Fcγ receptor II dependency of enhanced presentation of major histocompatibility complex class II peptides by a B cell lymphoma

Here we show that the B cell lymphoma A20.292 is capable of enhanced antigen presentation to CD4⁺ T cells in the presence of specific antibodies. This enhancement was inhibited by anti-Fcγ receptor (R) antibodies, suggesting that it might be due to preferential uptake of the antigen/antibody complex through the FcγRII receptor. However, immunoprecipitation studies revealed that the FcR of A20.292 cells was of the B cell type, FcγRIIb1, which is not thought to be able to internalize antigen/antibody complexes via clathrin-coated pits. It was considered unlikely that A20.292 had an altered form of the B cell FcγR (RIIb1) receptor that enabled internalization, since similar enhancing effects were also observed using an FcγRII^? cell line that had been transfected with FcγRIIb1. To reconcile these findings with the expression of FcγRIIb1, it is postulated that immune complexes are concentrated on the cell surface by the FcγRIIb1 and are thus available for preferential uptake by random fluid-phase endocytosis. This results in more efficient generation of the epitopes recognized by these T cell hybridomas.     

Membrane-Spanning Fcγ Receptor III Isoform Expressed on Human Placental Trophoblasts

PROBLEM: Fc receptor for immunoglobulin (FcγR) is an important mediator of immunological functions in the feto-maternal relationship. We have demonstrated by immunohistochemical means that three distinct classes of FcγRS are expressed in the different cell components of the human placenta. METHOD: In this study, FcγRIII isoform expressed on placental trophoblasts (PTs) was investigated by indirect immunofluorescence and cDNA cloning. PTs, isolated from human term placenta by digestion with proteolytic enzyme, were reacted with monoclonal antibodies (MAb) against the Fc-γRs and other surface markers of leukocytes and subjected to flow cytometric analysis. RESULTS: PTs were positively stained with 3G8 and Leul lb against FcγRIII, partially stained with MAb against MHC class I, but not with 32.2 (FcγRI), IV3 (FcγRII), or MAbs against CD4, CD19, or CD56, indicating that only low affinity receptor, FC7RIII, is γexpressed on PTs. The DNA sequence of cloned FcγRIII CDNA from PTs by PCR was identical to that of natural killer (NK) cell isoform, including the position of the stop codon that differs from the granulocyte isoform by several nucleotide substitutions. We further analyzed the susceptibility of PTs against phosphatidylinositol specific phospholipase C (PI-PLC) to determine the structural topology of PT isoform. While the reactivity with 3G8 on PTs was not influenced by treatment with PI-PLC, that on granulocytes was significantly diminished with PI-PLC. CONCLUSIONS: This result confirmed that FcγRIII on PTs is a membrane-spanning molecule, and that it is distinctive from PI anchoring FcγRIII on granulocytes.     

FcγRIII (CD16)‐mediated ADCC by NK cells is regulated by monocytes and FcγRII (CD32)

Monocytes are known to engage in reciprocal crosstalk with NK cells but their influence on NK‐cell‐associated antibody‐dependent cellular cytotoxicity (ADCC) is not well understood. We demonstrate that in humans FcγRIII (CD16)‐dependent ADCC by NK cells is considerably enhanced by monocytes, and that this effect is regulated by FcγRII (CD32) crosslinking in healthy individuals. It is known that during HIV‐1 infection, NK cells are known to express low levels of CD16 and exhibit reduced ADCC. We show that immune regulation of CD16‐mediated NK‐cell cytotoxicity by monocytes through CD32 engagement is substantially disturbed in chronic progressive HIV‐1 infection. Expression of activating isoform of CD32 represented a compensatory mechanism for reduced expression of CD16 on NK cells during HIV‐1 infection. As a result, the regulation of NK‐cell‐associated ADCC by monocytes is skewed and eventually constitutes a novel factor that contributes to HIV‐1‐associated immune deficiency, dysregulation and pathogenesis. Our data therefore provide evidence, for the first time, that in humans monocytes act as a rheostat for FcγRIII‐mediated NK‐cell functions maintaining a well‐balanced immune response.     

