High-affinity IgG Receptor Research Articles

Control of acute myeloid leukemia by a trifunctional NKp46-CD16a-NK cell engager targeting CD123

CD123, the alpha chain of the IL-3 receptor, is an attractive target for acute myeloid leukemia (AML) treatment. However, cytotoxic antibodies or T cell engagers targeting CD123 had insufficient efficacy or safety in clinical trials. We show that expression of CD64, the high-affinity receptor for human IgG, on AML blasts confers resistance to anti-CD123 antibody-dependent cell cytotoxicity (ADCC) in vitro. We engineer a trifunctional natural killer cell engager (NKCE) that targets CD123 on AML blasts and NKp46 and CD16a on NK cells (CD123-NKCE). CD123-NKCE has potent antitumor activity against primary AML blasts regardless of CD64 expression and induces NK cell activation and cytokine secretion only in the presence of AML cells. Its antitumor activity in a mouse CD123+ tumor model exceeds that of the benchmark ADCC-enhanced antibody. In nonhuman primates, it had prolonged pharmacodynamic effects, depleting CD123+ cells for more than 10 days with no signs of toxicity and very low inflammatory cytokine induction over a large dose range. These results support clinical development of CD123-NKCE.

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines—THP-1 and Mono Mac 6—Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation

Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.

Serum Amyloid P and a Dendritic Cell–Specific Intercellular Adhesion Molecule-3–Grabbing Nonintegrin Ligand Inhibit High-Fat Diet–Induced Adipose Tissue and Liver Inflammation and Steatosis in Mice

High-fat diet (HFD)-induced inflammation is associated with a variety of health risks. The systemic pentraxin serum amyloid P (SAP) inhibits inflammation. SAP activates the high-affinity IgG receptor Fcγ receptor I (FcγRI; CD64) and the lectin receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; CD209). Herein, we show that for mice on an HFD, injections of SAP and a synthetic CD209 ligand (1866) reduced HFD-increased adipose and liver tissue inflammation, adipocyte differentiation, and lipid accumulation in adipose tissue. HFD worsened glucose tolerance test results and caused increased adipocyte size; for mice on an HFD, SAP improved glucose tolerance test results and reduced adipocyte size. Mice on an HFD had elevated serum levels of IL-1β, IL-23, interferon (IFN)-β, IFN-γ, monocyte chemoattractant protein 1 [MCP-1; chemokine (C-C motif) ligand 2 (CCL2)], and tumor necrosis factor-α. SAP reduced serum levels of IL-23, IFN-β, MCP-1, and tumor necrosis factor-α, whereas 1866 reduced IFN-γ. Invitro, SAP, but not 1866, treated cells isolated from white fat tissue (stromal vesicular fraction) produced the anti-inflammatory cytokine IL-10. HFD causes steatosis, and both SAP and 1866 reduced it. Conversely, compared with control mice, SAP knockout mice fed on a normal diet had increased white adipocyte cell sizes, increased numbers of inflammatory cells in adipose and liver tissue, and steatosis; and these effects were exacerbated on an HFD. SAPand1866 may inhibit some, but not all, of the effects of a high-fat diet.

A distinct subset of FcγRI-expressing Th1 cells exert antibody-mediated cytotoxic activity.

While a high frequency of Th1 cells in tumors is associated with improved cancer prognosis, this benefit has been attributed mainly to support of cytotoxic activity of CD8+ T cells. By attempting to potentiate antibody-driven immunity, we found a remarkable synergy between CD4+ T cells and tumor-binding antibodies. This surprising synergy was mediated by a small subset of tumor-infiltrating CD4+ T cells that express the high-affinity Fcγ receptor for IgG (FcγRI) in both mouse and human patients. These cells efficiently lyse tumor cells coated with antibodies through concomitant crosslinking of their T cell receptor (TCR) and FcγRI. By expressing FcγRI and its signaling chain in conventional CD4+ T cells, we successfully employed this mechanism to treat established solid cancers. Overall, this discovery sheds new light on the biology of this T cell subset, their function during tumor immunity, and the means to utilize their unique killing signals in immunotherapy.

