data_sheet_2.docx

Introduction/objectives<p>In 2012, hypocomplementemia was included in the classification criteria of systemic lupus erythematosus (SLE). The suggested measurement of C3 or C4 often reflect disease activity poorly. Our objective was to establish an assay measuring C3dg, which is generated following complement activation, and to evaluate the assay in a cross-sectional SLE cohort.</p>Method<p>We included SLE patients (n = 169) and controls (n = 170) and developed a modified C3dg assay where C3dg fragments were separated from the large plasma proteins by polyethylene glycol (PEG), and the supernatant containing the C3dg fragment was used for analysis in an antibody-based sandwich-type assay. Gel permeation chromatography and western blotting were used to establish the optimal conditions for PEG precipitation.</p>Results<p>16% PEG was optimal for separating C3dg from C3 and the larger protein fragments. The assay showed a high degree of stability when using EDTA plasma, and measurements correlated well with commercially available complement activation assays. SLE patients had higher concentrations in plasma of C3dg than controls (p < 0.05). ROC analysis showed that the C3dg activation fragment of C3 with an AUC of 0.96 (CI 0.94–0.98) was superior to C3 (AUC 0.52) in differentiating between patients and controls.</p>Conclusion<p>Our results present a modified assay for the measurement of C3dg. We demonstrate that C3dg was superior to conventional C3 measurements in discriminating SLE patients from controls. We suggest that C3dg should be considered as a complement activation measurement in the SLE classification criteria.</p

presentation_1.PDF

Introduction/objectives<p>In 2012, hypocomplementemia was included in the classification criteria of systemic lupus erythematosus (SLE). The suggested measurement of C3 or C4 often reflect disease activity poorly. Our objective was to establish an assay measuring C3dg, which is generated following complement activation, and to evaluate the assay in a cross-sectional SLE cohort.</p>Method<p>We included SLE patients (n = 169) and controls (n = 170) and developed a modified C3dg assay where C3dg fragments were separated from the large plasma proteins by polyethylene glycol (PEG), and the supernatant containing the C3dg fragment was used for analysis in an antibody-based sandwich-type assay. Gel permeation chromatography and western blotting were used to establish the optimal conditions for PEG precipitation.</p>Results<p>16% PEG was optimal for separating C3dg from C3 and the larger protein fragments. The assay showed a high degree of stability when using EDTA plasma, and measurements correlated well with commercially available complement activation assays. SLE patients had higher concentrations in plasma of C3dg than controls (p < 0.05). ROC analysis showed that the C3dg activation fragment of C3 with an AUC of 0.96 (CI 0.94–0.98) was superior to C3 (AUC 0.52) in differentiating between patients and controls.</p>Conclusion<p>Our results present a modified assay for the measurement of C3dg. We demonstrate that C3dg was superior to conventional C3 measurements in discriminating SLE patients from controls. We suggest that C3dg should be considered as a complement activation measurement in the SLE classification criteria.</p

Table_2_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p

DataSheet_8_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p

DataSheet_1_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p

DataSheet_2_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p

DataSheet_4_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p

DataSheet_7_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p

DataSheet_5_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p

Table_1_The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus.docx

IntroductionMany autoimmune diseases are characterized by germinal center (GC)-derived, affinity-matured, class-switched autoantibodies, and strategies to block GC formation and progression are currently being explored clinically. However, extrafollicular responses can also play a role. The aim of this study was to investigate the contribution of the extrafollicular pathway to autoimmune disease development.MethodsWe blocked the GC pathway by knocking out the transcription factor Bcl-6 in GC B cells, leaving the extrafollicular pathway intact. We tested the impact of this intervention in two murine models of systemic lupus erythematosus (SLE): a pharmacological model based on chronic epicutaneous application of the Toll-like receptor (TLR)-7 agonist Resiquimod (R848), and 564Igi autoreactive B cell receptor knock-in mice. The B cell intrinsic effects were further investigated in vitro and in autoreactive mixed bone marrow chimeras.ResultsGC block failed to curb autoimmune progression in the R848 model based on anti-dsDNA and plasma cell output, superoligomeric DNA complexes, and immune complex deposition in glomeruli. The 564Igi model confirmed this based on anti-dsDNA and plasma cell output. In vitro, loss of Bcl-6 prevented GC B cell expansion and accelerated plasma cell differentiation. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output.DiscussionWe identified the extrafollicular pathway as a key contributor to autoimmune progression. We propose that therapeutic targeting of low quality and poorly controlled extrafollicular responses could be a desirable strategy to curb autoreactivity, as it would leave intact the more stringently controlled and high-quality GC responses providing durable protection against infection.</p