Expression Of Fc Gamma Receptors Research Articles

Broad Epitope Coverage of Therapeutic Multi-Antibody Combinations Targeting SARS-CoV-2 Boosts In Vivo Protection and Neutralization Potency to Corner an Immune-Evading Virus.

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.

Fc-gamma receptor expression and cytokine responses to intravenous human immunoglobulin in whole blood from non-pregnant and pregnant women and newborns.

Intravenous immunoglobulin (IVIG) can suppress the inflammatory response in adults, but its role in pregnant women and newborns is poorly studied. While the adult immune system is considered mature, it is immature in neonates and suppressed in pregnancy. Since the immune response differs in these 3 groups, the use of IVIG could differentially modulate the immune response. We aimed to explore the effect of IVIG on myeloid blood cells from non-pregnant women, pregnant women and newborns. Whole blood from healthy donors was incubated with lipopolysaccharide (LPS) and/or IVIG. After 0 h, 24 h and 48 h of culture, Fc-gamma receptor (CD16, CD32 and CD64) expression, monocyte and neutrophil bacterial phagocytosis, and cytokine and chemokine concentrations were determined in the supernatant. The baseline expression of monocyte CD16 was higher in newborns than in adult women, but the expression of CD32 and CD64 was similar between groups. Furthermore, LPS and IVIG stimulation, together or separately, did not change Fc-gamma receptor expression in monocytes or neutrophils and did not modify their phagocytosis capacity. On the other hand, IVIG did not downregulate the proinflammatory cytokine response induced by LPS in any group. Interestingly, IVIG induced a strong interleukin 8 (IL-8) response in neonates but not in non-pregnant or pregnant women. Our results show that IVIG did not induce changes in Fc-gamma receptor expression, phagocytic ability, or the cytokine response to LPS in blood cells from neonates, non-pregnant or pregnant women. However, IVIG induced a strong IL-8 response in neonates that could improve immunity.

An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody.

Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743ng/mL and can detect 1500ng/mL of a surrogate positive control antibody in the presence of 1500ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas. This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

A Comparison Between 1 Day versus 7 Days of Sepsis in Mice with the Experiments on LPS-Activated Macrophages Support the Use of Intravenous Immunoglobulin for Sepsis Attenuation.

BackgroundBecause survival and death after sepsis are partly due to a proper immune adaptation and immune dysregulation, respectively, survivors and moribund mice after cecal ligation and puncture (CLP) sepsis surgery and in vitro macrophage experiments were explored.MethodsCharacteristics of mice at 1-day and 7-days post-CLP, the representative of moribund mice (an innate immune hyper-responsiveness) and survivors (a successful control on innate immunity), respectively. In parallel, soluble heat aggregated immunoglobulin (sHA-Ig), a representative of immune complex, was tested in lipopolysaccharide (LPS)-activated macrophages together with a test of intravenous immunoglobulin (IVIG), a molecule of adaptive immunity, on CLP sepsis mice.ResultsExcept for a slight increase in alanine transaminase (liver injury), IL-10, endotoxemia, and gut leakage (FITC-dextran assay), most of the parameters in survivors (7-days post-CLP) were normalized, with enhanced adaptive immunity, including serum immunoglobulin (using serum protein electrophoresis) and activated immune cells in spleens (flow cytometry analysis). The addition of sHA-Ig in LPS-activated macrophages reduced supernatant cytokines, cell energy (extracellular flux analysis), reactive oxygen species (ROS), several cell activities (proteomic analysis), and Fc gamma receptors (FcgRs) expression. The loss of anti-inflammatory effect of sHA-Ig in LPS-activated macrophages from mice with a deficiency on Fc gamma receptor IIb (FcgRIIb-/-), the only inhibitory signaling of FcgRs family, when compared with wild-type macrophages, implying the FcgRIIb-dependent mechanism. Moreover, IVIG attenuated sepsis severity in CLP mice as evaluated by serum creatinine, liver enzyme (alanine transaminase), serum cytokines, spleen apoptosis, and abundance of dendritic cells in the spleen (24-h post-CLP) and survival analysis.ConclusionImmunoglobulin attenuated LPS-activated macrophages, partly, through the reduced cell energy of macrophages and might play a role in sepsis immune hyper-responsiveness. Despite the debate over IVIG’s use in sepsis, IVIG might be beneficial in sepsis with certain conditions.

