Article

FcR?? Chain Deletion results in Pleiotrophic Effector Cell Defects

March 1994
Cell 76(3):519-29

March 1994
76(3):519-29

DOI:10.1016/0092-8674(94)90115-5

Source
PubMed

Authors:

Toshiyuki Takai

Tohoku University

Raphael Clynes

Columbia University

Show all 5 authorsHide

The gamma subunit of immunoglobulin Fc receptors is an essential component of the high-affinity receptor for IgG (Fc gamma RIII) and is associated with the high-affinity receptor for IgG (Fc gamma RI) and the T cell receptor-CD3 complex. It is required for both receptor assembly and signal transduction. Targeted disruption of this subunit results in immunocompromised mice. Activated macrophages from gamma chain-deficient mice unexpectedly lack the ability to phagocytose antibody-coated particles, despite normal binding. Defects in NK cell-mediated antibody-dependent cytotoxicity and mast cell-mediated allergic responses are evident in these animals, establishing the indispensable role of FcRs in these responses. However, loss of gamma chain does not appear to perturb T cell development, since both thymic and peripheral T cell populations appear normal. These mice thus represent an important tool for evaluating the role of these receptors in humoral and cellular immune responses.

Bullous pemphigoid induced by IgG targeting type XVII collagen non-NC16A/NC15A extracellular domains is driven by Fc gamma receptor- and complement-mediated effector mechanisms and is ameliorated by neonatal Fc receptor blockade

Article

Full-text available

Nov 2023
J PATHOL

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies targeting type XVII collagen (Col17) with the noncollagenous 16A (NC16A) ectodomain representing the immunodominant site. The role of additional extracellular targets of Col17 outside NC16A has not been unequivocally demonstrated. In this study, we showed that Col17 ectodomain‐reactive patient sera depleted in NC16A IgG induced dermal–epidermal separation in a cryosection model indicating the pathogenic potential of anti‐Col17 non‐NC16A extracellular IgG. Moreover, injection of IgG targeting the murine Col17 NC14–1 domains (downstream of NC15A, the murine homologue of human NC16A) into C57BL/6J mice resulted in erythematous skin lesions and erosions. Clinical findings were accompanied by IgG/C3 deposits along the basement membrane and subepidermal blistering with inflammatory infiltrates. Disease development was significantly reduced in either Fc‐gamma receptor (FcγR)‐ or complement‐5a receptor‐1 (C5aR1)‐deficient mice. Inhibition of the neonatal FcR (FcRn), an atypical FcγR regulating IgG homeostasis, with the murine Fc fragment IgG2c‐ABDEG, a derivative of efgartigimod, reduced anti‐NC14–1 IgG levels, resulting in ameliorated skin inflammation compared with isotype‐treated controls. These data demonstrate that the pathogenic effects of IgG targeting the Col17 domain outside human NC16A/murine NC15A are partly attributable to antibody‐mediated FcγR‐ and C5aR1 effector mechanisms while pharmacological inhibition of the FcRn represents a promising treatment for BP. The mouse model of BP will be instrumental in further investigating the role of Col17 non‐NC16A/NC15A extracellular epitopes and validating new therapies for this disease. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A human immune system (HIS) mouse model that dissociates roles for mouse and human FcR+ cells during antibody-mediated immune responses

Article

Full-text available

Aug 2023
EUR J IMMUNOL

Human immune system (HIS) mice provide a model to study human immune responses in vivo . Currently available HIS mouse models may harbor mouse Fc Receptor (FcR)‐expressing cells that exert potent effector functions following administration of human Ig. Previous studies showed that ablation of the murine FcR gamma chain (FcRγ) results in loss of antibody‐dependent cellular cytotoxicity (ADCC) and antibody‐dependent cellular phagocytosis (ADCP) in vivo . We created a new FcRγ‐deficient HIS mouse model to compare host (mouse) versus graft (human) effects underlying antibody‐mediated immune responses in vivo . FcRγ‐deficient HIS recipients lack expression and function of mouse activating FcRs and can be stably and robustly reconstituted with human immune cells. By screening blood B cell depletion by Rituximab Ig variants, we found that human FcγRs mediated IgG1 effects, while mouse activating FcγRs were dominant in IgG4 effects. Complement played a role as an IgG1 variant (IgG1 K322A) lacking complement binding activity was largely ineffective. Finally, we provide evidence that FcγRIIIA on human NK cells could mediate complement‐independent B cell depletion by IgG1 K322A. We anticipate that our FcRγ‐deficient HIS model will help clarify mechanisms of action of exogenous administered human antibodies in vivo . This article is protected by copyright. All rights reserved

Association of IgA-Fc receptors (Fc alpha R) with Fc epsilon RI gamma 2 subunits in U937 cells. Aggregation induces the tyrosine phosphorylation of gamma 2.

Article

Oct 1994

We investigated the possibility that IgA-binding chains of Fc alpha R on monocytic cells are physically associated with gamma 2 subunits of Fc epsilon RI (Fc epsilon RI gamma 2 or gamma 2). Fc alpha R was precipitated from lysates of IFN gamma-treated U937 cells, subclone 10.6, and probed by immunoblotting with Ab against human gamma 2. Fc alpha R was precipitated through anti-Fc alpha R mAbs A59 or A62, through A62 from lysates that had been exhaustively precleared of high affinity IgG-Fc receptors (Fc gamma RI) and of low affinity Fc gamma RII, and through anti-Fc alpha R mAb A77 from Fc gamma RI-precleared lysates of untreated 10.6 cells. precipitation was also performed through F(ab')2 A77 and through the native ligand of the receptor, hlgA. In all cases, Fc alpha R precipitates contained co-isolated 22-kDa gamma 2 (unreduced). The Fc alpha R alpha-chain/gamma 2 complex did not readily dissociate in 1% Nonidet P-40 as did Fc gamma RI alpha-chain/gamma 2, suggesting a novel aspect to the Fc alpha R subunit interaction. Specific Fc alpha R aggregation on cells triggered a robust respiratory burst and the tyrosine phosphorylation of several proteins. Among them was phospho-gamma 2, which migrated as a 24- to 28-kDa gamma 2 phosphoprotein on gels and was detected as a 28-kDa phosphoprotein by anti-phosphotyrosine immunoblot. Aggregated Fc alpha Rs that were precipitated from Fc alpha R-triggered cells also contained a phosphoprotein of the same mobility and immunoreactivity, as did aggregated Fc gamma RI from which the 28-kDa phosphoprotein could be more readily eluted and identified (as phospho-gamma 2). We concluded that myelocytic Fc alpha Rs are multichain complexes containing gamma 2 subunits that are tyrosine phosphorylated upon Fc alpha R aggregation. As IgA is the predominant Ig on mucosal surfaces, gamma-subunits may play an important role in mucosal immunity involving leukocytic Fc alpha R.

Longevity and Mechanism of Heterosubtypic Protection Induced by M2SR (M2-Deficient Single-Replication) Live Influenza Virus Vaccine in Mice

Article

Full-text available

Dec 2022

Seasonal influenza and the threat of global pandemics present a continuing threat to public health. However, conventional inactivated influenza vaccines (IAVs) provide little cross-protective immunity and suboptimal efficacy, even against well-matched strains. Furthermore, the protection against matched strains has been shown to be of a short duration in both mouse models and humans. M2SR (M2-deficient single-replication influenza virus) is a single-replication vaccine that has been shown to provide effective cross-protection against heterosubtypic influenza viruses in both mouse and ferret models. In the present study, we investigated the duration and mechanism of heterosubtypic protection induced by M2SR in a mouse model. We previously showed that M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) significantly protected C57BL/6 mice against lethal challenge with both influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic), whereas the inactivated influenza vaccine provided no heterosubtypic protection. The homosubtypic protection induced by M2SR was robust and lasted for greater than 1 year, whereas that provided by the inactivated vaccine lasted for less than 6 months. The heterosubtypic protection induced by M2SR was of a somewhat shorter duration than the homosubtypic protection, with protection being evident 9 months after vaccination. However, heterosubtypic protection was not observed at 14 months post vaccination. M2SR has been shown to induce strong systemic and mucosal antibody and T cell responses. We investigated the relative importance of these immune mechanisms in heterosubtypic protection, using mice that were deficient in B cells or mice that were depleted of T cells immediately before challenge. Somewhat surprisingly, the heterosubtypic protection was completely dependent on B cells in this model, whereas the depletion of T cells had no significant effect on survival after a lethal heterosubtypic challenge. While antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated to be important in the response to some influenza vaccines, a lack of Fc receptors did not affect the survival of M2SR-vaccinated mice following a lethal challenge. We examined the influenza proteins targeted by the heterosubtypic antibody response. Shortly after the H1N1 M2SR vaccination, high titers of cross-reactive antibodies to heterosubtypic H3N2 nucleoprotein (NP) and lower titers to the stalk region of the hemagglutinin (HA2) and neuraminidase (NA) proteins were observed. The high antibody titers to heterosubtypic NP persisted one year after vaccination, whereas the antibody titers to the heterosubtypic HA2 and NA proteins were very low, or below the limit of detection, at this time. These results show that the intranasal M2SR vaccine elicits durable protective immune responses against homotypic and heterosubtypic influenza infection not seen with intramuscular inactivated vaccines. Both the homo- and heterosubtypic protection induced by the single-replication vaccine are dependent on B cells in this model. While the homosubtypic protection is mediated by antibodies to the head region of HA, our data suggest that the heterosubtypic protection for M2SR is due to cross-reactive antibodies elicited against the NP, HA2, and NA antigens that are not targeted by current seasonal influenza vaccines.

Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody

Article

Full-text available

Oct 2022

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro-and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting anti-bodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cyto-toxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxi-city (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition , we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.

The role of VISTA engagement in limiting neutrophil-mediated inflammation

Preprint

Full-text available

May 2024

A growing body of evidence suggests that VISTA, an immune checkpoint inhibitory receptor, plays a central role in the regulation of innate immunity in the settings of inflammatory diseases and cancer. Neutrophils are among the cells that have the highest membrane density of surface VISTA. Targeting VISTA on neutrophils with an agonist antibody resulted in a striking reduction in their LPS-induced peripheral accumulation. Fc receptor engagement was required for anti-VISTA antibody to mediate its effects on neutrophils. Concomitant with reduced peripheral neutrophil cell numbers, anti-VISTA antibody treatment increased neutrophil cell death in the liver. In a murine model of neutrophil-mediated arthritis, agonist anti-VISTA antibody treatment ameliorated disease severity, which was associated with reduced myeloperoxidase activity in the joints. These studies add to a growing spectrum of negative regulatory functions that VISTA performs in controlling inflammation through the innate and adaptive arms of the immune system that has implications for translation into the clinic.

ITAM‐based receptors in natural killer cells

Article

Full-text available

Feb 2024
IMMUNOL REV

The ability of cells of the immune system to acquire features such as increased longevity and enhanced secondary responses was long thought to be restricted to cells of the adaptive immune system. Natural killer (NK) cells have challenged this notion by demonstrating that they can also gain adaptive features. This has been observed in both humans and mice during infection with cytomegalovirus (CMV). The generation of adaptive NK cells requires antigen‐specific recognition of virally infected cells through stimulatory NK receptors. These receptors lack the ability to signal on their own and rather rely on adaptor molecules that contain ITAMs for driving signals. Here, we highlight our understanding of how these receptors influence the production of adaptive NK cells and propose areas in the field that merit further investigation.

Tissue niche occupancy determines the contribution of fetal- versus bone-marrow-derived macrophages to IgG effector functions

Article

Feb 2024

Effector functions are required for broad and potent protection of neonatal mice with antibodies targeting HSV glycoprotein D

Article

Full-text available

Feb 2024

Multiple failed herpes simplex virus (HSV) vaccine candidates induce robust neutralizing antibody (Ab) responses in clinical trials, raising the hypothesis that Fc-domain-dependent effector functions may be critical for protection. While neonatal HSV (nHSV) infection results in mortality and lifelong neurological morbidity in humans, it is uncommon among neonates with a seropositive birthing parent, supporting the hypothesis that Ab-based therapeutics could protect neonates from HSV. We therefore investigated the mechanisms of monoclonal Ab (mAb)-mediated protection in a mouse model of nHSV infection. For a panel of glycoprotein D (gD)-specific mAbs, neutralization and effector functions contributed to nHSV-1 protection. In contrast, effector functions alone were sufficient to protect against nHSV-2, exposing a functional dichotomy between virus types consistent with vaccine trial results. Effector functions are therefore crucial for protection by these gD-specific mAbs, informing effective Ab and vaccine design and demonstrating the potential of polyfunctional Abs as therapeutics for nHSV infections.

