Figure 6. Reduced development of iNKT cells in TN mice.

• A, B Thymocytes (A) or liver cells (B) from WT or tn/tn mice were stained with αGal-Cer/CD1d tetramer and anti-TCRβ antibody (left). The frequency and numbers of TCRβ⁺ αGal-Cer/CD1d tetramer⁺ cells from thymus (A), liver, or spleen (B) are shown (right) (n = 3–5).
• C The ratio of TCRβ⁺ tetramer⁺ iNKT cells to DP thymocytes (n = 5).
• D CD1d expression on DP thymocytes was equivalent between WT and tn/tn mice (n = 3–5).
• E DP thymocytes were isolated from WT or tn/tn mice and analyzed by quantitative RT–PCR (n = 3). The level of Vα14-Jα18 transcript was normalized to TCR-Cα mRNA, and those in WT were arbitrarily set to 1.
• F TCRβ⁺ tetramer⁺ thymic iNKT cells were further stained for Vβ2, Vβ7, Vβ8.1 + 2, or Vβ8.3 (n = 3–4).
• G TCRβ⁺ tetramer⁺ thymic iNKT cells were further stained for NK1.1 and CD44. Representative flow cytometry profiles (left) and the frequency among total iNKT cells (right) of NK1.1⁻CD44⁻ (stage 0–1), NK1.1⁻CD44⁺ (stage 2), or NK1.1⁺CD44⁺ (stage 3) cells are shown (n = 3).
• H TCRβ⁺ tetramer⁺ thymic iNKT cells were intracellularly stained for PLZF, T-bet, and RORγt. Representative flow cytometry profiles (left) and the frequency among total iNKT cells (right) of T-bet⁺PLZF⁻ (NKT1), RORγt⁻PLZF⁺ (NKT2), or RORγt⁺PLZF⁻ (NKT17) cells are shown (WT, n = 4; tn/tn, n = 5).

Data information: Data represent more than three independent experiments. Each circle represents an individual mouse, and horizontal bars indicate the mean (A, B, C, G, H). Mean ± SEM (D, E, F). The statistical significance was calculated with an unpaired one-tailed Student's t-test. *P < 0.05; **P < 0.01; NS, not significant.