IgG Subclasses Research Articles

S3106 N-Acetyl Cysteine Use in the Treatment of Ischemic Hepatitis in the Setting of COVID-19 Infection

Introduction: Acute liver injury occurs in 15-53% of COVID-19 patients. Most often, elevation in transaminases is mild, but severe liver injury has been described possibly secondary to hepatic ischemia. We present a case of ischemic hepatitis with concurrent COVID-19 infection, in which N-acetyl cysteine (NAC) administration led to clinical improvement and recovery. Case Description/Methods: A 58-year-old male with adult congenital heart disease (ACHD), on warfarin for atrial flutter and cardiac cirrhosis presented to the ER with generalized fatigue, chills, congestion, and cough. Patient tested positive for COVID-19. He was hemodynamically stable with an unremarkable exam. He was alert and oriented times three. He initially required 8 L of oxygen which continued to improve on a daily basis. His initial laboratory workup was notable for AST 1954 U/L, ALT 1905 U/L, INR 19.1, and total bilirubin 0.9 mg/dL. At baseline, patient’s liver enzymes were normal. He was admitted to the ICU due to the severity of his liver injury. Alcohol, acetaminophen, salicylate levels and urine drug screen were all within normal limits. Abdominal ultrasound revealed known liver cirrhosis and ascites. Acute hepatitis serologies including hepatitis E IgM, ANA, AMA, ASMA, anti-LKM, IGG subclasses, ceruloplasmin, alpha-1 antitrypsin, and celiac panel were done and unremarkable. In addition, HIV, VZV PCR, EBV PCR, HSV PCR and CMV PCR were also negative for acute infection. Patient was treated with a NAC protocol with decrease in elevated liver enzymes by half in the first 72 hours and >20-fold of the highest level upon discharge. Throughout his admission, his mental status remained normal. Discussion: Ischemic hepatitis (IH) results from insufficient blood flow volume and/or oxygen content to the hepatocytes. Acute hypoxic respiratory failure as a result of COVID-19 infection is one of the potential causes of this type of liver injury. Other possible causes of liver injury include direct viral cytopathic effect, cytokinesis and drug-induced liver injury. In the setting of COVID 19 infection, prompt diagnosis and recognition of IH is critical as studies describe mortality rates as high as 50%. We describe our clinical experience with NAC in the setting of COVID 19 with severe IH and propose that NAC is considered in the treatment of COVID-19 patients with ischemic hepatitis, however further research including prospective clinical trials is needed to better validate this.