Elevated Levels of CD64 MFI on Monocyte Subsets Are Associated with a History of Stroke in Sickle Cell Disease

Background: Ischemic stroke is a cause of morbidity and mortality in children and adults with sickle cell disease (SCD). Recent studies showed that non-classical monocytes may be protective against vaso-occlusive crisis. Monocyte subsets have been examined in non-SCD stroke, but little is known about the role of monocyte or their subsets in ischemic stroke in SCD. CD64 (FCγRI), a high affinity receptor for IgG, is a marker of endothelial adherence on immature neutrophils in SCD, however it is more classically exhibited on activated pro-inflammatory monocytes. We hypothesized that higher levels of CD64 mean fluorescence index (MFI) on monocyte subsets may be associated with ischemic stroke due to increased adherence.Methods: Adults (age ≥18) who presented for regularly scheduled clinic visits were enrolled. Blood samples were taken when subjects were at their healthy baseline and sent for complete blood count with differential. Monocytes were analyzed by flow cytometry and subsequently sub-classified by their CD14 and CD16 expression into classical (CD14high, CD16 low), intermediate (CD14high, CD16high), and non-classical (CD14low, CD16high). The CD64 MFI was determined for each monocyte subset. History of ischemic stroke was confirmed by neurologist or neuroimaging documentation in the medical record. Laboratory results between those with a history of stroke and those without history of stroke were compared using Student's T Tests. We modeled log-odds of stroke history for increasing levels of each monocyte subtype and CD64 MFI using both marginal and age-adjusted multivariate logistic regression.Results: We enrolled 49 adults, of whom 17 (35%) had a history of ischemic stroke. There was no difference in age, gender, or genotype between the two groups (Table 1). Patients with a history of stroke had higher total neutrophils, but there was otherwise no difference in complete blood counts (Table 1). When monocyte subsets were examined, patients with a history of stroke had a higher percent of intermediate monocytes and a lower percent of classical monocytes (Table 1). Patients with history of stroke also had a higher MFI (mean fluorescence index) both overall and on each monocyte subset. (Table 1). In univariate models increased levels of classical monocytes were associated with decreased odds of stroke history and increased levels of intermediate monocytes, CD64 MFI on each monocyte subtype, total monocyte CD64 MFI, and total neutrophil count were all associated with increased odds of stroke history (Table 2). However, in multivariate models adjusted for age and monocyte subtype percent only total monocyte CD64 MFI and total neutrophil counts remained significant and both had increased odds ratios for ischemic stroke history, (OR 3.8 per 1000 CD64 95% CI 1.2 - 11.4, p= 0.02, OR 1.2 per 1x10^9/L 95% CI 1.0 - 1.4, p=0.05 respectively, R-squared 0.45).Conclusion: Levels of monocyte subsets have been shown to correlate with clinical outcomes in non-SCD stroke, but to our knowledge this is the first study to examine their roles in ischemic stroke in SCD. The pathophysiology of stroke in SCD is unique and the role of monocytes in it deserves separate study from the role of monocytes in non-SCD stroke. We saw that while monocyte subsets were associated with a history of ischemic stroke, CD64 MFI on all monocyte subsets showed a strong association. We wonder if this may be due to CD64 MFI being a marker for cells which are more adherent to endothelium as has been shown in previous studies. This creates a need for future studies. In particular it would be valuable to know if increased CD64 MFI precedes stroke and could be used as a marker of future stroke risk. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FEATURES OF THE PHENOTYPE AND NAD(P)-DEPENDENT DEHYDROGENASES ACTIVITY IN NEUTROPHIL BY PATIENTS WITH WIDESPREAD PURULENT PERITONITIS IN PROGNOSIS FOR SEPSIS DEVELOPMENT

