MALDI-TOF MS glycan analysis of RhD-specific IgG antibodies (crude data)

<p>Series_A_09037-013_total_IgG.csv<br>MALDI-TOF MS glycan analysis of total IgG from Rhophylac, which was the starting material for the purification of Rhesus D (RhD)-specific IgG antibodies (purification A). The area under the peak is shown for each selected molecular mass (m/z) and presented as percentage of all the indicated area values. The glycan structure and abbreviation for each m/z is shown in Supplementary Figure 2 and the summary of all MALDI-TOF MS analyses is shown in Figure 1 in the main text.</p> <p>Series_A_09037-012_RhD-specific_IgG.csv<br>MALDI-TOF MS glycan analysis of RhD-specific IgG antibodies (purification A). The area under the peak is shown for each selected molecular mass (m/z) and presented as percentage of all the indicated area values. The glycan structure and abbreviation for each m/z is shown in Supplementary Figure 2 and the summary of all MALDI-TOF MS analyses is shown in Figure 1 in the main text.</p> <p>SeriesB_09037-192a total_IgG.csv<br>MALDI-TOF MS glycan analysis (measurement a of a duplicate measurement) of total IgG from Rhophylac, which was the starting material for the purification of RhD-specific IgG antibodies (purification B). The area under the peak is shown for each selected molecular mass (m/z) and presented as percentage of all the indicated area values. The glycan structure and abbreviation for each m/z is shown in Supplementary Figure 2 and the summary of all MALDI-TOF MS analyses is shown in Figure 1 in the main text.</p> <p>SeriesB_09037-192b total_IgG.csv<br>MALDI-TOF MS glycan analysis (measurement b of a duplicate measurement) of total IgG from Rhophylac, which was the starting material for the purification of RhD-specific IgG antibodies (purification B). The area under the peak is shown for each selected molecular mass (m/z) and presented as percentage of all the indicated area values. The glycan structure and abbreviation for each m/z is shown in Supplementary Figure 2 and the summary of all MALDI-TOF MS analyses is shown in Figure 1 in the main text.</p> <p>Series_B_09037-191a_RhD-specific_IgG.csv<br>MALDI-TOF MS glycan analysis (measurement a of a duplicate measurement) of RhD-specific IgG antibodies (purification B). The area under the peak is shown for each selected molecular mass (m/z) and presented as percentage of all the indicated area values. The glycan structure and abbreviation for each m/z is shown in Supplementary Figure 2 and the summary of all MALDI-TOF MS analyses is shown in Figure 1 in the main text.</p> <p>SeriesB_09037-191b RhD-specific_IgG .csv<br>MALDI-TOF MS glycan analysis (measurement b of a duplicate measurement) of RhD-specific IgG antibodies (purification B). The area under the peak is shown for each selected molecular mass (m/z) and presented as percentage of all the indicated area values. The glycan structure and abbreviation for each m/z is shown in Supplementary Figure 2 and the summary of all MALDI-TOF MS analyses is shown in Figure 1 in the main text.</p> <p> </p