SELECTIVE TARGETING OF FCRN AND IGG CLEARANCE BY NIPOCALIMAB PRESERVES KEY IMMUNE FUNCTIONS

Autoantibody disorders are caused by immune-system-produced antibodies that can damage the body's tissues or organs. In the case of warm autoimmune hemolytic anemia (wAIHA), pathogenic autoantibodies lead to the destruction of red blood cells. There is an unmet need for innovative targeted therapies that address the underlying pathogenesis of wAIHA in a safe and efficacious way. Nipocalimab is a fully human, aglycosylated, effectorless IgG1 monoclonal antibody that blocks the IgG binding site on the neonatal Fc receptor (FcRn) which prevents IgG recycling, leading to reduced serum levels of total IgGs, including pathogenic IgG autoantibodies. Nipocalimab is in clinical development for the treatment of multiple IgG-mediated autoantibody diseases, including wAIHA (NCT04119050). Rapid, sustained lowering of IgG was observed in the phase 2 VIVACITY study in generalized myasthenia gravis (gMG) and in phase 1 healthy volunteers. In gMG patients, nipocalimab induced rapid and sustained lowering of anti-AChR autoantibodies and MG-ADL scores, but no serious adverse events including clinically significant infections. Here, we characterize the effect of nipocalimab on immune function. Nipocalimab was evaluated extensively in vitro and in nonhuman primate-based chronic toxicology studies to evaluate selectivity, tolerability, safety and immunopharmacology. Safety, tolerability and immune-focused assessments in clinical phase 1 and Phase 2 MG studies were also completed (NCT02828046, NCT03772587). Nipocalimab binds specifically in vitro to FcRn without activation of effector function or inhibition of antigen presentation. In nonhuman primates administered up to 300 mg/kg nipocalimab QW for up to 6 months, sustained lowering of IgG was observed without adverse effects. Immunotoxicology identified no effect on immune cell phenotypes; CD8 T cell, NK or innate cell functions; T-dependent neoantigen IgM responses. Neoantigen IgG production was observed, but with lowered peak IgG titers consistent with the anticipated increase in IgG clearance with nipocalimab. In clinical studies, nipocalimab demonstrated a reproducible selective decrease in total serum IgG, including all subclasses of IgG, with no effect on IgM, IgA, IgE, CH50, C3, C4, inflammatory cytokines or acute phase proteins including, C-reactive protein (CRP). These data suggest that nipocalimab can selectively lower IgG and IgG autoantibodies while preserving cellular immunity, complete IgM response and IgG production after neoantigen challenge. Overall, nipocalimab's selective effect on IgG recycling provides a mechanistic rationale for potentially decreased infection risk despite substantial IgG lowering. LEL *, FY *, CJB *, JB *, RX *, AMBP are employed by the Janssen Pharmaceutical Companies of Johnson & Johnson and may hold stock or stock options in Johnson & Johnson; ST * is an employee of Orna Therapeutics; SK * is an employee of Checkmate Pharmaceuticals; JD * is an employee of Faze Medicines; WA * is an employee of Kisbee Therapeutics. *Former employees of Momenta Pharmaceuticals which was acquired by Janssen Pharmaceutical Companies of Johnson & Johnson.

S2996 Erdheim-Chester Disease Associated With Aggressive Sclerosing Cholangitis

Introduction: Erdheim-Chester disease (ECD) is a rare histiocytosis recently recognized as a neoplasm due to discovery of MAPK pathway mutations. ECD can involve multiple systems, but hepatic involvement is exceedingly rare. Case Description/Methods: A 64-year-old male presented with fatigue, pruritis, 15-pound weight loss, bilirubin 1.8 mg/dL (0.3-1), and alkaline phosphatase (ALP) 1671 IU/L (44-147). MRCP showed multifocal, short segment dilation of intrahepatic ducts only. Liver biopsy showed periportal and focal bridging fibrosis suggesting primary sclerosing cholangitis (PSC). Anti-smooth muscle, anti-mitochondrial, antineutrophil cytoplasmic antibodies (ANCA) were negative, IgG subclasses normal. A month later he developed emesis, weakness, and leukocytosis. CT scan showed perinephric and periaortic soft tissue stranding suggestive of ECD. 18FDG PET/CT showed a 2.1x2.9 cm highly FDG avid mediastinal mass, follicular lymphoma on biopsy. He improved with antibiotics but returned after a few days with emesis, leukocytosis, and bilirubin 10.7 mg/dL, ALP 1277 IU/L. CT scan showed progressive perinephric and periaortic soft-tissue encasement (Figure1A). Endoscopic retrograde cholangiopancreatography showed a common bile duct (CBD) stricture and non-filling right hepatic duct. Despite CBD stenting bilirubin continued to rise. He now had molluscum like skin lesions which on biopsy revealed dermal proliferation of CD68+, Factor XIIIa+, CD1a- histiocytes and multiple giant cells characteristic of ECD (Figure1B). Targeted therapy with BRAF or MEK pathway inhibitors was considered though there was concern that he may be too sick to receive these. Patient and family opted for comfort care. Discussion: Despite symptoms, cholestatic liver function and biopsy suggestive of PSC, several factors make us question this diagnosis; such as lack of inflammatory bowel disease or positive ANCA which are seen in 60-80% patients with PSC. PSC is usually indolent, time from diagnosis to death or liver transplant generally being 10-12 years. This patient was transitioned to hospice within 6 months. This makes us suspect hepatic involvement with ECD or an overlap syndrome, or that PSC associated with ECD runs a much more aggressive course. Though liver and CBD biopsies did not show ECD, it is not unusual for biopsy findings in ECD to be obscured by non-specific fibrosis. We want to highlight that a negative biopsy should not delay diagnosis of ECD if imaging is indicative, especially as effective therapy is now available.Figure 1.: 1A: CT abdomen showing perinephric soft tissue stranding and encasement. B: extensive histiocytic infiltration (majority of cells) seen on skin biopsy.