Innate signaling regulates cross‐priming at the level of DC licensing and not antigen presentation

Innate stimuli, such as TLR ligands, are known to greatly facilitate cross‐priming. Currently it is unclear whether innate stimuli enhance cross‐priming at the level of cross‐presentation or at the level of T‐cell priming. In this study, we addressed this question by measuring cross‐presentation as well as cross‐priming by virus‐like particles (VLP) displaying peptide p33 derived of lymphocytic choriomeningitis virus. Innate stimuli were varied by either packaging different TLR ligands into virus‐like particles or using mice deficient in two key molecules of TLR‐signaling, namely the adaptor molecule MyD88 as well as IFN‐α/β receptor. While efficient cross‐presentation occurred despite strongly reduced activation of DC in the absence of TLR ligand‐mediated signals, T‐cell priming was abolished. Thus, innate stimuli regulate cross‐priming at the level of DC licensing for T‐cell activation and not antigen presentation.     

Racially restricted contribution of immunoglobulin Fcγ and Fcγ receptor genotypes to humoral immunity to human epidermal growth factor receptor 2 in breast cancer

Tumour‐associated antigen human epidermal growth factor receptor 2 (HER2) is over‐expressed in 25–30% of breast cancer patients and is associated with poor prognosis. Naturally occurring anti‐HER2 antibody responses have been described in patients with HER2 over‐expressing tumours. There is significant interindividual variability in antibody responsiveness, but the host genetic factors responsible for this variability are poorly understood. The aim of the present investigation was to determine whether immunoglobulin genetic markers [GM (genetic determinants of γ chains)] and Fcγ receptor (FcγR) alleles contribute to the magnitude of natural antibody responsiveness to HER2 in patients with breast cancer. A total of 855 breast cancer patients from Japan and Brazil were genotyped for several GM and FcγR alleles. They were also characterized for immunoglobulin (Ig)G antibodies to HER2. In white subjects (n = 263), GM 23‐carriers had higher levels of anti‐HER2 antibodies than non‐carriers of this allele (p = 0·004). At the GM 5/21 locus, the homozygotes for the GM 5 allele had higher levels of anti‐HER2 antibodies than the other two genotypes (P = 0·0067). In black subjects (n = 42), FcγRIIa‐histidine/histidine homozygotes and FcγRIIIa‐phenylalanine/valine heterozygotes were associated with high antibody responses (P = 0·0071 and 0·0275, respectively). FcγR genotypes in white subjects and GM genotypes in black subjects were not associated with anti‐HER2 antibody responses. No significant associations were found in other study groups. These racially restricted contributions of GM and FcγR genotypes to humoral immunity to HER2 have potential implications for immunotherapy of breast cancer.     

Regulation of Fcγ receptors and immunoglobulin G-mediated phagocytosis in mouse microglia

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F_c receptors. In mice, both FcγRI and FcγRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-γ, IL-4, IL-13 and GM-CSF on expression of FcγRI and FcγRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-γ induced a 4-fold increase in the mRNA level of FcγRI but did not induce changes in FcγRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcγRIIb mRNA, but had no effect on FcγRI expression. GM-CSF increased both FcγRI and FcγRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-γ and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcγ receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.     

Vγ9Vδ2 TCR‐activation by phosphorylated antigens requires butyrophilin 3 A1 (BTN3A1) and additional genes on human chromosome 6

Pyrophosphorylated metabolites of isoprenoid‐biosynthesis (phosphoantigens, PAgs) activate Vγ9Vδ2 T cells during infections and trigger antitumor activity. This activation depends on expression of butyrophilin 3 A1 (BTN3A1) by antigen‐presenting cells. This report defines the minimal genetic requirements for activation of Vγ9Vδ2 T cells by PAgs and mAb 20.1. We compared PAg‐presentation by BTN3A1‐transduced CHO hamster cells with that of CHO cells containing the complete human chromosome 6 (Chr6). BTN3A1 expression alone was sufficient for activation of Vγ9Vδ2 T‐cell receptor transductants by mAb 20.1., while activation by PAgs also required the presence of Chr6. We take this finding as evidence that gene(s) on Chr6 in addition to BTN3A1 are mandatory for PAg‐mediated activation of Vγ9Vδ2 T cells. This observation is important for the design of animal models for PAg‐mediated immune responses and provokes speculations about the analogy between genes controlling PAg presentation and MHC‐localized genes controlling peptide‐antigen presentation.     