An Afucosylated Anti-CD32b Monoclonal Antibody Induced Platelet-Mediated Adverse Events in a Human Fcγ Receptor Transgenic Mouse Model and Its Potential Human Translatability.

To assess the safety and tolerability of NVS32b, a monoclonal, afucosylated, anti-CD32b (FCGR2B) antibody, we used a humanized transgenic (Tg) mouse model that expresses all human Fc gamma receptors (FCGRs) while lacking all mouse FCGRs. Prior to its use, we extensively characterized the model. We found expression of all human FCGRs in a pattern similar to humans with some exceptions, such as low CD32 expression on T cells (detected with the pan CD32 antibody but more notably with the CD32b-specific antibody), variation in the transgene copy number, integration of additional human genes, and overall higher expression of all FCGRs on myeloid cells compared to human. Unexpectedly, NVS32b induced severe acute generalized thrombosis in huFCGR mice upon iv dosing. Mechanistic evaluation on huFCGR and human platelets revealed distinct binding, activation, and aggregation driven by NVS32b in both species. In huFCGR mice, the anti-CD32b antibody NVS32b binds platelet CD32a via both Fc and/or complementarity determining region (CDR) causing their activation while in human, NVS32b binding requires platelet preactivation and interaction of platelet CD32a via the Fc portion and an unknown platelet epitope via the CDR portion of NVS32b. We deemed the huFCGR mice to be overpredictive of the NVS32b-associated human thrombotic risk.

Patient Data Supports the Rationale of Low Dose Cyclophosphamide to Potentiate the Anti-Myeloma Activity of Daratumumab through Augmentation of Macrophage-Induced ADCP