Skipping of FCER1G Exon 2 Is Common in Human Brain But Not Associated with the Alzheimer’s Disease Genetic Risk Factor rs2070902

Article

Full-text available

Nov 2023

Background Understanding the mechanisms whereby genetic variants influence the risk of Alzheimer’s disease (AD) may provide insights into treatments that could reduce AD risk. Objective Here, we sought to test the hypothesis that a single nucleotide polymorphism (SNP) associated with AD risk, rs2070902, influences splicing of FCER1G exon 2. Methods AD and non-AD brain samples were analyzed for FCER1G expression by genotyping, immunohistochemistry, immunofluorescence, and qPCR. Results The protein encoded by FCER1G, FcRγ, is robustly expressed in microglia in both AD and non-AD brain. The FCER1G isoform lacking exon 2 (D2-FCER1G) was readily detectable. Moreover, the proportion of FCER1G expressed as this isoform was increased in brains with high AD neuropathology. However, the proportion of FCER1G expressed as the D2-FCER1G isoform was not associated with rs2070902 genotype. Conclusions In summary, the proportion of FCER1G expressed as the D2-FCER1G isoform is increased with AD neuropathology but is not associated with rs2070902.

511 Activity of anti-CD47 antibodies is enhanced through Fc optimization

Conference Paper

Full-text available

Oct 2023

Background The CD47/SIRPa axis plays a crucial role in cancer immunosurveillance.1 2 While anti-CD47 antibodies have shown promise in several preclinical models,1 3–8 results from early phase clinical trials have shown limited clinical benefit,9–11 suggesting that the sole blockade of CD47 by the antibody Fab domain might not be sufficient for effective tumor control. A critical question that remains to be answered is whether interactions between the antibody Fc and Fc gamma receptors (FcgRs) also contribute to their antitumor activity.12 13 Our study aims to investigate the role of the Fc domain in the in vivo antitumor activity of anti-CD47 antibodies using immunocompetent species-matched models, overcoming limitations of previous studies conducted in immunocompromised models or with interspecies differences between mouse (m) and human (h) CD47, SIRPa and FcγRs. Methods We modified the Fc domain of the anti-mCD47 antibody MIAP301 to generate antibodies with varying affinity to mFcgRs: 1) MIAP301-mIgG2a Fc, binding to preferentially to activating mFcgRs, 2) MIAP301-mIgG1 Fc, binding to the inhibitory mFcgRIIB, and 3) MIAP301-mIgG1-D265A Fc, which lacks binding to any mFcgRs. We evaluated the antitumor activity of these antibodies in MC38 and B16 tumor models in immunocompetent C57BL/6J mice and mice lacking activating FcgRs.¹⁵ Additionally, we generated a mouse humanized for the expression of hCD47, hSIRPa and hFcgRs by CRISPR/Cas9-mediated gene-targeting strategy, and by backcrossing to our hFcgR mice.¹⁶ We used this model to compare the antitumor activities of the anti-hCD47 antibody Magrolimab (5F9-hIgG4).¹⁴ and an Fc-optimized variant that enhances binding for all the activating hFcgRs (5F9-GAALIE) generated in the lab. Results The MIAP301-mIgG2a Fc variant led to the most significant reduction in tumor burden when compared to the control or other Fc variants in WT mice in both MC38 and B16 models. This therapeutic effect was abrogated in mice lacking activating FcgRs (figure 1). The CD47/SIRPa/FcgR humanized mice recapitulate the expression profile of CD47 and SIRPa found in human cells (figure 2). Furthermore, increasing dosing concentrations of both 5F9-hIgG4 and 5F9-GAALIE antibodies led to on-target anemia and thrombocytopenia in hCD47/hSIRPa/hFcgR mice, recapitulating results from clinical trials (figure 3). Intratumoral administration of the Fc optimized 5F9-GAALIE results in enhanced long-term antitumor immunity, abscopal antitumor effect, and minimal on-target toxicity when compared to 5F9-hIgG4 or control alone or in combination with PD-1 blockade (figure 4). Conclusions The antitumor activities of anti-CD47 antibodies require interactions with activating FcgRs, highlighting the importance of Fc optimization in the development of effective anti-CD47 therapies. Acknowledgements We thank Maria L. Baez, Alessandra E. Marino, and Carlo M. Sevilla for their excellent technical assistance. We also thank all the members of the J.V.R. Laboratory of Molecular Genetics and Immunology for helping discussions and sharing experiment materials. References • Willingham SB, Volkmer JP, Gentles AJ, et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci U S A 2012; 109 :6662–7. • Adams S, van der Laan LJ, Vernon-Wilson E, et al. Signal-regulatory protein is selectively expressed by myeloid and neuronal cells. J Immunol 1998; 161 :1853–9. • Chao MP, Alizadeh AA, Tang C, et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell 2010; 142 :699–713. • Majeti R, Chao MP, Alizadeh AA, et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 2009; 138 :286–99. • Jaiswal S, Jamieson CH, Pang WW, et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 2009; 138 :271–85. • Weiskopf K, Jahchan NS, Schnorr PJ, et al. CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer. J Clin Invest 2016; 126 :2610–20. • Liu X, Pu Y, Cron K, et al. CD47 blockade triggers T cell-mediated destruction of immunogenic tumors. Nat Med 2015; 21 :1209–15. • Liu Q, Wen W, Tang L, et al. Inhibition of SIRPalpha in dendritic cells potentiates potent antitumor immunity. Oncoimmunology 2016; 5 :e1183850. • Bouwstra R, van Meerten T, Bremer E. CD47-SIRPalpha blocking-based immunotherapy: Current and prospective therapeutic strategies. Clin Transl Med 2022; 12 :e943. • Uger R, Johnson L. Blockade of the CD47-SIRPalpha axis: a promising approach for cancer immunotherapy. Expert Opin Biol Ther 2020; 20 :5–8. • Jalil AR, Andrechak JC, Discher DE. Macrophage checkpoint blockade: results from initial clinical trials, binding analyses, and CD47-SIRPalpha structure-function. Antib Ther 2020; 3 :80–94. • Zhao XW, Matlung HL, Kuijpers TW, van den Berg TK. On the mechanism of CD47 targeting in cancer. Proc Natl Acad Sci U S A 2012; 109 :E2843; author reply E4–5. • Zhao XW, Kuijpers TW, van den Berg TK. Is targeting of CD47-SIRPalpha enough for treating hematopoietic malignancy? Blood 2012; 119 :4333–4; author reply 4–5. • Takai T, Li M, Sylvestre D, Clynes R, Ravetch JV. FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell 1994; 76 :519–29. • Smith P, DiLillo DJ, Bournazos S, Li F, Ravetch JV. Mouse model recapitulating human Fcgamma receptor structural and functional diversity. Proc Natl Acad Sci U S A 2012; 109 :6181–6. • Advani R, Flinn I, Popplewell L, et al. CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin’s Lymphoma. N Engl J Med 2018;379:1711–21. • Download figure • Open in new tab • Download powerpoint Abstract 511 Figure 1 Engagement of activating FcgRs enhances in vivo antitumor activity of antibodies blocking mouse CD47. (A) Average growth ± SEM of sq. MC38 tumors in WT (right) or FcR-y chain mice (left), treated with Fc variants of anti-mCD47 ab (MIAP301) or control (50ug IT, d. 8,10,14 and 18). (B) Average of lung metastases (Min to Max) of WT mice inoculated IV with B16 tumor cells, treated with anti-mCD47 ab Fc variants (MIAP301) or control (20 mg/Kg IP, d. 1,4,7 and 11). • Download figure • Open in new tab • Download powerpoint Abstract 511 Figure 2 Generation of humanized mouse model for CD47, SIRPα, and FcyRs. (A) Schematic drawing showing the generation of the hCD47/hSIRPa/hFcyR mice. (B) Flow cytometry analysis of hCD47 and hSIRPα in red blood cells (RBC), platelets, CD3+ and CD11b+ leucocytes isolated from peripheral blood of human (top) and hCD47/hSIRPα KI mice (bottom). • Download figure • Open in new tab • Download powerpoint Abstract 511 Figure 3 Humanized mouse recapitulates toxicity profile of fully humanized anti-CD47 antibodies. (A) Peripheral RBC and platelet count from hCD47/hSIRPa/hFcyR mice after treatment with increasing doses of 5F9-hlgG4 or (B) 5F9- GAALIE ab (2.5, 5, and 10 mg/Kg d. 0, 4, 8 and 11) • Download figure • Open in new tab • Download powerpoint Abstract 511 Figure 4 Fc-optimized humanized anti-CD47 Ab promotes effective in vivo antitumor activity (A) Average growth ± SEM of injected (left) and non-injected contralateral (right) sq. MC38 hCD47 KI tumors in hCD47/hSIRPa/hFcyR, treated with 5F9-hlgG4, 5F9-GAALIE ab or control (50ug IT, d. 7, 9, and 12). (B) Serial measurements of peripheral RBC and platelets from hCD47/hSIRPa/hFcyR mice after treatment with 5F9-hlgG4 or 5F9-GAALIE (d. 0 indicates the day when treatment was started). (C) Average growth + SEM of sq. B16 hCD47 KI tumors in hCD47/hSIRPa/hFcyR, treated with 5F9-hlgG4, 5F9-GAALIE ab alone (50ug IT, d. 8,10,14 and 18), or in combination with anti-PD1 ab (RMP1–14, 200 ug IP d. 8, 10, 14 and 18), or control.

Uncovering the neuroprotective effect of vitamin B12 in pneumococcal meningitis: insights into its pleiotropic mode of action at the transcriptional level OPEN ACCESS EDITED BY

Article

Full-text available

Oct 2023

Background The interplay between bacterial virulence factors and the host innate immune response in pneumococcal meningitis (PM) can result in uncontrolled neuroinflammation, which is known to induce apoptotic death of progenitor cells and post-mitotic neurons in the hippocampal dentate gyrus, resulting in cognitive impairment. Vitamin B12 attenuates hippocampal damage and reduces the expression of some key inflammatory genes in PM, by acting as an epidrug that promotes DNA methylation, with increased production of S-adenosyl-methionine, the universal donor of methyl. Material and methods Eleven-day-old rats were infected with S. pneumoniae via intracisternal injection and then administered either vitamin B12 or a placebo. After 24 hours of infection, the animals were euthanized, and apoptosis in the hippocampal dentate gyrus, microglia activation, and the inflammatory infiltrate were quantified in one brain hemisphere. The other hemisphere was used for RNA-Seq and RT-qPCR analysis. Results In this study, adjuvant therapy with B12 was found to modulate the hippocampal transcriptional signature induced by PM in infant rats, mitigating the effects of the disease in canonical pathways related to the recognition of pathogens by immune cells, signaling via NF-kB, production of pro-inflammatory cytokines, migration of peripheral leukocytes into the central nervous system, and production of reactive species. Phenotypic analysis revealed that B12 effectively inhibited microglia activation in the hippocampus and reduced the inflammatory infiltrate in the central nervous system of the infected animals. These pleiotropic transcriptional effects of B12 that lead to neuroprotection are partly regulated by alterations in histone methylation markings. No adverse effects of B12 were predicted or observed, reinforcing the well-established safety profile of this epidrug. Conclusion B12 effectively mitigates the impact of PM on pivotal neuroinflammatory pathways. This leads to reduced microglia activation and inflammatory infiltrate within the central nervous system, resulting in the attenuation of hippocampal damage. The anti-inflammatory and neuroprotective effects of B12 involve the modulation of histone markings in hippocampal neural cells.

Autoantibody Diversity Is Augmented in Women with Breast Cancer and Is Related to the Stage of the Disease

Article

Full-text available

Sep 2023

Breast cancer (BC) is the most frequent malignant neoplasia and leading cause of cancer mortality for women. A timely diagnosis of BC is crucial to ensure the best chances of survival. Among the various screening tools for BC, antibodies directed towards self-antigens or tumor-associated anti-gens (autoantibodies) have emerged as an alternative to image-based screening modalities. However, little attention has been paid to the global diversity of autoantibodies. This work aimed to analyze the diversity of autoantibodies reactive to antigens expressed by the BC cell line T47D in the sera of Mexican women with BC, benign breast pathology (BBP), or without breast pathology (WBP). We found that the diversity of antibodies in the sera was higher in the BC and BBP groups than in the WBP group. Likewise, the diversity changed with the progression of BC. Our results show and measure the complexity of the antibody response in breast health and disease.