The aim of the study was to examine the features of phenotype and the levels of NAD(P)-dependent dehydrogenases activity of blood neutrophils in the prognosis of abdominal sepsis development in patients with widespread purulent peritonitis (WPP). 50 patients with WPP of community and hospital origin in the pre-operative period were examined. From the 5th to the 10th day of the postoperative period 35 patients (70%) had developed abdominal sepsis, 15 patients (30%) had absence of complications. As controls 67 respect healthy people were examined. The research blood neutrophils phenotype was performed by f low cytometry using a direct immunofluorescence whole peripheral blood. The levels of surface receptor expression was assessed by the mean fluorescence intensity. The NAD(P)-dependent dehydrogenases activity in the blood neutrophils studied using bioluminescence method. It was established that the inflammatory reaction in patients with WPP is characterized by neutrophilia and changes in the phenotype of blood neutrophils. The markers of the subsequent development of sepsis in WPP are less pronounced (in comparison with the indices of uncomplicated patients), an increase in the number of neutrophils, a decrease in the HLA-DR + -cell count against the background of a high level of neutrophils expressing a high affinity receptor for IgG. The patients without subsequent complications had the number of neutrophils with CD23 receptor expression is increased. Metabolism of neutrophils in patients with WPP is characterized by a decrease in the intensity of plastic processes due to low activity of glucose-6-phosphate dehydrogenase and an imbalance in the activity of the enzymes of the mitochondrial compartment. A feature of the neutrophil metabolism in patients with WPP without subsequent development of sepsis is a high activity of anaerobic lactate dehydrogenase reaction and a decrease in the activity of NADP-dependent decarboxylating malate dehydrogenase. The patients with WPP with subsequent development of sepsis had a high level of NAD-dependent out flow of substrates from the tricarboxylic acid cycle on the amino acid exchange reaction via glutamate dehydrogenase which can affect the activity of aerobic respiration of blood neutrophils. The established differences in the phenotype and activity of enzymes in the blood neutrophils in patients with WPP depending on the subsequent development of sepsis determine the possibility of creating a method for predicting the development of complications and developing immunoactive therapy in the postoperative period of WPP.

Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal.

Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)-mediated ablation of tissue-resident macrophages and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture-release-recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.

15 - G-CSF Treatment Further Impairs T-Cell Reconstitution in Patients with Residual Anti-Thymocyte Globulin Exposure after Hematopoietic Cell Transplantation: Implications for G-CSF Use?

Background: Residual anti-thymocyte globulin (ATG) exposure after allogeneic hematopoietic cell transplantation (HCT) negatively affects CD4+ T-cell immune reconstitution (IR). We previously described that IR after cord blood transplantation (CBT) is affected more by residual ATG exposure compared to bone marrow transplantation (BMT). In absence of residual ATG exposure, IR is at least as good or even better after CBT compared to BMT. We studied why residual ATG has a higher impact on CD4+ T-cell IR after CBT compared to BMT. Patients and Methods: We retrospectively analyzed the effect of residual ATG exposure on the recovery of CD4+ and CD8+ T-, B-, NK-cells, monocytes, and neutrophils in consecutive pediatric patients receiving an allogeneic CBT or BMT between January 2008 and September 2016. G-CSF was only given after CBT, from 7 days after transplant until neutrophil engraftment. We evaluated differences in ATG-binding and -cytotoxicity (by complement and effector cells; NK-cells, neutrophils, and monocytes) on target CD4+ and CD8+ T-, B-, NK-cells, and monocytes from CB- and BM-grafts obtained from healthy volunteers. Furthermore, we investigated the effect of G-CSF treatment on ATG-mediated cytotoxicity, by obtaining effector cells from volunteers who where or where not treated with G-CSF. Results: 275 Pediatric patients (median age 7.8 years; range .16-19.2), receiving either a CBT (n = 155; 56.4%) or a BMT (n = 120; 43.6%) were included. In patients who had no residual ATG (<10 active-IU*day/L), CD4+ T-cell reconstitution after CBT was faster compared to BMT (Figure 1; P = .037). However, residual ATG exposure >10 active-IU*day/ml significantly impairs CD4+ T-cell reconstitution after CBT (P = .002), but not after BMT (P = .68) (Figure 1). Residual ATG did not affect reconstitution of B-cells, NK-cells, monocytes, and neutrophils. No significant differences were observed in ATG-binding or -cytotoxicity between CB- and BM-derived CD4+ or CD8+ T-, B-, NK-cells, and monocytes. However, G-CSF treatment, standard after CBT, enhanced the expression of the high-affinity IgG-receptor CD64 on effector neutrophils (Figure 2; 1, 000 vs. 10, 000 MFI, P < .001) and increased neutrophil-mediated ATG-cytotoxicity of target cells by 40-fold (Figure 2; .5 vs. 20%, P = .002).Figure 2Higher ATG-mediated cytotoxicity by neutrophils after in vivo G-CSF treatment. (A-B) difference between ATG-mediated neutrophil target-cell killing from donors receiving (dark grey) or not receiving G-CSF (ligh grey). (C) expression of degranulation and activation markers on neutrophils. (D) confocal images from neutrophils (red) phagocytizing target-cells (blue) only when treated with G-CSF, in presence of ATG.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusion: These results suggest that G-CSF treatment further stimulates ATG-mediated cell clearance in patients with residual ATG exposure, leading to a delayed CD4+ T cell reconstitution in CB- compared to BM-recipients. Our findings imply that the use (or timing) of G-CSF, in context of ATG containing regimens, might be re-visited to improve CD4+ T-cell reconstitution after HCT.

