BYON4228 is a pan-allelic antagonistic SIRPα antibody that potentiates destruction of antibody-opsonized tumor cells and lacks binding to SIRPγ on T cells.

BACKGROUND: Preclinical studies have firmly established the CD47-signal-regulatory protein (SIRP)α axis as a myeloid immune checkpoint in cancer, and this is corroborated by available evidence from the first clinical studies with CD47 blockers. However, CD47 is ubiquitously expressed and mediates functional interactions with other ligands as well, and therefore targeting of the primarily myeloid cell-restricted inhibitory immunoreceptor SIRPα may represent a better strategy. METHOD: We generated BYON4228, a novel SIRPα-directed antibody. An extensive preclinical characterization was performed, including direct comparisons to previously reported anti-SIRPα antibodies. RESULTS: BYON4228 is an antibody directed against SIRPα that recognizes both allelic variants of SIRPα in the human population, thereby maximizing its potential clinical applicability. Notably, BYON4228 does not recognize the closely related T-cell expressed SIRPγ that mediates interactions with CD47 as well, which are known to be instrumental in T-cell extravasation and activation. BYON4228 binds to the N-terminal Ig-like domain of SIRPα and its epitope largely overlaps with the CD47-binding site. BYON4228 blocks binding of CD47 to SIRPα and inhibits signaling through the CD47-SIRPα axis. Functional studies show that BYON4228 potentiates macrophage-mediated and neutrophil-mediated killing of hematologic and solid cancer cells in vitro in the presence of a variety of tumor-targeting antibodies, including trastuzumab, rituximab, daratumumab and cetuximab. The silenced Fc region of BYON4228 precludes immune cell-mediated elimination of SIRPα-positive myeloid cells, implying anticipated preservation of myeloid immune effector cells in patients. The unique profile of BYON4228 clearly distinguishes it from previously reported antibodies representative of agents in clinical development, which either lack recognition of one of the two SIRPα polymorphic variants (HEFLB), or cross-react with SIRPγ and inhibit CD47-SIRPγ interactions (SIRPAB-11-K322A, 1H9), and/or have functional Fc regions thereby displaying myeloid cell depletion activity (SIRPAB-11-K322A). In vivo, BYON4228 increases the antitumor activity of rituximab in a B-cell Raji xenograft model in human SIRPαBIT transgenic mice. Finally, BYON4228 shows a favorable safety profile in cynomolgus monkeys. CONCLUSIONS: Collectively, this defines BYON4228 as a preclinically highly differentiating pan-allelic SIRPα antibody without T-cell SIRPγ recognition that promotes the destruction of antibody-opsonized cancer cells. Clinical studies are planned to start in 2023.

Neutrophils as immune effector cells in antibody therapy in cancer.

Tumor-targeting monoclonal antibodies are available for a number of cancer cell types (over)expressing the corresponding tumor antigens. Such antibodies can limit tumor progression by different mechanisms, including direct growth inhibition and immune-mediated mechanisms, in particular complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be mediated by various types of immune cells, including neutrophils, the most abundant leukocyte in circulation. Neutrophils express a number of Fc receptors, including Fcγ- and Fcα-receptors, and can therefore kill tumor cells opsonized with either IgG or IgA antibodies. In recent years, important insights have been obtained with respect to the mechanism(s) by which neutrophils engage and kill antibody-opsonized cancer cells and these findings are reviewed here. In addition, we consider a number of additional ways in which neutrophils may affect cancer progression, in particular by regulating adaptive anti-cancer immunity.

Targeting the CD47-SIRPα Innate Immune Checkpoint to Potentiate Antibody Therapy in Cancer by Neutrophils.

In the past 25 years, a considerable number of therapeutic monoclonal antibodies (mAb) against a variety of tumor-associated antigens (TAA) have become available for the targeted treatment of hematologic and solid cancers. Such antibodies opsonize cancer cells and can trigger cytotoxic responses mediated by Fc-receptor expressing immune cells in the tumor microenvironment (TME). Although frequently ignored, neutrophils, which are abundantly present in the circulation and many cancers, have demonstrated to constitute bona fide effector cells for antibody-mediated tumor elimination in vivo. It has now also been established that neutrophils exert a unique mechanism of cytotoxicity towards antibody-opsonized tumor cells, known as trogoptosis, which involves Fc-receptor (FcR)-mediated trogocytosis of cancer cell plasma membrane leading to a lytic/necrotic type of cell death. However, neutrophils prominently express the myeloid inhibitory receptor SIRPα, which upon interaction with the 'don't eat me' signal CD47 on cancer cells, limits cytotoxicity, forming a mechanism of resistance towards anti-cancer antibody therapeutics. In fact, tumor cells often overexpress CD47, thereby even more strongly restricting neutrophil-mediated tumor killing. Blocking the CD47-SIRPα interaction may therefore potentiate neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) towards cancer cells, and various inhibitors of the CD47-SIRPα axis are now in clinical studies. Here, we review the role of neutrophils in antibody therapy in cancer and their regulation by the CD47-SIRPα innate immune checkpoint. Moreover, initial results of CD47-SIRPα blockade in clinical trials are discussed.