Uncovering the Neuroprotective Effect of Vitamin B12 in Pneumococcal Meningitis: Insights into Its Pleiotropic Mode of Action at the Transcriptional Level

Preprint

Full-text available

Jun 2023

Background The interplay between bacterial virulence factors and the host innate immune response in pneumococcal meningitis (PM) can result in uncontrolled neuroinflammation, which is known to induce apoptotic death of progenitor cells and post-mitotic neurons in the hippocampal dentate gyrus, resulting in cognitive impairment. Vitamin B12 attenuates hippocampal damage and reduces the expression of some key inflammatory genes in PM, by acting as an epidrug that promotes DNA methylation, with increased production of S-adenosyl-methionine, the universal donor of methyl. Objective This study aimed to investigate the effects of adjuvant therapy with vitamin B12 on microglial activation, inflammatory infiltrate within the central nervous system, as well as the hippocampal transcriptome and histone markings in infant rats with PM Material and Methods Elven-day-old rats were infected with S. pneumoniae via intracisternal injection and then administered either vitamin B12 or a placebo. After 24 hours of infection, the animals were euthanized, and apoptosis in the hippocampal dentate gyrus, microglia activation, and the inflammatory infiltrate were quantified in one brain hemisphere. The other hemisphere was used for RNA-Seq and RT-qPCR analysis. Results In this study, adjuvant therapy with B12 was found to modulate the hippocampal transcriptional signature induced by PM in infant rats, mitigating the effects of the disease in canonical pathways related to the recognition of pathogens by immune cells, signaling via NF-kB, production of pro-inflammatory cytokines, migration of peripheral leukocytes into the central nervous system, and production of reactive species. Phenotypic analysis revealed that B12 effectively inhibited microglia activation in the hippocampus and reduced the inflammatory infiltrate in the central nervous system of the infected animals. These pleiotropic transcriptional effects of B12 that lead to neuroprotection are partly regulated by alterations in histone methylation markings. No adverse effects of B12 were predicted or observed, reinforcing the well-established safety profile of this epidrug. Conclusion B12 effectively mitigates the impact of PM on pivotal neuroinflammatory pathways. This leads to reduced microglia activation and inflammatory infiltrate within the central nervous system, resulting in the attenuation of hippocampal damage. The anti-inflammatory and neuroprotective effects of B12 involve the modulation of histone markings in hippocampal neural cells.

Blockade of trans PD-L1 interaction with CD80 augments antitumor immunity

Article

Full-text available

Apr 2023
P NATL ACAD SCI USA

PD-L1 has two receptors: PD-1 and CD80. Previous reports assumed that PD-L1 and CD80 interacted in trans, but recent reports showed that only cis PD-L1/CD80 interactions existed, and prevention of cis PD-L1/CD80 interactions on antigen-presenting cells (APCs) reduced antitumor immunity via augmenting PD-L1/PD-1 and CD80/CTLA4 interactions between T and APCs. Here, using tumor-bearing mice capable of cis and trans or trans only PD-L1/CD80 interactions, we show that trans PD-L1/CD80 interactions do exist between tumor and T cells, and the effects of trans PD-L1/CD80 interactions require tumor cell expression of MHC-I and T cell expression of CD28. The blockade of PD-L1/CD80 interactions in mice with both cis and trans interactions or with only trans interactions augments antitumor immunity by expanding IFN-γ-producing CD8+ T cells and IFN-γ-dependent NOS2-expressing tumor-associated macrophages. Our studies indicate that although cis and trans PD-L1/CD80 interactions may have opposite effects on antitumor immunity, the net effect of blocking PD-L1/CD80 interactions in vivo augments CD8+ T cell-mediated antitumor immunity.

Pili Subunit PilA Contributes to the Cytoadhesion of Glaesserella Parasuis to Host Cells and Provides Immunoprotection

Article

Full-text available

Mar 2023
APPL ENVIRON MICROB

Glaesserella parasuis (G. parasuis) is commonly located in the upper respiratory tract of pigs as an opportunistic pathogen. It can cause Glässer's disease, which leads to serious economic losses in the swine industry. The occurrence of the disease is often linked with the adhesion and colonization of the pathogen. The PilA pilus subunit is important for adhesion to the host, twitching motility, and biofilm formation in many bacteria. However, no research has focused on the function of PilA in G. parasuis. To further reveal the pathogenesis of G. parasuis and to search for subunit vaccine candidates, we investigated whether PilA could adhere to cells and provide immune protection. A bioinformatic analysis showed that the protein secondary structure of the G. parasuis PilA was similar to that of Haemophilus influenzae (HI). Cell adhesion, ELISA, and far-Western blotting showed that rPilA could bind porcine-derived, porcine kidney-15 (PK-15) cells, swine tracheal epithelial cells (STECs), and the extracellular matrix components fibronectin (FN) and laminin (LN). An immunogenicity analysis showed that recombinant PilA (rPilA) reacted specifically with convalescent and hyperimmune serum. Importantly, purified rPilA elicited a strong immune response and conferred robust protection against challenges with serovar 5 G. parasuis in mice. These results suggested that the PilA protein might help G. parasuis adhere to host cells by binding to FN and LN, and its immunogenicity establishes it as a promising, novel subunit vaccine candidate against infections with G. parasuis. IMPORTANCE G. parasuis is one of the most prevalent bacterial infections in swine production and can lead to huge economic losses around the world. A full understanding of colonization and immunity with G. parasuis infections will be essential in disease control. In this study, the PilA protein, which is a common virulence factor in other bacteria that mediates adherence to the host, was assessed. The results suggested that the PilA protein of G. parasuis can mediate adhesion to host cells through FN and LN, which provides a new idea for the study of the pathogenicity of G. parasuis. Furthermore, fimbriae usually have high immunogenicity. Immunogenicity and protective capacity results showed that the use of this recombinant PilA antigen might be a promising candidate vaccine antigen with which to prevent G. parasuis infections.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity

Article

Full-text available

Feb 2023
eLife

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

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

Article

Full-text available

Jan 2023
J LEUKOCYTE BIOL

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

Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor

Preprint

Full-text available

Dec 2022

DNAX-associated protein 12 kDa size (DAP12) is a dominant immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis. Although several DAP12-associated receptors (DARs) have been identified in osteoclasts, including triggering receptor expressed on myeloid cells 2 (TREM-2), C-type lectin member 5A (CLEC5A), and sialic acid-binding Ig-like lectin (Siglec)-15, their precise role in the development of osteoclasts and bone remodeling remain poorly understood. In this study, mice deficient in Trem-2 , Clec5a , Siglec-15 were generated. In addition, mice double deficient in these DAR genes and FcεRI gamma chain (FcR)γ, an alternative ITAM adaptor to DAP12, were generated. Bone mass analysis was conducted on all mice. Notably, Siglec-15 deficient mice and Siglec-15/FcRγ double deficient mice exhibited mild and severe osteopetrosis respectively. In contrast, other DAR deficient mice showed normal bone phenotype. Likewise, osteoclasts from Siglec- 15 deficient mice failed to form an actin ring, suggesting that Siglec-15 promotes bone resorption principally by modulating the cytoskeletal organization of osteoclasts. Our results further demonstrated that Siglec-15 may be involved in macrophage colony-stimulating factor (M-CSF) signaling; therefore, mediating cytoskeletal organization of osteoclasts via promoting activation of the Ras-associated protein-1 (RAP1)/Ras-related C3 botulinum toxin substrate 1 (RAC1) pathway, which is an essential component of the canonical osteoclast cytoskeletal organization complex. Furthermore, biochemical analysis revealed that Sigelc-15 activates M-CSF-induced Rac1 pathway through formation of a complex with p130CAS and CrkII, leading to cytoskeletal remodeling of osteoclasts. Our data provide genetic and biochemical evidence that Siglec-15 facilitates M-CSF-induced cytoskeletal remodeling of the osteoclasts.

RIG-I and TLR-7/8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine

Article

Full-text available

Nov 2022

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)‐PEG‐Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant. Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centers during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.

Mucosal Immunity and the Gut-Microbiota-Brain-Axis in Neuroimmune Disease

Article

Full-text available

Nov 2022
INT J MOL SCI

Recent advances in next-generation sequencing (NGS) technologies have opened the door to a wellspring of information regarding the composition of the gut microbiota. Leveraging NGS technology, early metagenomic studies revealed that several diseases, such as Alzheimer’s disease, Parkinson’s disease, autism, and myalgic encephalomyelitis, are characterized by alterations in the diversity of gut-associated microbes. More recently, interest has shifted toward understanding how these microbes impact their host, with a special emphasis on their interactions with the brain. Such interactions typically occur either systemically, through the production of small molecules in the gut that are released into circulation, or through signaling via the vagus nerves which directly connect the enteric nervous system to the central nervous system. Collectively, this system of communication is now commonly referred to as the gut-microbiota-brain axis. While equally important, little attention has focused on the causes of the alterations in the composition of gut microbiota. Although several factors can contribute, mucosal immunity plays a significant role in shaping the microbiota in both healthy individuals and in association with several diseases. The purpose of this review is to provide a brief overview of the components of mucosal immunity that impact the gut microbiota and then discuss how altered immunological conditions may shape the gut microbiota and consequently affect neuroimmune diseases, using a select group of common neuroimmune diseases as examples.

Impairment of IgG Fc functions promotes tumor progression and suppresses NK cell antitumor actions

Article

Full-text available

Sep 2022

Natural killer (NK) cells mediate antibody dependent cytotoxic killing of cancer cells via cross-linking FcγR on NK cells with IgG-Fc. Studies have shown that the single-hinge cleaved IgGs (scIgGs) have dysfunctional Fc and failed engagement with FcγRs on immune cells. However, little is known about how scIgGs impact on antitumor immunity in the tumor microenvironment. In this study, we revealed a significant association of tumor scIgGs with tumor progression and poor outcomes of breast cancer patients ( n = 547). Using multiple mouse tumor models, we demonstrated that tumor scIgGs reduced NK cell cytotoxic activities and resulted in aggressive tumor progression. We further showed that an anti-hinge specific monoclonal antibody (AHA) rescued the dysfunctional Fc in scIgGs by providing a functional Fc and restored NK cell cytotoxic activity. These findings point to a novel immunotherapeutic strategy to enhance Fc engagement with FcγRs for activation of anticancer immunity.

Genetic deletion and pharmacologic inhibition of FcER1 reduces renal injury in mouse models of diabetic nephropathy

Preprint

Sep 2022

FcER1 forms a high affinity multimeric cell-surface receptor for the Fc region of immunoglobulin E (IgE) and controls the activation of mast cells and basophils. Antigen binding and cross-linking of FcER1 associated IgE induces several downstream signaling pathways that result in diverse outcomes. Canonical signaling through IgE-FcER1 has been related to allergic responses, however, recent studies have identified that their function in mast cell and basophils contribute to other pathogenic conditions such as cancer and diabetes. Previous studies have demonstrated that FcER1 protein is upregulated in advanced diabetic kidney disease (DKD) making it a targetable molecule for the treatment of DKD. This study presents evidence that loss of FcER1 signaling reduces proteinuria and renal injury in two pre-clinical mouse models of diabetes. Mice deficient for fcer1 are protected from streptozotocin mediated induction of proteinuria and display reduced fibrosis and mast cell infiltration in kidney. Furthermore, inhibition of FcER1 signaling with an antibody directed against the γ-subunit reduces proteinuria in a spontaneous model of type II diabetes. Our results show significant reduction of proteinuria and tissue damage in pre-clinical DKD models demonstrating the potential of FcER1 inhibitory approaches for developing new therapies in DKD.

Distinct impact of IgG subclass on autoantibody pathogenicity in different IgG4-mediated diseases

Article

Full-text available

Aug 2022
eLife

IgG4 is the least potent human IgG subclass for the FcγR-mediated antibody effector function. Paradoxically, IgG4 is also the dominant IgG subclass of pathogenic autoantibodies in IgG4-mediated diseases. Here we show that the IgG subclass and Fc-FcγR interaction have a distinct impact on the pathogenic function of autoantibodies in different IgG4-mediated diseases in mouse models. While IgG4 and its weak Fc-FcγR interaction have an ameliorative role in the pathogenicity of anti-ADAMTS13 autoantibodies isolated from thrombotic thrombocytopenic purpura (TTP) patients, they have an unexpected exacerbating effect on anti-Dsg1 autoantibody pathogenicity in pemphigus foliaceus (PF) models. Strikingly, a non-pathogenic anti-Dsg1 antibody variant optimized for FcγR-mediated effector function can attenuate the skin lesions induced by pathogenic anti-Dsg1 antibodies by promoting the clearance of dead keratinocytes. These studies suggest that IgG effector function contributes to the clearance of autoantibody-Ag complexes, which is harmful in TTP, but beneficial in PF and may provide new therapeutic opportunity.

Antibody Diversity in Cancer: Translational Implications and Beyond

Article

Full-text available

Jul 2022

Patients with cancer tend to develop antibodies to autologous proteins. This phenomenon has been observed across multiple cancer types, including bladder, lung, colon, prostate, and melanoma. These antibodies potentially arise due to induced inflammation or an increase in self-antigens. Studies focusing on antibody diversity are particularly attractive for their diagnostic value considering antibodies are present at an early diseased stage, serum samples are relatively easy to obtain, and the prevalence of antibodies is high even when the target antigen is minimally expressed. Conversely, the surveillance of serum proteins in cancer patients is relatively challenging because they often show variability in expression and are less abundant. Moreover, an antibody’s presence is also useful as it suggests the relative immunogenicity of a given antigen. For these reasons, profiling antibodies’ responses is actively considered to detect the spread of antigens following immunotherapy. The current review focuses on expanding the knowledge of antibodies and their diversity, and the impact of antibody diversity on cancer regression and progression.