In vivo depletion of CD4+FOXP3+ Treg cells by the PC61 anti‐CD25 monoclonal antibody is mediated by FcγRIII+ phagocytes

Depletion of CD4⁺CD25⁺FoxP3⁺ Treg using PC61 mAb (anti‐murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated ～70% of CD4⁺FoxP3⁺ cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcγ receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcγ receptor (FcγR)III^?/? mice were resistant to Treg depletion. FcγRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab‐dependent‐cellular‐cytotoxicity and Ab‐dependent‐cellular‐phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab‐dependent‐cellular‐phagocytosis of CD25⁺ cells by WT or FcγRIIB^?/?, but not FcγRIII^?/?, macrophages. Altogether these data demonstrate the critical role of FcγRIII⁺ phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.     

Macrophage Fcγ Receptors Expression Is Altered by Treatment with Dopaminergic Drugs

Macrophage Fcγ receptors have an important role in host defense and the pathophysiology of immune mediated disorders. Alteration of splenic macrophage Fcγ receptors expression predisposes to severe infection. Inhibition or blockade of splenic macrophage Fcγ receptors is one of the mechanisms by which immune cytopenias improve. Dopaminergic drugs have clinically significant regulatory functions on the immune response. Using an experimental model in the guinea pig we assessed the effect of commonly used dopaminergic drugs on the expression of macrophage Fcγ receptors. Three dopa-antagonists, bromocryptine, leuprolide, and pergolide, and seven dopa-antagonists, chlorpromazine, SCH 23390, metochlopramide, sulpiride, veralipride, alizapride, and cisapride, were studied. Following guinea pig treatment with dopaminergic drugs, the clearance of IgG-sensitized RBCsin vivo,thein vitrobinding of IgG-sensitized RBCs by isolated splenic macrophages and flow cytometry with monoclonal antibodies were performed. Treatment with dopa-agonists enhanced the clearance of IgG-sensitized RBCs, thein vitrobinding of IgG-sensitized RBCs by isolated splenic macrophages, and the cell surface expression of both macrophage Fcγ receptors, and vice versa, dopa-antagonists impaired macrophage Fcγ receptors expression. Macrophage FcγR1,2 was more sensitive than FcγR2 to such dopaminergic effect. These alterations of macrophage Fcγ receptors expression are mediated by both D1 and D2 dopamine receptors, with a major participation of D2 receptors. Dopaminergic drugs alter the clearance of IgG-coated cells by an effect at the expression of splenic macrophage Fcγ receptors.     

Chimeric Fc receptors identify immunoglobulin-binding regions in human FcγRII and FcϵRI

FcγRII and Fc?RI are functionally distinct cell surface receptors for immunoglobulin (Ig); FcγRII binds IgG with low affinity, whereas Fc?RI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig-like domains with 38% amino acid identity. Chimeric receptors derived from human FcγRII and FcγRI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in FcγRII and IgE in Fc?RI. Firstly, a chimeric form of the Fc?RI α chain was produced by replacing the transmembrane region and cytoplasmic tail with that of FcγRII. This mutant α chain could be expressed on the cell surface independently of associated β and γ subunits, and retained high-affinity IgE binding, indicating that the extracellular region of the FcγRI α chain is sufficient for high-affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both FcγRII and FcγRI, chimeric receptors were generated by exchanging the first extracellular domains between FcγRII and the α chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of FcγRII and FcγRI; these identified a single region of IgG binding in FcγRII located between residues Ser¹³⁶ to Val¹⁶⁹, and at least three independent IgE binding regions in the FcγRI α chain, between residues Trp⁸⁷ to Lys¹²⁸, Tyr¹²⁹ to Asp¹⁴⁵, and Ser¹⁴⁶ to Val¹⁶⁹.     

Ectodomain shedding of Fcα receptor is mediated by ADAM10 and ADAM17

FcαR (CD89) plays important roles in immunoglobulin A (IgA)‐mediated immune responses. Soluble forms of FcαR (sFcαR) are found in the culture supernatants of FcαR‐expressing cells, in human serum and in the serum of FcαR transgenic mice, and have been suggested to be produced through a proteolytic process. However, little is known about the mechanism involved in the proteolytic release of sFcαR. In this study, we investigated the shedding mechanism of FcαR and determined the nature of the proteinase involved in FcαR shedding. In chemical inhibitor assays, shedding of FcαR was dramatically inhibited by EDTA, EGTA and a broad‐spectrum metalloproteinase inhibitor, GM6001, suggesting that a metalloproteinase was responsible for FcαR shedding. Overexpression of dominant‐negative mutants of ADAM (a disintegrin and metalloproteinase) 10 and ADAM17 markedly inhibited the production of sFcαR. Finally, knockdown of both endogenous ADAM10 and endogenous ADAM17 inhibited FcαR shedding, demonstrating that ADAM10 and ADAM17 were involved in the shedding of FcαR. The characterization of ADAM10 and ADAM17 as sFcαR‐releasing enzymes provides a novel insight into the molecular mechanism of sFcαR production and will help in further elucidation of the physiological and pathological roles of sFcαR.     