Neutrophil phenotypes and functions in cancer: A consensus statement.

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer.

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.

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.

C-Reactive Protein Enhances IgG-Mediated Cellular Destruction Through IgG-Fc Receptors in vitro.

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.

m6A methylation potentiates cytosolic dsDNA recognition in a sequence-specific manner.

Nucleic acid sensing through pattern recognition receptors is critical for immune recognition of microbial infections. Microbial DNA is frequently methylated at the N6 position of adenines (m6A), a modification that is rare in mammalian host DNA. We show here how that m6A methylation of 5'-GATC-3' motifs augments the immunogenicity of synthetic double-stranded (ds)DNA in murine macrophages and dendritic cells. Transfection with m6A-methylated DNA increased the expression of the activation markers CD69 and CD86, and of Ifnß, iNos and Cxcl10 mRNA. Similar to unmethylated cytosolic dsDNA, recognition of m6A DNA occurs independently of TLR and RIG-I signalling, but requires the two key mediators of cytosolic DNA sensing, STING and cGAS. Intriguingly, the response to m6A DNA is sequence-specific. m6A is immunostimulatory in some motifs, but immunosuppressive in others, a feature that is conserved between mouse and human macrophages. In conclusion, epigenetic alterations of DNA depend on the context of the sequence and are differentially perceived by innate cells, a feature that could potentially be used for the design of immune-modulating therapeutics.

Neutrophil specific granule and NETosis defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.

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.

Comparison of the PU.1 transcriptional regulome and interactome in human and mouse inflammatory dendritic cells.

Dendritic cells (DCs) are key immune modulators and are able to mount immune responses or tolerance. DC differentiation and activation imply a plethora of molecular and cellular responses, including transcriptional changes. PU.1 is a highly expressed transcription factor in DCs and coordinates relevant aspects of DC biology. Due to their role as immune regulators, DCs pose as a promising immunotherapy tool. However, some of their functional features, such as survival, activation, or migration, are compromised due to the limitations to simulate in vitro the physiologic DC differentiation process. A better knowledge of transcriptional programs would allow the identification of potential targets for manipulation with the aim of obtaining "qualified" DCs for immunotherapy purposes. Most of the current knowledge regarding DC biology derives from studies using mouse models, which not always find a parallel in human. In the present study, we dissect the PU.1 transcriptional regulome and interactome in mouse and human DCs, in the steady state or LPS activated. The PU.1 transcriptional regulome was identified by performing PU.1 chromatin immunoprecipitation followed by high-throughput sequencing and pairing these data with RNAsequencing data. The PU.1 interactome was identified by performing PU.1 immunoprecipitation followed by mass spectrometry analysis. Our results portray PU.1 as a pivotal factor that plays an important role in the regulation of genes required for proper DC activation and function, and assures the repression of nonlineage genes. The interspecies differences between human and mouse DCs are surprisingly substantial, highlighting the need to study the biology of human DCs.

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR-/-) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.

The association and functional relevance of genetic variation in low-to-medium-affinity Fc-gamma receptors with clinical platelet transfusion refractoriness.

BACKGROUND: Inadequate responses to platelet transfusions (i.e., platelet transfusion refractoriness [PLT refractoriness]) are a serious problem. Multiple factors contribute to low yields upon platelet transfusion, among which are platelet-reactive allo-antibodies. Platelet-reactive allo-antibodies occur in up to 30% of patients receiving multiple transfusions, and presumably lead to rapid destruction of the transfused platelets via receptors for IgG, the Fc-gamma receptors (FcγRs). Genetic variation in FcγRs is associated with susceptibility to immune thrombocytopenia, in which autoantibodies against platelets cause thrombocytopenia. OBJECTIVES: We hypothesized that genetic variation in FcγRs may also influence PLT refractoriness in allo-immunized patients and could help in identifying the patients at risk. PATIENTS/METHODS: Patients with severe PLT refractoriness for whom diagnostic testing for allo-immunization was requested in the period of 2005 to 2013 were retrospectively included. A case-control study was performed comparing patients in whom platelet-reactive antibodies were detected (n = 181) with ethnically matched healthy controls (n = 180) to determine differences in all known functional copy number variations and single nucleotide polymorphisms in FcγRs. RESULTS AND CONCLUSIONS: None of the tested FcγR genetic variations seemed associated with the development of severe PLT refractoriness. In contrast to observations in immune thrombocytopenia, genetic variation in FcγRs does not seem to influence the chance to develop PLT refractoriness. Our results do not support determination of FcγR genetic background as a means to identify patients most at risk for PLT refractoriness.

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.

Myeloid Ezh2 Deficiency Limits Atherosclerosis Development.