Heavy Chain Constant Region Usage in Antibodies to Peptidylarginine Deiminase 4 as a Marker of Disease Subsets in Rheumatoid Arthritis.

The study of autoantibody isotypes in autoimmune diseases is useful for identifying clinically relevant endotypes. This study was undertaken to study the prevalence and clinical significance of different isotypes and IgG subclasses of anti-peptidylarginine deiminase 4 (anti-PAD4) autoantibodies in individuals with rheumatoid arthritis (RA). In 196 RA subjects and 64 healthy controls, anti-PAD4 antibody types were determined using enzyme-linked immunosorbent assay. We investigated associations between anti-PAD4 antibodies and clinical outcomes, and relevant features were confirmed in an independent RA cohort. Anti-PAD4 IgG1, anti-PAD4 IgG2, anti-PAD4 IgG3, anti-PAD4 IgG4, anti-PAD4 IgA, and anti-PAD4 IgE antibodies were more frequent in RA patients than healthy controls (P < 0.001). Anti-PAD4 IgG1, anti-PAD4 IgG3, and anti-PAD4 IgE were associated with distinct clinical features. Anti-PAD4 IgG1 was predictive of progressive radiographic joint damage (odds ratio [OR] 4.88, P=0.005), especially in RA patients without baseline joint damage (40% versus 0%, P=0.003) or in those negative for anti-cyclic citrullinated peptide and/or rheumatoid factor (OR 32; P=0.009). IgG1 was also associated with higher levels of C-reactive protein (P=0.006) and interleukin-6 (P=0.021). RA patients with anti-PAD4 IgG3 had higher baseline joint damage scores (median Sharp/van der Heijde score 13 versus 7, P=0.046), while those with anti-PAD4 IgE had higher Disease Activity Score in 28 joints (median 4.0 versus 3.5, P=0.025), more frequent rheumatoid nodules (31% versus 16%, P=0.025), and more frequent interstitial lung disease (ground-glass opacification) (24% versus 9%, P=0.014). Anti-PAD4 IgG1 antibody associations with joint damage were corroborated in an independent RA cohort. Anti-PAD4 IgG1, anti-PAD4 IgG3, and anti-PAD4 IgE antibodies identify discrete disease subsets in RA, suggesting that heavy chain usage drives distinct effector mechanisms of anti-PAD4 antibodies in RA.

Modulation of urelumab glycosylation separates immune stimulatory activity from organ toxicity.

Checkpoint control and immunomodulatory antibodies have become important tools for modulating tumor or self-reactive immune responses. A major issue preventing to make full use of the potential of these immunomodulatory antibodies are the severe side-effects, ranging from systemic cytokine release syndrome to organ-specific toxicities. The IgG Fc-portion has been demonstrated to contribute to both, the desired as well as the undesired antibody activities of checkpoint control and immunomodulatory antibodies via binding to cellular Fcγ-receptors (FcγR). Thus, choosing IgG subclasses, such as human IgG4, with a low ability to interact with FcγRs has been identified as a potential strategy to limit FcγR or complement pathway dependent side-effects. However, even immunomodulatory antibodies on the human IgG4 background may interact with cellular FcγRs and show dose limiting toxicities. By using a humanized mouse model allowing to study the immunomodulatory activity of human checkpoint control antibodies in vivo, we demonstrate that deglycosylation of the CD137-specific IgG4 antibody urelumab results in an amelioration of liver toxicity, while maintaining T cell stimulatory activity. In addition, our results emphasize that antibody dosing impacts the separation of side-effects of urelumab from its therapeutic activity via IgG deglycosylation. Thus, glycoengineering of human IgG4 antibodies may be a possible approach to limit collateral damage by immunomodulatory antibodies and allow for a greater therapeutic window of opportunity.