FACS analyses of purified RhD-specific IgG antibodies

<p>unstained.001<br>FACS analysis with Rhesus D (RhD)-positive erythrocytes to verify the enrichment of RhD-specific IgGs from total IgG of Rhophylac. The cells in this file are unstained. The cells were gated on the main erythrocyte population in a FSC/SSC blot to exclude fragments and aggregates, analyzed in an anti-human IgG (APC) histogram and shown in the overlay histogram in Supplementary Figure 1 in the main text.</p> <p>only_secondary_antibody.004<br>FACS analysis with RhD-positive erythrocytes to verify the enrichment of RhD-specific IgGs from total IgG of Rhophylac.<br>The cells in this file are only stained with a secondary APC-coupled anti-human IgG antibody. The cells were gated on the main erythrocyte population in a FSC/SSC blot to exclude fragments and aggregates, analyzed in an anti-human IgG (APC) histogram and shown in the overlay histogram in Supplementary Figure 1 in the main text.</p> <p>260_µg_per_ml_total_IgG_from_Rhophylac.002<br>FACS analysis with RhD-positive erythrocytes to verify the enrichment of RhD-specific IgGs from total IgG of Rhophylac.<br>The cells in this file are stained with 260 µg/ml total IgG from Rhophylac and subsequently with a secondary APC-coupled anti-human IgG antibody.The cells were gated on the main erythrocyte population in a FSC/SSC blot to exclude fragments and aggregates, analyzed in an anti-human IgG (APC) histogram and shown in the overlay histogram in Supplementary Figure 1 in the main text.</p> <p>2µg_per_ml_purified_RhD_specific_IgG(from_purification_A).005<br>FACS analysis with RhD-positive erythrocytes to verify the enrichment of RhD-specific IgGs from total IgG of Rhophylac.<br>The cells in this file are stained with 2 µg/ml purified RhD-specific IgG from purification A and subsequently with a secondary APC-coupled anti-human IgG antibody. The cells were gated on the main erythrocyte population in a FSC/SSC blot to exclude fragments and aggregates, analyzed in an anti-human IgG (APC) histogram and shown in the overlay histogram in Supplementary Figure 1 in the main text.</p> <p>2µg_per_ml_purified_RhD_specific_IgG(from_purification_B).007<br>FACS analysis with RhD-positive erythrocytes to verify the enrichment of RhD-specific IgGs from total IgG of Rhophylac.<br>The cells in this file are stained with 2 µg/ml purified RhD-specific IgG from purification B and subsequently with a secondary APC-coupled anti-human IgG antibody.<br>These cells are not shown in the overlay histogram in Supplementary Figure 1 in the main text. However, the staining looks like the staining from the file before.</p> <p>2_µg_per_ml_total_IgG_from_Rhophylac.003<br>FACS analysis with RhD-positive erythrocytes to verify the enrichment of RhD-specific IgGs from total IgG of Rhophylac.<br>The cells in this file are stained with 2 µg/ml total IgG from Rhophylac and subsequently with a secondary APC-coupled anti-human IgG antibody. The cells were gated on the main erythrocyte population in a FSC/SSC blot to exclude fragments and aggregates, analyzed in an anti-human IgG (APC) histogram and shown in the overlay histogram in Supplementary Figure 1 in the main text.</p> <p> </p

Image_2_Potential of Murine IgG1 and Human IgG4 to Inhibit the Classical Complement and Fcγ Receptor Activation Pathways.tif

<p>IgG antibodies (Abs) mediate their effector functions through the interaction with Fcγ receptors (FcγRs) and the complement factors. The main IgG-mediated complement activation pathway is induced through the binding of complement C1q to IgG Abs. This interaction is dependent on antigen-dependent hexamer formation of human IgG1 and IgG3 to increase the affinity for the six-headed C1q molecule. By contrast, human IgG4 fails to bind to C1q. Instead, it has been suggested that human IgG4 can block IgG1 and IgG3 hexamerization required for their binding to C1q and activating the complement. Here, we show that murine IgG1, which functionally resembles human IgG4 by not interacting with C1q, inhibits the binding of IgG2a, IgG2b, and IgG3 to C1q in vitro, and suppresses IgG2a-mediated complement activation in a hemolytic assay in an antigen-dependent and IgG subclass-specific manner. From this perspective, we discuss the potential of murine IgG1 and human IgG4 to block the complement activation as well as suppressive effects of sialylated IgG subclass Abs on FcγR-mediated immune cell activation. Accumulating evidence suggests that both mechanisms seem to be responsible for preventing uncontrolled IgG (auto)Ab-induced inflammation in mice and humans. Distinct IgG subclass distributions and functionally opposite IgG Fc glycosylation patterns might explain different outcomes of IgG-mediated immune responses and provide new therapeutic options through the induction, enrichment, or application of antigen-specific sialylated human IgG4 to prevent complement and FcγR activation as well.</p

Image_1_Potential of Murine IgG1 and Human IgG4 to Inhibit the Classical Complement and Fcγ Receptor Activation Pathways.tif

