Figure 6. Presentation of LPC by cell surface CD1d.

(A) Human lymphoblastoid cell lines transfected with either wild-type (WT) or cytoplasmic tail-deleted (TD) human CD1d molecules, or the untransfected parental cells (i.e., CD1d-negative) were tested for the ability to stimulate cytokine secretion by human NKT cell clones. The cells were pulsed with 7.5 nM α-GalCer (αGC) or mock treated, then washed and incubated with NKT cell clones. Culture supernatants were collected after 24 h and assayed for GM-CSF (a cytokine produced by the NKT cells) using a standardized ELISA. The plot shows one representative experiment out of three, using clone Jc2.7. Similar results were observed with three other NKT cell clones. The asterisk indicates signal that was below the limit of detection. (B) Transfectants expressing wild-type or tail-deleted CD1d were pulsed with 1–10 µM LPC or 7.5 nM α-GalCer, and used to stimulate human NKT cell clones. Each dot represents an independent analysis, with the data expressed as the amount of cytokine secreted in response to antigen-pulsed APCs normalized by the response to mock-treated APCs. The horizontal line indicates the mean of the responses. (C) Transfected cells expressing tail-deleted CD1d (CD1d⁺ APCs) or the untransfected parent cells (CD1d⁻ APCs) were pulsed with the indicated concentrations of C18:1 or C16:0 LPC, and used to stimulate clone Jc2.7. Similar concentration-dependent LPC responses were observed in three independent experiments. (D) Freshly isolated human monocytes were incubated for 24 h in culture medium (“untreated”), or in culture medium containing anti-sPLA₂ IgY or negative control IgY, then washed and used to stimulate cytokine secretion by NKT cell clones J24L.17 and J3N.5. Culture supernatants were analyzed for GM-CSF and IL-13 concentration by ELISA; the plots show the means and standard deviations of triplicate samples. One representative experiment out of three is shown.