Introduction:Daratumumab (Dara) is the first CD38-directed monoclonal antibody approved for the treatment of Multiple Myeloma (MM). Dara is thought to have pleiotropic mechanisms of activity, including antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). We focused our attention on potential mechanisms of enhancing macrophage (Mφ)-mediated ADCP and have highlighted that exposure of MM cells to low doses of Cyclophosphamide (Cy) led to a secretory response, which greatly augments Mφ-induced ADCP of Dara-coated MM cells1. Here, we sought to confirm this phenomenon in MM and establish the in-vivo relevance of these findings as seen in newly diagnosed MM patients enrolled in CyBorD-DARA phase 1b clinical trial (NCT02951819).Materials and Methods:Bone Marrow (BM) and peripheral blood (PB) samples were collected in EDTA vacutainers from newly diagnosed MM patients recruited to the CyBorD-DARA clinical trial both prior to- and 24hrs after Cy (150-300mg/m2) treatment. Total lymphocyte, monocyte and neutrophil counts were quantified by flow cytometry. Mononuclear cells were prepared and analysed by 8-colour flow cytometry to quantify monocyte, lymphocyte and plasma subsets as well as determine changes in surface expression profiles following treatment. As previously described, MM cells were conditioned with low-dose Cy for 24hrs. Media containing chemotherapy agents were removed and replaced with fresh media for 24hrs (TCM)1. THP-1 Mφ were conditioned with TCM for up to 48hrs and either characterised or incubated at 2:1 effector target ratio with CFSE labelled MM1S cells ± Cytochalasin D for 18hrs in the presence or absence of Dara/Isotype control. The % of Dara-specific tumour cell clearance was calculated to determine if the mechanism of tumour cell clearance was mediated by Mφ-mediated ADCP.Results:In patients receiving Cy for 24hrs, total BM and PB neutrophil numbers were increased (P<0.01, n=11), lymphocyte numbers were decreased (P<0.01, n=11) while monocyte numbers remain unchanged (P>0.05, n=11). More specifically, an increase in CD19+ B cells (P<0.05, n=6) and a decrease in natural killer cells (NK) (P<0.05, n=6) were observed. Interestingly, the number of Mφ precursor monocytes did not change following Cy treatment in both BM and PB of these patients. As innate immune cells are essential in immune surveillance, coupled with our finding that NK cells are reduced, this highlights a potential role for Mφ in Cy induced anti-tumour activity. Additionally, data previously presented highlighted that exposure of Mφ to TCM from Cy treated MM cells significantly enhanced % of Dara-specific clearance of MM cells) p<0.01). We also confirmed ADCP and ADCC as a likely mechanisms of tumour cell clearance in vitro.Furthermore, we observed a marked reduction in expression of the “don't eat me” antigen, CD47, on chemotherapy-treated MM cells in vitro, which we have now confirmed in MM cells isolated from PB and BM of patients (n=2) following Cy treatment, which may enhance phagocytosis. We also observed increased expression of other targetable markers including BCMA (p<0.05), SLAMF7 (p=0.053), PD-L1 (p<0.001) and PD-L2 (p<0.001) in both RPMI8226 or MM1S MM cell lines following low dose Cy treatment in vitro. To date, we can confirm similar findings for both BCMA (p<0.05, n=5) and SLAMF7 (p<0.05, n=3) in patient samples. Furthermore, our in vitro observation of induced CD64 Fc gamma receptor expression on conditioned Mφ following single-agent Cy treatment has been confirmed in Mφ isolated from both BM (P<0.05, n=11) and PB (P<0.01, n=11) of newly diagnosed MM patients following Cy treatment. This finding emphasizes the potential physiological relevance of our in vitro data, implicating Cy in the potentiation of Mφ mediated ADCP in Dara specific anti-tumour immunity.Conclusions:Observations in Mφ precursors isolated from patients' BM and PB following 24hrs Cy treatment, confirm our previous in vitro findings which suggest a role for Cy potentiation of Mφ mediated ADCP in anti-tumour efficacy of Dara. These findings provide a rationale for the efficacy observed to date when combining Cy with Dara in the ongoing CyBorD-DARA clinical trial. Additionally, increases in BCMA and SLAMF7 highlight the potential of other combination immune therapies to further enhance therapeutic efficacy in these patients.1. Rigalou et al, Blood 2016 128:2101 [Display omitted] DisclosuresChiu:Janssen: Employment. Sasser:Janssen Pharmaceuticals: Employment. Ryan:Janssen: Research Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlycoMimetics Inc: Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Autoimmune regulation of chronic pain.

Chronic pain is an unpleasant and debilitating condition that is often poorly managed by existing therapeutics. Reciprocal interactions between the nervous system and the immune system have been recognized as playing an essential role in the initiation and maintenance of pain. In this review, we discuss how neuroimmune signaling can contribute to peripheral and central sensitization and promote chronic pain through various autoimmune mechanisms. These pathogenic autoimmune mechanisms involve the production and release of autoreactive antibodies from B cells. Autoantibodies-ie, antibodies that recognize self-antigens-have been identified as potential molecules that can modulate the function of nociceptive neurons and thereby induce persistent pain. Autoantibodies can influence neuronal excitability by activating the complement pathway; by directly signaling at sensory neurons expressing Fc gamma receptors, the receptors for the Fc fragment of immunoglobulin G immune complexes; or by binding and disrupting ion channels expressed by nociceptors. Using examples primarily from rheumatoid arthritis, complex regional pain syndrome, and channelopathies from potassium channel complex autoimmunity, we suggest that autoantibody signaling at the central nervous system has therapeutic implications for designing novel disease-modifying treatments for chronic pain.

The Current Landscape of Antibody-based Therapies in Solid Malignancies.

