Newly generated <i>Traj18</i>-deficient mice lack Vα14 NKT cells.

<p>(A) Flow cytometry profiles of thymocytes, splenocytes and liver mononuclear cells from WT, <i>Traj18</i>^-/- and <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- mice. Unloaded CD1d dimer staining was used as a staining control. Numbers depict percentage of αGC/CD1d dimer⁺ TCRβ⁺ NKT cells among viable CD8^-B220^- gated lymphocytes. The data are representative of three independent experiments. (B) <i>In vivo</i> cytokine production by NKT cells upon systemic activation with αGalCer administration. WT or <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- or <i>Traj18</i>^-/- mice were injected intravenously with 2 μg of αGalCer and blood plasma were collected after either 3 h and 24 h, and IFN-γ and IL-4 concentrations were measured using cytokine beads assay. Bars depict mean ± SEM of <i>n</i> = 3 mice per genotype analyzed. Data are representative of three experiments.</p

Generation of novel <i>Traj18</i>-deficient mice.

<p>Schematic representation of a <i>Traj18</i> region targeting construct, <i>Traj18</i> region before and after homologous recombination, and the genomic locus after FLP- and Cre-mediated deletions of the neomycin resistance gene and <i>Traj18</i>, respectively.</p

A validation of the adjuvant effect of Vα14 NKT cells using <i>Traj18</i>-deficient mice.

<p>(A) NKT cell-mediated adjuvant effect on the expansion of antigen-specific CD8 T cells. WT and <i>Traj18</i>^-/- mice were immunized with OVA antigen and αGalCer on day 0, and splenocytes were analyzed on day 7. Numbers on FACS plots represent percentage of OVA-tetramer positive cells among viable CD8 T cells. (B) Cell percentages and (C) numbers of OVA-tetramer positive cells gated as shown in A. Bars depict mean ± SEM for <i>n</i> = 9 mice per group. (D) NKT cell-mediated adjuvant effect on the activation of antigen-specific CD8 T cells. WT and <i>Traj18</i>^-/- mice were immunized with OVA antigen and αGalCer on day 0, and splenocytes were harvested on day 7. Cells then were cultured <i>in vitro</i> with or without OVA_257-264 peptide for 6 h in the presence of GolgiPlug Protein Transport Inhibitor, and were stained with an IFN-γ mAb using Cytofix/Cytoperm kit. Numbers on FACS plots represent percentage of IFN-γ positive cells among CD8 T cells. (E) Percentages and (F) numbers of IFN-γ positive cells shown in D. Bars graphs depict mean ± SEM for <i>n</i> = 5 mice per group. All data shown are representative from three independent experiments. ****, <i>P</i> < 0.0001 using unpaired <i>t</i> test.</p

Normal development of MAIT cells with an invariant Vα19Jα33 in <i>Traj18</i>-deficient mice.

<p>(A) Sorting strategy of αGC/CD1d^- TCRβ⁺ lung T lymphocytes from WT, <i>Traj18</i>^<i>-/-</i> and previously generated <i>Jα18</i>^<i>-/-</i> mice. Numbers on FACS plots depict percentage of gated cells among viable 7-AAD^- B220^- lung lymphocytes. (B) Relative expression of Vα19Jα33 mRNA by real-time quantitative RT-PCR in sorted lung cells shown in (A). Gene expression was normalized using <i>Trac</i> as internal control. Bars depict mean ± SEM, n.s., not significant using unpaired <i>t</i> test. All data are representative of three independent experiments with a combined total of three mice per genotype.</p

Undisturbed TCRα chain joining region usage in newly generated <i>Traj18</i>-deficient mice as revealed by next generation sequencing.

<p>Sequencing of TCRα chain joining region. PCR was carried out to amplify <i>Trav11-Trac</i> transcripts using cDNA prepared from sorted TCRβ^low CD4⁺CD8⁺ double-positive thymocytes from <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- (red bars) and <i>Traj18</i>^-/- (blue bars) mice. Bars depict mean ± SEM percentages of productive <i>Traj</i> gene segment rearrangements, and data are derived from three biologically independent samples per genotype. Numbers in parenthesis indicate the total number of sequences analyzed. The data are from one experiment.</p