Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal leukocidins in the pathogenesis of bovine S. aureus disease. We show that LukAB, in contrast to the γ-hemolysins, LukED, and LukMF′, was unable to kill bovine neutrophils, and identified CXCR2 as a bovine receptor for HlgAB and LukED. Furthermore, we assessed functional leukocidin secretion by bovine mastitis isolates and observed that, although leukocidin production was strain dependent, LukMF′ was most abundantly secreted and the major toxin killing bovine neutrophils. To determine the role of LukMF′ in bovine mastitis, cattle were challenged with high (S1444) or intermediate (S1449, S1463) LukMF′-producing isolates. Only animals infected with S1444 developed severe clinical symptoms. Importantly, LukM was produced in vivo during the course of infection and levels in milk were associated with the severity of mastitis. Altogether, these findings underline the importance of LukMF′ as a virulence factor and support the development of therapeutic approaches targeting LukMF′ to control S. aureus mastitis in cattle.

The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14 + DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system.

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component.

Cancer immunotherapies have induced long-lasting responses in cancer patients including those with melanoma and head and neck squamous cell carcinoma (HNSCC). However, the majority of treated patients does not achieve clinical benefit from immunotherapy because of systemic tumor-induced immunosuppression. Monocytic myeloid-derived suppressor cells (M-MDSCs) are implicated as key players in inhibiting anti-tumor immune responses and their frequencies are closely associated with tumor progression. Tumor-derived signals, including signaling via STAT3-COX-2, induce the transformation of monocytic precursors into suppressive M-MDSCs. In a retrospective assessment, we observed that survival of melanoma patients undergoing dendritic cell vaccination was negatively associated with blood M-MDSC levels. Previously, it was shown that platinum-based chemotherapeutics inhibit STAT signaling. Here, we show that cisplatin and oxaliplatin treatment interfere with the development of M-MDSCs, potentially synergizing with cancer immunotherapy. In vitro , subclinical doses of platinum-based drugs prevented the generation of COX-2 + M-MDSCs induced by tumor cells from melanoma patients. This was confirmed in HNSCC patients where intravenous cisplatin treatment drastically lowered M-MDSC frequency while monocyte levels remained stable. In treated patients, expression of COX-2 and arginase-1 in M-MDSCs was significantly decreased after two rounds of cisplatin, indicating inhibition of STAT3 signaling. In line, the capacity of M-MDSCs to inhibit activated T cell responses ex vivo was significantly decreased after patients received cisplatin. These results show that platinum-based chemotherapeutics inhibit the expansion and suppressive activity of M-MDSCs in vitro and in cancer patients. Therefore, platinum-based drugs have the potential to enhance response rates of immunotherapy by overcoming M-MDSC-mediated immunosuppression.

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