Single Nucleotide Polymorphisms of the High Affinity IgG Receptor FcγRI Reduce Immune Complex Binding and Downstream Effector Functions.

Binding of IgG Abs to FcγRs on immune cells induces FcγR cross-linking that leads to cellular effector functions, such as phagocytosis, Ab-dependent cellular cytotoxicity, and cytokine release. However, polymorphisms in low affinity FcγRs have been associated with altered avidity toward IgG, thereby substantially impacting clinical outcomes of multimodular therapy when targeting cancer or autoimmune diseases with mAbs as well as the frequency and severity of autoimmune diseases. In this context, we investigated the consequences of three nonsynonymous single nucleotide polymorphisms (SNPs) for the high affinity receptor for IgG, FcγRI. Only SNP V39I, located in the extracellular domain of FcγRI, reduces immune-complex binding of FcγRI whereas monomeric IgG binding is unaffected. This leads to reduced FcγRI effector functions, including Fc receptor γ-chain signaling and intracellular calcium mobilization. SNPs I301M and I338T, located in the transmembrane or intracellular domain, respectively, have no influence on monomeric IgG or immune complex binding, but FcRγ signaling is decreased for both SNPs, especially for I338T. We also found that the frequency of these SNPs in a cohort of healthy Dutch individuals is very low within the population. To our knowledge, this study addresses for the first time the biological consequences of SNPs in the high affinity FcγR, and reveals reduction in several FcγRI functions, which have the potential to alter efficacy of therapeutic Abs.

In vivo effector functions of high-affinity mouse IgG receptor FcγRI in disease and therapy models

Two activating mouse IgG receptors (FcγRs) have the ability to bind monomeric IgG, the high-affinity mouse FcγRI and FcγRIV. Despite high circulating levels of IgG, reports using FcγRI−/− or FcγRIV−/− mice or FcγRIV-blocking antibodies implicate these receptors in IgG-induced disease severity or therapeutic Ab efficacy. From these studies, however, one cannot conclude on the effector capabilities of a given receptor, because different activating FcγRs possess redundant properties in vivo, and cooperation between FcγRs may occur, or priming phenomena. To help resolve these uncertainties, we used mice expressing only FcγRI to determine its intrinsic properties in vivo. FcγRIonly mice were sensitive to IgG-induced autoimmune thrombocytopenia and anti-CD20 and anti-tumour immunotherapy, but resistant to IgG-induced autoimmune arthritis, anaphylaxis and airway inflammation. Our results show that the in vivo roles of FcγRI are more restricted than initially reported using FcγRI−/− mice, but confirm effector capabilities for this high-affinity IgG receptor in vivo.

High-Affinity Fc Receptor Expression Indicates Relative Immaturity in Human Monocytes.

