Figure 2: Lipid reactivity of atypical TRBV25-1⁻ NKT cell lines.

(a) CD1d tetramer staining of CD1d–α-GalCer-reactive cells enriched and expanded from PBMCs from four healthy human donors. Plots show TRBV25-1 versus CD1d tetramers loaded with α-GalCer (C24:1), ‘endogenous’ antigen, α-GlcCer, 3′-deoxy-α-GalCer, 4′-deoxy-α-GalCer or OCH. Data show one of two representative experiments. (b) Histograms depicting human CD1d–lipid antigen tetramer staining (white histograms) of CD3⁺ Jurkat T-cell lines transduced with the 9C1, 9B1, 9B2, 9B3 atypical NKT cell TCRs or with the NKT15 type I NKT cell TCR or an irrelevant pHLA-specific TCR control, overlaid with ‘endogenous’ tetramers (grey histograms). Numbers in each histogram represent CD1d–lipid tetramer mean fluorescence intensity. Data are representative of two separate experiments. (c) Graphs depict the mean IFN-γ, IL-2, IL-4 and IL-13 concentrations in culture supernatants of 4–5 × 10³ in vitro-expanded/purified CD1d–α-GalCer tetramer⁺ TRBV25-1⁺ (type I NKT, white bars), CD1d–α-GalCer tetramer⁺ TRBV25-1⁻ (atypical NKT, black bars), CD1d–α-GalCer tetramer⁺ TRDV1⁺ γδTCR⁻ (δ/αβ NKT, grey bars), and CD1d–α-GalCer tetramer⁻ (control T cells, hashed bars), with different lipid Ag (0.5 μg ml⁻¹) in the presence of K562.CD1d APCs or PMA/ionomycin for 24 h. Data are representative of n=5–7 donors, with each symbol depicting a separate donor (each symbol derived from n=1–2 technical replicates). Data are pooled from two independent experiments.