Circulating CD4⁺CD161⁺ Th17 cells are not NKT, but NKT-like, cells. (A) The TCR Vα24 and TCR Vβ repertoires were determined by CDR3 length analysis evaluated by spectratyping. The TCR Vα24 and TCR Vβ repertoires were assessed on freshly isolated (total CD4⁺, CD4⁺CD161⁺, and CD4⁺CD161⁻ T cells) and on 1-wk in vitro–expanded CD4⁺CD161⁺ and CD4⁺CD161⁻ T cells. Representative families of different Vβ chains assessed (BV3, BV7, BV8, BV9, BV11, BV14, BV21, BV22, and BV23) are depicted. One representative out of three different experiments is shown. CDR3 length analysis of Vα24 and Vβ11 in iNKT cells is also shown. (B) CD4⁺CD161⁺ and CD4⁺CD161⁻ T cells were stimulated for 8 h with allogeneic DCs in the presence of an anti–MHC class II or an isotype control mAb, and were then assessed for CD154 expression. Bars represent mean values ± SE of percentages of CD154⁺ cells obtained in four different experiments. (C) A PPD-specific short-term T cell line highly enriched in IL-17–producing cells was stimulated for 8 h with PPD-loaded autologous DCs in the presence of an anti–MHC class II or an isotype control mAb. CD154 and cytokine expression were assessed by flow cytometry. Percentages of gated cells are shown.