IgG Autoantibodies Research Articles

Inflammatory and Autoimmune Aspects of Multisystem Inflammatory Syndrome in Children (MIS-C): A Prospective Cohort Study.

Multisystem Inflammatory Syndrome in Children (MIS-C) is a potentially life-threatening complication of COVID-19. The pathophysiological mechanisms leading to severe disease are poorly understood. This study leveraged clinical samples from a well-characterized cohort of children hospitalized with COVID-19 or MIS-C to compare immune-mediated biomarkers. Our objective was to identify selected immune molecules that could explain, in part, why certain SARS-CoV-2-infected children developed MIS-C. We hypothesized that type-2 helper T cell-mediated inflammation can elicit autoantibodies, which may account for some of the differences observed between the moderate-severe COVID-19 (COVID+) and MIS-C cohort. We enumerated blood leukocytes and measured levels of selected serum cytokines, chemokines, antibodies to COVID-19 antigens, and autoantibodies in children presenting to an academic medical center in Connecticut, United States. The neutrophil/lymphocyte and eosinophil/lymphocyte ratios were significantly higher in those in the MIS-C versus COVID+ cohort. IgM and IgA, but not IgG antibodies to SARS-CoV-2 receptor binding domain were significantly higher in the MIS-C cohort than the COVID+ cohort. The serum levels of certain type-2 cytokines (interleukin (IL)-4, IL-5, IL-6, IL-8, IL-10, IL-13, and IL-33) were significantly higher in children with MIS-C compared to the COVID+ and SARS-CoV-2-negative cohorts. IgG autoantibodies to brain antigens and pentraxin were higher in children with MIS-C compared to SARS-CoV-19-negative controls, and children with MIS-C had higher levels of IgG anti-contactin-associated protein-like 2 (caspr2) compared to the COVID+ and SARS-CoV-19-negative controls. We speculate that autoimmune responses in certain COVID-19 patients may induce pathophysiological changes that lead to MIS-C. The triggers of autoimmunity and factors accounting for type-2 inflammation require further investigation.

#1261 A small-molecule inhibitor of Bruton's tyrosine kinase reduces production of galactose-deficient IgA1 in IgA nephropathy

Abstract Background and Aims IgA nephropathy (IgAN) is a chronic kidney disease characterized by glomerular immunodeposits that contain aberrantly glycosylated IgA1, i.e., IgA1 with some hinge-region O-glycans deficient in galactose (galactose-deficient IgA1; Gd-IgA1). These immunodeposits originate from circulating immune complexes that contain Gd-IgA1 bound by IgG autoantibodies. Consistent with this hypothesis, serum levels of Gd-IgA1 and IgG autoantibodies independently predict disease progression in IgAN patients. Biochemical studies using IgA1-secreting cell lines derived from peripheral blood of IgAN patients and healthy controls have revealed that Gd-IgA1 production is driven by altered biosynthetic pathways of IgA1 O-glycans. Specifically, cell lines from IgAN patients have altered expression and activity of several glycosyltransferases, C1GalT1 and ST6GalNAc2, and a C1GalT1-specific chaperone, C1GalT1C1. Genetic studies have shown that serum levels of Gd-IgA1 are genetically co-determined and genome-wide association studies (GWAS) identified genetic variations that impact the expression C1GALT1 and C1GALT1C1 genes. Moreover, macroscopic hematuria during an upper-respiratory-tract infection is common at the clinical onset of IgAN and during episodes of accentuated disease activity, indicating a connection between IgAN and mucosal inflammation. Notably, a pro-inflammatory cytokine interleukin-6 (IL-6), enhances production of Gd-IgA1 exclusively in the cell lines derived from IgAN patients. Leukemia inhibitory factor (LIF) has similar effects. LIF-induced effects are mediated by abnormal STAT1 signaling and pathways that include Lyn, a Src-family kinase. The corresponding gene, LYN, was recently identified in one of the 16 new IgAN-associated GWAS loci. Lyn, together with spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk), is involved in B-cell-receptor (BCR)-signaling pathways that include activation and inhibitory signals. The aim of this study was to assess if Btk may be involved in Gd-IgA1 production in IgAN. Method IgA1-producing cell lines were cloned by limiting dilution of Epstein-Barr-virus-immortalized peripheral-blood B cells from patients with IgAN (n = 5) and healthy controls (n = 5). Cell signaling, induced by LIF or by BCR crosslinking with anti-IgA antibody, was assessed by flow cytometry or SDS-PAGE/immunoblotting using antibodies for specific phosphorylated sites of selected proteins. Data were normalized using antibodies for specific proteins. Total IgA and Gd-IgA1 were quantified in cell-culture medium using standard ELISA and lectin-ELISA, respectively. Gd-IgA1, normalized to IgA, was expressed in Units (U) of standard Gd-IgA1 protein. Results IgA1-producing cell lines from patients with IgAN and healthy controls secreted similar amounts of total IgA (17.2 ± 9.3 ug/ml vs. 14.6 ± 9.5 ug/ml), but the cells from IgAN patients secreted more Gd-IgA1 (24.2 ± 7.5 U vs. 15.5 ± 2.7 U; p < 0.05). Baseline phosphorylation of Btk (Y223), but not Syk (Y551), was higher in the cells from patients with IgAN than in those from healthy controls. We used an IgA1-secreting cell line from an IgAN patient to confirm that BCR crosslinking with F(ab’)2 anti-IgA antibody in IgA + CD19+ cells lines induced phosphorylation of Btk (Y223). Conversely, anti-IgM antibody, used as a negative control, did not activate Btk. LIF stimulation did not affect phosphorylation of Btk or Syk. A Btk inhibitor, PCI-32765 (ibrutinib), reduced Gd-IgA1 production in a dose-dependent fashion exclusively in the IgAN-derived cells. Notably, 1 uM PCI-32765 reduced Gd-IgA1 production to that for healthy controls (25.7 ± 1.2 U to 17.3 ± 1.8 U; p < 0.01). Conclusion Inhibition of Btk with a small-molecule inhibitor normalized Gd-IgA1 production in IgA1-secreting cells from patients with IgAN, revealing a previously unknown role of Btk in aberrant O-glycosylation of IgA1. Btk may thus represent a potential new target for disease-specific therapy of IgAN.