Lung cancer patients with nephropathy as the first manifestation: Literature review and clinical study report

BackgroundTo investigate the relationship between membranous nephropathy (MN) and lung cancer.MethodsTo report patients with lung cancer detected by follow-up after the diagnosis of MN by renal biopsy in China-Japan Friendship Hospital from January 2010 to December 2019, and to study the prognosis of lung cancer-associated MN and have a review of the literature.ResultsLung cancer was detected in six patients followed for 1–27 months (median 8 months) after the diagnosis of MN: including four cases of lung adenocarcinoma, one case of carcinoma in situ, and one case of small cell lung cancer with multiple metastases. Five cases were in remission after surgical resection, and one case was remitted after chemotherapy. Six patients were negative for serum anti-PLA2R antibodies, and glomerular IgG subclass deposition detected by immunofluorescence was positive for IgG1 and IgG2. Glomerular PLA2R, THSD7A, and NELL-1 stainings were assessed in all six patients; one patient was positive for glomerular PLA2R staining, two patients were positive for glomerular THSD7A staining, and all patients were negative for NELL-1 staining. A literature review of the relationship between MN and lung cancer was performed: seven articles about cancer-associated MN were searched, reporting 32 cases of MN associated with lung cancer, among which 14 cases had nephropathy as the first manifestation and only five patients had remission of MN after treatment of lung cancer.ConclusionsA few lung cancer patients have nephropathy as the first clinical manifestation, and MN can also be remitted after treatment of lung cancer.

P42 Primary Sjogren’s Syndrome (pSS) in a seronegative adolescent female with family history of BehÇet’s disease: A challenging disease to diagnose Case-based discussion

Introduction/BackgroundSjogren’s Syndrome (SS) is heterogenous chronic autoimmune disease with symptoms ranging from organ-specific to variable non-specific systemic features. It is a rare disease in childhood and can be very difficult to diagnose, especially in seronegative patients, with the absence of any specific diagnostic investigation or criteria. We are presenting a seronegative juvenile Primary Sjogren’s syndrome (pSS) patient with all the challenges we had to confirm the diagnosis and a highlight on the role of MDT as the most sensitive tool.Description/MethodA 14-year-old female patient was first referred to our Paediatric and adolescent rheumatology department at the age of 10 with history of recurrent bilateral painful parotid swellings since the age of 3 with absence of ANA, RF, Anti-SSA-Ro and Anti-SSB-La antibodies repeatedly. From 2016 till 2019 she was under the care of ENT with recurrent complicated bilateral ear infections. Episodes tend to occur every 2-3 weeks and last between a few days to 2 weeks. Her events were associated with fever, fatigue, shivering, joint and leg pain, cervical lymphadenopathy, infrequent folliculitis, and headaches. No history to indicate mouth or eye dryness or significant repeated ulcers. Both of our patient’s mother and maternal grandmother have Behçet’s disease. Further blood investigation only showed mild hypergammaglobulinemia with mildly elevated ESR and CRP, normal LFT, U&Es, complements, muscle enzymes and thyroid function. HIV serology was negative and serum IgG4 subclass was normal. Many USSs confirmed parotitis. Parotid gland biopsy was not specific and indicated chronic parotitis with diffuse infiltration of mature