Figure 2.

T322D and S323D mutations in the human CD1d cytoplasmic tail cause a cumulative negative effect on its cell surface expression. (a) Amino acid sequence of the cytoplasmic tail mutants used in the experiments. Wild-type (WT). (b) CD1d WT- and mutant-expressing HEK293 cells were fixed and stained with the CD1d-β₂m-specific monoclonal antibody (mAb) 42.1 (white bars), followed by a phycoerythrin-conjugated anti-mouse immunoglobulin antiserum. For the CD1d heavy chain (HC)-specific mAb C3D5 (black bars), the cells were permeabilized with 0·1% saponin following fixation. Analysis was by flow cytometry. The data are displayed as the mean fluorescence intensity (MFI) relative to WT (WT = 1). *P < 0·05; **P < 0·01. CD1d WT and the indicated CD1d tail mutant-expressing cells were co-cultured with human NKT cells (E:T = 1 : 1) for 48 hr in the presence of vehicle (c) or the indicated concentrations of α-GalCer (d). Interleukin-4 (IL-4) production in the supernatants was measured by ELISA.