#2086 Understanding B cell memory autoreactivity in idiopathic membranous nephropathy for tailored therapy

Abstract Background and Aims Idiopathic membranous nephropathy (iMN) is an autoantibody-mediated glomerular disease, and one of the leading causes of nephrotic syndrome in adults. iMN is caused by the formation of immune complexes composed by IgG autoantibodies and target antigens expressed on podocytes. The main autoantigen is the phospholipase A2 receptor1 (PLA2R1) and the antibodies are mainly belonging to the IgG4 isotype. Those immune complexes are responsible for profound changes in the structure and function of the podocytes and disruption of the glomerular barrier. Nevertheless, despite complement components at the site of glomerular injury have been found in biopsies from patients with MN, many factors involved in the pathogenesis are still unclear. We overall aim at characterizing both the serological and the memory cellular component in iMN patients, with identification of dominant B cell epitopes recognized by circulating autoantibodies and autoreactive memory B cells, which could be potentially used to deplete PLA2R1 specific memory B cells and plasma blasts. Method We analyzed the serum from a cohort of 159 iMN patients collected at diagnosis without any previous or concomitant treatment as well as a similar number of healthy controls’ samples. An ELISA-based quantification method has been used to estimate the concentration of circulating anti-PLA2R1 antibodies. Furthermore, we processed 27 additional blood samples from independent iMN patients as well as age and gender matched healthy controls to extract and analyze peripheral blood mononuclear cells by flow cytometry (BD FACS Fortessa) and identify the major B and T cell populations via multi-color staining. In order to identify and clone human anti-PLA2R1 antibodies from primary memory B cells we reproduced the experimental workflow exactly as described in Lindner J.M. et al. Immunity 2019. The isolated antibodies were then characterized with analytical methods (affinity, binding, epitope determination and complement activation ability) and used in competition assays. Results A deep analysis of the serum reactivity has been performed and iMN patients showed a high prevalence of autoantibodies against PLA2R1, mostly of high affine IgG4 subclass with a broad concentration range (1-20ug/ml). A concomitant IgG1 response versus PLA2R1 has been also detected in the serum in different proportion with IgG4. An extended immunophenotyping of circulating peripheral blood mononuclear cells revealed an altered B and T cell compartment in iMN patients. In particular, we described an expansion of circulating CD27neg IgG4 memory B cells, IgG4 plasma blasts, CD4+ T follicular helper cells and CX3CR1+ T peripheral helper cells. Moreover, we isolated and cloned five very specific, highly affine, and somatically hypermutated patient's derived anti-PLA2R1 antibodies. Competition experiments between patient sera and patients’ derived monoclonal antibodies showed a strong inhibitory effect, indicating the presence of a shared epitope recognized on the surface of the CysR domain of PLA2R1. The identified epitope has also shown an important role in complement activation, despite a component patient-specific seems to be involved as well. Conclusion Overall, we performed a comprehensive characterization of the autoreactive B cell memory compartment in iMN, shedding lights on the complexity of the autoantibody repertoire. Indeed, the investigation of the granularity of the anti-PLA2R1 response in term of IgG1 versus IgG4 isotypes provided a potential new way to stratify the patients’ population and potentially tailored the therapy or follow the disease progression. Moreover, the isolation of patient's derived anti-PLA2R1 antibodies opened the possibility of using them the as tools to establish new models directly from the in vivo-selected specificities. Finally, the identification of an immunodominant epitope within PLA2R1 shared among the patients is crucial to develop molecules aimed at depletion of PLA2R1 specific memory B cells and plasma blasts in the patients.