Durable Immunity to Ricin Toxin Elicited by Intranasally Administered Monoclonal Antibody–Based Immune Complexes

Article

Full-text available

Jun 2022

Inhalation of ricin toxin (RT) elicits profuse inflammation and cell death within the upper and lower airways, ultimately culminating in acute respiratory distress syndrome. We previously reported that the effects of pulmonary RT exposure in mice are nullified by intranasal administration of an mAb mixture consisting of PB10, directed against ricin's enzymatic subunit (RTA), and SylH3, directed against ricin's binding subunit (RTB). We now report that delivery of PB10 and SylH3 as an RT-mAb immune complex (RIC) to mice by the intranasal or i.p. routes stimulates the rapid onset of RT-specific serum IgG that persists for months. RIC administration also induced high-titer, toxin-neutralizing Abs. Moreover, RIC-treated mice were immune to a subsequent 5 × LD50 RT challenge on days 30 or 90. Intranasal RIC administration was more effective than i.p. delivery at rendering mice immune to intranasal RT exposure. Finally, we found that the onset of RT-specific serum IgG following RIC delivery was independent of FcγR engagement, as revealed through FcγR knockout mice and RICs generated with PB10/SylH3 LALA (leucine to alanine) derivatives. In conclusion, a single dose of RICs given intranasally to mice was sufficient to stimulate durable protective immunity to RT by an FcγR-independent pathway.

RIG-I and TLR-7/8 agonists as combination adjuvant shape unique antibody and cellular vaccine responses to seasonal influenza vaccine

Preprint

Full-text available

May 2022

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)‐PEG‐Chol) as adjuvants, when co-administered with a licenced quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant. Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centres during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.

RIG-I and TLR-7/8 agonists as combination adjuvant shape unique antibody and cellular vaccine responses to seasonal influenza vaccine

Preprint

Full-text available

May 2022

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)‐PEG‐Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant. Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centres during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.

RIG-I and TLR-7/8 agonists as combination adjuvant shape unique antibody and cellular vaccine responses to seasonal influenza vaccine

Preprint

Full-text available

May 2022

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)‐PEG‐Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant. Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centres during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.

Comparison of Immune Responses Elicited by SARS-CoV-2 mRNA and Recombinant Protein Vaccine Candidates

Article

Full-text available

May 2022

After the outbreak of COVID-19, billions of vaccines with different types have been administrated, including recombinant protein vaccines and mRNA vaccines. Although both types of SARS-CoV-2 vaccine can protect people from viral infection, their differences in humoral and cellular immune responses are still not clearly understood. In this study, we made a head-to-head comparison between an mRNA vaccine candidate and a recombinant protein vaccine we developed previously. Results demonstrated that both vaccine candidates could elicit high specific binding and neutralizing antibody titers in BALB/c mice, but with bias towards different IgG subtypes. Besides, the mRNA vaccine candidate induces higher cellular immune responses than the recombinant protein vaccine. To date, this is the first reported study to directly compare the immune responses of both arms between SARS-CoV-2 mRNA and recombinant vaccines.

An Updated Review and Meta Analysis of Lipoprotein Glomerulopathy

Article

Full-text available

May 2022

More than 200 cases of lipoprotein glomerulopathy (LPG) have been reported since it was first discovered 30 years ago. Although relatively rare, LPG is clinically an important cause of nephrotic syndrome and end-stage renal disease. Mutations in the APOE gene are the leading cause of LPG. APOE mutations are an important determinant of lipid profiles and cardiovascular health in the population and can precipitate dysbetalipoproteinemia and glomerulopathy. Apolipoprotein E-related glomerular disorders include APOE2 homozygote glomerulopathy and LPG with heterozygous APOE mutations. In recent years, there has been a rapid increase in the number of LPG case reports and some progress in research into the mechanism and animal models of LPG. We consequently need to update recent epidemiological studies and the molecular mechanisms of LPG. This endeavor may help us not only to diagnose and treat LPG in a more personized manner but also to better understand the potential relationship between lipids and the kidney.

IgE+, Kit–, I-A I-E myeloid cells are the

Article

Full-text available

Apr 2002

FcγRIIB potentiates differentiation of myeloid-derived suppressor cells to mediate tumor immunoescape

Article

Full-text available

Jan 2022
thno

Background: FcγRIIB, the sole inhibitory receptor of the Fc gamma receptor family, plays pivotal roles in innate and adaptive immune responses. However, the expression and function of FcγRIIB in myeloid-derived suppressor cells (MDSCs) remains unknown. This study aimed to investigate whether and how FcγRIIB regulates the immunosuppressive activity of MDSCs during cancer development. Methods: The MC38 and B16-F10 tumor-bearing mouse models were established to investigate the role of FcγRIIB during tumor progression. FcγRIIB-deficient mice, adoptive cell transfer, mRNA-sequencing and flow cytometry analysis were used to assess the role of FcγRIIB on immunosuppressive activity and differentiation of MDSCs. Results: Here we show that FcγRIIB was upregulated in tumor-infiltrated MDSCs. FcγRIIB-deficient mice showed decreased accumulation of MDSCs in the tumor microenvironment (TME) compared with wild-type mice. FcγRIIB was required for the differentiation and immunosuppressive activity of MDSCs. Mechanistically, tumor cell-derived granulocyte-macrophage colony stimulating factor (GM-CSF) increased the expression of FcγRIIB on hematopoietic progenitor cells (HPCs) by activating specificity protein 1 (Sp1), subsequently FcγRIIB promoted the generation of MDSCs from HPCs via Stat3 signaling. Furthermore, blockade of Sp1 dampened MDSC differentiation and infiltration in the TME and enhanced the anti-tumor therapeutic efficacy of gemcitabine. Conclusion: These results uncover an unrecognized regulatory role of the FcγRIIB in abnormal differentiation of MDSCs during cancer development and suggest a potential therapeutic target for anti-tumor therapy.

Nanoparticle-assisted Targeting Delivery Technologies for Preventing Organ Rejection

Article

Apr 2024
TRANSPLANTATION

Although organ transplantation is a life-saving medical procedure, the challenge of posttransplant rejection necessitates safe and effective immune modulation strategies. Nanodelivery approaches may have the potential to overcome the limitations of small-molecule immunosuppressive drugs, achieving efficacious treatment options for transplant tolerance without compromising overall host immunity. This review highlights recent advances in biomaterial-assisted formulations and technologies for targeted nanodrug delivery with transplant organ- or immune cell–level precision for treating graft rejection after transplantation. We provide an overview of the mechanism of transplantation rejection, current clinically approved immunosuppressive drugs, and their relevant limitations. Finally, we discuss the targeting principles and advantages of organ- and immune cell–specific delivery technologies. The development of biomaterial-assisted novel therapeutic strategies holds considerable promise for treating organ rejection and clinical translation.

Supplementation of seasonal vaccine with multi-subtype neuraminidase and M2 ectodomain virus-like particle improves protection against homologous and heterologous influenza viruses in aged mice

Article

Mar 2024
ANTIVIR RES

Microbiota-dependent activation of CD4+ T cells induces CTLA-4 blockade-associated colitis via Fcγ receptors

Article

Jan 2024
SCIENCE

Immune checkpoint inhibitors can stimulate antitumor immunity but can also induce toxicities termed immune-related adverse events (irAEs). Colitis is a common and severe irAE that can lead to treatment discontinuation. Mechanistic understanding of gut irAEs has been hampered because robust colitis is not observed in laboratory mice treated with checkpoint inhibitors. We report here that this limitation can be overcome by using mice harboring the microbiota of wild-caught mice, which develop overt colitis following treatment with anti-CTLA-4 antibodies. Intestinal inflammation is driven by unrestrained activation of IFNγ-producing CD4 ⁺ T cells and depletion of peripherally induced regulatory T cells through Fcγ receptor signaling. Accordingly, anti-CTLA-4 nanobodies that lack an Fc domain can promote antitumor responses without triggering colitis. This work suggests a strategy for mitigating gut irAEs while preserving antitumor stimulating effects of CTLA-4 blockade.

Fcγ receptors and immunomodulatory antibodies in cancer

Article

Dec 2023
NAT REV CANCER

The discovery of both cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) as negative regulators of antitumour immunity led to the development of numerous immunomodulatory antibodies as cancer treatments. Preclinical studies have demonstrated that the efficacy of immunoglobulin G (IgG)-based therapies depends not only on their ability to block or engage their targets but also on the antibody's constant region (Fc) and its interactions with Fcγ receptors (FcγRs). Fc-FcγR interactions are essential for the activity of tumour-targeting antibodies, such as rituximab, trastuzumab and cetuximab, where the killing of tumour cells occurs at least in part due to these mechanisms. However, our understanding of these interactions in the context of immunomodulatory antibodies designed to boost antitumour immunity remains less explored. In this Review, we discuss our current understanding of the contribution of FcγRs to the in vivo activity of immunomodulatory antibodies and the challenges of translating results from preclinical models into the clinic. In addition, we review the impact of genetic variability of human FcγRs on the activity of therapeutic antibodies and how antibody engineering is being utilized to develop the next generation of cancer immunotherapies.

The antitumor activities of anti-CD47 antibodies require Fc-FcγR interactions

Article

Nov 2023
CANCER CELL

An immunomodulatory role of Fc receptor γ chain independent of FcγR ligation by IgG in acute neuroinflammation triggered by MPTP intoxication

Article

Nov 2023
NEUROCHEM INT

Antibody effector functions are required for broad and potent protection of neonates from herpes simplex virus infection

Preprint

Full-text available

Aug 2023

The failure of multiple herpes simplex virus (HSV) vaccine candidates that induce neutralizing antibody responses raises the hypothesis that other activities, such as Fc domain-dependent effector functions, may be critical for protection. While neonatal HSV (nHSV) infection result in mortality and lifelong neurological morbidity in humans, it is uncommon among neonates with a seropositive birthing parent, suggesting the potential efficacy of antibody-based therapeutics to protect neonates. We therefore investigated the mechanisms of monoclonal antibody (mAb)-mediated protection in a mouse model of nHSV infection. Both neutralization and effector functions contributed to robust protection against nHSV-1. In contrast, effector functions alone were sufficient to protect against nHSV-2, exposing a functional dichotomy between virus types that is consistent with vaccine trial results. Together, these results emphasize that effector functions are crucial for optimal mAb-mediated protection, informing effective Ab and vaccine design, and demonstrating the potential of polyfunctional Abs as potent therapeutics for nHSV infections. Graphical abstract. Mechanistic dissection of antibody-mediated protection from HSV Monoclonal antibodies (mAbs) with varying neutralizing potencies and Fc modifications that impact effector function were evaluated in wildtype (WT) and FcγR-/- mice to define mechanisms of antibody-mediated protection from HSV infection. To model human vulnerability to HSV disease during the neonatal period, neonatal mice were challenged with HSV, treated with mAb, and then assessed for morbidity and mortality. We observed that polyfunctional mAbs provide broader and more potent protection than antibodies with either low neutralization or low effector function. Moreover, while sufficient for protection against HSV-1, neutralization activity alone was unable to protect from HSV-2 infection.

Equine β-defensin 1 regulates cytokine expression and phagocytosis in S. aureus-infected mouse monocyte macrophages via the Paxillin-FAK-PI3K pathway

Article

Aug 2023

β-defensin-1 (BD-1) is a rich source of disulfide bonds and antibacterial peptides that exhibit direct bactericidal function. The expression of BD-1 is primarily induced by external stimulation and is known to correlate with TLR-mediated inflammation, suggesting its association with innate immune responses. Equine β-defensin-1 (eBD-1) belongs to the BD-1 family. Our previous study demonstrated that eBD-1 enhances cytokine expression and promotes macrophage phagocytosis of S. aureus, although the underlying mechanism remains unknown. In this study, we utilized a PI-3K inhibitor (PKI-402) to treat eBD-1 -treated S. aureus-infected macrophages in vitro. Our results revealed that PKI-402 decreased the expression of eBD-1-promoted TNF-α, IL-6, CXCL10, CD40, RANTES, and p65 mRNA. To further investigate the relationship between eBD-1 and phagocytosis, we examined the expression of paxillin and FcγRIII (CD16 receptor) using western blot and immunofluorescence techniques. Our findings demonstrated that eBD-1 enhanced CD16 and paxillin expression in S. aureus -infected macrophages. Considering the correlation between paxillin expression and focal adhesion kinase (FAK), we transfected FAK siRNA into macrophages and evaluated paxillin expression using western blot analysis. Additionally, we quantified the number of S. aureus phagocytosed by macrophages. The results indicated a reduction in both paxillin expression and the number of S. aureus phagocytosed by macrophages upon FAK siRNA treatment. Our study showed the eBD-1 promotes cytokine mRNA expression in S. aureus-infected macrophages regulated by PI-3K-NF-κB pathway, and it increases macrophage phagocytosis of S. aureus associated with the FAK-paxillin signaling pathway.

