Dysfunctional Plasmacytoid Dendritic Cells but not NK cells in the Peripheral Blood of Stage IV Melanoma Patients

Dendritic cells (DCs) and Natural Killer (NK) cells are two key innate immune effectors that are of importance in the initiation and linkage of both innate and adaptive immune responses for preventing growth and spreading of malignant tumours. Immune dysfunction of DCs and NK cells in cancer patients apparently has a critical role in promoting tumour progression and limiting the efficacy of various immunotherapies. Therefore, identification of the mechanisms underlying this impairment and the molecules involved could facilitate the design of effective cancer therapies. In the current work, the integrity of the number and functions of peripheral blood DC subsets, mDC1, mDC2 and pDCs from stage IV melanoma patients were analysed. We found a significant decrease in the absolute numbers of circulating pDCs and mDC2 in melanoma patients compared to aged-matched healthy controls. This change led to alterations of the mDC1/pDC and mDC2/mDC1 balance. Co-incubation experiments revealed an impairment of the mDC/pDC interaction upon CpG A stimulation, with little or no enhancement of surface CD40 expression on mDC1 from melanoma patients. This was in contrast to an up-regulation of CD40 expression on mDC1 from healthy donors under similar condition. The activation of TLR9 signalling in pDCs by CpG A typically induces the production of various pro-inflammatory cytokines and chemokines. Interestingly, purified pDCs and PBMCs from melanoma patients when stimulated with CpG A produced lesser amounts of IFN-alpha than healthy controls. Furthermore, PBMCs from melanoma patients showed lower CCL5, CCL3 and CCL4 fold change compared to healthy controls, suggesting an impairment of TLR9 signalling in pDCs in response to CpG A stimulation. Interestingly, a dysregulated profile of cytokines and chemokines including IL-6, IL-8, CXCL10, CCL2, CCL4, CCL5 and IL-10 was also observed in the sera of patients. Further analysis revealed a significantly stronger up-regulation of MyD88 and both IRF7 mRNA and protein expression in pDCs from healthy controls compared to patients upon TLR9 activation. Using flow cytometry and immunoblot analysis, it was demonstrated that phosphorylation levels of the 4E-BP1 protein, controlling IRF7 mRNA translation, remained constant in CpG A stimulated pDCs and PBMCs from melanoma patients in contrast to healthy donors. Besides, the impairment of IRF7 up-regulation in pDCs from melanoma patients was accompanied by a reduced IRF7 translocation into the nucleus. Taken together, these data indicate that an impairment of the TLR9 downstream signalling cascade is responsible for the reduced IFN-alpha production by pDCs from melanoma patients. Next, enumeration of NK cells in whole blood from melanoma patients revealed a significant reduction in CD56+CD16+ NK cell numbers. Interestingly, we found a significantly higher percentage of NKp30 and NKp46 receptor expressing NK cells accompanied with a higher expression level of both activating receptors on the peripheral blood NK cells from melanoma patients. Furthermore, patient NK cells exhibited comparable functional activity to healthy controls. In conclusion, the data of this study provide evidences for an alteration in the function of circulating pDCs but not of NK cells from melanoma patients. An aberrant IFN-alpha production by pDCs is due to an impaired TLR9 signaling. This provides new insights in designing and improving melanoma therapy.