Profiling autoreactive IgG, IgM, IgA and IgE antibodies in system lupus erythematosus patients using antigen microarrays

Abstract OmicsArray™ antigen microarrays bearing 120 antigens were used for profiling IgG, IgM, IgA and IgE autoantibodies in 240 well defined SLE patients and matched healthy controls (HCs). Our analysis show that the average number of positive IgG autoantibodies in SLE is 28.5 with over 95% of SLE patients harbor 5 or more autoantibodies, comparing with 3 positive IgG Autoantibodies in HCs with 25% have 5 or more autoantibodies (p<0.05). A group of 19 IgG autoantibodies targeting to various nuclear antigens (dsDNA, chromatin, histone, Sm/RNP, PCNA, CENP-B, etc.) were significantly enriched in SLE by clustering analysis. The presence of IgG autoantibodies against DNA-associated antigens were most significantly associated with disease activity (SLEDAI) and lupus nephritis (r=0.56, p<0.001) in SLE. The IgG autoantibodies which are positive in HCs were mostly those targeting non-nuclear antigens, such as collagens, alpha-actinin, heparin and cardiolipin. However, some ANA positive HCs exhibited higher IgM specificities targeting to both non-nuclear and nuclear antigens whereas in SLE the IgM autoAb reaction were relatively lower. IgA autoantibodies were detected in over 50% of SLE patients, preferentially targeting to DNA- and RNA-associated antigens indicating the high prevalence of IgA autoantibodies may play a role in SLE. IgE autoantibodies were not detectable for most autoantigens except about 15% of SLE patients showed positive IgE autoantibodies to a small subset of DNA antigens.

Re-evaluating the prevalence of anti-desmocollin-1 IgA autoantibodies in canine pemphigus foliaceus

Pemphigus foliaceus (PF) is an autoimmune skin disease of dogs characterized by intraepidermal pustules containing neutrophils and dissociated keratinocytes that develop in association with circulating and tissue-bound IgG autoantibodies. A subset of IgG autoantibodies in canine PF target desmocollin-1 (DSC1), a component of intercellular adhesion complexes within the epidermis. Passive transfer of IgG autoantibodies from canine PF sera to mice was previously shown to induce skin disease in the absence of infiltrating neutrophils. In attempts to identify a mechanism responsible for neutrophil recruitment, past studies evaluated the prevalence of IgA autoantibodies in canine PF sera where they were found in <20% of affected dogs. We re-evaluated the prevalence of anti-DSC1 IgA in canine PF due to concerns regarding the sensitivity of previously used methods. We hypothesized that anti-DSC1 IgA are present in most dogs with PF but have been under-detected due to competition with concurrent anti-DSC1 IgG for binding to their mutual antigenic target. Despite removing approximately 80% of IgG from patient sera using affinity chromatography, we did not detect an increase in anti-DSC1 IgA by performing indirect immunofluorescence on canine DSC1-transfected HEK293T cells. Taken together, our results do not support a role for pathogenic IgA in canine PF.

Membrane Proteome-Wide Screening of Autoantibodies in CIDP Using Human Cell Microarray Technology.

Autoantibody discovery in complex autoimmune diseases is challenging. Diverse successful antigen identification strategies are available, but, so far, have often been unsuccessful, especially in the discovery of protein antigens in which conformational and post-translational modification are critical. Our study assesses the utility of a human membrane and secreted protein microarray technology to detect autoantibodies in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). A cell microarray consisting of human embryonic kidney-293 cells expressing >5,000 human proteins was used. First, a validation step was performed with 4 serum samples from patients with autoimmune nodopathy (AN) to assess the ability of this technology to detect circulating known autoantibodies. The ability of the cell microarray technology to discover novel IgG autoantibodies was assessed incubating the array with 8 CIDP serum samples. Identified autoantibodies were subsequently validated using cell-based assays (CBAs), ELISA, and/or tissue immunohistochemistry and analyzed in a cohort of CIDP and AN (n = 96) and control (n = 100) samples. Serum anti-contactin-1 and anti-neurofascin-155 were detected by the human cell microarray technology. Nine potentially relevant antigens were found in patients with CIDP without other detectable antibodies; confirmation was possible in six of them: ephrin type-A receptor 7 (EPHA7); potassium-transporting ATPase alpha chain 1 and subunit beta (ATP4A/4B); leukemia-inhibitory factor (LIF); and interferon lambda 1, 2, and 3 (IFNL1, IFNL2, IFNL3). Anti-ATP4A/4B and anti-EPHA7 antibodies were detected in patients and controls and considered unrelated to CIDP. Both anti-LIF and anti-IFNL antibodies were found in the same 2 patients and were not detected in any control. Both patients showed the same staining pattern against myelinating fibers of peripheral nerve tissue and of myelinating neuron-Schwann cell cocultures. Clinically relevant correlations could not be established for anti-LIF and anti-IFNL3 antibodies. Our work demonstrates the utility of human cell microarray technology to detect known and discover unknown autoantibodies in human serum samples. Despite potential CIDP-associated autoantibodies (anti-LIF and anti-IFNL3) being identified, their clinical and pathogenic relevance needs to be elucidated in bigger cohorts.

