RESUMO
Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models.
Assuntos
Antígenos Ly/imunologia , Neutrófilos/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Antígenos Ly/genética , Medula Óssea/imunologia , Morte Celular , Modelos Animais de Doenças , Expressão Gênica , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
OBJECTIVES: Malignant pleural mesothelioma (MPM) is a deadly disease with limited treatment options. Approaches to enhance patient immunity against MPM have been tested but shown variable results. Previously, we have demonstrated interesting vascular modulating properties of low-dose photodynamic therapy (L-PDT) on MPM. Here, we hypothesized that L-PDT vascular modulation could favour immune cell extravasation in MPM and improve tumour control in combination with immune checkpoint inhibitors. METHODS: First, we assessed the impact of L-PDT on vascular endothelial E-selectin expression, a key molecule for immune cell extravasation, in vitro and in a syngeneic murine model of MPM. Second, we characterized the tumour immune cell infiltrate by 15-colour flow cytometry analysis 2 and 7 days after L-PDT treatment of the murine MPM model. Third, we determined how L-PDT combined with immune checkpoint inhibitor anti-CTLA4 affected tumour growth in a murine MPM model. RESULTS: L-PDT significantly enhanced E-selectin expression by endothelial cells in vitro and in vivo. This correlated with increased CD8+ T cells and activated antigen-presenting cells (CD11b+ dendritic cells and macrophages) infiltration in MPM. Also, compared to anti-CTLA4 that only affects tumour growth, the combination of L-PDT with anti-CTLA4 caused complete MPM regression in 37.5% of animals. CONCLUSIONS: L-PDT enhances E-selectin expression in the MPM endothelium, which favours immune infiltration of tumours. The combination of L-PDT with immune checkpoint inhibitor anti-CTLA4 allows best tumour control and regression.