lymphocytes, periductal lymphocytic inflammation and exocytosis of lymphocytes into the ductal epithelium. Focus score nearly reaching 50 cells. Plasma cell staining ruled out IgG4 related disease. Ophthalmic assessment ruled out eye involvement by the appearance of her eyes. The Oral Medicine Team confirmed the appearance of dry mucosa with minimal salivary flow from the parotid ducts, though it has not been subjectively reported to cause any problem before. Failed trial of colchicine, never been off-prednisolone for at least the last 2 years, tried Methotrexate which resulted in significant but not full improvement. Now she is on azathioprine and Hydroxychloquine, but still reporting flares. Escalation in treatment is required though there are some concerns about previous medication compliance, the actual severity of symptoms reported and the lack of current suitable accommodation.Discussion/ResultsDiagnosing pSS is quite challenging especially in seronegative patients due to the possibility of other non-autoimmune diseases causing recurrent parotid gland swellings, especially Recurrent Juvenile Idiopathic Parotitis with very young age of onset. With the persistence of symptoms and lack of an underlying infection, our patient ended up having a parotid biopsy. This was generally not specific, with a focus score close to the 50 mononuclear cells required as evidence of exocrine inflammation. This further added to the challenge of confirming an underlying autoimmune disease. Nine years into the disease course, our patient was confirmed to have objective evidence of dry mucosa and decreased salivary flow from the parotid ducts. This shows how important it is to assess salivary and lacrimal gland dysfunction in a child with suspected pSS, despite the absence of typical xerophthalmia and xerostomia. Considering the non-typical features (sero-negativity and the non-specific histopathology findings), our patient clinical picture was discussed in an MDT meeting including ENT, Histopathology and Rheumatology and the diagnoses of pSS was confirmed. The chronicity of the disease, significant improvement of the flare frequency and severity while on immunosuppressants, the reoccurence of significant flares when the patient was off-treatment (personal decision), and the objective evidence of poor salivary flow all supported the diagnosis of pSS later in the disease course. This case shows us how the typical adult features of pSS should not be strictly applied to the juvenile form of the disease. It also highlights the importance of having an MDT as the most sensitive available tool to confirm the diagnosis.Regarding lack of subjective full response, how do you think we can prove this objectively and what would be the most appropriate next step in treatment escalation?Key learning points/ConclusionA high index of suspicion is crucial to reach the diagnosis and to avoid irreversible damage in children with SS.Quadrable sero-negativity should not be looked at as an absolute exclusion criterion of juvenile pSS.Exclusion of other more common and diverse causes of parotitis is crucial.The typical adult features of SS should not be strictly applied to the juvenile form of the disease.MDT approach is the most sensitive tool to diagnose SS.When there is no subjectively reported ophthalmic or oral dryness, we would advocate sending suspected children of pSS for formal assessment of it- we understand the age-related challenges.Considering its rarity, we need to improve the general awareness of the juvenile form of the disease among the population and the medical practitioners.