Macrophages define a key component of immune cells present in atherosclerotic lesions and are central regulators of the disease. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways in macrophages might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by polycomb repressive complex with EZH2 as the catalytic subunit. EZH2 is described to increase macrophage inflammatory responses by supressing the suppressor of cytokine signaling, Socs3. We previously showed that myeloid deletion of Kdm6b, an enzymes that in contrast to EZH2 removes repressive histone H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function and importance of EZH2 in macrophage inflammatory responses, we here studied the role of myeloid EZH2 in atherosclerosis. A myeloid-specific Ezh2 deficient mouse strain (Ezh2del) was generated (LysM-cre+ x Ezh2fl/fl) and bone marrow from Ezh2del or Ezh2wt mice was transplanted to Ldlr-/- mice which were fed a high fat diet for 9 weeks to study atherosclerosis. Atherosclerotic lesion size was significantly decreased in Ezh2del transplanted mice compared to control. The percentage of macrophages in the atherosclerotic lesion was similar, however neutrophil numbers were lower in Ezh2del transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased in Ezh2del mice. Moreover, peritoneal Ezh2del foam cells showed a reduction in the inflammatory response with reduced production of nitric oxide, IL-6 and IL-12. In Conclusion, myeloid Ezh2 deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.

Activated neutrophils exert myeloid-derived suppressor cell activity damaging T cells beyond repair.

Myeloid-derived suppressor cells (MDSCs) have the capacity to suppress T-cell-mediated immune responses and impact the clinical outcome of cancer, infections, and transplantation settings. Although MDSCs were initially described as bone marrow-derived immature myeloid cells (either monocytic or granulocytic MDSCs), mature neutrophils have been shown to exert MDSC activity toward T cells in ways that remain unclear. In this study, we demonstrated that human neutrophils from both healthy donors and cancer patients do not exert MDSC activity unless they are activated. By using neutrophils with genetically well-defined defects, we found that reactive oxygen species (ROS) and granule-derived constituents are required for MDSC activity after direct CD11b-dependent interactions between neutrophils and T cells. In addition to these cellular interactions, neutrophils are engaged in the uptake of pieces of T-cell membrane, a process called trogocytosis. Together, these interactions led to changes in T-cell morphology, mitochondrial dysfunction, and adenosine triphosphate depletion, as indicated by electron microscopy, mass spectrometry, and metabolic parameters. Our studies characterize the different steps by which activated mature neutrophils induce functional T-cell nonresponsiveness and irreparable cell damage.

Tissue-specific expression of IgG receptors by human macrophages ex vivo.

Recently it was discovered that tissue-resident macrophages derive from embryonic precursors, not only from peripheral blood monocytes, and maintain themselves by self-renewal. Most in-vitro studies on macrophage biology make use of in-vitro cultured human monocyte-derived macrophages. Phagocytosis of IgG-opsonized particles by tissue-resident macrophages takes place via interaction with IgG receptors, the Fc-gamma receptors (FcγRs). We investigated the FcγR expression on macrophages both in-vivo and ex-vivo from different human tissues. Upon isolation of primary human macrophages from bone marrow, spleen, liver and lung, we observed that macrophages from all studied tissues expressed high levels of FcγRIII, which was in direct contrast with the low expression on blood monocyte-derived macrophages. Expression levels of FcγRI were highly variable, with bone marrow macrophages showing the lowest and alveolar macrophages the highest expression. Kupffer cells in the liver were the only tissue-resident macrophages that expressed the inhibitory IgG receptor, FcγRIIB. This inhibitory receptor was also found to be expressed by sinusoidal endothelial cells in the liver. In sum, our immunofluorescence data combined with ex-vivo stainings of isolated macrophages indicated that tissue-resident macrophages are remarkably unique and different from monocyte-derived macrophages in their phenotypic expression of IgG receptors. Tissue macrophages show distinct tissue-specific FcγR expression patterns.

Identification of genetic biomarkers for alloimmunization in sickle cell disease.

Most sickle cell disease (SCD) patients rely on blood transfusion as their main treatment strategy. However, frequent blood transfusion poses the risk of alloimmunization. On average, 30% of SCD patients will alloimmunize while other patient groups form antibodies less frequently. Identification of genetic markers may help to predict which patients are at risk to form alloantibodies. The aim of this study was to evaluate whether genetic variations in the Toll-like receptor pathway or in genes previously associated with antibody-mediated conditions are associated with red blood cell (RBC) alloimmunization in a cohort of SCD patients. In this case-control study, cases had a documented history of alloimmunization while controls had received ≥20 RBC units without alloantibody formation. We used a customized single nucleotide polymorphism (SNP) panel to genotype 690 SNPs in 275 (130 controls, 145 cases) patients. Frequencies were compared using multiple logistic regression analysis. In our primary analysis, no SNPs were found to be significantly associated with alloimmunization after correction for multiple testing. However, in a secondary analysis with a less stringent threshold for significance we found 19 moderately associated SNPs. Among others, SNPs in TLR1/TANK and MALT1 were associated with a higher alloimmunization risk, while SNPs in STAM/IFNAR1 and STAT4 conferred a lower alloimmunization risk.