In vitro and in vivo proof-of-concept studies of extracellular protein degradation using novel M6PR-targeting chimeras (MTACs).

Abstract The mannose-6-phosphate-receptor (M6PR) traffics proteins from extracellular and intracellular locales to late endosomes/lysosomes for degradation and is ubiquitously expressed. This natural endogenous function of the mannose-6-phosphate-receptor (M6PR) may be harnessed for degradation of soluble circulating proteins and cell specific degradation of membrane proteins. Here we describe the design and development of novel M6PR targeting chimeras (MTACs). MTACs are heterobifunctional molecules containing Avilar’s novel, potent, small molecule M6PR-binding ligands conjugated to ligands that bind a target protein of interest for degradation. For proof-of-concept studies, MTACs were designed to target the extracellular protein IgG, the second most abundant circulating protein with a long plasma half-life. Reduction of pathogenic IgG autoantibodies is a clinically validated strategy for treating multiple debilitating autoimmune diseases. In vitro characterization of tridentate and monodentate MTACs revealed potent biochemical binding to M6PR and IgG, and MTAC-mediated cellular uptake and degradation of IgG via the endolysosomal pathway in K562 and LNCaP cells. In vivo MTAC-mediated depletion of human IgG was demonstrated using a heterologous rat PK/PD model.

Investigating the novel role of sarcospan in modulating immune function in mice

Objective: The tetraspanin-like protein sarcospan (SSPN) has been shown to have an important in stabilizing muscle cell membranes and promoting cell adhesion. SSPN has documented expression in immune cells with the highest level of expression in B cells. Examination of SSPN-deficient (SSPN−/−) mice revealed alterations in important immune pathways, which remain uncharacterized. This led us to hypothesize that SSPN may play a role in modulating immune cell development and/or function. Methods: Flow cytometry was used to perform immunophenotyping of spleens obtained from SSPN−/− and WT mice. Cytokine analysis was performed using Luminex to assess cytokines that serve as important signals for immune cell maturation. Hematopoietic stem cells were isolated from red bone marrow and stimulated with phorbol myristate acetate (PMA), a mitogen and polyclonal activator of B cells, to assess whether SSPN deficiency affected their proliferative capacity. To determine whether SSPN plays a role in immunoglobulin isotype switching — antibody isotyping was performed. The presence of immune (antibody-antigen) complexes was investigated in kidney sections obtained from WT and SSPN−/− mice and visualized using biotinylated IgG and IgM antibodies. Autoantigen screening was also performed to assess whether SSPN−/− mice generated abnormal levels of autoantibodies that could be responsible for circulating antibody-antigen complexes. Results: The presence of SSPN on major immune cell types was confirmed using flow cytometry. In addition, flow cytometry analysis showed insignificant baseline differences in the percentage of B cells, T cells, plasmacytoid dendritic cells in SSPN−/− compared to WT spleens. One difference was noted in SSPN−/− females with increased counts of dendritic cells. Spleens in female SSPN−/− mice were found to be slightly larger compared to WT. Levels of B-cell activating factor (BAFF) and B-cell maturation antigen (BCMA) were elevated in SSPN−/− mice, therefore it is expected that SSPN−/− B cells received appropriate signals. Overall B cell proliferation was assessed by CD19+ and appeared unaffected by the absence of SSPN. Subtle increases in IgG1 and IgG2b were detected in SSPN−/− mice, however immunoglobulin isotypes were within normal ranges. SSPN−/− mice were also assessed for signs of dysregulated immune reactivity, evidenced by the presence of immune (antibody-antigen) complexes within filtration units of the kidney. Overall, male SSPN−/− mice were found to have a larger subset of increased IgG autoantibodies compared to male WT mice while female SSPN−/− mice exhibited a larger subset of increased IgM autoantibodies compared to female WT mice. Conclusions: This study examines potentially important functions of SSPN in the immune system. Overall, deletion of SSPN has not been found to affect maturation of major immune cell classes. However, SSPN does appear to influence formation of antigen-antibody complexes in mice, which may contribute to kidney dysfunction. These studies are ongoing and directed toward understanding the role SSPN plays in immune function. FSU Funding, Florida Department of Health, James and Esther King Biomedical Research Program #21K12. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Isotype-Specific Autoantibody Signatures in Cancer and Sepsis Samples through Multiplex Profiling