Over the past three decades, monoclonal antibodies (mAbs) have revolutionized the landscape of cancer therapy. Still, this benefit remains restricted to a small proportion of patients due to moderate response rates and resistance emergence. The field has started to embrace better mAb-based formats with advancements in molecular and protein engineering technologies. The development of a therapeutic mAb with long-lasting clinical impact demands a prodigious understanding of target antigen, effective mechanism of action, gene engineering technologies, complex interplay between tumor and host immune system, and biomarkers for prediction of clinical response. This review discusses the various approaches used by mAbs for tumor targeting and mechanisms of therapeutic resistance that is not only caused by the heterogeneity of tumor antigen, but also the resistance imposed by tumor microenvironment (TME), including inefficient delivery to the tumor, alteration of effector functions in the TME, and Fc-gamma receptor expression diversity and polymorphism. Further, this article provides a perspective on potential strategies to overcome these barriers and how diagnostic and prognostic biomarkers are being used in predicting response to mAb-based therapies. Overall, understanding these interdependent parameters can improve the current mAb-based formulations and develop novel mAb-based therapeutics for achieving durable clinical outcomes in a large subset of patients.

Pharmacokinetics, Pharmacodynamics and Drug-Drug Interactions of New Anti-Migraine Drugs-Lasmiditan, Gepants, and Calcitonin-Gene-Related Peptide (CGRP) Receptor Monoclonal Antibodies.

In the last few years, there have been significant advances in migraine management and prevention. Lasmiditan, ubrogepant, rimegepant and monoclonal antibodies (erenumab, fremanezumab, galcanezumab, and eptinezumab) are new drugs that were launched on the US pharmaceutical market; some of them also in Europe. This publication reviews the available worldwide references on the safety of these anti-migraine drugs with a focus on the possible drug–drug (DDI) or drug–food interactions. As is known, bioavailability of a drug and, hence, its pharmacological efficacy depend on its pharmacokinetics and pharmacodynamics, which may be altered by drug interactions. This paper discusses the interactions of gepants and lasmiditan with, i.a., serotonergic drugs, CYP3A4 inhibitors, and inducers or breast cancer resistant protein (BCRP) and P-glycoprotein (P-gp) inhibitors. In the case of monoclonal antibodies, the issue of pharmacodynamic interactions related to the modulation of the immune system functions was addressed. It also focuses on the effect of monoclonal antibodies on expression of class Fc gamma receptors (FcγR).

Impact of Glycosylation and Species Origin on the Uptake and Permeation of IgGs through the Nasal Airway Mucosa.

Although we have recently reported the involvement of neonatal Fc receptor (FcRn) in intranasal transport, the transport mechanisms are far from being elucidated. Ex vivo porcine olfactory tissue, primary cells from porcine olfactory epithelium (OEPC) and the human cell line RPMI 2650 were used to evaluate the permeation of porcine and human IgG antibodies through the nasal mucosa. IgGs were used in their wild type and deglycosylated form to investigate the impact of glycosylation. Further, the expression of FcRn and Fc-gamma receptor (FCGR) and their interaction with IgG were analyzed. Comparable permeation rates for human and porcine IgG were observed in OEPC, which display the highest expression of FcRn. Only traces of porcine IgGs could be recovered at the basolateral compartment in ex vivo olfactory tissue, while human IgGs reached far higher levels. Deglycosylated human IgG showed significantly higher permeation in comparison to the wild type in RPMI 2650 and OEPC, but insignificantly elevated in the ex vivo model. An immunoprecipitation with porcine primary cells and tissue identified FCGR2 as a potential interaction partner in the nasal mucosa. Glycosylation sensitive receptors appear to be involved in the uptake, transport, but also degradation of therapeutic IgGs in the airway epithelial layer.

Fc gamma receptor expression serves as prognostic and diagnostic factor in AML

Risk assessment in acute myeloid leukemia (AML) mainly relies on (cyto-)genetic and morphologic features. Nonetheless, further markers are needed to allow for accurate risk stratification. Type I Fc gamma receptors (FcγRs) such as CD16, CD32, and CD64 play an important role in mediating immunomodulatory functions in different myeloid cell types as well as NK and B cells. We here evaluated expression of the three FcγR on peripheral blood AML blasts. Using flow cytometry, we found heterogeneous expression of the FcγR throughout the patient cohort. Correlation of expression levels with disease outcome revealed significantly shorter OS in patients with CD16+ blasts at first diagnosis. CD32 and CD64 expression showed no association with survival but correlated with a mature phenotype and FAB M6. Our data provide clear evidence for the value of immunophenotyping FcγR expression on leukemic cells using peripheral blood, which is rapidly available and improves risk stratification in AML.

