Abstract A84: Altered monocyte-derived dendritic cell differentiation in the presence of tumor supernatant: Possible involvement the p38MAPK pathway

Introduction: Dendritic cells (DC) are the most efficient antigen-presenting cells in the immune system. In cancer patients, however, these cells present functional and phenotypic disturbances that could be related to a stressful environment induced by the tumor. Heat Shock Proteins (HSP) and the p38MAPK pathway are involved in cell responses against stress and, interestingly, we noted that mature monocyte-derived dendritic cells (Mo-DC) from breast cancer patients present, indeed, higher levels of HSP27 mRNA. Furthermore, HSP27 is a phosphorylation target of the p38 pathway and high HSP27 levels have been associated with impaired Mo-DC differentiation. To investigate these phenomena we exposed monocytes from healthy donors to a tumor cell line supernatant and evaluated their differentiation into DCs and the activation of HSP27 and p38 in the differentiating cells. Methods and Results: Monocytes from healthy donors were negatively selected by magnetic beads and induced to differentiate into DCs (by treatment with GM-CSF and IL-4) either in the presence of 20% conditioned medium (CM) from the breast adenocarcinoma cell line SK-BR-3 (tDC) or not (cDC). The surface phenotype, the presence of total p38, total HSP27, phospho-p38, phospho-HSP27 and the levels of IL-1beta, IL-6, IL-8, IL-10, TNF-alpha and IL-12p70 were evaluated at different time points of the culture, by flow cytometry. At 24h tDC cells expressed less HLA-DR, CD11c and CD86 and showed higher levels of phospho-p38, but less phospho-HSP27. At 72h of culture tDC contained a higher frequency of PD-L1+ cells. Furthermore, during differentiation, tDCs expressed higher levels of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha, but were produced less IL-12p70 in response to lipopolysaccharide (LPS) in comparison with cDCs. Conclusions: CM from SK-BR-3 cells affected the differentiation of Mo-DCs, inducing a phenotype with lesser expression of molecules associated with the induction of immune responses (HLA-DR, CD11c and CD86) but higher expression of PD-L1, a molecule associated with the induction of immune suppression. Dendritic cells exposed to CM also produced high levels of cytokines since the first day of differentiation. This cytokine production exposes the cells simultaneously to signals of differentiation (GM-CSF and IL-4, added to the culture) and maturation (TNF-alpha and IL-1beta produced by the cells), what might impair their differentiation. This was concomitant to an early increase in p38 phosph