Antibody apparent solubility prediction from sequence by transfer learning

SummaryDeveloping therapeutic monoclonal antibodies (mAbs) for the subcutaneous administration requires identifying mAbs with superior solubility that are amenable for high-concentration formulation. However, experimental screening is often material and labor intensive. Here, we present a strategy (named solPredict) that employs the embeddings from pretrained protein language modeling to predict the apparent solubility of mAbs in histidine (pH 6.0) buffer. A dataset of 220 diverse, in-house mAbs were used for model training and hyperparameter tuning through 5-fold cross validation. solPredict achieves high correlation with experimental solubility on an independent test set of 40 mAbs. Importantly, solPredict performs well for both IgG1 and IgG4 subclasses despite the distinct solubility behaviors. This approach eliminates the need of 3D structure modeling of mAbs, descriptor computation, and expert-crafted input features. The minimal computational expense of solPredict enables rapid, large-scale, and high-throughput screening of mAbs using sequence information alone during early antibody discovery.

Association of gestational diabetes mellitus and negative modulation of the specific humoral and cellular immune response against Toxoplasma gondii.

In order to evaluate and compare the specific immune response of pregnant women (PW) chronically infected with Toxoplasma gondii, with and without gestational diabetes mellitus (GDM), and the humoral response of their respective newborns (NB), the study was carried out on 81 PW (34 GDM and 47 controls) from whose medical records the results of the oral glucose tolerance test (OGTT) were obtained, and blood samples were collected at the third trimester of pregnancy; also, on 45 NBs (20 GDM and 25 controls) from whom umbilical cord blood samples were obtained. Humoral immunity was analyzed by measuring anti-T. gondii total IgG, IgG subclasses and IgG avidity. To evaluate cellular immunity, peripheral blood mononuclear cells (PBMC) from 32 PW (16 GDM and 16 controls) were cultured, supernatant cytokines were determined, and flow cytometry was performed to analyze the expression at lymphocytes of surface molecules, cytokines and transcription factors. All PW and NBs were positive for total IgG, and the prevalent subclass was IgG1. There was a negative correlation between the OGTT glycemia of PW and the levels of total IgG, IgG1 and IgG avidity. The IgG avidity of the GDM group was significantly lower than the control group. Patients from the GDM group had a higher number of T lymphocytes expressing markers of cell activation and exhaustion (CD28 and PD-1). In the presence of T. gondii soluble antigen (STAg) the amount of CD4+ T cells producing IFN-γ, IL-10 and IL-17 was significantly lower in the GDM group, while there was no difference between groups in the number of CD4+ CD25HighFOXP3+LAP+ functional Treg cells. Additionally, under STAg stimulus, the secretion of IL-17, IL-4, TNF and IL-2 cytokines at PBMCs culture supernatant was lower in the GDM group. In conclusion, there was a correlation between the increase in blood glucose and the decrease in levels of anti-T. gondii antibodies, associated with the decreased IgG avidity in patients who develop GDM. Also, the GDM group had decreased immune responses in Th1, Th2 and Th17 profiles, suggesting an association between GDM and the negative modulation of the humoral and cellular immune responses against T. gondii.

The combination of very-small size proteoliposomes and alum is a safe adjuvant alternative for inducing anti-EGF antibodies: a preclinical study

Immunization with human recombinant EGF chemically bound to the P64k protein of Neisseria meningitides (hrEGF-P64k) and adjuvanted in Montanide ISA 51 VG (Montanide) is an efficient strategy to induce polyclonal antibodies (PAbs) response targeting this self -antigen in cancer patients, which is the basis of the CIMAvax-EGF vaccine. The neutralizing potential of EGF-specific induced PAbs supports promising clinical data obtained to date with this vaccine. Herein, we evaluated a combination of very small-size proteoliposomes (VSSP) and aluminum hydroxide (Alum) as a novel adjuvant to induce specific PAbs with neutralizing and anti-proliferative properties on tumor cells, considering EGF as a model antigen. Toxicity at the injection site was not detected for the vaccine formulation containing VSSP/Alum, and it was immunogenic in BALB/c mice, as evidenced by the induction of high titers of EGF-specific polyclonal antibodies (PAbs). While schedule optimization increased the magnitude of the PAbs response induced by VSSP/Alum, induced PAbs’s avidity and intrinsic neutralizing potential were comparable to the humoral response induced by Montanide. Also, VSSP addition switched IgG subclasses distribution into a Th1-like pattern, as obtained with Montanide and desirable for a cancer vaccine. Finally, equivalent PAbs titers were induced by the vaccine formulations adjuvanted in VSSP/Alum or Montanide in tumor-bearing-mice, and immunosuppressed mice, suggesting the feasibility of the VSSP/Alum combined adjuvant for inducing anti-EGF antibodies in cancer patients at advanced stages of the disease.

Disease Association of Anti‒Carboxyethyl Lysine Autoantibodies in Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurring suppurating lesions of the intertriginous areas, resulting in a substantial impact on patients' QOL. HS pathogenesis remains poorly understood. An autoimmune component has been proposed, but disease-specific autoantibodies, autoantigens, or autoreactive T cells have yet to be described. In this study, we identify a high prevalence of IgM, IgG, and IgA antibodies directed against Nε-carboxyethyl lysine (CEL), a methylglyoxal-induced advanced glycation end-product, in the sera of patients with HS. Titers of anti-CEL IgG and IgA antibodies were highly elevated in HS compared with those in healthy controls and individuals with other inflammatory skin diseases. Strikingly, the majority of anti-CEL IgG was of the IgG2 subclass and correlated independently with both disease severity and duration. Both CEL and anti-CEL‒producing plasmablasts could be isolated directly from HS skin lesions, further confirming the disease relevance of this autoimmune response. Our data point to an aberration of the methylglyoxal pathway in HS and support an autoimmune axis in the pathogenesis of this debilitating disease.

A colloidal gold immunochromatography for the detection of flumioxazin residues in fruits.

