FcγR Expression Research Articles

Interferon-γ regulates growth and controls Fcγ receptor expression and activation in human intestinal mast cells.

BackgroundDevelopment and function of tissue resident mast cells (MCs) is tightly controlled by various cytokines, most of which belong to the typical T helper (Th) 2-type cytokines such as IL-3 and IL-4. The effects of the Th1-type cytokine IFN-γ on human MCs is less clear.ResultsHere, we analyzed the effects of IFN-γ on tissue-derived, mature human MCs. We found that INF-γ decreases proliferation, without affecting apoptosis in human intestinal MCs cultured in the presence of optimal concentrations of stem cell factor (SCF) or SCF and IL-4. However, in the absence of growth factors or at suboptimal concentrations of SCF, INF-γ promotes survival through inhibition of MC apoptosis. Interestingly, we found that INF-γ has no effect on FcϵRI expression and FcϵRI-mediated release of histamine and leukotriene (LT)C4, while it has profound effects on FcγR expression and activation. We show that intestinal MCs express FcγRI, FcγRIIa, and FcγRIIc, whereas FcγRIIb expression was found in only 40% of the isolates and FcγRIII was never detectable. INF-γ strongly increases FcγRI and decreases FcγRIIa expression. INF-γ-naïve MCs produce LTC4 but fail to degranulate upon crosslinking of surface-bound monomeric IgG. In contrast, INF-γ-treated MCs rapidly release granule-stored histamine in addition to de novo-synthesized LTC4.ConclusionIn summary, we identify INF-γ as an important regulator of tissue-resident human MCs. IFN-γ displays a dual function by blocking extensive MC proliferation, while decreasing apoptosis in starving MCs and enhancing FcγRI expression and activation. These results emphasize the involvement of mucosal MCs in Th1-mediated disorders.

Itraconazole, a Commonly Used Antifungal, Inhibits Fcγ Receptor–Mediated Phagocytosis

Itraconazole (ICZ) is commonly used for the treatment of fungal infections, particularly in immunocompromised patients. In addition, ICZ has been recently found to have antiangiogenic effects and is currently being tested as a new chemotherapeutic agent in several cancer clinical trials. We have previously shown that ICZ impaired complex N-linked glycosylation processing, leading to the accumulation of high-mannose glycoproteins on the surface of macrophages (Møs). This investigation was directed at determining the effects of ICZ on phagocytosis as a major function of Møs. We found a significant decrease in the phagocytosis of opsonized bacterial particles in ICZ-treated murine Møs in comparison with nontreated Møs. Furthermore, the impairment of phagocytosis was associated with a decrease in cell surface expression of Fcγ receptors (FcγRs) as well as alteration of their glycosylation pattern. Concomitantly, a reduction in all three isoforms of the FcγR family (i.e., Fcgr1, Fcgr2, and Fcgr3) mRNA levels was observed after incubation with ICZ. The effect of ICZ on phagocytosis and FcγR expression was reversed by addition of low-density lipoprotein. These studies indicate that ICZ treatment certainly has a dramatic effect on macrophage function, which could result in a potential impairment of the immune system';s ability to respond to pathogens and may lead to an elevated incidence of infections.

TLR7/8 agonists enhance FcγR in CLL patient monocytes (TUM7P.958)

Abstract Monoclonal antibody therapy has been used to treat cancer since 1997, but there is a continued need to improve its effectiveness. We have previously shown that the TLR7/8 dual agonist R-848 could modulate monocyte FcγR expression and function, and that it synergized with antibody to reduce tumor growth rate in vivo. Hence, the purpose of this study was to test whether these agonists could enhance antibody-mediated effects in patient samples and to determine whether it was TLR7, TLR8 or both that drove these FcγR responses. Using peripheral blood samples from Chronic Lymphocytic Leukemia (CLL) patients we found that treatment with the TLR7/8 dual agonist R-848 significantly enhanced monocyte FcγR-mediated TNFα production. In patient Peripheral Blood Mononuclear Cells (PBMC) treated with the anti-CD20 antibody Rituximab, R-848 increased both cytokine production and CLL cell clearance. We next compared the effects of TLR7- versus TLR8-selective agonists on monocytes and found that TLR8 agonists led to greater FcγR-mediated cytokine production, higher levels of miR-155 and lower levels of SHIP-1. Unexpectedly, while all agonists upregulated the activating FcγR, only TLR8 agonists downregulated the inhibitory FcγRIIb. These results suggest that TLR8 agonists are superior to those for TLR7 with regard to enhancing monocyte FcγR expression and function, and may be better adjuvants for antibody therapy.