Insights into FcγR involvement in RA-associated autoantibody-induced pain-like behavior

Article

Jul 2023
BRAIN BEHAV IMMUN

Joint pain is one of the most debilitating symptoms of rheumatoid arthritis (RA) and patients frequently rate improvements in pain management as their priority. RA is hallmarked by the presence of autoantibodies against several post-translationally modified proteins (AMPAs) such as citrullinated, carbamylated and acetylated proteins. It has been suggested that autoantibody-mediated processes represent distinct mechanisms contributing to pain in RA. In this study we investigated the pronociceptive properties of monoclonal AMPA 1325:01B09 (B09 mAb) derived from plasma cells of a RA patient. We found that B09 mAb induces pain-like behavior in mice that is not associated with any visual, histological or transcriptional signs of inflammation in the joints, and not alleviated by non-steroidal anti-inflammatory drugs (NSAIDs). Instead, we found that B09 mAb is retained in dorsal root ganglia (DRG) and alters the expression of several satellite glia cell (SGC), neuron and macrophage-related factors in DRGs. Using mice that lack activating FcγRs, we uncovered that FcγRs are critical for the development of B09-induced pain-like behavior, and partially drive the transcriptional changes in the DRGs. Finally, we observed that B09 mAb binds SGC in vitro and in combination with external stimuli like ATP enhances transcriptional changes and protein release of pronociceptive factors from SGCs. We propose that certain RA antibodies bind epitopes in the DRG, here on SGCs, form immune complexes and activate resident macrophages via FcγR cross-linking. Our work supports the growing notion that autoantibodies can alter nociceptor signaling via mechanisms that are at large independent of local inflammatory processes in the joint.

The Antitumor Activities of Anti-CD47 Antibodies Require Fc-FcγR interactions

Preprint

Full-text available

Jun 2023

While anti-CD47 antibodies hold promise for cancer immunotherapy, early phase clinical trials have shown limited signs of clinical benefit, suggesting that blockade of CD47 alone may not be sufficient for effective tumor control. Here, we investigate the contributions of the Fc domain of anti-CD47 antibodies required for optimal in vivo antitumor activity across multiple species-matched models, providing new insights into the mechanisms underlying the efficacy of this emerging class of therapeutic antibodies. Using a novel mouse model humanized for CD47, SIRPα and FcγRs, we demonstrate that local administration of an Fc-engineered anti-CD47 antibody with enhanced binding to activating FcγRs modulates myeloid and T-cell subsets in the tumor microenvironment, resulting in improved long-term systemic antitumor immunity and minimal on-target off-tumor toxicity. Our results highlight the importance of Fc optimization in the development of effective anti-CD47 therapies and provide a novel approach for enhancing the antitumor activity of this promising immunotherapy. Highlights - Engagement of activating FcγRs augments the in vivo antitumor activity of CD47 blocking antibodies - Humanized mice for CD47, SIRPα and FcγRs allow assessment of hFcγRs contribution to the activity of anti-hCD47 Abs - Fc-optimized anti-hCD47 ab promotes systemic antitumor immunity with abscopal effect and minimal on-target toxicity Graphical Abstract

Fc epsilonRI gamma can support T cell development and function in mice lacking endogenous TCR zeta-chain.

Article

Jul 1997

Fc epsilonRI gamma (Fc gamma) is a member of the zeta family of signal transducing molecules that function as components of both the TCR and Fc receptors (FcR). While the majority of thymocytes and T cells express TCRs containing zeta-chain homodimers, certain unique populations of T cells express TCRs that contain both zeta and Fc gamma. To examine the ability of Fc gamma to substitute for zeta-chain in T cell development and function, we introduced a transgene encoding Fc gamma into mice made genetically deficient for zeta-chain (zeta(e)-/-). Analysis of thymocyte development in zeta(e)-/-;Fc gamma Tg mice demonstrated that Fc gamma was able to support the maturation of both gammadelta TCR+ and alphabeta TCR+ T cells. However, positive selection of alphabeta TCR+ thymocytes was less efficient in zeta(e)-/-;Fc gamma Tg mice than in zeta(e)-/- mice reconstituted with zeta-chain. This difference may be due to the fact that Fc gamma contains a single immunoreceptor tyrosine-based activation motif (ITAM) whereas zeta-chain contains three ITAMs. Interestingly, the peripheral T cells that develop in zeta(e)-/- mice reconstituted with Fc gamma are functional and respond to TCR-specific stimuli. These data suggest that Fc gamma and zeta are interchangeable in their ability to mediate T cell development and function, however zeta-chain is more efficient at promoting positive selection and T cell maturation. The difference in efficiency between zeta and Fc gamma may be responsible in part for the unusual developmental and functional properties of T cells that constitutively express Fc gamma as a signaling component of their TCRs.

Phagocytosis: receptors and biology

Chapter

Sep 2006

This book provides up-to-date information on the crucial interaction of pathogenic bacteria and professional phagocytes, the host cells whose purpose is to ingest, kill, and digest bacteria in defense against infection. The introductory chapters focus on the receptors used by professional phagocytes to recognize and phagocytose bacteria, and the signal transduction events that are essential for phagocytosis of bacteria. Subsequent chapters discuss specific bacterial pathogens and the strategies they use in confronting professional phagocytes. Examples include Helicobacter pylori, Streptococcus pneumoniae, and Yersinae, each of which uses distinct mechanisms to avoid being phagocytosed and killed. Contrasting examples include Listeria monocytogenes and Mycobacterium tuberculosis, which survive and replicate intracellularly, and actually cooperate with phagocytes to promote their entry into these cells. Together, the contributions in this book provide an outstanding review of current knowledge regarding the mechanisms of phagocytosis and how specific pathogenic bacteria avoid or exploit these mechanisms.

Die molekulare Grundlage für die höhere Sensitivität regulatorischer CD4\(^+\) T-Zellen im Vergleich zu konventionellen CD4\(^+\) T-Zellen gegenüber der Stimulation mit CD28 Superagonisten

Thesis

Jan 2022

Tobias Simon Gulde

In Ratten und Mäusen aktiviert der superagonistische anti-CD28 monoklonale Antikörper (CD28SA) vorzugsweise regulatorische T-Zellen. In niedriger Dosierung führt CD28SA zu einer fast ausschließlichen Aktivierung von regulatorischen T-Zellen (Tregs). Diese Beobachtung konnte inzwischen auch für menschliche Zellen in Zellkultur bestätigt werden. In gesunden und freiwilligen Testpersonen deutet die Zytokin-Antwort nach Applikationen von niedrigen CD28SA-Dosen darauf hin, dass sich diese Beobachtung auch in-vivo bewahrheitet. Eine Gabe von CD28SA in niedriger Dosierung, die zu einer exklusiven Aktivierung von regulatorischen T-Zellen führt, könnte somit in der Behandlung von Autoimmunkrankheiten oder von entzündlichen Erkrankungen eingesetzt werden. Eine mechanistische Erklärung für dieses Phänomen blieb lange Zeit unklar. Die CD28SA-vermittelte T-Zell-Aktivierung ist abhängig von der Verstärkung von basalen tonischen Signalen, die T-Zellen über ihren T-Zell-Rezeptor erhalten. Diese Tatsache führte zu der Hypothese, dass die schwachen, tonischen Signale, die konventionelle CD4+ T-Zellen in Abwesenheit ihrer spezifischen Antigene über den T-Zell-Rezeptor erhalten, ein stärkeres CD28 Signal für ihre Aktivierung benötigen als die selbstreaktiven regulatorischen T-Zellen, die ein stärkeres Selbstpeptid-TCR Signal erhalten. In dieser Arbeit konnte gezeigt werden, dass die Blockade von MHC-Klasse-II-Molekülen in Mäusen, in-vitro und in-vivo, den Vorteil der regulatorischen T-Zellen gegenüber den konventionellen T-Zellen bezüglich der Antwort auf niedrige CD28SA Dosierungen, aufhebt.

Autoantibodies targeting malondialdehyde-modifications in rheumatoid arthritis regulate osteoclasts via inducing glycolysis and lipid biosynthesis

Article

Dec 2022
J AUTOIMMUN

Proteins subjected to post-translational modifications, such as citrullination, carbamylation, acetylation or malondialdehyde (MDA)-modification are targeted by autoantibodies in seropositive rheumatoid arthritis (RA). Epidemiological and experimental studies have both suggested the pathogenicity of such humoral autoimmunity, however, molecular mechanisms triggered by anti-modified protein antibodies have remained to be identified. Here we describe in detail the pathways induced by anti-MDA modified protein antibodies that were obtained from synovial B cells of RA patients and that possessed robust osteoclast stimulatory potential and induced bone erosion in vivo. Anti-MDA antibodies boosted glycolysis in developing osteoclasts via an FcγRI, HIF-1α and MYC-dependent mechanism and subsequently increased oxidative phosphorylation. Osteoclast development required robust phosphoglyceride and triacylglyceride biosynthesis, which was also enhanced by anti-MDA by modulating citrate production and expression of the glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate acyltransferase 2 (GPAT2) genes. In summary, we described novel metabolic pathways instrumental for osteoclast differentiation, which were targeted by anti-MDA antibodies, accelerating bone erosion, a central component of RA pathogenesis.

Unraveling the Arthus Mystery: Fc Receptors and the Holy Grail of Inflammation

Article

Apr 2022

This Pillars of Immunology article is a commentary on “Fc receptors initiate the Arthus reaction: redefining the inflammatory cascade,” a pivotal article written by D. L. Sylvestre and J. V. Ravetch, and published in Science, in 1994. https://www.science.org/doi/10.1126/science.8066448.

Blocking the inhibitory receptor PD-1 prevents allergic immune response and anaphylaxis in mice

Article

Jan 2022
J ALLERGY CLIN IMMUN

Background Food allergy and acute anaphylaxis can be life-threatening. While T follicular helper (Tfh) cells play a pivotal role in the allergic immune responses, the immunologic mechanisms that regulate the production of antibodies that mediate anaphylaxis are not fully understood. Objective The aim of this study was to investigate the role of the inhibitory receptor programmed cell death protein 1 (PD-1), which is highly expressed on Tfh cells, in allergic immune responses using an animal model of peanut allergy and anaphylaxis. Methods Naïve wild-type mice were exposed to peanut flour intranasally and then challenged with peanut extract to induce systemic anaphylaxis. The roles of PD-1 were examined by blocking antibodies and using gene-deficient animals. A hapten model and passive cutaneous anaphylaxis were used to characterize allergen-specific antibodies. Results Treatment with anti-PD-1 enhanced development of Tfh cells and germinal center B cells in mice exposed to peanut flour. Nonetheless, anti-PD-1 or anti-PD-L1 fully protected mice from developing anaphylaxis. Anti-PD-1 treatment or genetic deficiency of PD-1 in CD4⁺ T cells inhibited production of peanut-specific IgE and increased the levels of IgG. The passive cutaneous anaphylaxis showed that peanut-specific antibodies generated in anti-PD-1-treated animals prevented, rather than provoked, anaphylaxis when transferred to naïve animals. Anti-PD-1 promoted production of antibodies with low affinity for an antigen in the hapten model. Conclusion Blockade of the PD-1/PD-L1 pathway is protective against allergic immune responses. The direct interaction between Tfh cells and B cells may play a pivotal role in controlling antibody quality and clinical manifestation of allergic diseases.

Die Auswirkung von Sialinsäuren auf die Entwicklung, Aktivierung und das Überleben von B- und T-Zellen in Mäusen

Thesis

Full-text available

Jan 2021

Alexandra T. Linder

Die Oberfläche von Lymphozyten ist dicht mit Glykokonjugaten bedeckt, der sogenannte Glykokalyx. Darunter befindet sich die Sialinsäure (Sia), ein abundant exprimierter Zucker, welcher die Oligosaccharidketten terminiert und deshalb häufig als Ligand für verschiedene Rezeptoren dient. Durch die endständige Lage und der Varianz in den glykosidischen Verknüpfungen wird den Sia verschiedene Funktionen im Immunsystem zugeteilt. Unter anderem spielen sie eine Rolle bei Zell-Zell-Kontakten, bei der Migration von Leukozyten und bei der Zell-Aktivierung. Um die Relevanz von Sias auf B- und T-Zellen zu untersuchen, haben wir spezifische Knockout-Mäuse für die CMP-Sialinsäure-Synthetase (Cmas) generiert. Cmas ist essentiell für die Aktivierung der Sia und damit für deren Transfer auf Glykane notwendig. Die Auswirkungen des Sia-Defekts auf die Entwicklung, Funktion und das Überleben von B- und T-Zellen wurden in diesen Mäusen untersucht. Interessanterweise, zeigten sowohl desialylierte B-Zellen als auch T-Zellen große Ähnlichkeiten in den Ergebnissen, wobei hauptsächlich eine Eliminierung der Sialinsäure-freien Zellen detektiert werden konnte. Beide Genotypen zeigten den größten Verlust der desialylierten Zellen beim Übertritt der Zellen vom primären Organ ins vaskuläre System. Im B-Zell-spezifischen Knockout konnte dieser Phänotyp hauptsächlich auf die Komplementaktivierung zurückgeführt werden, wie der Schutz vor Zelldelektion nach adoptivem Transfer in C3-defiziente Mäuse zeigt. Die verbleibenden B-Zellen in der Peripherie zeigten einen aktivierten Phänotyp, während eine erhöhte Apoptose bei naiven, reifen B- und T-Zellen in Cmas-defizienten Mäusen nachgewiesen werden konnte. Zusammenfassend lässt sich anhand der Daten die Aussage treffen, dass eine Desialylierung von B- bzw. T-Lymphozyten negative Folgen für die Homöostase der Zellen hat und schlussendlich zum Verlust dieser Zellen führt. Dabei sind vorwiegend Komplement- und Apoptosemechanismen für das Verschwinden der Cmas-defizienten Zellen verantwortlich, wobei ein Verlust der Sialinsäuren auch eine Aktivierung der Zelle begünstigen kann.

Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal

Article

Full-text available

Jan 1987

A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.

A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB modulates B-cell receptor signalling

Article

Full-text available

Mar 1994
NATURE

THE Fc receptor on B lymphocytes, FcγRIIB (β1 isoform), helps to modulate B-cell activation triggered by the surface immunoglobulin complex1,2. Crosslinking of membrane immunoglobulin by antigen or anti-Ig F(ab')2 antibody induces a transient increase in cytosolic free Ca2+, a rise in inositol-3-phosphate, activation of protein kinase C, and enhanced protein tyrosine phosphorylation3-5. Crosslinking FcγRIIB with the surface immunoglobulin complex confers a dominant signal that prevents or aborts lymphocyte activation triggered through the ARH-1 motifs of the signal transduction subunits Ig-α and Ig-β. Here we show that FcγRIIB modulates membrane immunoglobulin-induced Ca2+ mobilization by inhibiting Ca2+ influx, without changing the pattern of tyrosine phosphorylation. A 13-amino-acid motif in the cytoplasmic domain of FcγRIIB is both necessary and sufficient for this effect. Tyrosine at residue 309 in this motif is phosphorylated upon co-crosslinking with surface immunoglobulin; mutation of this residue aborts the inhibitory effect of FcγRIIB. This inhibition is directly coupled to signalling mediated through Ig-α and Ig-β as evidenced by chimaeric IgM/α and IgM/β molecules. The 13-residue motif in FcyRIIB controls lymphocyte activation by inhibiting a Ca2+ sig-nalling pathway triggered through ARH-1 motifs as a result of recruitment of novel SH2-containing proteins that interact with this FcγRIIB cytoplasmic motif.

Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors

Article

Full-text available

Oct 1979

Jay Unkeless

To investigate the antigenic relationship between the macrophage and lymphocyte Fc receptors (FcR), a monoclonal antibody capable of blocking mouse macrophage Fc receptors was selected. Hybrids were formed by fusing the P3U1 mouse myeloma and spleen cells from a rat immunized with the mouse macrophage-like cell lines J774 and P388D1. The Fab fragment of the monoclonal IgG secreted by clone 2.4G2, inhibited the trypsin-resistant Fc receptor II (FcRII), which is specific for immune aggregates of mouse IgG1 and IgG2b, but had no inhibitory effect on the trypsin-sensitive Fc receptor I (FcRI), which binds monomeric IgG2a and erythrocytes coated with IgG2a. Thus, the monoclonal 2.4G2 IgG appeared to be specific for macrophage FcRII. Further evidence that the 2.4G2 IgG was directed against FcRII came from binding studies of the monoclonal antibody to J774 cells and a series of independently isolated variants which do not express FcRII. These variants of J774 bound 5% as much of the monoclonal antibody as the parent line, which bound 600,000 molecules of 2.4G2 IgG per cell. The antigenic relatedness of mouse lymphocyte FcR to mouse macrophage FcRII was demonstrated by the binding of 2.4G2 IgG to FcR-bearing lymphoid cell lines and the inhibition of the lymphocyte FcR by the monoclonal antibody. Preincubation of spleen cells and peritioneal cells with 2.3G2 IgG likewise inhibited rosette formation with ox erythrocytes coated with rabbit IgG. The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex. The 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human FcR-bearing cells.

Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line

Article

Full-text available

Jun 1977

To evaluate subclass specificity and aggregate size requirements of IgG receptors on mouse cells, we measured binding of radiolabeled monomeric and BDB-aggregated mouse myeloma proteins fractionated into various sizes by means of gel filtration. Monomers, tetramers, and high molecular weight (approximately 10(7) daltons) aggregates were used. The various cells and cell lines studied could be segregated into three patterns of reactivity: (a) Macrophage and macrophage-like cell lines bound monomer IgG2a preferentially; high molecular weight IgG aggregates bound as follows: IgG1 = IgG2b = IgG2a. (b) Lymphoid lines D2N and S49 showed no capacity to bind monomer IgG2a; high molecular weight aggregates bound as follows: IgG1 = IgG2b less than IgG2a. (c) Other Thy-1-positive lymphoid cell lines (EL4 and L5178) and normal T and B cells showed no capacity to bind monomer IgG; high molecular weight IgG aggregates bound to a lesser extent than to cells of the first two categories in the following manner: IgG1 less than IgG2b greater than or equal to IgG2a. The variable pattern of reactivity of the macrophage-like cell lines with monomer and aggregated IgG suggested that two distinct receptors for IgG were present: one capable of binding IgG2a and another capable of binding all aggregates. Further evidence for this hypothesis was obtained by analysis of the inhibitory capacity of different IgG subclasses on the binding of aggregated IgG and monomer IgG2a to P388 cells. Inhibition of monomer IgG2a binding was effected only by monomer or aggregated IgG2a, whereas inhibition of binding of aggregated IgG1 or IgG2b was noted with aggregates of all three subclasses with some preferential inhibition by monomer IgG2b being observed. Furthermore, monomer IgG2b binding was preferentially inhibitable by monomer IgG2b. It is postulated from these data that two receptor sites are present on this macrophage-like cell line, one reactive with aggregates of all three subclasses as well as monomer IgG2b, and another receptor specific for monomer IgG2a which also binds aggregated IgG2a. Support of this concept was obtained by trypsinization experiments in which the binding of monomer IgG2a was markedly decreased by trypsin treatment of cells, whereas the binding of aggregated IgG2b was unaffected by this treatment.

Signal transduction by FcγRIII (CD16) is mediated through the γ chain

Article

Full-text available

Jun 1992

To determine the functional role of the two isoforms of Fc gamma RIII (CD16) (IIIA, IIIB), the signal transduction capabilities of wild-type and mutant forms of these receptors were analyzed in transfected lymphoid, myeloid, and fibroblastic cell lines. Functional reconstitution of receptor signalling was observed in hematopoietic T and mast cells, and was absent in nonhematopoietic (CHO) cells. Fc gamma RIIIA, a hetero-oligomeric receptor composed of a ligand-binding subunit alpha and dimeric gamma chains, generated both proximal and distal responses in Jurkat and P815 cells, typical of what is seen in natural killer cells and macrophages upon receptor activation. In contrast, Fc gamma RIIIB, which is normally attached to the cell surface via a glycosyl-phosphatidylinositol anchor, was incapable of transducing signals. After crosslinking, Fc gamma RIIIA signalling was dependent only upon the gamma chain. Fc gamma RIIIA chimeras in which the alpha subunit transmembrane and cytoplasmic domains were substituted with the corresponding gamma chain sequences functioned as well as wild-type hetero-oligomeric receptors. These data indicate that the ability of the Fc gamma RIIIA complex to activate the appropriate pathways for cell activation is cell-type restricted and independent of the transmembrane and cytoplasmic domains of the alpha subunit. The presence of the gamma chain is responsible for the assembly of, as well as the signal transduction by, the functional cell surface complex.

Single-Step Method Of RNA Isolation By Acid Guanidinium Thiocyanate-Phenol-Chlorform Extraction

Article

Full-text available

May 1987

Murine natural killer cells express functional Fcγ receptor II encoded by the FcγRα gene

Article

Full-text available

Aug 1989

We report evidence that murine NK cells express a functional Fc gamma RII encoded by the Fc gamma RII alpha gene. Several lines of indirect evidence indicate that freshly obtained NK cells from mice of several strains bear a functional Fc gamma RII: (a) anti-Fc gamma RII antibody 2.4G2 detects a small but significant proportion of sIg- cells and a small proportion of the 2.4G2+ cells are included in the Thy-1+ population; (b) sIg- lymphocytes contain 2.4G2+ and Fc gamma R-bearing cells in similar proportions; (c) binding of particulate immune complexes by sIg- lymphocytes is completely inhibited by 2.4G2; (d) 2.4G2+ cells mediate greater than 50% of the spontaneous cytotoxicity in sIg- splenic lymphocytes. Direct evidence for the presence of Fc gamma RII on murine NK cells is provided by the results of two-color immunofluorescence studies performed on splenic lymphocytes from C57BL/6 mice showing coexpression of NK-1.1 and 2.4G2. Studies of in vitro propagated homogeneous NK cell populations confirm that murine NK cells express only Fc gamma RII and that this Fc gamma R is functional, as shown in experiments of inhibition of ADCC by the anti-Fc gamma RII antibody 2.4G2. The results of studies at the molecular level show that an Fc gamma RII alpha transcript identical to that expressed in macrophages is the only molecule encoding Fc gamma RII in murine NK cells.

Complete structure of the mouse mast cell receptor for IgE (FcεRI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells

Article

Full-text available

Oct 1989

The high affinity receptor for IgE (Fc ϵ RI) found on mast cells and basophils is a tetrameric complex of a single α subunit, a single β subunit, and two identical γ subunits. The genes for the three subunits of mouse Fc ϵ RI have now been cloned from the mast cell line, PT18. When compared at the DNA level, the rat and mouse subunits are similarly conserved. However, at the protein level the homology between mouse and rat α is surprisingly low (71% identities) especially in the cytoplasmic regions (57% identities) which are of different length (25 and 20 residues, respectively). By contrast the β and γ are homogeneously conserved between mouse and rat (83 and 93% identities, respectively). The consensus amino acid sequence of the α subunit derived from three species (rat, mouse, and human) shows that the cytoplasmic tail diverges to the same extent as the leader peptide. Conversely, the transmembrane domain of the α is highly conserved and contains 10 consecutive residues that are identical. Comparisons between mouse Fc ϵ RI and other mouse proteins reveal regions of high homology between the α subunit and Fc γ RIIa and between the γ subunit and the ζ chain of the T cell receptor. Cells transfected with the α gene express the α subunit on their surface very inefficiently. Efficient expression is only achieved after co-transfection of the three rodent genes or of the human α gene together with the rodent γ without apparent need for β. The subunits are completely interchangeable upon transfection so that various chimeric mouse-rat-human receptors can be expressed.

Structure and expression of human IgG (FcRII(CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes

Article

Full-text available

Nov 1989

The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.

Human IgG Fc receptor (hFcRII; CD32) exists as multiple isoforms in macrophages, lymphocytes and IgG-transporting placental epithelium

Article

Full-text available

Jan 1990

We previously isolated cDNA clones from a human monocyte library that encoded one member of a family of low-affinity surface receptors for the Fc domain of IgG (hFcRII-A). To investigate possible structural and functional heterogeneity among these receptors, we have now isolated two additional cDNAs (hFcRII-B and hFcRII-C) from a human placental library, placenta being a good source of FcR-bearing macrophages and epithelial cells. Three cDNAs encoded related but distinct transmembrane glycoproteins containing two immunoglobulin-like domains; however, transfected cells produced receptors that were indistinguishable on the basis of ligand binding or reactivity with anti-hFcRII monoclonal antibodies. The sequences of hFcRII-A and -B were most closely related and were identical except for several amino acid substitutions and one small internal deletion. While the ectodomain of hFcRII-C was identical to hFcRII-B, its cytoplasmic tail was unrelated but highly homologous to the corresponding domain of the receptor isoform (mFcRII-B2) found in murine macrophages. Thus, human FcRII may be derived from at least two alternatively spliced genes. Northern blots revealed little difference in the pattern of expression of hFcRII isoforms among various myeloid and lymphoid cells or cell lines. However, the blots--as well as in situ hybridization and immunohistochemistry--demonstrated that hFcRII-C (along with a second monocyte marker, the c-fms encoded CSF-1 receptor) was expressed in placental syncytiotrophoblasts. Since syncytiotrophoblasts comprise the IgG-transporting epithelium of the placental villus, these findings suggest that FcR found in the immune system and in certain epithelia may be structurally or functionally related.

