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.

Neutrophils orchestrate the innate immune response against microorganisms and are increasingly recognized to modulate cancer development in primary tumors and metastases.To address their function in vivo, different approaches are used, the most common ones relying on antibody-mediated neutrophil depletion. By comparing the effects of two widely used antibodies, we demonstrate a strong efficacy but a lack of specificity for anti-Gr1. In contrast, anti-Ly6G lacks neutrophil-depletion capacity in C57BL/6 mice, which can be explained by an insufficient celerity of neutrophil clearance that is counterbalanced by exacerbated mobilization of immature cells. When combined with a secondary antibody, anti-Ly6G treatment results in specific and efficient neutrophil depletion. Using a mouse model of lung adenocarcinoma, we demonstrate the efficacy of this new approach to diminish primary tumor growth and propose the existence of a local intercellular communication between neutrophils and alveolar macrophages that fosters regulatory T cell proliferation in lung cancer.3

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.

Introduction: The management of malignant pleural mesothelioma (MPM) remains challenging with poor patient survival. Local therapies such as hyperthermic intrathoracic cisplatin (HITOC) have shown good tumor control in selected patients. HITOC was shown to increase MPM drug exposure while limiting systemic side effects but alternative mechanisms for HITOC are still lacking. Here, we hypothesized that HITOC induces an immune response directed against MPM which decreases cancer related mortality. Methods: We implanted AB12-luc MPM cells in the pleural cavity of BALB/c mice. A chemotherapy perfusion circuit was downsized to administer cisplatin (80mg/m2 equivalent dose) in the thoracic cavity at normo (37°C, ITOC) or hyperthermic (39°C, HITOC) conditions for 30 minutes. Tumor growth (visualized by bioluminescence) and mouse survival were then assessed. We determined tumor platinum content and distribution by inductively coupled plasma mass spectrometry (ICP-MS) and by laser ablation ICP-MS (LA-ICP-MS) respectively. We also questioned the impact of (H)ITOC on the MPM immune microenvironment (innate and adaptive immune cells, activity and checkpoint expression) by 16-colour flow cytometry and immunohistochemistry. Finally, tumor response to (H)ITOC was assessed in BALB/c athymic mice implanted with AB12-luc cells. Results: MPM tumor control and mouse survival were significantly improved by HITOC compared to controls (ITOC, saline 37 and 39°C). Tumor platinum content was significantly higher in HITOC compared to ITOC but was majorly located at the surface of tumors. HITOC enhanced MPM infiltration by CD8+Granzyme B+ T-cells and decreased the levels of MCHII−/CD80− (M2-like) macrophages compared to controls at day 7. Interestingly, immune checkpoint expression of PD1 and CTLA4 was significantly enhanced in CD8+ lymphocytes in HITOC treated MPM compared to controls at day 7. Finally, the lack of T lymphocytes (BALB/c athymic mice) abrogated the impact of HITOC on MPM control and mouse survival. Conclusion: HITOC improves MPM control through a T lymphocyte immune mediated response. The enhanced immune checkpoint (PD1/CTLA4) expression in CD8+ lymphocytes opens perspectives for the combination of HITOC with immune checkpoint inhibitors. Citation Format: Yameng Hao, Aspasia Gkasti, Louis-Emmanuel Chriqui, Damien Marie, Michel Gonzalez, Thorsten Krueger, Solange Peters, Paul J. Dyson, Etienne Meylan, Johanna Joyce, Sabrina Cavin, Jean Y. Perentes. Hyperthermic intrathoracic chemotherapy (HITOC) improves malignant pleural mesothelioma control through a tumor specific cytotoxic immune response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6399.