Quantification of neutrophil and monocyte CD64 expression: a predictive biomarker for active tuberculosis.

SETTING: QuantiFERON TB assay (QFT) is used to screen tuberculosis (TB) infection, but it cannot distinguish active TB from latent TB infection (LTBI).OBJECTIVE: To evaluate the quantitative expression of the high-affinity FCgamma receptor I (CD64) on neutrophils (NE) and monocytes (MO) in peripheral blood using flow cytometry, measured in antibody binding capacity (ABC) units as a predictive biomarker of TB.DESIGN: Fifty-two patients were enrolled (45 QFT-positive and 7 QFT-indeterminate). Cultures and molecular analyses were performed.RESULTS: Of the 45 QFT-positive patients, 29 were culture-positive (active TB) and 16 were negative (LTBI). The median NE CD64 ABC and MO CD64 ABC expression was significantly higher (P < 0.001) in culture-positive patients. The NE CD64 and MO CD64 area under the receiver operating characteristic curve values were respectively 0.948 (95%CI 0.838-0.992) and 0.989 (0.901-1.000). By setting the cut-off NE CD64 value at >2400 ABC or MO CD64 value >25 800 the assay sensitivity increased to 95.5% with 100% specificity and 100% positive predictive value. In the QFT-indeterminate group, five culture-positive cases had NE CD64 >2400 ABC or MO CD64 value >25 800; two culture-negative cases had lower values.CONCLUSION: The CD64 quantitative expression on peripheral blood cells may be used as a predictive biomarker for active TB.

Understanding Inter-Individual Variability in Monoclonal Antibody Disposition.

Monoclonal antibodies (mAbs) are currently the largest and most dominant class of therapeutic proteins. Inter-individual variability has been observed for several mAbs; however, an understanding of the underlying mechanisms and factors contributing to inter-subject differences in mAb disposition is still lacking. In this review, we analyze the mechanisms of antibody disposition and the putative mechanistic determinants of inter-individual variability. Results from in vitro, preclinical, and clinical studies were reviewed evaluate the role of the neonatal Fc receptor and Fc gamma receptors (expression and polymorphism), target properties (expression, shedding, turnover, internalization, heterogeneity, polymorphism), and the influence of anti-drug antibodies. Particular attention is given to the influence of co-administered drugs and disease, and to the physiological relevance of covariates identified by population pharmacokinetic modeling, as determinants of variability in mAb pharmacokinetics.

CD31 acts as a checkpoint molecule and is modulated by FcγR-mediated signaling in monocytes

Abstract Monocytes and macrophages express Fc-gamma Receptors (FcγR) that engage IgG immune complexes such as antibody-opsonized pathogens or cancer cells to destroy them by various mechanisms including phagocytosis. FcγR-mediated phagocytosis is regulated by the concerted actions of activating FcγR and inhibitory receptors such as FcγRIIb and SIRPα. Here, we report that another ITIM-containing receptor, Platelet Endothelial Adhesion Molecule-1 (PECAM1/CD31), regulates FcγR function, and is itself regulated by FcγR activation. First, qRT-PCR and flow cytometry analyses revealed that monocyte FcγR activation leads to significant downregulation of CD31 expression, both at the message level and at surface expression. Interestingly, the kinetics of downregulation between the two varied, with surface expression reducing earlier than the message. Experiments to analyze the mechanism behind this discrepancy revealed that loss of surface expression was due to internalization, which depended predominantly on the PI3 Kinase pathway. Finally, functional analyses showed that downregulation of CD31 expression in monocytes by siRNA enhanced FcγR-mediated phagocytic ability. Together, these results suggest that CD31 functions as a checkpoint receptor that could be targeted to enhance FcγR functions in antibody-mediated therapies.