Haplotypes of FcγRIIa and FcγRIIIb Polymorphic Variants Influence IgG-Mediated Responses in Neutrophils

Human blood neutrophils normally express two FcγRs (FcγRIIa and FcγRIIIb) that, upon multivalent binding of IgG in immune complexes or on opsonized targets, mediate responses such as phagocytosis, Ab-dependent cellular cytotoxicity, and respiratory burst. Allelic variants have been described for both FcγRIIa (131H/R) and FcγRIIIb (NA1/NA2/SH), with different binding affinity for IgG subclasses. Because neither of these variants acts alone, we have set out to systematically analyze in a large cohort of healthy FCGR2/3-genotyped volunteers how the different haplotypes of neutrophil FcγRs functionally interact. Maximal IgG-induced H2O2 production by neutrophils from individuals with different (homozygous) haplotypes was observed in the following order: 131HH-NA2NA2 > 131RR-NA1NA1 > 131HH-NA1NA1 > 131RR-NA2NA2. Although FcγRIIa 131H is known to bind IgG1 and IgG2 more avidly, no such differences in affinity are known for FcγRIIIb variants. Nonetheless, a remarkable impact of the FcγRIIIb variants on IgG-mediated neutrophil activity was thus demonstrated, which was not explained by differences in FcγR surface expression. The FcγR expression profile was changed by overnight G-CSF/IFN-γ activation of the neutrophils and eliminated any haplotypic impact on the respiratory burst. To our knowledge, our results are the first to provide an integrated functional analysis of neutrophil FcγR haplotypes and suggest that particularly the early phase of IgG-mediated neutrophil reactivity is influenced by FCGR2/3 genotypic variation.

Humanized mice to study FcγR function.

Passive immunotherapy represents a promising therapeutic intervention for a number of neoplastic, chronic inflammatory, and infectious diseases, with several monoclonal antibodies currently under development or already in use in the clinic. While Fab-antigen interactions play a crucial role in the activity of an antibody, it has become clear that Fc-mediated effector functions are involved during antibody-mediated activities in vivo. A complete understanding of the contributions of effector activities mediated by an antibody during its in vivo function is required for the development of antibodies with improved therapeutic efficacies. Animal models that are commonly used for the preclinical evaluation of antibodies include murine and non-human primate species, whose FcγRs present substantial structural, functional, and genetic variation compared with their human counterparts. Therefore, the use of such animal models provides limited information on the role of human IgG Fc-FcγR interactions during the in vivo activities of antibodies intended for human therapeutics. In this chapter, we describe the development and evaluation of an FcγR-humanized mouse model for the study of human FcγR function in vivo. In this model, endogenous mouse FcγR genes have been deleted and human FcγRs are expressed as transgenes that faithfully recapitulate the unique pattern of human FcγR expression. Evaluation of the in vivo activities of a number of cytotoxic or therapeutic antibodies using FcγR-humanized mice provided useful insights into human IgG Fc effector function. This mouse model has become a vital preclinical model for testing therapeutic human antibodies to treat malignancies, autoimmunity, inflammation, and infectious disease.

Toll‐like Receptor 2 Controls Acute Immune Complex–Driven Arthritis in Mice by Regulating the Inhibitory Fcγ Receptor IIB

Previous studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis. Serum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcγ receptor (FcγR) expression, and response to immune complexes was determined. While TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcγR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcγR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcγR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcγRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcγRIIB, but not the activating FcγR, on macrophages. TLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcγR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.