Abstract Many diseases can be characterized by specific autoantibodies. The Luminex™ multi-analyte profiling platform enables simultaneous detection a panel of autoantibodies in a single assay with minimal sample volume. MILLIPLEX™ multiplex assays include the 15-plex Human Cancer Autoantibody Panel, 23-plex Human Cytokine Autoantibody Panel, and 20-plex Human Autoimmune Autoantibody Panel, allowing qualitative profiling of autoantibodies in serum and plasma samples. In this study, we focused on detecting cancer-associated autoantibodies and anti-cytokine autoantibodies in breast cancer, colorectal cancer, sepsis, and healthy control samples (n ≥ 20 in each group). We used the Luminex™ 200™ system and the xMAP™ INTELLIFLEX DRSE instrument for analysis. Preliminary data revealed anti-cytokine IgG autoantibodies against multiple cytokines (e.g., IL-2, IL-4, IFNγ) in cancer, sepsis, and healthy individuals. Additionally, certain cytokine autoantibodies (e.g., IL-1α, IL-12p40, IL-6) were detected in the tested disease samples but not in healthy controls. Isotype profiling demonstrated increased detection of cancer-associated autoantibodies by combining IgG and IgM signatures in cancer samples (e.g., p53, CCNB1/Cyclin B1, SOX2, CTAG1B/NY-ESO-1, Her2/ErbB2). We believe this robust MILLIPLEX™ autoantibody detection and comprehensive isotype profiling can guide further research on autoantibodies as translational biomarkers in a clinical setting.

Decrypting the Pathological Pathways in IgA Nephropathy.

IgAN is the most common form of glomerulonephritis affecting 2000000 people annually. The disease ultimately progresses to chronic renal failure and ESRD. In this article, we focused on a comprehensive understanding of the pathogenesis of the disease and thus identifying different target proteins that could be essential in therapeutic approaches in the management of the disease. Aberrantly glycosylated IgA1 produced by the suppression of the enzyme β-1, 3 galactosyltransferase ultimately triggered the formation of IgG autoantibodies which form complexes with Gd-IgA1. The complex gets circulated through the blood vessels through monocytes and ultimately gets deposited in the glomerular mesangial cells via CD71 receptors present locally. This complex triggers the inflammatory pathways activating the alternate complement system, various types of T Cells, toll-like receptors, cytokines, and chemokines ultimately recruiting the phagocytic cells to eliminate the Gd-IgA complex. The inflammatory proteins cause severe mesangial and podocyte damage in the kidney which ultimately initiates the repair process following chronic inflammation by an important protein named TGFβ1. TGF β1 is an important protein produced during chronic inflammation mediating the repair process via various downstream transduction proteins and ultimately producing fibrotic proteins which help in the repair process but permanently damage the glomerular cells.

PD-BAT: A novel approach of pooling basophil donors for expansion of commercial laboratory testing of Chronic Spontaneous Urticaria

The type II autoimmune subtype of Chronic Spontaneous Urticaria (CSU) is characterized by the presence of IgG autoantibodies targeting IgE or the IgE high-affinity receptor (FcεRI) on mast cells and basophils. In evaluation of CSU patients, indirect basophil activation testing (BAT), has been utilized, involving the mixing of patient serum with heterologous peripheral blood donors, followed by flow cytometric assessment of basophil markers. However, the reliability of the indirect BAT results hinges on the quality of the donor basophils utilized. In this study, we introduce an innovative approach where multiple potential basophil donors undergo rigorous BAT characterization alongside control samples. By selecting and pooling donors with optimal performance, we significantly enhance the inter-assay reproducibility of the indirect BAT test.

A rare case of acquired hemophilia A

Acquired hemophilia A (AHA) is a rare bleeding disorder characterized by IgG autoantibodies directed against circulating coagulation factor (F) VIII neutralizing its coagulant function. The usual clinical presentation of AHA is spontaneous or provoked bleeding in a person with a negative personal or family history of a coagulopathy and can lead life threatening bleeding especially in older patients. Here we report a case of 54-year-old male patient presenting with blood and passage of clots in urine. He was treated with recombinant factor VII and VIII with steroid as eradication therapy. Underlying cause remained unclear in this case.

