-Tumor infiltrating mononuclear cells derived from tumors induced with LPS-activated B16 cells express higher levels of IFNƒÁ and reduced levels of IL10

VIRGINIA ANDREANI, NICOLAS NUÑEZ, MARÍA LAURA BRESER, VIRGINIA RIVERO; MARIANA MACCIONI

Congreso; FIRST FRENCH-ARGENTINE IMMUNOLOGY CONGRESS. FAIC 2010; 2010

Sociedad Argentina Inmunologia

B16 murine melanoma cells stimulated in vitro with a TLR4- ligand during 48h prior to their inoculation into TLR4 deficient mice (TLR4lps-del), induce tumors significantly smaller than controls. The apoptosis-proliferation balance of LPS-stimulated B16 cells is not modified and inhibition of tumor growth was not observed in nude mice; thus we hypothesized that TLR4 triggering on B16 cells themselves could induce the expression of proinflammatory mediators, that even if they are transitory, could dramatically alter the function of dendritic cells (DCs) present at the site of inoculation and switch the type of immune response elicited against the tumor. TLR4lps-del DCs matured with CpG in the presence of supernantant (SN) from LPS-stimulated (SN B16+LPS) B16 cells were capable of overcoming the inhibition of activation observed when they were matured in the presence of non-stimulated B16-SN. To further analyze the DCs and T cell function in vivo, infiltrating mononuclear cells (TILs) from tumors induced with B16 cells stimulated (B16 + LPS) or not (B16 Basal) with LPS in TLR4lps-del mice obtained at day 20 and 32 post injection (p.i), were cultured in vitro with PMA-Ionomycin and their cytokine expression was evaluated by intracellular flow cytometry. Interestingly, we observed an increase in CD11c+ IL-12+ cells (2.64%・}0.45 vs 0.43・}0.02%), higher levels of IFNg+ cells (11.18・}2.81% vs 3.46・}0.54%) and reduced levels of IL10+ cells (2.11・}0.45% vs 4.35・}0.42%, p<0.05) in TILs from B16+LPS tumors, in comparison with those induced by B16 cells at day 20 p.i. Similar results were observed when we analyzed TILs isolated at day 32 p.i (IFNg+ cells: 5.24・}3.19% vs 1.66・}1.34%, p<0.05; IL-10+ cells: 0.19%・}0.06 vs 0.31・}0.05%). Therefore, stimulation of murine melanoma cells with LPS in vitro before inoculating them into TLR4lps-del, promotes an improvement of the immune response, activating DCs and enhancing IFNg+ TILs in vivo, at early and later times p.i of tumor cells. and their cytokine expression was evaluated by intracellular flow cytometry. Interestingly, we observed an increase in CD11c+ IL-12+ cells (2.64%・}0.45 vs 0.43・}0.02%), higher levels of IFNg+ cells (11.18・}2.81% vs 3.46・}0.54%) and reduced levels of IL10+ cells (2.11・}0.45% vs 4.35・}0.42%, p<0.05) in TILs from B16+LPS tumors, in comparison with those induced by B16 cells at day 20 p.i. Similar results were observed when we analyzed TILs isolated at day 32 p.i (IFNg+ cells: 5.24・}3.19% vs 1.66・}1.34%, p<0.05; IL-10+ cells: 0.19%・}0.06 vs 0.31・}0.05%). Therefore, stimulation of murine melanoma cells with LPS in vitro before inoculating them into TLR4lps-del, promotes an improvement of the immune response, activating DCs and enhancing IFNg+ TILs in vivo, at early and later times p.i of tumor cells. of IL10+ cells (2.11・}0.45% vs 4.35・}0.42%, p<0.05) in TILs from B16+LPS tumors, in comparison with those induced by B16 cells at day 20 p.i. Similar results were observed when we analyzed TILs isolated at day 32 p.i (IFNg+ cells: 5.24・}3.19% vs 1.66・}1.34%, p<0.05; IL-10+ cells: 0.19%・}0.06 vs 0.31・}0.05%). Therefore, stimulation of murine melanoma cells with LPS in vitro before inoculating them into TLR4lps-del, promotes an improvement of the immune response, activating DCs and enhancing IFNg+ TILs in vivo, at early and later times p.i of tumor cells. p<0.05; IL-10+ cells: 0.19%・}0.06 vs 0.31・}0.05%). Therefore, stimulation of murine melanoma cells with LPS in vitro before inoculating them into TLR4lps-del, promotes an improvement of the immune response, activating DCs and enhancing IFNg+ TILs in vivo, at early and later times p.i of tumor cells. vivo, at early and later times p.i of tumor cells.