Characterization of plasmacytoid dendritic cells compartment in advanced melanoma

Plasmacytoid Dendritic Cells (PDC) are a rare innate immune cells subpopulation .After development, PDC are realised by the bone marrow into the blood stream and migrate from bone marrow to lymph nodes, mucosal associated lymphoid tissue and spleen in steady state conditions. Properly activated PDC may exert antitumor activity through TLR7/9 dependent IFN-α production. IFN-ɑ affects tumor cell proliferation, tumor lymph/angiogenesis, tumor metastasis and enhances the NK cell citoxicity against tumor cells. Autocrine IFNα/β signalling in PDC also modulates a set of interferon-inducible genes such CXCL10/IP-10. IP-10 is a pro-inflammatory chemokine involved in leucocytes recruitment and responsible of Th1 and Th17 polarization of T-cell. PDC recruitment in peripheral tissue has been reported during inflammation and in several types of tumor such as melanoma, head and neck cancer, ovarian carcinoma and breast cancer. Despite of infiltration in many cancers, the PDC anti-tumoral function is still debating. Recent data suggest that PDC could exert a negative regulator role of antitumor response. Emerging hypothesis explains the controversial function of PDC with the tumour microenvironment involvement in subversion of their anti-tumoral activity. Our previous unpublished data on a large cohort of Primary Cutaneous Melanoma (PCM) showed that PDC density within the tumor fails to correlate with a good prognosis in term of IFN-α production. Analysing a cohort of Metastatic Melanoma (MM) patients, we reported a striking decrease of PDC in MM tissues compared to PCM suggesting a severe defect of this population during advanced disease stage. This observation might indicate a reduced PDC bone marrow output or, alternatively, defective migratory properties of PDC. Analysis of Chemokine Receptor (CKR) repertoire of circulating PDC of MM patients suggested lack of activation of these cells. In addition, a significant reduction of circulating PDC and Myeloid Dendritic Cell (MDC) frequencies was detected in blood of MM patients. Interestingly, PDC and MDC reduction was correlated with the tumor burden of MM patients. Moreover, stimulation of TLR7/9 agonists resulted in PDC impairment of MM patients in IFN-α and IP-10 production. Constitutive hyper-activation of the RAF/MEK/ERK pathway is common in melanoma. Combination of BRAF (BRAFi: Vemurafenib and Dabrafenib) and MEK (MEKi: Trametinib and Cometinib) inhibitors represents the standard of care for BRAF mutated MM patients. It is interesting to note that clinical response to Vemurafenib is partially mediated by the immune system. Recently, it has been demonstrated that exposure to MEKi and BRAFi might modulate dendritic cell function. So, we extended the analysis to patients treated with target therapies (i.e. Vemurafenib, Dabrafenib, Trametinib and Cometinib). In our cohort, PDC and MDC frequencies remained unchanged during treatment and single patient analysis revealed no recovery in blood MDC and PDC pool during the treatment. Similarly, PDC capability to produce IFN-α and IP-10 was not restored. This might indicate that microscopic residual disease interferes with PDC function. Alternatively, BRAFi and MEKi might exert some direct effect on PDC function. Our preliminary data obtained on purified PDC suggested that at the therapeutic dose of Vemurafenib cell viability was not affected. Experiments based on an in vitro culture model have enabled us to mimick the direct impact of melanoma cells. PDC viability was affected by melanoma cell supernatant (SN-mel) after 24h of exposure and the death rate was different among different melanoma cell lines. In our experiments, we could observed a downregulation of surface BDCA-2 on PDC after SN-mel exposure. In addition, SN-mel significantly reduced IFN-α and IP-10 production by TLR 7/9 agonists stimulation. These finding suggest that biochemical compounds and ligands produced by melanoma cells affect the survival and function of PDC.