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