Within monocyte heterogeneity, subsets represent discrete, well-characterized phenotypes. Although many studies have highlighted differences between subsets, there is evidence that subpopulations represent contiguous stages in a maturational series. As CD14(hi)CD64(hi) monocytes have higher proliferative potential than CD14(hi)CD64(lo) monocytes, the surface marker profile on 4 subsets defined by CD14 and CD64 was measured. The profiles were compared to that of subsets defined by the high-affinity IgE receptor (FcɛRIα), CD16, and CD14; further differences in size, granularity, and buoyancy were measured in subsets delineated by these markers. There was a positive correlation between proliferative monocyte (PM) prevalence and CD64 expression on the classical monocyte subset, and also between PM prevalence and circulating FcɛRIα(+) monocytes. The expression of CD64, the high-affinity IgG receptor, on canonical human monocyte subsets was determined before and after short-term culture, and in response to interleukin (IL)-6, IL-10, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and interferon-γ; the influence of these cytokines on monocyte subset transition was also measured. The loss of FcɛRIα expression preceded an increase in CD16 expression in whole blood cultures. These data indicate that high-affinity Fc receptors are expressed on less mature monocytes and that FcɛRIα(+) monocytes are developmentally antecedent to the canonical classical and intermediate monocyte subsets.

The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

The high-affinity human IgG receptor Fc gamma receptor I (FcγRI) is not associated with vascular leakage of dengue.

BackgroundDengue is a major public health problem in many tropical and sub-tropical countries. Vascular leakage and shock are identified as the major causes of deaths in patients with severe dengue. Studies have suggested the potential role of Fc gamma receptors I (FcγRI) in the pathogenesis of dengue. We hypothesized that the circulating level of Fcγ receptor I could potentially be used as an indicator in assisting early diagnosis of severe dengue.ResultsA selected cohort of 66 dengue patients including 42 dengue with signs of vascular leakage, and 24 dengue without signs of vascular leakage were identified and were afterwards referred to as ‘cases’ and ‘controls’ respectively. Thirty seven normal healthy controls were also recruited in this study. The circulating level of FcγRI was quantified from the serum using enzyme-link immunosorbent assay (ELISA). The levels of FcγRI in both groups of patients with and without vascular leakage were found to be significantly higher than the normal healthy controls (P < 0.001). However, there was no significant difference found between patients with vascular leakage and those without vascular leakage (p = 0.777).ConclusionWe suggest that FcγRI is not associated with the vascular leakage in dengue. However, further studies are necessary to delineate the role of FcγRI in antibody-dependent enhancement (ADE) mechanism.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding

Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop-glycan interaction resulted in an ∼ 20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

Tyk2 and Stat3 Regulate Brown Adipose Tissue Differentiation and Obesity

Mice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2(-/-) brown preadipocytes. Furthermore, Tyk2(-/-) mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

Engineering of recombinant human Fc receptor I by directed evolution

Human FcγRI is a high-affinity receptor for human IgG. On the basis of its binding activity, recombinant human FcγRI (rhFcγRI) has several possible applications, including as a therapeutic reagent to treat immune complex-mediated disease and as a ligand in affinity chromatography for purification of human IgG. As the stability and production rate of rhFcγRI are low, it would need to be engineered for use in such applications. In this study, we demonstrated engineering of rhFcγRI by directed evolution through random mutagenesis and integration of mutations. Engineered rhFcγRI was expressed by Escherichia coli. Screening identified 19 amino acid mutations contributing to the thermal stability and production rate of rhFcγRI. By integration of these mutations, engineered rhFcγRI containing all 19 amino acid mutations (enFcRd) was constructed and showed markedly enhanced thermal stability (transition midpoint temperature [Tm] = 65.6°C) and production rate (3.27 mg L-medium(-1) OD(600)(-1)) compared with wild-type rhFcγRI (Tm = 48.5°C; production rate, 0.07 mg L-medium(-1) OD(600)(-1)) without a change in the specificities of binding to human IgG subclasses. Moreover, the binding affinity of enFcRd for human IgG1/к (equilibrium dissociation constant [K(D)] = 0.80 × 10(-10) M) was higher than that of wild-type rhFcγRI (K(D) = 1.23 × 10(-10) M). Our study showed that substantial engineering of rhFcγRI is possible.