Inflammatory monocytes and Fcγ receptor IV on osteoclasts are critical for bone destruction during inflammatory arthritis in mice

Destruction of bone tissue by osteoclasts represents a severe pathological phenotype during inflammatory arthritis and results in joint pain and bone malformations. Previous studies have established the essential role of cytokines including TNFα and receptor-ligand interactions, such as the receptor activator of nuclear factor-kappa B-receptor activator of nuclear factor-kappa B ligand interaction for osteoclast formation during joint inflammation. Moreover, autoantibodies contribute to joint inflammation in inflammatory arthritis by triggering cellular fragment crystallizable (Fc)γ receptors (FcγR), resulting in the release of proinflammatory cytokines and chemokines essential for recruitment and activation of innate immune effector cells. In contrast, little is known about the expression pattern and function of different FcγRs during osteoclast differentiation. This would allow osteoclasts to directly interact with autoantibody immune complexes, rather than being influenced indirectly via proinflammatory cytokines released upon immune complex binding to other FcγR-expressing innate immune cells. To address this question, we studied FcγR expression and function on osteoclasts during the steady state and during acute joint inflammation in a model of inflammatory arthritis. Our results suggest that osteoclastogenesis is directly influenced by IgG autoantibody binding to select activating FcγRs on immature osteoclasts, resulting in enhanced osteoclast generation and, ultimately, bone destruction.

FRI0009 Il4-10 synerkine: a novel anti-inflammatory drug to inhibit immunopathology in rheumatic diseases

BackgroundA considerable percentage of patients shows a limited response to biologics targeting one specific inflammatory mediator, largely because of redundancy of these mediators. An attractive alternative is inhibition of multiple...

TLR2 ligands modulate Fcγ receptor expression and function in monocytes (P4397)

Abstract Fcγ receptor (FcγR) clustering on monocytes/macrophages leads to phagocytosis, cytokine release and other responses designed to clear the antibody-opsonized targets. Toll-like Receptor 2 activation elicits strong proinflammatory responses, so we explored the possibility that ligands for TLR2 could indirectly strengthen FcγR activity. We treated human peripheral blood monocytes (PBM) with the TLR2 agonist Pam2CSK4 and found significantly enhanced FcγR-mediated cytokine production and phagocytosis. Further examination showed that the agonist increased the mRNA and protein levels of FcγRIIa and the common γ-subunit. Interestingly however, the inhibitory receptor FcγRIIb was also upregulated. We also found a concurrent decrease in SHIP and an increase in miR-155, which may help explain the enhanced FcγR function despite upregulated inhibitory FcγRIIb. Next, using a murine Her2/neu solid-tumor model of antibody therapy we found that Pam2CSK4 significantly enhanced antibody-mediated reduction of tumor growth, although the effects were not superadditive. Finally, we tested whether the effect was acylation-specific by treating PBM with the tri-acylated Pam3CSK4 and measuring FcγR expression and function. Results showed that Pam3CSK4 was equally effective at all dosages tested. Collectively, these findings show that TLR2 ligands can positively modulate FcγR expression and function, and hence may warrant further investigation as putative adjuvants for antitumor antibody therapy.

Toll-like Receptor 2 Ligands Regulate Monocyte Fcγ Receptor Expression and Function

Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy.

C-reactive protein exacerbates renal ischemia-reperfusion injury

Renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), occurring with hypotension and cardiovascular surgery and inevitably during kidney transplantation. Mortality from AKI is high due to incomplete knowledge of the pathogenesis of IRI and the lack of an effective therapy. Inflammation accompanies IRI and increases the blood level of C-reactive protein (CRP), a biomarker of worsened outcomes in AKI. To test if CRP is causal in AKI we subjected wild-type mice (WT) and human CRP transgenic mice (CRPtg) to bilateral renal IRI (both pedicles clamped for 30 min at 37°C then reperfused for 24 h). Serum human CRP level was increased approximately sixfold after IRI in CRPtg (10.62 ± 1.31 μg/ml at baseline vs. 72.01 ± 9.41 μg/ml at 24 h) but was not elevated by sham surgery wherein kidneys were manipulated but not clamped. Compared with WT, serum creatinine, urine albumin, and histological evidence of kidney damage were increased after IRI in CRPtg mice. RT-PCR analysis of mRNA isolated from whole kidneys of CRPtg and WT subjected to IRI revealed that in CRPtg kidneys 1) upregulation of markers of macrophage classical activation (M1 markers) was blunted, 2) downregulation of markers of macrophage alternative activation (M2 markers) was more robust, and 3) expression of the activating receptor FcγRI was increased. Our finding that CRP exacerbates IRI-induced AKI, perhaps by shifting the balance of macrophage activation and FcγR expression towards a detrimental portfolio, might make CRP a promising therapeutic target for the treatment of AKI.