<p>IgG antibodies (Abs) mediate their effector functions through the interaction with Fcγ receptors (FcγRs) and the complement factors. The main IgG-mediated complement activation pathway is induced through the binding of complement C1q to IgG Abs. This interaction is dependent on antigen-dependent hexamer formation of human IgG1 and IgG3 to increase the affinity for the six-headed C1q molecule. By contrast, human IgG4 fails to bind to C1q. Instead, it has been suggested that human IgG4 can block IgG1 and IgG3 hexamerization required for their binding to C1q and activating the complement. Here, we show that murine IgG1, which functionally resembles human IgG4 by not interacting with C1q, inhibits the binding of IgG2a, IgG2b, and IgG3 to C1q in vitro, and suppresses IgG2a-mediated complement activation in a hemolytic assay in an antigen-dependent and IgG subclass-specific manner. From this perspective, we discuss the potential of murine IgG1 and human IgG4 to block the complement activation as well as suppressive effects of sialylated IgG subclass Abs on FcγR-mediated immune cell activation. Accumulating evidence suggests that both mechanisms seem to be responsible for preventing uncontrolled IgG (auto)Ab-induced inflammation in mice and humans. Distinct IgG subclass distributions and functionally opposite IgG Fc glycosylation patterns might explain different outcomes of IgG-mediated immune responses and provide new therapeutic options through the induction, enrichment, or application of antigen-specific sialylated human IgG4 to prevent complement and FcγR activation as well.</p

Presentation_1_Sialylated Autoantigen-Reactive IgG Antibodies Attenuate Disease Development in Autoimmune Mouse Models of Lupus Nephritis and Rheumatoid Arthritis.PDF

<p>Pro- and anti-inflammatory effector functions of IgG antibodies (Abs) depend on their subclass and Fc glycosylation pattern. Accumulation of non-galactosylated (agalactosylated; G0) IgG Abs in the serum of rheumatoid arthritis and systemic lupus erythematosus (SLE) patients reflects severity of the diseases. In contrast, sialylated IgG Abs are responsible for anti-inflammatory effects of the intravenous immunoglobulin (pooled human serum IgG from healthy donors), administered in high doses (2 g/kg) to treat autoimmune patients. However, whether low amounts of sialylated autoantigen-reactive IgG Abs can also inhibit autoimmune diseases is hardly investigated. Here, we explore whether sialylated autoantigen-reactive IgG Abs can inhibit autoimmune pathology in different mouse models. We found that sialylated IgG auto-Abs fail to induce inflammation and lupus nephritis in a B cell receptor (BCR) transgenic lupus model, but instead are associated with lower frequencies of pathogenic Th1, Th17 and B cell responses. In accordance, the transfer of small amounts of immune complexes containing sialylated IgG Abs was sufficient to attenuate the development of nephritis. We further showed that administration of sialylated collagen type II (Col II)-specific IgG Abs attenuated the disease symptoms in a model of Col II-induced arthritis and reduced pathogenic Th17 cell and autoantigen-specific IgG Ab responses. We conclude that sialylated autoantigen-specific IgG Abs may represent a promising tool for treating pathogenic T and B cell immune responses in autoimmune diseases.</p

table_1.docx

<p>Anti-neutrophil cytoplasmic autoantibodies (ANCA) targeting proteinase 3 (PR3) and myeloperoxidase expressed by innate immune cells (neutrophils and monocytes) are salient diagnostic and pathogenic features of small vessel vasculitis, comprising granulomatosis with polyangiitis (GPA), microscopic polyangiitis, and eosinophilic GPA. Genetic studies suggest that ANCA-associated vasculitides (AAV) constitute separate diseases, which share common immunological and pathological features, but are otherwise heterogeneous. The successful therapeutic use of anti-CD20 antibodies emphasizes the prominent role of ANCA and possibly other autoantibodies in the pathogenesis of AAV. However, to elucidate causal effects in AAV, a better understanding of the complex interplay leading to the emergence of B lymphocytes that produce pathogenic ANCA remains a challenge. Different scenarios seem possible; e.g., the break of tolerance induced by a shift from non-pathogenic toward pathogenic autoantigen epitopes in inflamed tissue. This review gives a brief overview on current knowledge about genetic and epigenetic factors, barrier dysfunction and chronic non-resolving inflammation, necro-inflammatory auto-amplification of cellular death and inflammation, altered autoantigen presentation, alternative complement pathway activation, alterations within peripheral and inflamed tissue-residing T- and B-cell populations, ectopic lymphoid tissue neoformation, the characterization of PR3-specific T-cells, properties of ANCA, links between autoimmune disease and infection-triggered pathology, and animal models in AAV.</p