Humanized MISTRG as a preclinical in vivo model to study human neutrophil-mediated immune processes.

Introduction: MISTRG mice have been genetically modified to allow development of a human myeloid compartment from engrafted human CD34+ haemopoietic stem cells, making them particularly suited to study the human innate immune system in vivo. Here, we characterized the human neutrophil population in these mice to establish a model that can be used to study the biology and contribution in immune processes of these cells in vivo. Methods and results: We could isolate human bone marrow neutrophils from humanized MISTRG mice and confirmed that all neutrophil maturation stages from promyelocytes (CD11b-CD16-) to end-stage segmented cells (CD11b+CD16+) were present. We documented that these cells possessed normal functional properties, including degranulation, reactive oxygen species production, adhesion, and antibody-dependent cellular cytotoxicity towards antibody-opsonized tumor cells ex vivo. The acquisition of functional capacities positively correlated with the maturation state of the cell. We found that human neutrophils were retained in the bone marrow of humanized MISTRG mice during steady state. However, the mature segmented CD11b+CD16+ human neutrophils were released from the bone marrow in response to two well-established neutrophil-mobilizing agents (i.e., G-CSF and/or CXCR4 antagonist Plerixafor). Moreover, the neutrophil population in the humanized MISTRG mice actively reacted to thioglycolate-induced peritonitis and could infiltrate implanted human tumors, as shown by flow cytometry and fluorescent microscopy. Discussion: These results show that functional human neutrophils are generated and can be studied in vivo using the humanized MISTRG mice, providing a model to study the various functions of neutrophils in inflammation and in tumors.

CD47-SIRPα Checkpoint Inhibition Enhances Neutrophil-Mediated Killing of Dinutuximab-Opsonized Neuroblastoma Cells.

High-risk neuroblastoma, especially after recurrence, still has a very low survival rate. Immune checkpoint inhibitors targeting T cells have shown remarkable clinical efficacy in adult solid tumors, but their effects in pediatric cancers have been limited so far. On the other hand, targeting myeloid immune checkpoints, such as CD47-SIPRα, provide the opportunity to enhance antitumor effects of myeloid cells, including that of neutrophils, especially in the presence of cancer-opsonizing antibodies. Disialoganglioside (GD2)-expressing neuroblastoma cells targeted with anti-GD2 antibody dinutuximab are in part eradicated by neutrophils, as they recognize and bind the antibody targeted tumor cells through their Fc receptors. Therapeutic targeting of the innate immune checkpoint CD47-SIRPα has been shown to promote the potential of neutrophils as cytotoxic cells in different solid tumor indications using different cancer-targeting antibodies. Here, we demonstrate that the capacity of neutrophils to kill dinutuximab-opsonized neuroblastoma cells is also controlled by the CD47-SIRPα axis and can be further enhanced by antagonizing CD47-SIRPα interactions. In particular, CD47-SIRPa checkpoint inhibition enhanced neutrophil-mediated ADCC of dinutuximab-opsonized adrenergic neuroblastoma cells, whereas mesenchymal neuroblastoma cells may evade immune recognition by a reduction of GD2 expression. These findings provide a rational basis for targeting CD47-SIRPα interactions to potentiate dinutuximab responsiveness in neuroblastomas with adrenergic phenotype.

G-CSF as a suitable alternative to GM-CSF to boost dinutuximab-mediated neutrophil cytotoxicity in neuroblastoma treatment.

BACKGROUND: Current immunotherapy for patients with high-risk neuroblastoma involves the therapeutic antibody dinutuximab that targets GD2, a ganglioside expressed on the majority of neuroblastoma tumors. Opsonized tumor cells are killed through antibody-dependent cellular cytotoxicity (ADCC), a process mediated by various immune cells, including neutrophils. The capacity of neutrophils to kill dinutuximab-opsonized tumor cells can be further enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which has been shown in the past to improve responses to anti-GD2 immunotherapy. However, access to GM-CSF (sargramostim) is limited outside of Northern America, creating a high clinical need for an alternative method to stimulate dinutuximab responsiveness in the treatment of neuroblastoma. In this in vitro study, we have investigated whether clinically well-established granulocyte colony-stimulating factor (G-CSF) can be a potentially suitable alternative for GM-CSF in the dinutuximab immunotherapy regimen of patients with neuroblastoma. METHODS: We compared the capacity of neutrophils stimulated either in vitro or in vivo with GM-CSF or G-CSF to kill dinutuximab-opsonized GD2-positive neuroblastoma cell lines and primary patient tumor material. Blocking experiments with antibodies inhibiting either respective Fc gamma receptors (FcγR) or neutrophil integrin CD11b/CD18 demonstrated the involvement of these receptors in the process of ADCC. Flow cytometry and live cell microscopy were used to quantify and visualize neutrophil-neuroblastoma interactions. RESULTS: We found that G-CSF was as potent as GM-CSF in enhancing the killing capacity of neutrophils towards neuroblastoma cells. This was observed with in vitro stimulated neutrophils, and with in vivo stimulated neutrophils from both patients with neuroblastoma and healthy donors. Enhanced killing due to GM-CSF or G-CSF stimulation was consistent regardless of dinutuximab concentration, tumor-to-neutrophil ratio and concentration of the stimulating cytokine. Both GM-CSF and G-CSF stimulated neutrophils required FcγRIIa and CD11b/CD18 integrin to perform ADCC, and this was accompanied by trogocytosis of tumor material by neutrophils and tumor cell death in both stimulation conditions. CONCLUSIONS: Our preclinical data support the use of G-CSF as an alternative stimulating cytokine to GM-CSF in the treatment of high-risk neuroblastoma with dinutuximab, warranting further testing of G-CSF in a clinical setting.

Plasticity in Pro- and Anti-tumor Activity of Neutrophils: Shifting the Balance.

Over the last decades, cancer immunotherapies such as checkpoint blockade and adoptive T cell transfer have been a game changer in many aspects and have improved the treatment for various malignancies considerably. Despite the clinical success of harnessing the adaptive immunity to combat the tumor, the benefits of immunotherapy are still limited to a subset of patients and cancer types. In recent years, neutrophils, the most abundant circulating leukocytes, have emerged as promising targets for anti-cancer therapies. Traditionally regarded as the first line of defense against infections, neutrophils are increasingly recognized as critical players during cancer progression. Evidence shows the functional plasticity of neutrophils in the tumor microenvironment, allowing neutrophils to exert either pro-tumor or anti-tumor effects. This review describes the tumor-promoting roles of neutrophils, focusing on their myeloid-derived suppressor cell activity, as well as their role in tumor elimination, exerted mainly via antibody-dependent cellular cytotoxicity. We will discuss potential approaches to therapeutically target neutrophils in cancer. These include strategies in humans to either silence the pro-tumor activity of neutrophils, or to activate or enhance their anti-tumor functions. Redirecting neutrophils seems a promising approach to harness innate immunity to improve treatment for cancer patients.