Chronic HIV-1 Infection Alters the Cellular Distribution of FcγRIIIa and the Functional Consequence of the FcγRIIIa-F158V Variant.

Chronic HIV-infection modulates the expression of Fc gamma receptors (FcγRs) on immune cells and their antibody-dependent effector function capability. Given the increasingly recognized importance of antibody-dependent cellular cytotoxicity (ADCC) in HIV-specific immunity, we investigated the cellular distribution of FcγRIIIa on cytotoxic lymphocytes—natural killer cells and CD8+ T cells—and the effect of the FcγRIIIa-F158V variant on ADCC capacity in HIV-infected individuals (n = 23) and healthy controls (n = 23). Study participants were matched for F158V genotypes, carried two copies of the FCGR3A gene and were negative for FcγRIIb expression on NK cells. The distribution of CD56dimFcγRIIIabright and CD56negFcγRIIIabright NK cell subsets, but not FcγRIIIa surface expression, differed significantly between HIV-1 negative and HIV-1 positive donors. NK cell-mediated ADCC responses negatively correlated with the proportion of the immunoregulatory CD56brightFcγRIIIadim/neg cells and were lower in the HIV-1 positive group. Intriguingly, the FcγRIIIa-F158V variant differentially affected the NK-mediated ADCC responses for HIV-1 negative and HIV-1 positive donors. Healthy donors bearing at least one 158V allele had higher ADCC responses compared to those homozygous for the 158F allele (48.1 vs. 34.1%), whereas the opposite was observed for the HIV-infected group (26.4 vs. 34.6%), although not statistically significantly different. Furthermore, FcγRIIIa+CD8bright and FcγRIIIa+CD8dim T cell subsets were observed in both HIV-1 negative and HIV-1 positive donors, with median proportions that were significantly higher in HIV-1 positive donors compared to healthy controls (15.7 vs. 8.3%; P = 0.016 and 18.2 vs. 14.1%; P = 0.038, respectively). Using an HIV-1-specific GranToxiLux assay, we demonstrate that CD8+ T cells mediate ADCC through the delivery of granzyme B, which was overall lower compared to that of autologous NK cells. In conclusion, our findings demonstrate that in the presence of an HIV-1 infection, the cellular distribution of FcγRIIIa is altered and that the functional consequence of FcγRIIIa variant is affected. Importantly, it underscores the need to characterize FcγR expression, cellular distribution and functional consequences of FcγR genetic variants within a specific environment or disease state.

FCGR2B B2.4 Haplotype Predicts Increased Risk of Red Blood Cell Alloimmunization in Sickle Cell Disease Patients