Co-association of CD3C with a receptor (CDI6) for IgG Fc on human natural killer cells

Article

Full-text available

Jan 1990

Natural killer (NK) cells are a subset of lymphocytes that mediate major histocompatibility complex (MHC)-nonrestricted cytotoxicity against tumours and virus-infected cells and secrete numerous cytokines on activation. NK cells are distinct from mature T lymphocytes, because they do not rearrange or productively transcribe T-cell receptor alpha-, beta-, gamma- or delta-chain genes and do not express the CD3 gamma- or delta-subunits. But recent studies indicate that NK cells do express CD3 zeta, co-associated with other membrane proteins. Here we report that CD16, the receptor for the Fc (constant) region of IgG, specifically associates with the CD3 zeta homodimer on the membrane of human NK cells, and that co-transfection with CD3 zeta complementary DNA permits expression of a transmembrane-linked CD16 complex on COS-7 cells. These findings indicate that CD3 zeta can co-associate with membrane receptors of diverse cell types and function as a common structure for signal transduction.

Activation-driven programmed cell death and T cell receptor expression

Article

Full-text available

Jan 1990

Activation of spontaneously dividing T cell hybridomas induces interleukin-2 (IL-2) production, a cell cycle block, and programmed cell death. T cell hybridomas that express the T cell antigen receptor (TCR) zeta homodimer (zeta 2), but not the TCR zeta eta heterodimer, were studied. The zeta eta- cells produced little or no inositol phosphates (IP) when stimulated with antigen. In most cases the hydrolysis of phosphoinositides was also impaired after stimulation with antibody to CD3, although one zeta eta- cell produced normal concentrations of IP. The zeta eta- cells slowed their growth and secreted IL-2 in response to both stimuli. However, the zeta eta- cells did not die after activation with antigen. Since activated thymocytes also undergo programmed cell death, these results may have important implications for the role of the zeta eta.TCR in negative selection.

Function and regulation of a murine macrophage-specific IgG Fc receptor, Fc??R-??

Article

Full-text available

Jul 1988

Ligand binding specificities of two cloned murine Fc gamma Rs (Fc gamma R-alpha, Fc gamma R-beta [9]) were determined by gene transfer into Fc gamma R negative cell lines. Both receptors were expressed as full-length molecules capable of IgG immune complex binding that was inhibitable by the mAb 2.4G2. The ligand binding profiles of these receptors were indistinguishable whereby both bound immune-complexed mouse IgG1, IgG2a, and IgG2b, but not IgG3. Neither receptor could bind monomeric IgG2a, indicating these receptors to be low-affinity IgG Fc receptors. Accumulation of the Fc gamma R-alpha mRNA can be induced with murine IFN-gamma at a concentration of 200 U/ml in the macrophage-like cell lines RAW 264.7 and J774a. The time course for induction indicates that the mRNA accumulation is transient but does not return to the uninduced level even after 50 h of treatment. Fc gamma R-beta mRNA was not induced by IFN-gamma, rather its expression was down modulated in mouse peritoneal macrophages. Both RAW and J774a cells lines exhibited increased receptor levels after IFN-gamma stimulation as measured by 125I-2.4G2 and ligand binding. In the absence of IFN-gamma, the RAW and J774a cell lines were minimally phagocytic, while P388D1 cells were actively phagocytic. In the presence of IFN-gamma, however, RAW 264.7 and J774a cells were induced to become actively phagocytic. Induction of Fc gamma R-alpha mRNA and protein by IFN-gamma may be part of the process by which macrophages become activated to engulf antibody-coated particles.

HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells

Article

Full-text available

Mar 1987

Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts. Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.

Fc??R family: Proteins, transcripts and genes

Article

Jan 1990

Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain

Article

May 1992

Urs Wirthmueller

Function and regulation of a murine macrophage-specific IgG Fc receptor, Fc gamma R-alpha

Article

Jun 1988

R L Weinshank

Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line

Article

May 1977

Christoph Heusser

The PIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inac-tivation

Article

Jan 1985
GENE

The versatility of insertional inactivation of β-galactosidase activity for subcloning and sequencing has been enhanced by combining a chemically synthesized oligonucleotide which specifies nine 6-bp-cutter restriction sites including BglII, XhoI, NruI, ClaI, SacI and EcoRV in various configurations with existing polylinkers to create a set of highly versatile cloning sites. These improved polylinkers have been inserted into plasmids (the pICs) for routine cloning of double-stranded DNA, and into chimeric phage/plasmids (the pICEMs) for biological production of single stranded DNA. The most versatile Polylinker specifies 17 restriction sites in the β-galactosidase α-complementing gene fragment. One of the new polylinkers was inserted into M 13 DNA to produce a vector (M13mIC7) with nine cloning sites.

Signal transduction by Fc receptors: the Fc&RI case

Article

May 1993
Immunol Today

The family of proteins collectively known as Fc receptors (FcR) plays a variety of roles both in the initiation of the immune response and in its consequences. During the past five years the structure of these proteins and the genes that code for them have been largely elucidated. The most unexpected finding has been their extensive diversity. Considerable efforts are now being expended to define the molecular events initiated by these various FcR and these events are the focus of our review.

The Fc Receptors of Primary and Cultured Phagocytic Cells Studied with Homogeneous Antibodies

Article

Nov 1978

Mouse spleen cell-myeloma hybrids were produced that secreted monoclonal antibodies to SRBC of the IgG2a and IgG2b subclass. These homogeneous antibodies were used to examine the Fc receptors for mouse IgG2a and IgG2b on resident and thioglycollate-induced macrophages and on two continuous macrophage-like cell lines (J774.2 and FC-1). By cross-inhibition experiments, we have shown that all four of these cell types have separate receptors for aggregated IgG2a and IgG2b. Both receptors bind heterogeneous rabbit antibody complexed to antigen. Large amounts of soluble proteins do not inhibit the binding of antigen-antibody complexes to either receptor. The rosetting of IgG2a-coated SRBC, but not of IgG2b-coated SRBC, is inhibited by treating all four cell types with trypsin or cytochalasin and is decreased at 4°C. Both the IgG2a and IgG2b receptors will mediate phagocytosis in all four cell types. Mutants of the FC-1 cell line were obtained that can bind both IgG2a and IgG2b antibody-antigen complexes but phagocytize only through the IgG2b receptor. We conclude from these studies that naturally occurring complexes between antigen and mouse IgG2a and IgG2b antibodies are bound to mouse macrophages and phagocytized through functionally distinguishable Fc receptors. The use of homogeneous monoclonal antibodies facilitates such studies and avoids some of the ambiguities that arise when artificially aggregated immunoglobulins or heterogeneous antibodies are used.

Murine recombinant FcγRIII, but not FcγRII, trigger serotonin release in rat basophilic leukemia cells

Article

Sep 1992

Murine Fc gamma RII and Fc gamma RIII have highly homologous extracellular domains, but unrelated transmembrane and intracytoplasmic (IC) domains. Murine Fc gamma RIIb1 and b2 are two isoforms of single-chain receptors which differ only by 47 aa in their IC domain. Murine Fc gamma RIII are composed of an IgG-binding alpha-chain, the intracellular portion of which is unrelated to that of Fc gamma RII, and of a homodimeric gamma-chain which also associates with Fc epsilon RI. Murine mast cells express Fc gamma RII, Fc gamma RIII, and Fc epsilon RI. They can be induced to degranulate by murine IgG immune complexes or by F(ab')2 fragments of the rat anti-murine Fc gamma RII/III mAb 2.4G2, complexed to mouse anti-rat (MAR) F(ab')2. In order to determine which murine Fc gamma R can activate mast cells, cDNA encoding murine Fc gamma RIIb1, Fc gamma RIIb2 or Fc gamma RIII alpha were stably transfected into RBL-2H3 cells. Murine Fc gamma RIII but not Fc gamma RIIb1 or Fc gamma RIIb2 induced serotonin release when aggregated by (2.4G2-MAR) F(ab')2 complexes. The respective roles of the IC domains of murine Fc gamma RIII subunits in signal transduction were investigated by stably transfecting cDNA encoding IC-deleted or chimeric murine Fc gamma R into RBL-2H3 cells. The substitution of the IC domain of murine Fc gamma RII for that of murine Fc gamma RIII gamma, but not that of murine Fc gamma RIII alpha, conferred the ability to trigger serotonin release. The deletion of IC sequences of the alpha subunit did not alter the ability of murine Fc gamma RIII to trigger serotonin release. It follows that 1) murine Fc gamma RIII, but not Fc gamma RII, can induce RBL cells to release serotonin, 2) the aggregation of the IC domain of the murine Fc gamma RIII gamma subunit is sufficient, but 3) the IC domain of the murine Fc gamma RIII alpha subunit is neither sufficient nor necessary for triggering serotonin release.

A population of early fetal thymocytes expressing FcγRII/III contains precursors of T lymphocytes and natural killer cells

Article

May 1992
CELL

We have identified a dominant fetal thymocyte population at day 14.5 of gestation in the mouse that lacks CD4 and CD8 but expresses Fc gamma RII/III several days prior to acquisition of the T cell receptor (TCR) in vivo. If maintained in a thymic microenvironment, this population of CD4-CD8-TCR-Fc gamma RII/III+ thymocytes differentiates first into CD4+CD8+TCRlowFc gamma RII/III- thymocytes and subsequently CD4+CD8-TCRhighFc gamma RII/III- and CD4-CD8+TCRhighFc gamma RII/III- mature Ti alpha-beta lineage T cells. However, if removed from the thymus, the CD4-CD8-TCR-Fc gamma RII/III+ thymocyte population selectively generates functional natural killer (NK) cells in vivo as well as in vitro. These findings show that a cellular pool of Fc gamma RII/III+ precursors gives rise to T and NK lineages in a microenvironment-dependent manner. Moreover, they suggest a hitherto unrecognized role for Fc receptors on primitive T cells.

A subunit common to an IGG FC-receptor and the T-cell receptor mediates assembly through different interactions

Article

Jun 1991

The zeta subunit is a component of the Fc gamma receptor of natural killer cells (Fc gamma RIII or CD16), as well as the multimeric T-cell receptor/CD3 complex, and is required for assembly of both native receptors. The role of the zeta subunit in human Fc gamma RIIIA assembly differs from its role in T-cell receptor/CD3 complex assembly. The transmembrane domain of the Fc gamma RIIIA alpha subunit forms noncovalent interactions with the comparable domain of the zeta subunit and is sufficient for surface expression of the Fc gamma RIIIA complex. In the absence of these interactions, sequences in the transmembrane domain of the Fc gamma RIIIA alpha subunit signal its degradation. Leu-46, present in the transmembrane domain of the human zeta subunit, is important for assembly with the Fc gamma RIIIA alpha subunit. Substitution of this leucine with an isoleucine, as found in the mouse zeta subunit, significantly reduces this interaction. In contrast, the mouse and human zeta subunits interact with the pentameric T-cell receptor/CD3 complex, resulting in surface expression of this receptor.

Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides

Article

Apr 1991
CELL

We describe functional simplified T cell and Fc receptor chimeras that are capable of directing CD8+ cytotoxic T lymphocytes (CTLs) to specifically recognize and lyse cells expressing HIV envelope proteins. Target cells bearing HLA-DR molecules are not recognized by CTL armed with the chimeras. The variety of cell types in which the native receptors are active suggests multiple possibilities for antiviral intervention through genetic means.

Fc Receptors

Article

Feb 1991

Recent advances in the structural analysis of the genes and proteins for immunoglobulin Fc domain receptors have provided a molecular characterization of this complex family. The wide cellular distribution of these receptors and their functional heterogeneity are reflected in the diversity of molecules which bind antibody and immune complexes. The detailed analysis of the IgG and IgE Fc receptors has indicated that these molecules have evolved from a common precursor through gene duplication. Similarities among these receptors, in both structure and function have emerged. Thus, the Fc receptors provide an example of a class of molecules in which conserved domains are combined with divergent sequences to yield a diversity of function.

Organization of the Human and Mouse Low-affinity FcγR Genes: Duplication and Recombination

Article

Jun 1990

Receptors for immunoglobulin G immune complexes (Fc gamma RII and Fc gamma RIII) are expressed on most hematopoietic cells and show much structural and functional diversity. In order to determine the genetic basis for this diversity, a family of genes encoding the human and mouse receptors was isolated and characterized. Humans have five distinct genes for low-affinity Fc gamma Rs, in contrast to two in the mouse. With the use of yeast artificial chromosomes, the genes encoding the human receptors were oriented and linked, which established the structure of this complex locus. Comparison of the human and mouse genes generated a model for the evolutionary amplification of this locus.

Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells

Article

Jan 1991

Specific genes can be inactivated or mutated in the mouse germ line. The phenotypic consequences of the mutation can provide pivotal information on the function of the gene in development and maintenance of the mammalian organism. The procedure entails homologous recombination in embryonic stem cells, which, on fusion to recipient blastocysts, give rise to chimaeric mice that can transmit the mutant gene to their offspring. Inbreeding can then yield mice carrying the mutation in both alleles allowing the phenotypic analysis of recessive mutations. In addition to mice lacking a particular gene function, cell lines carrying null alleles of normally expressed genes can be instrumental in assessing the function of the gene. These cell lines can either be obtained from homozygous animals or, should the mutation be lethal early in embryonic development, be generated by consecutive inactivation of both alleles by homologous recombination in cultured cells. Here we illustrate the feasibility of this latter approach by the efficient consecutive inactivation of both alleles of the pim-1 proto-oncogene in embryonic stem cells.

Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice

Article

Feb 1990

Genetically mast cell-deficient WBB6F1-W/Wv mice showed an apparent defect in manifestation of the resistance against larval Haemaphysalis longicornis ticks, but their serum IgE levels increased more than 100-fold after the second tick infestation. Immune sera obtained from the WBB6F1-W/Wv mice were adoptively transferred to the other WBB6F1-W/Wv mice which had received intracutaneous injections of WBB6F1-+/+ mouse-derived cultured mast cells. Because the resistance against ticks was detectable only when both mast cells and IgE antibodies were available, immediate hypersensitivity reaction appeared to have a physiologic role in the manifestation of the resistance against H. longicornis ticks.

Mast cell lines produce lymphokines in response to cross linkage of FCERI or to calcium ionophore

Article

Jun 1989

The cross-linkage of high affinity Fc epsilon receptors (Fc epsilon RI) on mast cells and basophils is central to the induction of allergic inflammatory responses. As a result of such cross-linkage, mast cells secrete a variety of preformed biologically active substances, such as histamine, and newly synthesized arachidonic acid metabolites. Here we show that cross-linkage of Fc epsilon RI on a series of nontransformed murine mast cell lines, or treatment of these cells with calcium ionophores, stimulates increased messenger RNA levels and secretion of a group of lymphokines classically produced by a subset of murine T cell lines (TH2 cells). These factors include interleukin-3 (a mast cell growth factor)s interleukin-4 (an IgE 'switch factor'), interleukin-5 (an eosinophil differentiation factor) and interleukin-6 (a factor controlling immunoglobulin secretion). The production of these polypeptide factors by mast cells may have great importance in the induction of allergic and anti-parasite inflammatory responses.

A macrophage Fc?? receptor and the Mast-cell receptor for IgE share an identical subunit

Article

Nov 1989

Fc receptors for immunoglobulins are found on many immune cells and trigger essential functions of the immune defence system. With the exception of the high-affinity receptor for immunoglobulin E (Fc epsilon RI), these receptors were thought to consist of single polypeptides. Fc epsilon RI is a tetrameric complex of one alpha-subunit, one beta-subunit and two gamma-subunits. Here we report the cloning of a polypeptide identical to the gamma-chains of Fc epsilon RI, from mouse macrophages that do not express this receptor. Biosynthetic labelling and gene transfer together show that these gamma-chains associate with one of the macrophage receptors (Fc gamma RIIa). The human homologue, Fc gamma RIII (CD16), from natural killer cells is also expected to associate with gamma-chains. It is possible that these gamma-chains and the homologous zeta-chains of the T-cell antigen receptor belong to a new family of related proteins which share a common role in the signal transducing pathway.

A single amino acid in the glycosyl phosphatidylinositol attachment domain determines the membrane topology of Fc??RIII

Article

Jan 1990

Cell-surface proteins are associated with the lipid bilayer either as membrane-spanning molecules or as glycosyl phosphatidylinositol (GPtdIns)-linked proteins. Proteins destined for GPtdIns anchoring are synthesized as precursors with a hydrophobic C-terminal transmembrane domain, which is removed during the processing of these proteins in the endoplasmic reticulum (ref. 1). We have investigated the structural requirements for GPtdIns anchoring through the study of two closely related proteins which exhibit alternative membrane attachment. The IgG Fc receptor, Fc gamma RIII, is GPtdIns-linked on neurophils (III-1) whereas on natural killer (NK) cells and macrophages it is found as a transmembrane-anchored molecule (III-2), able to mediate antibody-dependent cellular cytotoxicity and phagocytosis. At the primary structural level, the III-1 gene differs from that encoding III-2 by only nine nucleotide substitutions, which result in six amino-acid differences, and the absence of 21 amino acids at the C terminus. We have analysed a series of III-1 and III-2 mutants in transient expression assays, and show that Ser 203 in the GPtdIns attachment domain is the dominant residue in determining whether the molecule can be GPtdIns-anchored. As in the case of its murine homologue, Fc gamma RII alpha, surface expression of the III-2 molecule is dependent on co-expression of a second subunit, the gamma chain of F epsilon RI. Our data also suggest that gamma chain can associate with the III-1 precursor, preventing GPtdIns attachment, favouring instead a transmembrane form.

Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity

Article

Aug 1989
CELL

Mouse macrophages and lymphocytes express two distinct isoforms of a single class of Fc receptor for IgG. The macrophage isoform (FcRII-B2) is identical to the lymphocyte isoform (FcRII-B1) except for an inframe insertion in the cytoplasmic tail of FcRII-B1 that increases its length from 47 to 94 amino acids. To determine the functional significance of this cytoplasmic domain variation, presumably the result of alternative mRNA splicing, we expressed both isoforms in receptor-negative fibroblasts. While FcRII-B2 mediated the efficient ligand internalization and delivery to lysosomes, endocytosis via FcRII-B1--and via a tailminus mutant--was relatively inefficient. This difference reflected the inability of FcRII-B1 (and the tailminus mutant) to accumulate in clathrin-coated pits. Thus, the FcRII-B2 cytoplasmic tail contains a domain needed for accumulation in coated pits, and this domain is disrupted by the 47 amino acid insertion in FcRII-B1.

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells

Article

Dec 1987
CELL

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.

Isolation and Expression of Functional High-Affinity Fc Receptor Complementary DNAs

Article

Feb 1989

Human and murine mononuclear phagocytes express a high-affinity receptor for immunoglobulin G that plays a central role in macrophage antibody-dependent cellular cytotoxicity and clearance of immune complexes. The receptor (FcRI) may also be involved in CD4-independent infection of human macrophages by human immunodeficiency virus. This report describes the isolation of cDNA clones encoding the human FcRI by a ligand-mediated selection technique. Expression of the cDNAs in COS cells gave rise to immunoglobulin G binding of the expected affinity and subtype specificity. RNA blot analysis revealed expression of a 1.7-kilobase transcript in macrophages and in cells of the promonocytic cell line U937 induced with interferon-gamma. The extracellular region of FcRI consists of three immunoglobulin-like domains, two of which share homology with low-affinity receptor domains.

Antigen-antibody complexes bound to B-lymphocyte Fc receptors regulate B-lymphocyte differentiation

Article

Nov 1985

We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab')2 anti-mu and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55-70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2-1.0 microgram/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fc gamma receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fc gamma receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fc gamma receptors of B lymphocytes regulate B-lymphocyte differentiation.

Structural Heterogeneity and Functional Domains of Murine Immunoglobulin G Fc Receptors

Article

Dec 1986

Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule E beta. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.

Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

Article

Jun 1987

The alpha, beta, and gamma subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the alpha chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for alpha and beta. However, these putative homologies were not apparent when tryptic maps of the biosynthetically ([3H]leucine) labeled subunits were analyzed.

Cloning of cDNA encoding the murine IgG1 induction factor by a novel strategy using SP6 promoter

Article

Feb 1986

Complementary DNA encoding the IgG1 induction factor, the first lymphokine directed to B lymphocytes, from a murine T-cell line has been cloned using a new strategy. The putative primary amino-acid sequence was deduced from the nucleotide sequence determined. The lymphokine synthesized by the direction of this cloned cDNA has many other functions, such as production of B-cell growth factor-1 and induction of Ia on B cells.

Lipid Mediators Produced Through The Lipoxygenase Pathway

Article

Feb 1987

C W Parker

Isolation and Characterization of a Mouse Interleukin cDNA Clone That Expresses B-Cell Stimulatory Factor 1 Activities and T-Cell-and Mast-Cell-Stimulating Activities

Article

May 1986

A cDNA sequence coding for a unique mouse interleukin that expresses B-cell-, T-cell, and mast-cell-stimulating activities has been isolated from a mouse helper T-cell cDNA library. The library, constructed in the pcD expression vector, was screened by transfecting COS monkey cells with DNA pools to express the products encoded by full-length cDNA inserts. By assaying the transfected cell supernatants, we identified clones encoding a factor that stimulates T-cell and mast cell lines. This factor also induces Ia expression on resting B cells and enhances IgG1 and IgE production by B cells, two properties of B-cell-stimulatory factor 1. The DNA sequence codes for a polypeptide of 140 amino acid residues including a putative signal peptide. These results demonstrate that a single cDNA clone distinct from interleukin 2 and interleukin 3 encodes a polypeptide with multiple biological activities.

Molecular cloning of the ζ chain of the T cell antigen receptor

Article

Mar 1988

The T cell antigen receptor is a multi-subunit receptor complex present on the surface of all mature and many developing T cells. It consists of clonotypic heterodimers noncovalently linked to five invariant chains that are encoded by four genes and referred to as the CD3 complex. The CD3 gamma, delta, and epsilon chains have been molecularly characterized. In this report the molecular cloning of a complementary DNA encoding the zeta chain of the murine T cell antigen receptor is described. The predicted protein sequence of the zeta chain suggests a structure distinct from those of any of the previously described receptor subunits.

Cytophilic antibodies in mice contact sensitized with oxazolone. Immunochemical characterization and preferential binding to a trypsin sensitive macrophage receptor

Article

Nov 1974

The macrophage as effector cell. N Engl J Med

Article

Oct 1980

The emphasis in this review has been on the growing awareness of the complexity of the effector functions of the macrophage. However, patterns are emerging that help to simplify the present picture. It now appears that the macrophage uses some of the same biochemical processes in modifying its extracellular environment and in dealing with phagocytized particles. For example, oxidative mechanisms appear prominent not only in the inhibition of intracellular parasites but also in the damage inflicted by macrophages on extracellular tumor cells and probably other tissues at inflammatory sites. We need to learn much more about how such processes are regulated.

T Cell Development in Mice that Lack the ζ Chain of the T Cell Antigen Receptor Complex

Article

Sep 1993

The zeta subunit of the T cell antigen receptor complex is required for targeting nascent receptor complexes to the cell surface and for receptor-mediated signal transduction. To examine the significance of the zeta subunit in T cell development, mice deficient for zeta expression were generated by gene targeting. These zeta-/- mice had few CD4+CD8+ thymocytes, and the generation of CD4+ and CD8+ single positive T cells was impaired but not completely abrogated. Peripheral T cells were present but were unusual in that they expressed small amounts of CD5 and few T cell receptors. Thus, zeta chain expression influences thymocyte differentiation but is not absolutely required for the generation of single positive T cells.

Association of the high-affinity receptor for IgG (FcγRI) with the γ subunit of the IgE receptor

Article

Aug 1993

How receptors for the Fc portion of IgG antibodies (Fc gamma R) trigger a variety of immune functions by clustering upon engagement of ligand is largely unknown. Of the three distinct classes of human Fc gamma R, only Fc gamma RIIIA has been shown to associate with a potential signal-generating subunit, either the gamma chain or the zeta chain. With these studies we show that Fc gamma RI, the high-affinity receptor for IgG, also is found in association with gamma chain. This association can be demonstrated by using anti-Fc gamma RI antibodies, Fc gamma RI ligand, and anti-gamma-chain antibodies that coadsorb the proteins from detergent lysates of Fc gamma RI-expressing cells. The association of Fc gamma RI and gamma chain can be reconstituted by cotransfection of cDNAs for both proteins into COS cells.

Signal transduction by Fc receptors: The FcεRI case

Article

Jun 1993
Immunol Today

The low-affinity Fc receptor for human IgG (hFcRII) exists as multiple isoforms Consecutive inactivation of both alleles of the pin?-1 proto-oncogene by homologous recombination in embryonic stem cells

Jan 1989
3657-3666

S G Stuart
N E Simister
S B Clarkson
M Shapiro
I Te Riele
H Maandag
E R Clarke
A Hooper
M Bens

Stuart, S. G., Simister, N. E., Clarkson, S. B., Shapiro, M., and Mell-man, I. (1989). The low-affinity Fc receptor for human IgG (hFcRII) exists as multiple isoforms. EMBO J. 8, 3657-3666. te Riele, H., Maandag, E. R., Clarke, A., Hooper, M., and Bens, A. (1990). Consecutive inactivation of both alleles of the pin?-1 proto-oncogene by homologous recombination in embryonic stem cells, Na-ture 348, 649-651.

Structure and expression of human IgG FcRll (CD32)

Jan 1989
1369-388

D G Brooks
W Qiu
Ct
A D Luster
J V Ravetch

Brooks, D. G., Qiu, W. CT., Luster, A. D., and Ravetch, J. V. (1989). Structure and expression of human IgG FcRll (CD32). J. Exp. Med. 170, 1369-l 388.

The low-affinity Fc receptor for human IgG (hFcRII) exists as multiple isoforms

Jan 1989
3657

Stuart

FcR?? Chain Deletion results in Pleiotrophic Effector Cell Defects

Abstract

No full-text available

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