CD64 Expression Distinguishes Monocyte-Derived and Conventional Dendritic Cells and Reveals Their Distinct Role during Intramuscular Immunization

Although most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α(+)- and CD11b(+)-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ-producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study.

Allergen Induces Dual Upregulation of TSLP Receptor on Circulating Basophils and Dendritic Cells in Atopic Dermatitis

We previously reported a link between the high affinity IgG receptor, FcγRI, and thymic stromal lymphopoietin (TSLP) pathways in monocyte-derived dendritic cells from patients with atopic dermatitis (AD). Our findings indicated a propensity for dendritic cells (DCs) from atopic donors to upregulate TSLP receptor (TSLPR). We aimed to rigorously assess the capacity of diverse circulating immune cell types from AD patients to upregulate TSLPR in response to allergens and elucidate the mechanism involved. PBMCs isolated from AD and non-AD subjects were cultured with and without allergens, or cross-linking anti-IgE antibody (24 hours), and phenotyped by multi-color flow cytometry. Stimulation with diverse allergens (Fel d 1, Der p 1 and Der p 2) induced TSLPR+ cells that were highly enriched within a lineage-negative population (CD3-CD14-CD16-CD19-CD20-CD56-) and this phenomenon was restricted to AD patients. While TSLPR+ cells in the absence of any stimulus were exclusively CD11c+ myeloid DCs, allergen-induced TSLPR+ cells comprised predominantly of basophils (FcεRIhiHLA-DR-CD123+). Wright-Giemsa staining of flow cytometry sorted TSLPR+ basophils confirmed their characteristic nuclear morphology and cytoplasmic granular content. Lesser TSLPR+ populations included a heterogeneous population of FcεRI+HLA-DR+ myeloid DCs which expressed variable levels of CD11c, CD1c, CD123 and CD141, as well as rare CD34+ cells that were consistent with DC precursors. Cross-linking FcεRI with anti-IgE antibody produced a TSLPR+ cellular profile comparable to that induced by allergen. Our findings demonstrate allergen-triggered IgE-dependent upregulation of TSLPR on circulating basophils and DCs in AD, implying a dual role for these cells in promoting TSLP-driven Th2 responses.

Serine phosphorylation of FcγRI cytoplasmic domain directs lipid raft localization and interaction with protein 4.1G

The high-affinity IgG receptor (CD64, FcγRI) has several special capacities, including the receptor-stimulated cleavage of the cell surface B cell-activating factor of the TNF superfamily (TNFSF13B). With the use of the yeast two-hybrid system, we and others have shown that FcγRI interacts with protein 4.1G (EPB41L2). Our mutational analyses identified two required 4.1G-interacting regions in the FcγRI CY and one FcγRI-interacting site in the C-terminus of protein 4.1G. Herein, we explore mechanism(s) that may regulate the interaction between protein 4.1G and FcγRI CY and influence FcγRI membrane mobility and function. We show that FcγRI CY interacts with protein 4.1G in vitro and that FcγRI coimmunoprecipitates protein 4.1G in freshly isolated human PBMC. With the use of immunostaining, we show that FcγRI colocalizes with protein 4.1G in unstimulated U937 cells, in which the FcγRI CY is constitutively serine-phosphorylated, but significant uncoupling occurs following FcγRI cross-linking, suggesting phosphoserine-regulated interaction. In vitro, protein 4.1G interacted preferentially with CK2-phosphorylated FcγRI CY, and compared with WT FcγRI, a nonphosphorylatable FcγRI mutant receptor was excluded from lipid rafts, suggesting a key role for protein 4.1G in targeting phosphorylated FcγRI to rafts. These data are consistent with a phosphoserine-dependent tethering role for protein 4.1G in maintaining FcγRI in lipid rafts and provide insight into the unique phosphoserine-based regulation of receptor signaling by FcγRI CY.