Application of knockout mouse models to investigate the influence of FcγR on the pharmacokinetics and anti-platelet effects of MWReg30, a monoclonal anti-GPIIb antibody

This work evaluates the influence of FcγR on the pharmacokinetics and pharmacodynamics of a rat anti-integrin-αIIb IgG1 monoclonal antibody, MWReg30, in mice. The pharmacokinetics and pharmacodynamics of MWReg30 were investigated in C57BL/6 control mice, FcγRI/RIII knockout mice, and FcγRIIb knockout mice, following intravenous doses of 0.04-0.4mg/kg. MWReg30 treatment resulted in a dose-dependent induction of thrombocytopenia in all strains, but sensitivity to MWReg30 was increased in FcγRIIb knockout mice and decreased in FcγRI/RIII knockout mice, relative to results found in control mice. Expressed as a percentage of pre-treatment platelet counts, nadir platelet counts were 28.6±5.0, 88.7±16.6 and 25.3±6.1% at 0.05mg/kg, 28.4±13.7, 56.7±5.1, and 20.6±9.5% at 0.2mg/kg, and 24.9±7.2, 38.7±7.5, and 7.4±2.2% at 0.4mg/kg in control, FcγRI/RIII(-/-) and FcγRIIb(-/-) mice. However, knocking out FcγR did not affect MWReg30 pharmacokinetics. Plasma areas under the concentration vs. time curves (AUC0-10 days) ±SD for MWReg30 were: 5.24±0.68, 5.51±0.24, and 5.39±1.05 nM×d at 0.04mg/kg, and 12.7±0.5, 13.6±1.1, and 14.5±2.0nM×d at 0.1mg/kg in control, FcγRI/RIII(-/-) and FcγRIIb(-/-) mice. The findings further emphasize the role of activating vs. inhibitory FcγR in processing immune complexes (i.e., MWReg30-platelets), while also providing an example where monoclonal antibody pharmacokinetics are not substantially influenced by FcγR expression.

Mechanism of Fcγ Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

The whole blood erythrocyte lysis method is the most common protocol of sample preparation for flow cytometry (FCM). Although this method has many virtues, our recent study has demonstrated false-positive results when surface markers of monocytes were examined by this method due to the phenomenon called Fcγ receptor (FcγR)-mediated trogocytosis. In the present study, similar FcγR-mediated trogocytosis-based false-positive results have been demonstrated when granulocytes were focused on instead of monocytes. These findings indicated that not only monocytes but also granulocytes, the largest population with FcγR expression in peripheral blood, could perform FcγR-mediated trogocytosis. Since the capacity of FcγR-mediated trogocytosis was different among blood samples, identification of factors that could regulate the occurrence of FcγR-mediated trogocytosis should be important for the quality control of FCM. Our studies have suggested that such factors are present in the serum. In order to identify the serum factors, we employed the in vitro model of FcγR-mediated trogocytosis using granulocytes. Investigation with this model determined the serum factors as heat-labile molecules with molecular weight of more than 100 kDa. Complements in the classical pathway were initially assumed as candidates; however, the C1 inhibitor did not yield an obvious influence on FcγR-mediated trogocytosis. On the other hand, although immunoglobulin ought to be resistant to heat inactivation, the inhibitor of human anti-mouse antibodies (HAMA) effectively blocked FcγR-mediated trogocytosis. Moreover, the inhibition rates were significantly higher in HAMAhigh serum than HAMAlow serum. The collective findings suggested the involvement of heterophilic antibodies such as HAMA in the mechanism of false-positive results in FCM due to FcγR-mediated trogocytosis.