In this study, an excellent monoclonal antibody (mAb) was produced that could sensitively and specifically recognize Flu in fruit samples. The mAb belonged to the IgG2 subclass and had a half-inhibitory concentration (IC50 ) of 0.59ng/ml and a limit of detection (LOD) of 0.13ng/ml. A cross-reactivity test revealed that the mAb had good specificity for Flu. A gold nanoparticle-based lateral-flow immunochromatographic (ICA) strip was used for the rapid screening of Flu in fruit samples. The method had a visual cut-off value of 10µg/kg for Flu in three different fruit samples when evaluated with the naked eye. The LODs were 5.25, 3.13, and 4.64µg/kg for Flu in apples, grapes, and oranges, respectively, as obtained with the aid of a strip scan reader. Therefore, this ICA strip assay represented a potential tool for the detection of Flu in fruit samples.

Laboratory characteristics of IgG4-related disease: A retrospective study from a single tertiary medical center.

Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition with unique histopathological features that can affect most organs, making diagnosis challenging. This study characterized detailed laboratory characteristics of IgG4-RD. Baseline clinical and laboratory features of 33 patients with IgG4-RD were reviewed, including serum IgG4 concentrations, serum free light chains (sFLCs), IgGĸ- and IgGλ-heavy/light chains (HLCs), capillary serum protein electrophoresis (SPE), and immunofixation electrophoresis (IFE) of IgG4 subclass. The cohort of 33 patients showed male predominance (94%), with 8 (24%) exhibiting multiple organ involvement. Most patients (88%) had an elevated IgG4 concentration, and 67% had elevated erythrocyte sedimentation rate and IgE levels. Median IgG4 concentration at baseline was significantly higher in patients with >2 organs involved than those with ≤2. Furthermore, erythrocyte sedimentation rate was significantly correlated with serum IgG4 concentrations at baseline. SPE results demonstrated polyclonal gammopathy in most patients. Half of the patients had an increased κ/λ sFLC ratio, 42% had an increased IgGκ/IgGλ HLC ratio. Most patients exhibited hypergammaglobulinemia in the anodal end of the ɤ region on SPE. This study describes detailed laboratory features of IgG4-RD. Although none of these tests are considered diagnostically sufficient by itself, the provided laboratory characteristics can increase awareness of this disorder and help distinguish it from other IgG4-RD mimics.

Finding the Right Heavy Chains for Immunostimulatory Antibodies

For twelve years, the oncology field has been revolutionized by antibodies targeting immune checkpoints. They must be considered as a heterogenous family of immunostimulatory antibodies displaying very different mechanisms of action, not only depending on the target or on the cells expressing it, but also on the IgG subclass or IgG variant that has been chosen. To dissect this complex landscape, the clinical experience has been confronted with a precise analysis of the heavy chain isotypes, referred as new Ge nomenclature. For antibodies targeting inhibitory receptors, anti-CTLA-4 antibodies (whose main effect is to kill regulatory T cells) will be distinguished from anti-PD-1 antibodies and other true antagonistic antibodies. Antibodies targeting ligands of inhibitory receptors (PD-L1, CD47) represent another different category, due to the antigen expression on tumors and a possible beneficial killing effect. The case of agonistic antibodies targeting lymphocyte activatory receptors, such as CD40 or 4-1BB, is still another “under construction” category because these products are less advanced in their clinical development. Altogether, it appears that choosing the right heavy chain is crucial to obtain the desired pharmacological effect in patients.

Decoding human-macaque interspecies differences in Fc-effector functions: The structural basis for CD16-dependent effector function in Rhesus macaques.

Fc mediated effector functions of antibodies play important roles in immunotherapies and vaccine efficacy but assessing those functions in animal models can be challenging due to species differences. Rhesus macaques, Macaca mulatta (Mm) share approximately 93% sequence identity with humans but display important differences in their adaptive immune system that complicates their use in validating therapeutics and vaccines that rely on Fc effector functions. In contrast to humans, macaques only have one low affinity FcγRIII receptor, CD16, which shares a polymorphism at position 158 with human FcγRIIIa with Ile158 and Val158 variants. Here we describe structure-function relationships of the Ile/Val158 polymorphism in Mm FcγRIII. Our data indicate that the affinity of the allelic variants of Mm FcγRIII for the macaque IgG subclasses vary greatly with changes in glycan composition both on the Fc and the receptor. However, unlike the human Phe/Val158 polymorphism in FcγRIIIa, the higher affinity variant corresponds to the larger, more hydrophobic side chain, Ile, even though it is not directly involved in the binding interface. Instead, this side chain appears to modulate glycan-glycan interactions at the Fc/FcγRIII interface. Furthermore, changes in glycan composition on the receptor have a greater effect for the Val158 variant such that with oligomannose type glycans and with glycans only on Asn45 and Asn162, Val158 becomes the variant with higher affinity to Fc. These results have implications not only for the better interpretation of nonhuman primate studies but also for studies performed with human effector cells carrying different FcγRIIIa alleles.