IgG-mediated anaphylaxis via Fcγ receptor in CD40-deficient mice

Anaphylaxis denotes an immediate hypersensitivity reaction to allergen, exclusively mediated by IgE antibodies. However, IgE antibodies do not explain all the syndromes that are encountered. We investigated potent IgG-mediated anaphylaxis in CD40-deficient mice that lack the immunoglobulin class switching for T cell-dependent antigens. Immunization with ovalbumin did not induce either humoral responses of IgG, IgA, and IgE, or systemic anaphylaxis in CD40-deficient mice. Although systemic anaphylaxis by active immunization was not observed in CD40-deficient mice, both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis assessed by mouse blood pressure monitoring with cervical artery catheterization did take place when antigen-specific IgG was transferred and then antigen challenge given. Further, to investigate the inflammatory pathway of IgG-mediated immediate hypersensitivity reactions, we focused on the Fcγ receptor (FcγR) function. Pretreatment of the mice with the anti-FcγRII/FcγRIII MoAb clearly blocked the response of PCA and passive systemic anaphylaxis, suggesting that they were initiated through FcγR. In conclusion, we directly demonstrate the IgG-mediated anaphylaxis and its triggering mechanism through FcγR in in vivo conditions. In addition to IgE-mediated anaphylaxis, IgG-mediated anaphylaxis should be considered and the blocking of FcγR would provide one of the therapeutic targets for the control of IgG-mediated hypersensitivity diseases.     

Differential uptake and cross‐presentation of soluble and necrotic cell antigen by human DC subsets

Cross‐presentation is the mechanism by which exogenous Ag is processed for recognition by CD8⁺ T cells. Murine CD8α⁺ DCs are specialized at cross‐presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross‐presentation of soluble and cellular Ag by human blood CD141⁺ DCs, the human equivalent of mouse CD8α⁺ DCs, and compared them with human monocyte‐derived DCs (MoDCs) and blood CD1c⁺ DC subsets. MoDCs were superior in their capacity to internalize and cross‐present soluble protein whereas CD141⁺ DCs were more efficient at ingesting and cross‐presenting cellular Ag. Whilst cross‐presentation by CD1c⁺ DCs and CD141⁺ DCs was dependent on the proteasome, and hence cytosolic translocation, cross‐presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross‐presentation by CD1c⁺ DCs and MoDCs but not by CD141⁺ DCs. These data demonstrate that CD1c⁺ DCs, CD141⁺ DCs, and MoDCs are capable of cross‐presentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141⁺ DCs, like their murine CD8α⁺ DC counterparts, are specialized at cross‐presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway.     

Neutrophil Fcγ and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fcγ receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fcγ and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

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

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

Effect of interferon-γ on antigen processing in human monocytes

The effect of interferon-γ (IFN-γ) on the ability of human monocytic cells to process exogenous (major histocompability complex class II) antigens was investigated. The processing (i.e. protein degradation) of antigens that were internalized via Fcγ receptor (FcγR) was followed for various times after treatment of cells with IFN-γ. THP-1 cells that had been treated with IFN-γ for 4 h degraded antigen, internalized as an immune complex, at an enhanced rate. After 24 h of IFN-γ treatment the rate of processing was similar to untreated cells. Unexpectedly, in cells which had been treated for 48–72 h there was a significant decrease in the rate of processing of the exogenous antigen. These effects were not due to changes in the rate of internalization of immune complex. The inhibition of the rate of processing was independent of the type of antigen, was dependent on the dose of IFN-γ, and also occurred with normal human peripheral monocytes. Analysis of the degraded peptides by high-pressure liquid chromatography indicated that some of the peptides generated in the IFN-γ-treated cells were both quantitatively and qualitatively different from the peptides generated in untreated cells. These data suggest that IFN-γ modulates the way in which antigens, internalized through Fc receptors as immune complexes, are processed. Additionally, the results imply that decreases in the rate of antigen processing may lead to more efficient antigen presentation.