Human intravenous immunoglobulin (hIVIG) inhibits anti-CD32 antibody binding to canine DH82 cells and canine monocytes in vitro

The IgG receptors CD16 and CD32 (FcγRIII and FcγRII) link the humoral immune response to effector cell immune responses by binding immune complexes. Human intravenous immunoglobulin (hIVIG) consisting of immunoglobulin from pooled donors is reported to block FcγRs and has been used to treat a variety of canine autoimmune disorders. FcγRs have been poorly described for canine monocytes; therefore, the objectives of this study were to: (1) identify canine monocyte/macrophage FcγR (CD16 and CD32) expression and (2) demonstrate in vitro hIVIG binding to these receptors. The canine monocyte/macrophage-like cell line (DH82) and monocytes isolated from peripheral blood of healthy dogs were evaluated by flow cytometry (FACS) for CD16 and CD32 expression using commercially available anti-CD16 and anti-CD32 antibodies directed against the human isoforms. The mean percentage of cells expressing CD16 was 55% of DH82 cells and 13% of blood monocytes and the mean percentage of cells expressing CD32 was 85% of DH82 cells and 73% of blood monocytes. Immunoprecipitation of canine DH82 cells lysate using the same anti-CD16 or anti-CD32 antibodies suggested that these anti-human antibodies recognize the canine homologues. To demonstrate FcγR blockade, cells were incubated with increasing concentrations of hIVIG and then incubated with anti-CD16 or anti-CD32 antibodies. The percentage of CD32 expression decreased in a concentration dependent fashion in DH82 cells and blood monocytes after incubation with increasing concentrations of IVIG, suggesting that hIVIG was binding to CD32 and inhibiting anti-CD32 antibody binding. The same results were not demonstrated with anti-CD16 antibody. We believe this is the first report to demonstrate Fcγ receptors CD16 and CD32 expression on canine monocytes and in vitro CD32 binding by human IgG, which may represent one of the immunomodulatory mechanisms of hIVIG.

TLR7/8 Agonists Overcome the Suppression of FcγR Activity in Monocytes From Chronic Lymphocytic Leukemia Patients

