Human RP105 monoclonal antibody enables the production of antigen-specific antibody
Ontology highlight
ABSTRACT: Antibody detection is a critical approach in clinical contexts. Detecting antibodies, particularly those directed against donor HLA in organ transplantation and self-antigens in autoimmune diseases, is crucial in diagnosis and therapy. Radioprotective 105 (RP105, CD180 ), a type I transmembrane protein with structural similarities to Toll-like receptor 4, is expressed in immune-competent cells, such as B cells. Studies involving mice have reported that the antimouse RP105 antibody strongly activates B cells and triggers an adjuvant effect against viral infections. However, the antihuman RP105 antibody (ɑhRP105) weakly activates human B cells. In this study, we established new culture conditions under which human B cells are strongly activated by ɑhRP105. When combined with CpGDNA, this activation induced a heightened production of interleukin 6. Under these culture conditions, specific antibody production against blood group carbohydrates, ɑGal, and SARS-CoV-2 within supernatants from human B-cell cultures was successfully detected. Furthermore, comprehensive analysis employing LC/MS/MS analysis and single-cell RNA sequencing revealed that ɑhRP105 triggered a different activation stimulus from CpGDNA. These findings could be applied to developing a straightforward approach for identifying antibody-producing B cells in cases involving transplant rejection and autoimmune diseases.
Project description:Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, mechanisms linking blood-brain barrier (BBB) disruption with neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8 targeted against the cryptic fibrin epitope γ377-395 to selectively inhibit fibrin-induced inflammation. 5B8 suppressed fibrin-induced proinflammatory gene expression, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, and oxidative stress. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced innate immune activation and neurodegeneration without interfering with clotting. Thus, fibrin-targeting immunotherapy inhibits autoimmune- and amyloid-driven neurotoxicity and may have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.
Project description:Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer’s disease (AD). However, mechanisms linking blood-brain barrier (BBB) disruption with neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8 targeted against the cryptic fibrin epitope γ377-395 to selectively inhibit fibrin-induced inflammation. 5B8 suppressed fibrin-induced proinflammatory gene expression, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, and oxidative stress. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced innate immune activation and neurodegeneration without interfering with clotting. Thus, fibrin-targeting immunotherapy inhibits autoimmune- and amyloid-driven neurotoxicity and may have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.
Project description:Antibody responses, involving B cells, CD4+ T cells, and macrophages, are implicated in autoimmune diseases and organ transplant rejection. We have previously shown that inhibiting FROUNT with disulfiram (DSF) suppresses macrophage activation and migration, effectively treating inflammatory diseases. In this study, we investigated the effectiveness of DSF in antibody-producing reactions. Using a heart transplantation mouse model with antibody-mediated rejection, we administered anti-CD8 antibody to exclude cellular rejection. DSF directly inhibited B cell responses in vitro and significantly reduced plasma donor-specific antibodies and graft antibody deposition in vivo, resulting in prolonged survival of the heart graft. DSF also mediated various effects, including decreased macrophage infiltration and increased Foxp3+ regulatory T-cells in the grafts. Additionally, DSF inhibited pyrimidine metabolism-related gene expression induced by B-cell stimulation. These findings demonstrate that DSF modulates antibody production in the immune response complexity by regulating B-cell and macrophage responses.
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS. Fig. 1A. Lipid-array profiling of IgG+IgM antibody reactivity in cerebrospinal fluid (CSF) samples from MS patients (relapsing remitting MS; secondary progressive MS; primary progressive MS), healthy controls, and other neurological disease controls. Lipid hits with the lowest FDR (q=0.048) were clustered according to their reactivity profiles. 48 different lipids were custom-spotted in duplicate using the CAMAG Automatic TLC Sampler (ATS4) robot to spray 200 nl of 10 to 100 pmol of lipids onto PVDF membranes affixed to the surface of microscope slides. The slides were probed with cerebrospinal fluid (CSF) from 59 human patient samples. 60 slides total: 18 relapsing-remitting MS, 14 secondary-progressive MS, 1 primary-progressive MS, 21 other neurological disease, 5 healthy control, 1 secondary Ab alone (not included in this submission). CSF diluted 1/10. HRP-conjugated secondary Ab (goat anti-human IgM/IgG) diluted 1/8000. ECL for 3 minutes.
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS. Fig. 2A. Mini-Array I: IgG antibody reactivity to various glycero-3-phosphocholine lipids in CSF samples from patients with relapsing remitting MS and from control patients with other neurological disease. Lipid hits with the lowest FDR (q=0.029) were clustered according to their reactivity profiles. 47 different lipids were custom-spotted in duplicate using the CAMAG Automatic TLC Sampler (ATS4) robot to spray 200 nl of 10 to 100 pmol of lipids onto PVDF membranes affixed to the surface of microscope slides. The slides were probed with cerebrospinal fluid (CSF) from 24 human patient samples. 25 slides total: 13 relapsing-remitting MS, 11 other neurological disease, and 1 secondary Ab alone (not included in this submission). CSF diluted 1/20. HRP-conjugated secondary Ab (goat anti-human IgM/IgG) diluted 1/175. ECL for 3 minutes.
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS.
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS.
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS.
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS. Fig. 2C. Mini-Array II: IgG antibody reactivity to lipids constituting polar head-group and side-chain modifications of PGPC in CSF samples from relapsing remitting MS patients and other neurological disease controls. Lipid hits with the lowest FDR (q=0.016) were clustered according to their reactivity profiles. 19 different lipids were custom-spotted in duplicate using the CAMAG Automatic TLC Sampler (ATS4) robot to spray 200 nl of 10 to 100 pmol of lipids onto PVDF membranes affixed to the surface of microscope slides. The slides were probed with cerebrospinal fluid (CSF) from 26 human patient samples. 27 slides total: 12 relapsing-remitting MS, 13 other neurological disease, 1 healthy control (not included in this submission), and 1 secondary Ab alone (not included in this submission). CSF diluted 1/20. HRP-conjugated secondary Ab (donkey anti-human IgG) diluted 1/8000. ECL for 3 minutes.