Introduction: Red blood cell (RBC) alloimmunization is an important transfusion complication which is very prevalent among patients with sickle cell disease (SCD). Only part of patients is able to develop alloantibodies following antigen-mismatched transfusions, named the ‘immune responders’, who should be prospectively transfused with antigen-matched RBC units, comprising the most immunogenic erythrocyte antigens. Auto-immune diseases are a known risk factor for RBC alloimmunization, suggesting that auto-immunity and post-transfusion alloantibody development happen through similar physiopathological pathways. In this context, polymorphisms in FCGR2B gene have already been associated with several auto-immune disorders and, hypothetically, could be associated with RBC alloimmunization and help to identify the ‘immune responders’ in transfusion practice.Methods: In this case-control study, we enrolled 277 transfused SCD patients with retrospective data on alloimmunization status and transfusion exposure from two Brazilian centers. DNA samples (collected from March, 2017 to June, 2018) were extracted from whole blood samples using commercial kits (PureLink™ Genomic DNA®, Invitrogen, CA), and used for PCR amplification of promoter region of FCGR2B gene containing both -386G/C and -120T/A single nucleotide polymorphisms (SNPs) using gene-specific primer. SNPs of the FCGR2B were genotyped by direct Sanger sequencing (ABI3730XL, Applied Biosystems, CA). Cases had a positive history of alloimmunization with a clinically significant alloantibody and a minimum transfusion exposure of 1 RBC unit. Controls had a negative history of alloimmunization and having received ≥ 2 RBC units. Patients with autoimmune diseases or poor quality of DNA were excluded. Unadjusted and adjusted analyses were conducted by using Logistic Regression models to determine the association of covariates with binary outcome. Since the assumption of linearity in the logit was not supported, continuous covariates were categorized based on optimum cut-off point derived from ROC analysis. All statistical analyses were performed using SAS University Edition. A p-value of <0.05 was considered to be statistically significant.Results: A total of 237 patients met the eligibility criteria, 121 cases (alloimmunized) and 117 controls (non-alloimmunized). Majority of patients were female in cases (61%) and male in controls (86%), with mean age of 27.9 years (SD 15.3). Table 1 list the characteristics between the groups. RBC alloimmunization was associated with female sex (p<0.001), lifetime number of RBC units transfused (p=0.002) and FCGR2B 120T/A SNP (p=0.031). No statistically differences in the allele frequency of c.386C were observed between groups (p=0.602). FCGR2B -386G/C and -120T/A SNPs were in intense linkage disequilibrium. Three haplotypes of promoter region were identified as 2B.1 (-386G-120T), 2B.2 (-386C-120T) and 2B.4 (-386C-120A) with statistical significance (p=0.045). Logistic regression model was performed to assess the effects of age, gender, lifetime number of RBC units transfused and FCGR2B 2B.4 haplotype on the likelihood of RBC alloimmunization in patients with SCD. The analysis identified that female sex (OR 10.03, CI95% 5.16-19.49; p<0.001) and FCGR2B 2B.4 haplotype (OR 4.55, CI95% 1.11-18.65; p=0.035) predict increased risk of RBC alloimmunization in SCD patients as show in Table 2.Conclusions: SCD patients with the FCGR2B 2B.4 haplotype had over a 4-fold higher risk for RBC alloimmunization in comparison with patients without this haplotype and probably may be helpful to identify the ‘immune responders’. The presence of FCGR2B 2B.4 haplotype have been associated with increased expression of this inhibitory Fc gamma receptor in immune system cells, that was strongly associated with capacity of immune tolerance, regulating the development of antibodies. Future studies are needed to understand more about the functional and immune mechanism behind alloimmunization in SCD patients and ultimately improve our current preventive strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of Fc < gamma > Rs on monocytes among Kawasaki disease patients with coronary artery lesions

ObjectiveTo study expression of Fc gamma receptors (Fc<gamma>Rs) on monocytes in Kawasaki disease (KD) patients with coronary artery lesions (CAL). Methods160 newly diagnosed KD patients and 80 health children were enrolled in this study. All patients were scheduled to receive both aspirin and intravenous immunoglobulin (IVIG). Serial blood samples were obtained before and 3days after completing IVIG therapy. The first two-dimensional echocardiographic examination was performed for all KD patients within 10days, and was repeated at 3weeks. CAL was defined by coronary artery Z-scores≥2.5 by echocardiography. Expression of inhibitory and activating Fc<gamma>Rs on CD14+ monocytes (MCs) was assessed by flow cytometry. Cytokine expression in MC was evaluated by PCR. ResultsOf the 160KD patients enrolled in this study, 36 had coronary artery lesions (KD-CAL+ group), while 124 did not (KD-CAL− group). There was no significant difference in Fc<gamma>RI expression on MCs from KD patients and that of Ctrls. Although Fc<gamma>RIII and Fc<gamma>RIIa levels were significantly higher in KD patients compared with those in Ctrls, there were no significant differences between the KD-CAL+ and KD-CAL− groups. Fc<gamma>RIIb expression in the KD patients was lower than that of Ctrls, meanwhile expression in the KD-CAL+ group was lower than that in the KD-CAL− group. After IVIG therapy, Fc<gamma>RIIb expression increased in KD-CAL+, but did not reach the normal range. A negative correlation was observed between the levels of IL-6, TNF-α and Fc<gamma>RIIb expression. ConclusionDecreased Fc<gamma>RIIb expression on MCs may contribute to the development of coronary artery lesions in patients with KD.

