Extended Data Fig. 2: VHH1D12 selectively activates type I NKT cells.
a, Fold change in CD69 expression on gated Jurkat-76 (J76) or SKW3 cell lines transduced with the CD1d(α-GalCer)-restricted NKT15-, 9B3- or 9C1-TCRs after overnight co-culture with either C1R.WT (CD1d negative) or C1R.GFP.CD1d plus NC, α-GalCer (100 ng/ml), VHH1D5 (1 µM), VHH1D12 (1 µM), VHH1D22 (1 µM) or a combination thereof (n = 4 (NKT15) or n = 3 (9B3-, and 9C1 TCR) independent experiments), analysed by flow cytometry. MF, median fluorescence. b, Fold change in CD69 expression on gated Jurkat J.RT3-T3.5 (JRT3) cell line transduced with the CD1d(sulfatide)-restricted DP10.7 TCR after overnight co-culture with either C1R.WT (CD1d negative) or C1R.CD1d plus NC, sulfatide (25 µg/ml), VHH1D22 (1 µM) or a combination thereof (n = 3 independent experiments), analysed by flow cytometry. c, Histograms depicting binding of lipid loaded CD1d-tetramers, pre-incubated with (red histograms) or without (dark grey histograms) VHH1D12 (~5 times molar excess), to JRT3.DP10.7 cell line. Number in each histogram indicates MF. Data are representative of 3 separate experiments. d, Type I NKT cell binding of lipid loaded CD1d tetramers, simultaneously (+) or after 60 minutes (>) incubated with or without VHH1D12 and/or 1D22 (~5 times molar excess) as indicated (n = 3 independent experiments with type I NKT cells obtained from individual donors). a,b,d, The bars indicate the mean +s.d. a,b,d, One-way analysis of variance (ANOVA) with Tukey multiple comparisons test.
