Defective NKT Cell Activation by CD1d+ TRAMP Prostate Tumor Cells Is Corrected by Interleukin-12 with alpha-Galactosylceramide (original) (raw)
Figure 4
TRAMP-C2 cells partially activate iNKT cells by up-regulating activation markers and cell-contact dependently block STAT4 phosphorylation in iNKT cells.
A. TRAMP-C2 and BM-DC were pulsed with α-GalCer or vehicle and used as stimulators for liver MNC as responder cells. Cell culture supernatants were measured 24 hrs (IL-4) and 48 hrs thereafter (IFNγ) by ELISA. B. WT hepatic MNC were exposed overnight to TRAMP-C2 cells, BM-DC, or left alone. Non-adherent cells were stained with indicated mAbs and analyzed by FACS. Histograms show expression of gated iNKT cells. C. Liver MNC were cultured overnight alone or in the presence of TRAMP-C2 cells, pretreated with saturating amounts of anti-mouse CD1d blocking mAbs (3C11) or separated by 0.4 µm transwell membranes. Cells were then stimulated for 30 mins. with 10 ng/ml rIL-12, immediately fixed and stained with mAbs against αβTCR and NK1.1. Cells were then permeabilized using methanol and intracellularly stained against phospho-STAT4 (Ser721). Histograms show phospho-STAT4 staining of electronically gated NKT cells (αβTCR +NK1.1+) shown in left plot. Data shown are representative results of two experiments.