AbstractAbstract 4595It has been documented that monocytes from Chronic Lymphocytic Leukemia (CLL) patients are immunosuppressed (Flieger et al., 1990), and this may play a significant role in attenuating responses to antibody therapy. Fc-gamma receptors (FcγR) are critical for binding therapeutic antibody and effecting tumor cell destruction. Both monocytes and Natural Killer (NK) cells have been shown to be critical for this process. FcγR cluster upon binding opsonized targets and this results in activation of downstream mediators such as Ras and PI3K. These activation events are tempered, however, both at the receptor level and within the signaling pathways. The inhibitory FcγR, FcγRIIb, clusters along with the activating FcγR and dampens signaling. This receptor recruits SHIP, an inositol phosphatase that antagonizes PI3K. Other phosphatases such as SHIP2 and SHP-1 also serve to negatively regulate FcγR activity.Toll-like receptors (TLR) are innate immune receptors that recognize characteristic components of pathogens and other potentially harmful agents. Recently we discovered that treatment of human monocytes with the TLR7/8 agonist R-848 led to enhanced antitumor activity of therapeutic antibody in vitro, and in a murine solid-tumor model of antibody therapy.Hence, we began to examine the extent of suppression within the FcγR pathway of CLL patient monocytes, with the goal of testing whether TLR7/8 activation could at least in part reverse this suppression. We first tested FcγR-mediated cytokine responses and found they were significantly attenuated in freshly isolated monocytes from CLL patients. However, real-time PCR showed equivalent levels of FcγR in monocytes from patients and healthy donors, suggesting that the suppression was occurring in one or more portions of the downstream signaling pathways. We then examined phosphatase expression and found increased levels of SHIP, SHIP2 and SHP-1 in CLL patient monocytes.Next, we tested the functional effects of TLR7/8 agonist treatment on monocytes from CLL patients. Overnight treatment with 1 μM R-848 led to dramatic and significant increases in FcγR-mediated cytokine production. To determine whether this would still occur in the presence of the immunosuppressive B-CLL cells, we treated patient PBMC with R-848 and rituximab. ELISA results showed significant enhancement of antibody-mediated cytokine production with R-848 treatment. To determine whether TLR7/8 activation could increase the antibody-mediated clearance of B-CLL cells we treated patient PBMC overnight with or without rituximab and with either R-848 or vehicle. Results showed an approximately twofold increase in B-CLL cell clearance with R-848. These findings suggest that TLR7/8 activation can serve to overcome the monocyte immunosuppression seen with CLL.We then examined changes in FcγR expression and found that R-848 increased the expression of activating FcγR while decreasing expression of the inhibitory FcγRIIb in patient monocytes. In conjunction with this receptor modulation, we found significant decreases in transcripts of SHIP, SHIP2 and SHP-1. These results show that TLR7/8 activation enhances FcγR expression and drives concomitant decreases in inhibitory phosphatase expression in CLL patient monocytes.Because we used an agonist that activated both TLR7 and TLR8, it was unclear which TLR was primarily responsible for the changes observed. Hence, we treated monocytes with 3M–055 (TLR7-selective), R-848 (TLR7/8 dual-agonist) or a TLR8-selective agonist and measured FcγR expression. Results showed that activation of both TLR7 and TLR8 was sufficient to drive upregulation of the activating FcγR. However, TLR7 activation alone had no effect on the inhibitory receptor FcγRIIb. Conversely, the TLR8-selective agonist led to an almost complete abolishment of FcγRIIb expression. Dual TLR7/8 activation decreased FcγRIIb to levels between TLR7- and TLR8-selective agonist treatment. We also found that all 3 agonists reduced phosphatase expression, with the TLR8-selective agonist leading to the greatest decrease.In summary, these results show that FcγR function but not expression is compromised in CLL patient monocytes. However, treatment with TLR7/8 agonists, in particular TLR8-selective agonists, can reverse this suppression and promote stronger FcγR responses. Hence, these compounds may be particularly useful as adjuvants for antibody therapy. Disclosures:Vasilakos:3M: Employment.

The Fcγ receptor expression profile on porcine dendritic cells depends on the nature of the stimulus

Nowadays, vaccine research focuses on the development and implementation of subunit vaccines into existing or future vaccination platforms. However, recombinant proteins are often poor immunogens, necessitating adjuvants to activate and direct the immune response. Alternatively, the immunogenicity of antigens can be enhanced by targeting antigens to Fcγ receptors on antigen-presenting cells and as dendritic cells (DCs) are the most potent antigen-presenting cells, orchestrating innate and adaptive immune responses, they are attractive for this selective targeting of vaccine antigens. However, DCs express both inhibitory and activating Fcγ receptors and regulating DC function is pivotal to ensure the induction of effective immune responses and to prevent exaggerated immune responses causing inflammation. Previously, we demonstrated that immature porcine MoDC express FcγRII and FcγRIII on their cell surface, which mediate a functional DC maturation upon activation through immune complexes. In the present study, we clearly demonstrate that immune complexes are primarily internalised via FcγRIII, resulting in DC maturation and that depending on the DC maturation stimulus the FcγR expression profile is differentially regulated. These results could not only expedite the development of FcγR-targeting based vaccines, but also provide insights into FcγR-mediated autoimmune diseases.

Bacterial reprogramming of PBMCs impairs monocyte phagocytosis and modulates adaptive T cell responses

Septic diseases are characterized by an initial systemic, proinflammatory phase, followed by a period of anti-inflammation. In the context of the latter, monocytes have been described to display altered functions, including reduced TNF secretion and T cell-stimulating capacities in response to recall antigens. This hyporesponsiveness is supposed to be detrimental for coping with secondary infections. We here characterize bacterially reprogrammed PBMC-derived monocytes with special focus on their phagocytic activity. Hence, we have implemented a surrogate model of the early, postinflammatory period by exposing PBMCs to Escherichia coli on d0 and rechallenging them with bacteria on d2. This induced the emergence of a distinct monocytic phenotype with profound phagocytic impairments but a preserved ability for naïve T cell stimulation. The compromising effects on phagocytosis required the presence of bacteria and were not mimicked by TLR4 ligation or exposure to isolated cytokines alone. Moreover, the impairments were specific for the engulfment of bacteria and were coupled to a selective down-regulation of FcγR and SR expression. Intriguingly, this monocytic phenotype contributed to the stimulation of a T(H)17-polarized adaptive immune response in the context of secondary infection. Our findings extend the current knowledge of monocytic reprogramming and identify the phagocytic capacity of monocytes as a putative sepsis biomarker.

