Transcriptional analysis of bone marrow derived dendritic cells from TLR4 deficient mice matured in the presence of soluble factors secreted by LPS-stimulated B16 melanoma cells

NICOLAS NU&2411EZZ MAR 23737;A LAURA BRESER, VIRGINIA RIVERO; MARIANA MACCIONI

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

Sociedad Argentina Inmunologia

B16 cells stimulated in vitro with a TLR4-ligand 48h prior to their inoculation into TLR4 deficient mice (TLR4lps-del), induce tumors significantly smaller than controls. Apoptosis-proliferation levels of LPS-stimulated B16 cells are not modified and inhibition of tumor growth was not observed in nude mice. LPS-stimulated B16 cells supernatant (LPS-B16 CM) can significantly improve the maturation and function of TLR4lps-del bone marrow derived dendritic cells (BMDC). Here, we analyze the molecular changes that TLR4lps-delDCs experience when they are incubated with B16-CM or LPS-B16 CM for 20h before inducing their maturation with CpG for 4 hs. Transcriptional analysis was performed by a quantitative PCR array and the results were analysed with the 2-ÄÄCT method. TLR4lps-del BMDCs exposed only to CpG for 4 hs, increase the transcription of IL12a (x 8); Csf2 (x 15); Csf3 (x 16.65); IL1a (x 12); IL1b (x 7); IL6 (x 11) and TNFalpha (x 20) genes. The expression of IL1a and b, TNFalpha and IL6 genes was not altered in TLR4lps-del BMDCs matured with B16-CM or LPS-B16 CM, while Csf2 and Csf3 transcription was extremely down regulated. NF-kB complex expression showed a significant increase (x380 NF-kB1, x468 Rela) only in TLR4lps-del BMDCs matured in the presence of LPS-B16 CM. Interestingly, the expression of IL12a that was inhibited in DCs incubated with B16-CM was partially restored when B16-LPS CM was present at the time of maturation. When a neutralizing antibody against IFNbeta was added to LPS-B16 CM the improvement in the expression of coestimulatory molecules analyzed by flow cytometry was lost. We hypothesize that soluble molecules secreted by LPS-stimulated B16 cells can positively modulate the maturation state of DCs, which are