Type I Interferon beta is highly up-regulated 24 hours after stimulation of murine melanoma B16 cells with a Toll Like Receptor 4 ligand

N NUÑEZ , V ANDREANI , V RIVERO , M MACCIONI

Mar del Plata

Congreso; LVII Reunión Científica Anual de la Sociedad Argentina de Inmunología; 2009

Sociedad Argentina de Inmunología

Toll like receptors (TLRs) are key sensor of microbes and are expressed on sentinel cells of the immune system. TLRs agonists are potent activators of innate immune responses, activating antigen presenting cells and promoting adaptive immune response. Their use as adjuvants to reinforce the immune response against tumors, is among the first and oldest strategies used in anticancer immunity, even before these receptors were described. Recently, functional TLRs have been shown to be expressed in numerous cancer cells, but their significance has only recently begun to be explored. Our lab is interested in understanding the possible role that TLR4 could be playing in a sterile environment, like tumors. Stimulation of murine melanoma B16 cells with LPS via TLR4 for 48h in-vitro inhibits subsequent tumor growth in-vivo. This inhibition is not due to a direct effect of TLR4 signaling on the proliferation or apoptosis of tumor cells. Using mice deficient for TLR4 (TLR4 lps-del), we showed that this in vivo inhibition of tumor growth depends exclusively on TLR4 present on tumor cell themselves and not on antigen presenting cells from the host. Our data indicates that the T cell compartment is somehow involved in the described phenomenon since the inhibitory effect observed is not seen in athymic nude mice and the phenotype and function of tumor infiltrating lymphocytes purified from tumors induced by TLR4-activated cells is also modified. We hypothesize that TLR4 signaling in tumor cells in vitro induces the expression of proinflammatory mediators, which could dramatically alter the maturation state of dendritic cells present at the site of inoculation, switching the type of immune response elicited against the tumor. To test this hypothesis, relative mRNA expressions of genes related to TLR-mediated signal transduction were analyzed with a NFkB-Signaling-Pathway RT-ProfilerPCRArray. cDNA obtained from B16 cells stimulated with ultrapure LPS at different times (0, 6 and 24h) was amplified by qPCR and results were analyzed following the 2–ΔΔCT method. Whereas more than 6 fold-induction was observed for the NFkB mediator genes after 6h stimulation, most of the effector molecules were induced after 24h post-stimulation. Interestingly, more than a 100 fold-induction of genes from the IRFpathway, mainly IFNb was observed. Since IFNI has been suggested to be a modulator of DC activity, we hypothesize that its induction could positively modulate the anti-tumoral response.