Autoimmune nodopathy

AbstractAutoimmune nodopathy (AN) is characterized by the presence of autoantibodies targeting molecules essential for saltatory conduction in myelinated nerves. Clinical manifestations of AN show similarities with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), including progressive and symmetrical sensorimotor deficits with electrophysiological demyelinating features in nerve conduction studies. Although no common autoantibodies have yet been identified in CIDP, AN is characterized by autoantibodies that primarily target specific molecular complex structures represented by the Ranvier node and paranode. Furthermore, these autoantibodies, such as neurofascin‐155 (NF155), contactin‐1 (CNTN1), contactin‐related protein 1 (Caspr1), and the CNTN1/Caspr1 complex, are illustrative examples of such autoantibodies. They can disrupt the septal‐like junction of paranodes without triggering cellular immune responses. AN manifests uniquely with symptoms such as ataxia, tremors, and markedly high cerebrospinal fluid (CSF) protein levels, often accompanied by nerve root and cranial nerve hypertrophy. Notably, this condition is resistant to immunotherapies typically effective against CIDP, including intravenous immunoglobulin therapy (IVIg). Current evidence suggests that B‐cell depletion therapies, such as rituximab, could benefit AN treatment. Since it has been suggested that existing treatments for CIDP may be effective in cases of autoantibody positivity of subclasses other than IgG4, CIDP that is resistant to conventional therapy requires novel therapeutic strategies that take into account the possibility of IgG4 autoantibodies.

P140 IgG Autoantibodies with Increased Affinity for Epithelial Cells in Severe Systemic Sclerosis

Abstract Background/Aims In health IgG immunoglobulins form a network through interaction with cell surface proteins and controlled by regulatory cells, whereas in autoimmune diseases IgG responses become skewed towards higher affinity and potentially damaging responses against self-antigens. Moreover, recent studies have demonstrated activation of the epithelial layer of skin in systemic sclerosis (SSc), implicating keratinocytes as a target of the autoimmune process. We investigated the presence of plasma IgG autoantibodies directed against keratinocytes and sought correlation with clinical parameters in well-characterized patients. Methods Diffuse SSc (dcSSc), limited SSc (lcSSc) and healthy control (HC) plasma samples (each n = 30), were assayed in an anti-epithelial cell antibody assay (AEpCA), using fixed keratinocytes to evaluate binding of IgG . Clinical characteristics of patients with positive versus negative AEpCA were compared. The capacity for SSc IgG to activate epithelial cells was assessed using keratinocytes in vitro. Results Preliminary serial dilution assay of SSc and HC plasma (n = 10) showed enhanced affinity of SSc IgG for keratinocytes (ANOVA p &amp;lt; 0.001 for overall effect SSc vs HC IgG p &amp;lt; 0.0048 for dose effect)(Figure 1A). Dilution of 1/80 gave optimal differential binding in the AEpCA assay (corrected p &amp;lt; 0.027 for SSc vs HC at 1/80). Further HC, dcSSc, and lcSSc plasma samples (each n = 30) were screened, demonstrating increased AEpCA titre in SSc (Figure 1B) (Kruskall-Wallace test p &amp;lt; 0.014 for lcSSc vs HC, p &amp;lt; 0.041 for dcSSc vs HC). SSc patients with positive AEpCA had higher skin scores (mRSS) (AEPCA neg 5(0-24); AEpCA pos 9(0-40) p &amp;lt; 0.0032), higher frequency of ATA antibody (AEpCA neg ATA pos 6/30; AEpCA pos ATA pos 13/30, chi squared p = 0.052) more interstitial lung disease (AEPCA neg 7/30, AEpCA pos 13/30, p NS)(Figure 1C) and trend towards higher use of immunosuppression. SSc but not HC IgG activated keratinocytes in vitro, triggering IL-1α release. Conclusion In this study, we have shown evidence of IgG autoantibodies with increased affinity for keratinocytes in the plasma of SSc patients. Positive testing for the AEpCA was associated with more severe skin disease and lung involvement. The perturbed IgG network in such patients could be activating through common epithelial cells antigens in the skin and lung. Disclosure C. Moezinia: None. V. Wong: None. J. Watson: None. L. Nagib: None. S. Lopez Garces: None. H. Lopez: None. G. Martin: None. S. Zhang: None. B. Ahmed Abdi: None. C. Denton: None. D. Abraham: None. R.J. Stratton: None.

Notch1 hyperactivity drives ubiquitination of NOX2 and dysfunction of CD8+ regulatory T cells in patients with systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) display heightened immune activation and elevated IgG autoantibody levels, indicating compromised regulatory T cell (Tregs) function. Our recent findings pinpoint CD8+ Tregs as crucial regulators within secondary lymphoid organs, operating in a NOX2-dependent mechanism. However, the specific involvement of CD8+ Tregs in SLE pathogenesis and the mechanisms underlying their role remain uncertain. SLE and healthy individuals were enlisted to assess the quantity and efficacy of Tregs. CD8+CD45RA+CCR7+ Tregs were generated ex vivo, and their suppressive capability was gauged by measuring pZAP70 levels in targeted T cells. Notch1 activity was evaluated by examining activated Notch1 and HES1, with manipulation of Notch1 accomplished with Notch inhibitor DAPT, Notch1 shRNA, and Notch1-ICD. To create humanized SLE chimeras, immune-deficient NSG mice were engrafted with PBMCs from SLE patients. We observed a reduced frequency and impaired functionality of CD8+ Tregs in SLE patients. There was a downregulation of NOX2 in CD8+ Tregs from SLE patients, leading to a dysfunction. Mechanistically, the reduction of NOX2 in SLE CD8+ Tregs occurred at a post-translational level rather than at the transcriptional level. SLE CD8+ Tregs exhibited heightened Notch1 activity, resulting in increased expression of STUB1, an E3 ubiquitin ligase that binds to NOX2 and facilitates its ubiquitination. Consequently, restoring NOX2 levels and inhibiting Notch1 activity could alleviate the severity of the disease in humanized SLE chimeras. Notch1 is the cell-intrinsic mechanism underlying NOX2 deficiency and CD8+ Treg dysfunction, serving as a therapeutic target for clinical management of SLE.