Toll-like receptor 2 ligands enhance monocyte Fcγ receptor function (68.16)

Abstract Fcγ receptors (FcγR) are major effectors of antibody therapy so it is critical to seek new ways to enhance their effectivness. Toll-like receptors (TLR) recognize a wide range of microbial components, eliciting cellular responses such as cytokine production and generation of antimicrobial factors. Among these receptors, TLR2 recognizes both bacterial and fungal components. Given the strong cellular proinflammatory responses to TLR2 activation, we explored the possibility that TLR2 agonists could strengthen FcγR activity. Human peripheral blood monocytes (PBM) were treated with or without the TLR2 agonist PAM2CSK4 and tested for FcγR expression and function. Results showed that overnight treatment with PAM2CSK4 led to an increase in the expression of both FcγRIIa and the common γ-subunit, while expression of the inhibitory receptor FcγRIIb remained unchanged. Along with this, PAM2CSK4 significantly increased IgG-mediated TNFα production in plate-bound assays. We then tested its effect on phagocytosis, using fluorescently-labeled opsonized sheep red blood cells as targets. PAM2CSK4 significantly enhanced the phagocytic ability of PBM. To test the effect of PAM2CSK4 in vivo, we injected it in combination with anti-CD20 antibody and measured B-cell depletion. Results showed that PAM2CSK4 led to greater depletion of splenic B cells. These findings suggest that TLR2 agonists modulate FcγR expression and function and should be investigated as possible adjuvants for antibody therapy.

Mouse model recapitulating human Fcγ receptor structural and functional diversity

The in vivo biological activities of IgG antibodies result from their bifunctional nature, in which antigen recognition by the Fab is coupled to the effector and immunomodulatory diversity found in the Fc domain. This diversity, resulting from both amino acid and glycan heterogeneity, is translated into cellular responses through Fcγ receptors (FcγRs), a structurally and functionally diverse family of cell surface receptors found throughout the immune system. Although many of the overall features of this system are maintained throughout mammalian evolution, species diversity has precluded direct analysis of human antibodies in animal species, and, thus, detailed investigations into the unique features of the human IgG antibodies and their FcγRs have been limited. We now report the development of a mouse model in which all murine FcγRs have been deleted and human FcγRs, encoded as transgenes, have been inserted into the mouse genome resulting in recapitulation of the unique profile of human FcγR expression. These human FcγRs are shown to function to mediate the immunomodulatory, inflammatory, and cytotoxic activities of human IgG antibodies and Fc engineered variants and provide a platform for the detailed mechanistic analysis of therapeutic and pathogenic IgG antibodies.

Intravenous Immunoglobulins Suppress Antibody-Dependent Effector Functions of Human Peripheral Blood Cells

