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Abstract

Innate immune cells represent the first line of defense against infections and cancer and therefore play a pivotal role in determining the fate of anti-tumor immune responses. As a critical aspect of melanoma biology is represented by the ability to evade immune destruction, modern therapies can benefit from breaking immune suppression, as demonstrated by checkpoint inhibitor therapy. However, many patients acquire defects in antigen presentation and thereby remain unresponsive to T cell mediated immune clearance. For those patients, therapies enabling innate immune responses might prove to be effective in preventing cancer progression and metastasis. Therefore, this study investigated the anti-melanoma effects of two innate immune cell types, slan+ monocytes (slanMo) and natural killer (NK) cells, in combination with stimulation by R848, a synthetic TLR 7/8 ligand for topical application that is currently investigated regarding therapeutic efficacy in melanoma. Here, we addressed the question whether slanMo actively recruit NK cells in order to initiate an innate immune response that limits tumor growth and induces senescence in melanoma cells. We showed that slanMo are frequently detected in melanoma tissue, but rarely in healthy skin. More invasive forms of melanoma exhibited reduced numbers of infiltrating slanMo that were accompanied by correlating numbers of NK cells. To elucidate whether the correlating infiltration patterns represent a connected migration mechanism, we investigated chemokine production by slanMo. In vitro culture of slanMo resulted in the secretion of various chemokines such as IL-8/CXCL8, which was further increased by stimulation with R848. Stimulated slanMo conditioned medium (slanMo CM) induced chemotaxis of NK cells and led to the downregulation of the IL-8 receptors CXCR1 and CXCR2 on NK cells, indicative of receptor engagement. Neutralization experiments revealed IL-8 as the relevant NK cell chemoattractant in slanMo CM. To investigate the influence of the slanMo/NK cell crosstalk on melanoma cells, we transferred supernatants from R848-stimulated slanMo/NK cell co-cultures to melanoma cells, resulting in strongly attenuated cell growth. We found that R848-stimulated slanMo/NK cell co-culture CM contained high levels of TNF-α and IFN-γ, not reached when NK cells were cultured with CD14+ monocytes or slanMo were cultured with T cells. Neutralization experiments revealed that TNF-α and IFN-γ present in slanMo/NK cell co-culture CM induced senescence in melanoma cells as indicated by reduced proliferation, increased senescence-associated β-Galactosidase expression, p21 upregulation, and induction of a senescence-associated secretory phenotype (SASP) in the tumor cells. Taken together, this study describes the potential of innate immunity to generate a senescence inducing microenvironment after stimulation with TLR ligands. We provide 4 insight into the therapeutical mechanism of TLR ligands such as the TLR 7/8 ligand Imiquimod for topical melanoma therapy. Accordingly, our results argue for increased NK cell recruitment as a foundation for eliciting slanMo/NK cell crosstalk induced melanoma cell senescence.