Figure 6. Ovarian cancer cells can present antigen to NKT cells.

(A) LCD1dwt cells were treated with control medium or with ovarian cancer ascites fluid from patients for 4 h, then washed extensively and cocultured with a panel of NKT cell hybridomas (DN32.D3, N38-3C3 and N37-1A12). After 20-24 h, IL-2 was measured as an indication of NKT cell activation using standard cytokine ELISA. (B) Ascites pretreatment induces MAPK signaling. LCD1d1wt cells were incubated with ascites for 4 hours, the cells were lysed and then equal amounts of protein were loaded per well for the detection of phosphorylated and total p38, phosphorylated JNK, and ERK1/2 expression by Western blot analysis. (C) Fixation of OV-CAR-3 and SK-OV-3 cells restored their antigen presentation capabilities. The ovarian cancer cells were fixed in 0.05% paraformaldehyde, pulsed with α-GC and co-cultured with NKT cells. Supernatants were harvested after 16 hours. (D) Proposed model of crosstalk between the VEGF, MAPK, and GD3 signaling pathways. We postulate that activation of VEGF receptor signaling leads to the activation of MAPK signaling. MAPK signaling can induce GM3 synthase, which will lead to the production of GD3. Fixation of the ovarian cancer cells will prevent the induction of these signaling cascades, thereby removing these immunosuppressive factors from the microenvironment and allowing the presentation of glycolipid antigen to NKT cells.