Predicting the Speed of Response to Omalizumab in Chronic Spontaneous Urticaria

Introduction: Two distinct chronic spontaneous urticaria (CSU) endotypes, IgE-mediated autoallergic and IgG-mediated autoimmune, were defined based on the response patterns to omalizumab. However, the coexistence of IgE and IgG autoantibodies in a subset of patients might complicate the prediction of the treatment outcomes. This study aimed to evaluate the effectiveness and safety of omalizumab in CSU patients, focusing on the factors predicting the response patterns. Methods: This was a retrospective cross-sectional single-center study investigating CSU patients treated with omalizumab for at least 6 months between September 2015 and February 2023. Patients were evaluated regarding demographics, clinical findings, baseline laboratory parameters, treatment outcomes, and side effects. Early and late responders were defined depending on the time for response, within or after 3 months, respectively. Results: Among 82 patients, 75 (91.5%) responded to omalizumab during the first 6 months, classified as early (n = 51) and late responders (n = 24). The IgG anti-thyroid peroxidase (anti-TPO)/total IgE ratio was an independent predictor for determining the speed of response (p < 0.05). Of 29 patients who discontinued omalizumab, 19 (65.5%) experienced relapse with a good response to retreatment (n = 18/19, 94.7%). Early responders relapsed more frequently than late responders (77.3% vs. 28.6%) (p < 0.05). Only mild side effects were observed in a minority of patients (n = 8/82, 9.8%). Conclusion: Omalizumab is an effective and safe treatment in CSU. The IgG anti-TPO/total IgE ratio seems a valuable tool to predict the early and late responders, the former having a higher possibility of relapse upon drug withdrawal.

A protocol for anti-CD4 IgG antibody purification using plasma samples from people with HIV and antibody-mediated cytotoxicity

Background. Up to 20% of people with HIV (PWH) fail to recover their CD4+ T cell counts to levels similar to healthy controls after suppressive antiretroviral therapy (ART). Immune non-responders (INRs) are PWH on suppressive ART with CD4+ T cell counts lower than 350 cells/mL, whereas their CD8+ T cell counts are higher than healthy controls. We are the first group to report that increased anti-CD4 autoantibody IgGs in INRs are responsible for blunted CD4+ T cell reconstitution in PWH with ART and viral suppression through anti-CD4 IgG-induced antibody-mediated cytotoxicity (ADCC) against CD4+ T cells in vitro. Notably, anti-CD4 IgG-mediated poor CD4+ T cell recovery from suppressive ART is the only mechanism targeting CD4+ T cells, specifically. Results. We provide a detailed one-by-one step protocol from antigen-specific antibody isolation using plasma samples, to ADCC assay. Conclusions. To promote reproducible research, a detailed protocol for isolating anti-CD4 IgG autoantibodies from plasma samples of PWH and evaluating ADCC effects is reported here.•Antigen-specific antibody isolation using human plasma samples•Antibody-mediated cytotoxicity (ADCC)

Autoimmune Hemolytic Anemias: Classifications, Pathophysiology, Diagnoses and Management.

Autoimmune hemolytic anemias (AIHAs) are conditions involving the production of antibodies against one's own red blood cells (RBCs). These can be primary with unknown cause or secondary (by association with diseases or infections). There are several different categories of AIHAs recognized according to their features in the direct antiglobulin test (DAT). (1) Warm-antibody AIHA (wAIHA) exhibits a pan-reactive IgG autoantibody recognizing a portion of band 3 (wherein the DAT may be positive with IgG, C3d or both). Treatment involves glucocorticoids and steroid-sparing agents and may consider IVIG or monoclonal antibodies to CD20, CD38 or C1q. (2) Cold-antibody AIHA due to IgMs range from cold agglutinin syndrome (CAS) to cold agglutin disease (CAD). These are typically specific to the Ii blood group system, with the former (CAS) being polyclonal and the latter (CAD) being a more severe and monoclonal entity. The DAT in either case is positive only with C3d. Foundationally, the patient is kept warm, though treatment for significant complement-related outcomes may, therefore, capitalize on monoclonal options against C1q or C5. (3) Mixed AIHA, also called combined cold and warm AIHA, has a DAT positive for both IgG and C3d, with treatment approaches inclusive of those appropriate for wAIHA and cold AIHA. (4) Paroxysmal cold hemoglobinuria (PCH), also termed Donath-Landsteiner test-positive AIHA, has a DAT positive only for C3d, driven upstream by a biphasic cold-reactive IgG antibody recruiting complement. Although usually self-remitting, management may consider monoclonal antibodies to C1q or C5. (5) Direct antiglobulin test-negative AIHA (DAT-neg AIHA), due to IgG antibody below detection thresholds in the DAT, or by non-detected IgM or IgA antibodies, is managed as wAIHA. (6) Drug-induced immune hemolytic anemia (DIIHA) appears as wAIHA with DAT IgG and/or C3d. Some cases may resolve after ceasing the instigating drug. (7) Passenger lymphocyte syndrome, found after transplantation, is caused by B-cells transferred from an antigen-negative donor whose antibodies react with a recipient who produces antigen-positive RBCs. This comprehensive review will discuss in detail each of these AIHAs and provide information on diagnosis, pathophysiology and treatment modalities.

Electrochemical Biosensing Strategy for Ultrasensitive Detection of Aquaporin-4 Antibody as Neuromyelitis Optica Autoimmune Disease Biomarker Using Aquaporin-4 Extracellular Loop

Neuromyelitis optica (NMO) is a severe and disabling neurodegenerative disorder of the central nervous system (CNS). Neuromyelitis optica-Immunoglobulin G (NMO-IgG) is a serum IgG autoantibody almost exclusively present in NMO patients, which helps to differentiate NMO from other CNS disorders. Developing standardized and user-friendly assays remains a significant challenge in making NMO-IgG testing widely available. Label-free methods are simpler and faster, without additional reagents and procedures. Here, we present a peptide-based label-free electrochemical biosensor for detecting aquaporin-4 antibodies (AQP4-Abs) using extracellular AQP4 to diagnose NMO disease via the DPV electrochemical method. We have developed a novel approach in which the E loop of extracellular AQP4 is bemployed to detect NMO. 3 phenylalanines (Phe) were annexed to the C terminal, and because phenylalanine has a benzene ring, it can have π-π interaction with the benzene ring of carbon nanotube (CNT). In the designated platform, instead of using functional groups with complex and multi-step processes for immobilizing on the electrode surface, we used Nickel-Metal−organic framework /CNT as a novel modifier for measuring AQP4 antibodies with a simple, cheap, and accessible synthesis method. The developed sensor can detect antibodies with detection limit and quantification of 6.2 and 10.0 pg ml−1, respectively (S/N = 3). Also, superb sensitivity of the biosensor was attained as 28.8 μA mL ng−1 cm−2, confirming that the sensor has great potential for clinical application as a diagnostic test.

Clinical and laboratory characteristics of early-onset and delayed-onset lupus nephritis patients: A single-center retrospective study

BackgroundLupus nephritis (LN) manifests systemic lupus erythematosus (SLE) and is characterized by various clinical and laboratory features. This study aimed to comprehensively evaluate the characteristics of LN patients according to the time of LN diagnosis: early-onset (LN diagnosed within one year from SLE diagnosis) vs. delayed-onset (LN diagnosed more than one year after SLE diagnosis).MethodsWe conducted a retrospective analysis of medical records from all SLE patients treated at the University Hospital in Kraków, Poland, from 2012 to 2022. We collected data on demographic, clinical, and laboratory characteristics, including histological findings, treatment modalities, and disease outcomes. Statistical analyses were performed to identify factors impacting LN development and prognosis.ResultsAmong 331 LN patients, early-onset was diagnosed in 207 (62.54%) and delayed-onset was documented in 122 cases (36.86%). In 2 (0.6%) LN cases, the time of first kidney manifestation in the SLE course was unknown. Delayed-onset LN had a higher female-to-male ratio and younger age at SLE diagnosis. This group was associated with more severe clinical manifestations. In turn, studied subgroups did not differ in internist comorbidities, kidney histopathology, and family history regarding autoimmune diseases. Delayed-onset LN exhibited a higher frequency of anti-dsDNA, anti-Smith, anti-Ro, anti-RNP, and anti-cardiolipin IgG autoantibodies. During a 14-year follow-up period, 16 patients died. Mortality rate and causes of death were comparable in both analyzed subgroups.ConclusionsMore severe clinical manifestations in delayed-onset LN prompt strict monitoring of non-LN SLE patients to diagnose and treat kidney involvement early. Also, recognizing the higher frequency of autoantibodies such as anti-dsDNA or anti-Smith in delayed-onset LN underscores the potential value of autoantibody profiling as a diagnostic and prognostic tool.