OR32 Binding of ILT3 to its ligand inhibits signaling pathways involved in cell proliferation and migration.

Aim To identify target cells with high levels of Immunoglobulin Transcript 3 (ILT3) ligand on their membrane in order to identify the signaling mechanism of this strongly inhibitory protein. Methods We used the U2O osteosarcoma cell line which binds ILT3.Fc on 100% of the cell population. Cells were pre-incubated for 24 to 48 h with 50 μg/ml of ILT3.Fc or left untreated. Next, the cells were washed twice with cold PBS and fixed in 80% ethanol at 4C overnight. Cells were washed once before staining with Propidium Iodide/RNase Staining Buffer (BD Pharmingen) according to the manufacturer’s protocol. Cell doublets were gated out based on pulse width versus pulse area. The data was accumulated and analyzed by FACS using Cell Quest software. Cell growth in the presence of 0, 12.5, 25, and 50 μg/ml of ILT3.Fc was monitored at 24 h interval using the trypan blue exclusion method. The wound healing scratch assay was used according to the protocol described in PMID: 17406593 to assess the effect of ILT3.Fc on cell migration. Western Blot analysis of cells treated or not treated with ILT3.Fc was performed using antibodies to various kinases. Results We have discovered that cells from the U2OS cell line bind ILT3.FITC which inhibits their growth. Cell cycle analysis using Propidium iodide staining showed that ILT3.Fc induced cell cycle arrest in the transition from G1 to S phase.Wound healing cell culture experiments demonstrated inhibition of this process up to 48 h of U2OS exposure to ILR3.Fc, confirming morphological observation on apoptotic changes of the cells. Western blot analysis of PI3K/AKT and MAPK/ERK showed that ILT3.Fc induced dephosphorylation of these proteins inhibiting their signaling pathway. Conclusions Binding of ILT3.Fc to its ligand on the membrane of malignant cells blocks cell cycle progression and inhibits cell migration. These studies suggest that ILT3.Fc may serve as a potent inhibitor of tumor cell growth for cancer therapy. This opens new perspectives to the increasing immunotherapy field of check point blockade through ligand or receptor inhibition. [ABSTRACT FROM AUTHOR]