Immunoreceptors on neutrophils.

Neutrophils play a critical role in the host defense against infection, and they are able to perform a variety of effector mechanisms for this purpose. However, there are also a number of pathological conditions, including autoimmunity and cancer, in which the activities of neutrophils can be harmful to the host. Thus the activities of neutrophils need to be tightly controlled. As in the case of other immune cells, many of the neutrophil effector functions are regulated by a series of immunoreceptors on the plasma membrane. Here, we review what is currently known about the functions of the various individual immunoreceptors and their signaling in neutrophils. While these immunoreceptors allow for the recognition of a diverse range of extracellular ligands, such as cell surface structures (like proteins, glycans and lipids) and extracellular matrix components, they commonly signal via conserved ITAM or ITIM motifs and their associated downstream pathways that depend on the phosphorylation of tyrosine residues in proteins and/or inositol lipids. This allows for a balanced homeostatic regulation of neutrophil effector functions. Given the number of available immunoreceptors and their fundamental importance for neutrophil behavior, it is perhaps not surprising that pathogens have evolved means to evade immune responses through some of these pathways. Inversely, some of these receptors evolved to specifically recognize these pathogens. Finally, some interactions mediated by immunoreceptors in neutrophils have been identified as promising targets for therapeutic intervention.

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody-dependent cellular cytotoxicity towards cancer cells.

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti-cancer antibodies act through different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47-SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab-coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa-131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa-131R. Furthermore, ADCC was consistently enhanced by targeting CD47-SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47-SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47-SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.

Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components.

Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of antibacterial activity, but less is known about their fungal killing capacity. We investigated the neutrophil-mediated cytotoxic response against C. albicans and A. fumigatus in detail. Whereas G-CSF/dexamethasone-mobilized neutrophils appeared less mature as compared to neutrophils from untreated controls, these cells exhibited normal ROS production by the NADPH oxidase system and an unaltered granule mobilization capacity upon stimulation. G-CSF/dexamethasone-mobilized neutrophils efficiently inhibited A. fumigatus germination and killed Aspergillus and Candida hyphae, but the killing of C. albicans yeasts was distinctly impaired. Following normal Candida phagocytosis, analysis by mass spectrometry of purified phagosomes after fusion with granules demonstrated that major constituents of the antimicrobial granule components, including major basic protein (MBP), were reduced. Purified MBP showed candidacidal activity, and neutrophil-like Crisp-Cas9 NB4-KO-MBP differentiated into phagocytes were impaired in Candida killing. Together, these findings indicate that G-CSF/dexamethasone-mobilized neutrophils for transfusion purposes have a selectively impaired capacity to kill Candida yeasts, as a consequence of an altered neutrophil granular content.

Sodium stibogluconate and CD47-SIRPα blockade overcome resistance of anti-CD20-opsonized B cells to neutrophil killing.

Anti-CD20 antibodies such as rituximab are broadly used to treat B-cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC toward solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils seem to be incapable of killing rituximab-opsonized B-cell lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B-cell lymphoma cells, but this only reduces CD20 surface expression and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B-cell lymphoma cells toward neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmaniasis drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells but enabled trogoptotic killing of B-cell lymphoma cells by turning trogocytosis from a mechanism that contributes to resistance into a cytotoxic anti-cancer mechanism. Tumor cell killing in the presence of SSG required both antibody opsonization of the target cells and disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B-cell malignancies.

Cancer cells resist antibody-mediated destruction by neutrophils through activation of the exocyst complex.

BACKGROUND: Neutrophils kill antibody-opsonized tumor cells using trogocytosis, a unique mechanism of destruction of the target plasma. This previously unknown cytotoxic process of neutrophils is dependent on antibody opsonization, Fcγ receptors and CD11b/CD18 integrins. Here, we demonstrate that tumor cells can escape neutrophil-mediated cytotoxicity by calcium (Ca2+)-dependent and exocyst complex-dependent plasma membrane repair. METHODS: We knocked down EXOC7 or EXOC4, two exocyst components, to evaluate their involvement in tumor cell membrane repair after neutrophil-induced trogocytosis. We used live cell microscopy and flow cytometry for visualization of the host and tumor cell interaction and tumor cell membrane repair. Last, we reported the mRNA levels of exocyst in breast cancer tumors in correlation to the response in trastuzumab-treated patients. RESULTS: We found that tumor cells can evade neutrophil antibody-dependent cellular cytotoxicity (ADCC) by Ca2+-dependent cell membrane repair, a process induced upon neutrophil trogocytosis. Absence of exocyst components EXOC7 or EXOC4 rendered tumor cells vulnerable to neutrophil-mediated ADCC (but not natural killer cell-mediated killing), while neutrophil trogocytosis remained unaltered. Finally, mRNA levels of exocyst components in trastuzumab-treated patients were inversely correlated to complete response to therapy. CONCLUSIONS: Our results support that neutrophil attack towards antibody-opsonized cancer cells by trogocytosis induces an active repair process by the exocyst complex in vitro. Our findings provide insight to the possible contribution of neutrophils in current antibody therapies and the tolerance mechanism of tumor cells and support further studies for potential use of the exocyst components as clinical biomarkers.

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.

Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis.

Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions.