Spleen cells presented exogenous sulfatide to XV19 T hybridoma cells. (A) A titration of splenocytes was cultured together with the XV19 NKT-cell hybridoma, and IL-2 secretion assayed by CTLL-2 cells. Autoreactivity of the NKT-cell hybridoma XV19 was demonstrated to C57BL/6 (squares), but not CD1d−/− (CD1d KO, triangles) splenocytes. Data shown are representative of more than five experiments (mean of duplicate cultures). (B) Sulfatide pulsed CD1d+ splenocytes could stimulate XV19 cells above background autoreactivity. Splenocytes (12 × 103 cells/well) from C57BL/6 (black bars) or CD1d−/− (CD1d KO, white bars) mice were cultured together with sulfatide isoforms (30 nmol/mL) for 4 h before co-culture with the XV19 NKT-cell hybridoma (40 × 103 cells per well). Data shown are one representative experiment of three (mean of duplicate cultures). Lyso, lysosulfatide.