Potentiation of Anti-Myeloma Activity of Daratumumab with Combination of Cyclophosphamide, Lenalidomide or Bortezomib Via a Tumor Secretory Response That Greatly Augments Macrophage-Induced ADCP

IntroductionDaratumumab (Dara) is the first CD38-directed monoclonal antibody (moAb) approved anywhere in the world as well as the first moAb approved for the treatment of Multiple Myeloma (MM). While currently approved as monotherapy, recent results from randomized trials have shown dramatic activity in relapsed MM patients in combination with both lenalidomide (Len) and bortezomib (Bor) and Dara is likely to become an intrinsic component of MM regimens in the future. Based on pre-clinical data Dara is thought to have pleiotropic mechanisms of activity, killing myeloma cells via a direct anti-CD38 effect, induction of complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Therefore, we focused our attention on potential mechanisms of enhancing macrophage (Mϕ)-mediated ADCP. Previously, Pallasch reported that resistance to Mϕ-mediated killing in lymphoid malignancy can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, Cyclophosphamide (Cy) was shown to induce an acute secretory activating phenotype from treated tumor cells, which led to Mϕ infiltration and phagocytic activity in the bone marrow. Given this background we sought to establish the existence of this phenomenon in MM in the context of drugs likely to be used in combination with Dara, i.e. Cy, Len and Bor, respectively.Materials and Methods:MM1S MM cells were conditioned with low dose single agents, Cy, Len and Bor for 24 hours. Media containing single agent chemotherapy was removed and replaced with fresh media for 24 hours (TCM). THP-1 Mϕ were conditioned with TCM for up to 48 hours and then incubated at 2:1 effector target ratio with CFSE labelled MM1S cells for 18 hours in the presence or absence of Dara or isotype control antibody. The % of Dara-specific tumour cell clearance was calculated as the number of live CFSE labelled tumour cells in Dara-treated cells/Isotype-treated cells. Additionally, Mϕ were pre-incubated with Cytochalasin D (CytoD), an inhibitor of actin polymerization, to determine if the mechanism of tumour cell clearance is mediated by Mϕ mediated ADCP. MM1S expression of CD47, as well as Mϕ expression of FC receptor expression, CD32 and CD64 was analysed by flow cytometry. Mϕ migration was assessed using transwell assays assessment of fluorescently labelled Mϕ migration to Cy, Len and /or Bor TCM.Results:Exposure of Mϕ to TCM from Cy, Len and Bor treated MM1S cells significantly enhanced % Dara specific clearance of MM1S cells (p<0.01). The effect was also maintained when combinations of the Cy, Len and Bor were used. This effect was partially reversed by incubation of Mϕ co-cultures with CytoD (p<0.01), indicating ADCP as one of the likely mechanisms of tumour cell clearance. We observed reduced expression of the "don’t eat me" antigen, CD47, on chemotherapy treated MMIS cells after 24 hours, which could enhance phagocytosis. In addition, we detected induction of CD32 and CD64 Fc gamma receptor expression on conditioned Mϕ as well as altered migratory capacity, which was more evident in Cy and Bor TCM conditioned Mϕ.Conclusions:Exposure of MM cells to both low doses of Cy, Len and Bor leads to a secretory response, which, along with downregulation of CD47, greatly augments Mϕ induced ADCP of Dara coated MM cells. This effect may explain in part the dramatic synergy observed between Len, Bor and Dara in clinical trials and provides a rationale for combining Cy with Dara in the clinic. Future research, including use of patient samples, will focus on understanding the mechanisms responsible for this anti-tumour effect.Reference:Pallasch CP et al. Sensitizing protective tumor microenvironments to antibody-mediated therapy. Cell. 2014 Jan 30;156(3):590-602. DisclosuresChiu:Janssen: Employment. Sasser:Janssen Pharma: Employment. O'Dwyer:Glycomimetics: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.