+; CTR, <i>CD4-Cre</i> or <i>Vα14i^StopF/wt</i>.