RationaleSeveral studies have shown the importance of self-reactive antibodies in the maintenance of autoimmune diseases. Therefore, we asked whether IGIV, which is therapeutically effective in some of those diseases, directly modulates antibody-mediated effector functions.MethodsWe used a human in vitro system to analyze potential modulatory effects of IGIV on effector functions in antibody-dependent cellular cytotoxicity (ADCC). Human peripheral blood mononuclear cells (PBMC) from 47 healthy volunteers were pre-incubated with IGIV and then added to human breast cancer cells (SK-BR3) opsonized with a specific antibody. The cytotoxic damage to SK-BR3 cells was determined by measuring lactate dehydrogenase release into supernatants. IGIV-mediated effects on viability and Fc gamma receptor (FcγR) expression of PBMC were analyzed by flow cytometry.ResultsPre-incubation of PBMC with IGIV for 48h resulted in a marked inhibition of ADCC, ranging from 15% to 74% (mean 42%) which could not be mimicked with human monoclonal IgG1 and IgG2 antibodies. Flow cytometric analysis indicated that IGIV induced natural killer (NK) cell death, which significantly correlated with ADCC inhibition. The IGIV-induced decrease in NK cell viability was not triggered by FcγR ligation since the addition of FcγR blocking antibodies could not prevent cell death induction. Furthermore, IGIV enriched for sialic acid bearing glycans by lectin affinity chromatography with Sambucus nigra agglutinin showed no differences in the effects on NK cell viability and ADCC compared with non-enriched IGIV.ConclusionsIGIV modulates antibody-dependent effector functions of human immune cells, which could contribute to the modulatory activities of IGIV in inflammatory and autoimmune diseases. RationaleSeveral studies have shown the importance of self-reactive antibodies in the maintenance of autoimmune diseases. Therefore, we asked whether IGIV, which is therapeutically effective in some of those diseases, directly modulates antibody-mediated effector functions. Several studies have shown the importance of self-reactive antibodies in the maintenance of autoimmune diseases. Therefore, we asked whether IGIV, which is therapeutically effective in some of those diseases, directly modulates antibody-mediated effector functions. MethodsWe used a human in vitro system to analyze potential modulatory effects of IGIV on effector functions in antibody-dependent cellular cytotoxicity (ADCC). Human peripheral blood mononuclear cells (PBMC) from 47 healthy volunteers were pre-incubated with IGIV and then added to human breast cancer cells (SK-BR3) opsonized with a specific antibody. The cytotoxic damage to SK-BR3 cells was determined by measuring lactate dehydrogenase release into supernatants. IGIV-mediated effects on viability and Fc gamma receptor (FcγR) expression of PBMC were analyzed by flow cytometry. We used a human in vitro system to analyze potential modulatory effects of IGIV on effector functions in antibody-dependent cellular cytotoxicity (ADCC). Human peripheral blood mononuclear cells (PBMC) from 47 healthy volunteers were pre-incubated with IGIV and then added to human breast cancer cells (SK-BR3) opsonized with a specific antibody. The cytotoxic damage to SK-BR3 cells was determined by measuring lactate dehydrogenase release into supernatants. IGIV-mediated effects on viability and Fc gamma receptor (FcγR) expression of PBMC were analyzed by flow cytometry. ResultsPre-incubation of PBMC with IGIV for 48h resulted in a marked inhibition of ADCC, ranging from 15% to 74% (mean 42%) which could not be mimicked with human monoclonal IgG1 and IgG2 antibodies. Flow cytometric analysis indicated that IGIV induced natural killer (NK) cell death, which significantly correlated with ADCC inhibition. The IGIV-induced decrease in NK cell viability was not triggered by FcγR ligation since the addition of FcγR blocking antibodies could not prevent cell death induction. Furthermore, IGIV enriched for sialic acid bearing glycans by lectin affinity chromatography with Sambucus nigra agglutinin showed no differences in the effects on NK cell viability and ADCC compared with non-enriched IGIV. Pre-incubation of PBMC with IGIV for 48h resulted in a marked inhibition of ADCC, ranging from 15% to 74% (mean 42%) which could not be mimicked with human monoclonal IgG1 and IgG2 antibodies. Flow cytometric analysis indicated that IGIV induced natural killer (NK) cell death, which significantly correlated with ADCC inhibition. The IGIV-induced decrease in NK cell viability was not triggered by FcγR ligation since the addition of FcγR blocking antibodies could not prevent cell death induction. Furthermore, IGIV enriched for sialic acid bearing glycans by lectin affinity chromatography with Sambucus nigra agglutinin showed no differences in the effects on NK cell viability and ADCC compared with non-enriched IGIV. ConclusionsIGIV modulates antibody-dependent effector functions of human immune cells, which could contribute to the modulatory activities of IGIV in inflammatory and autoimmune diseases. IGIV modulates antibody-dependent effector functions of human immune cells, which could contribute to the modulatory activities of IGIV in inflammatory and autoimmune diseases.