SIGLEC-5/14 Inhibits CD11b/CD18 Integrin Activation and Neutrophil-Mediated Tumor Cell Cytotoxicity.

Since the successful introduction of checkpoint inhibitors targeting the adaptive immune system, monoclonal antibodies inhibiting CD47-SIRPα interaction have shown promise in enhancing anti-tumor treatment efficacy. Apart from SIRPα, neutrophils express a broad repertoire of inhibitory receptors, including several members of the sialic acid-binding receptor (SIGLEC) family. Here, we demonstrate that interaction between tumor cell-expressed sialic acids and SIGLEC-5/14 on neutrophils inhibits antibody-dependent cellular cytotoxicity (ADCC). We observed that conjugate formation and trogocytosis, both essential processes for neutrophil ADCC, were limited by the sialic acid-SIGLEC-5/14 interaction. During neutrophil-tumor cell conjugate formation, we found that inhibition of the interaction between tumor-expressed sialic acids and SIGLEC-5/14 on neutrophils increased the CD11b/CD18 high affinity conformation. By dynamic acoustic force measurement, the binding between tumor cells and neutrophils was assessed. The interaction between SIGLEC-5/14 and the sialic acids was shown to inhibit the CD11b/CD18-regulated binding between neutrophils and antibody-opsonized tumor cells. Moreover, the interaction between sialic acids and SIGLEC-5/14-consequently hindered trogocytosis and tumor cell killing. In summary, our results provide evidence that the sialic acid-SIGLEC-5/14 interaction is an additional target for innate checkpoint blockade in the tumor microenvironment.

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.

Kindlin3-Dependent CD11b/CD18-Integrin Activation Is Required for Potentiation of Neutrophil Cytotoxicity by CD47-SIRPα Checkpoint Disruption.

The CD47-signal regulatory protein-alpha (SIRPα) immune checkpoint constitutes a therapeutic target in cancer, and initial clinical studies using inhibitors of CD47-SIRPα interactions in combination with tumor-targeting antibodies show promising results. Blockade of CD47-SIRPα interaction can promote neutrophil antibody-dependent cellular cytotoxicity (ADCC) toward antibody-opsonized targets. Neutrophils induce killing of antibody-opsonized tumor cells by a process identified as trogoptosis, a necrotic/lytic type of cancer cell death that involves trogocytosis, the antibody-mediated endocytic acquisition of cancer membrane fragments by neutrophils. Both trogocytosis and killing strictly depend on CD11b/CD18-(Mac-1)-mediated neutrophil-cancer cell conjugate formation, but the mechanism by which CD47-SIRPα checkpoint disruption promotes cytotoxicity has remained elusive. Here, by using neutrophils from patients with leukocyte adhesion deficiency type III carrying FERMT3 gene mutations, hence lacking the integrin-associated protein kindlin3, we demonstrated that CD47-SIRPα signaling controlled the inside-out activation of the neutrophil CD11b/CD18-integrin and cytotoxic synapse formation in a kindlin3-dependent fashion. Our findings also revealed a role for kindlin3 in trogocytosis and an absolute requirement in the killing process, which involved direct interactions between kindlin3 and CD18 integrin. Collectively, these results identified a dual role for kindlin3 in neutrophil ADCC and provide mechanistic insights into the way neutrophil cytotoxicity is governed by CD47-SIRPα interactions.

The Gardos effect drives erythrocyte senescence and leads to Lu/BCAM and CD44 adhesion molecule activation.

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)-containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel-mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

MKL1 deficiency results in a severe neutrophil motility defect due to impaired actin polymerization.

Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.

IgA-Mediated Killing of Tumor Cells by Neutrophils Is Enhanced by CD47-SIRPα Checkpoint Inhibition.

Therapeutic monoclonal antibodies (mAb), directed toward either tumor antigens or inhibitory checkpoints on immune cells, are effective in cancer therapy. Increasing evidence suggests that the therapeutic efficacy of these tumor antigen-targeting mAbs is mediated-at least partially-by myeloid effector cells, which are controlled by the innate immune-checkpoint interaction between CD47 and SIRPα. We and others have previously demonstrated that inhibiting CD47-SIRPα interactions can substantially potentiate antibody-dependent cellular phagocytosis and cytotoxicity of tumor cells by IgG antibodies both in vivo and in vitro IgA antibodies are superior in killing cancer cells by neutrophils compared with IgG antibodies with the same variable regions, but the impact of CD47-SIRPα on IgA-mediated killing has not been investigated. Here, we show that checkpoint inhibition of CD47-SIRPα interactions further enhances destruction of IgA antibody-opsonized cancer cells by human neutrophils. This was shown for multiple tumor types and IgA antibodies against different antigens, i.e., HER2/neu and EGFR. Consequently, combining IgA antibodies against HER2/neu or EGFR with SIRPα inhibition proved to be effective in eradicating cancer cells in vivo In a syngeneic in vivo model, the eradication of cancer cells was predominantly mediated by granulocytes, which were actively recruited to the tumor site by SIRPα blockade. We conclude that IgA-mediated tumor cell destruction can be further enhanced by CD47-SIRPα checkpoint inhibition. These findings provide a basis for targeting CD47-SIRPα interactions in combination with IgA therapeutic antibodies to improve their potential clinical efficacy in tumor patients.

FcγRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils.

The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface FcγRIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects.

Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.