C1q or IgA deposition in glomeruli of children with primary membranous nephropathy

Objective: To assess the correlation of glomerular C1q or IgA deposition with clinical and pathological features of primary membranous nephropathy (PMN) in children. Methods: The clinical and pathological manifestations including (phospholipase A2 receptor, PLA2R) and IgG subclasses staining in renal biopsies, serum anti-PLA2R antibody and therapeutic response of 33 children diagnosed with PMN in Peking University First Hospital from December 2012 to December 2020 were retrospectively summarized and analyzed. According to results of PLA2R test and findings renal pathological, the patients were divided into PLA2R-related group and non-PLA2R-related group, typical MN group and atypical MN group, C1q deposit group and non-C1q deposit group, as well as IgA deposit group and non-IgA deposit group respectively. T-test, Mann-Whitney U test and Fisher's exact probability test were used for comparison between the groups. Results: Among the 33 children with PMN, there were 20 males and 13 females, of that the age of onset was 11 (8, 13) years, and 32 patients had nephrotic level proteinuria. Renal biopsies were performed at 4.6 (2.1, 11.6) months after onset, and 28 patients (85%) received glucocorticoid or immunosuppressive therapy prior to renal biopsy. There were 20 cases (61%) with PLA2R-related MN and 13 cases (39%) with non-PLA2R-related MN. Compared with the non-PLA2R-related group, the PLA2R-related group had an older age of onset (12 (10, 13) vs. 7 (3, 12) years, Z=-2.52, P=0.011), a lower preceding infection rate (45% (9/20) vs. 11/13, P=0.032) and lower spontaneous remission rate (0 vs. 4/13, P=0.017). Renal PLA2R positivity was significantly associated with predominant or co-deposition of IgG4 (13/17 vs. 5/15, P=0.031) and low albumin levels at renal biopsy ((25±6) vs. (29±7) g/L, t=2.14, P=0.041). There were 12 patients with typical PMN and 21 patients with atypical PMN, and no significant difference in clinical and pathological manifestations was found between these 2 groups (all P>0.05). There were 10 cases (32.3%) with glomerular C1q deposition, and their disease course before renal biopsy was significantly shorter than those without C1q deposition (1.8 (0.8, 5.9) vs. 6.0 (2.5, 22.3) months, Z=-2.27, P=0.023). Twelve cases (36.4%) had glomerular IgA deposition, and their course of disease,clinical and pathological manifestations were not significantly different from those without IgA deposition (all P>0.05). Conclusion: Glomerular C1q or IgA deposition may not affect the clinical manifestations, glomerular PLA2R and IgG subclasses staining pattern, or the response to treatment of PMN in children.

Pulmonary Delivery of Levamisole Nanoparticles as an Immunomodulator Affecting Th and a Potential ADAM10 Inhibitor to Ameliorate Severe Allergic Asthma.

Asthma is a common chronic lung disease without absolute treatment, and hypersensitivity reactions and type 2 immune responses are responsible for asthma pathophysiology. ADAM10 as a metalloproteinase transmembrane protein is critical for development of Th2 responses, and levamisole as an anthelmintic drug has immunomodulatory effects, which not only regulates ADAM10 activity but also can suppress the bone marrow and neutrophil production. Therefore, in the present study, nanoparticles were used as a levamisole delivery system to reduce bone marrow suppression, and the immunomodulatory and ADAM10 inhibitory effects of levamisole were studied in allergic asthma. Asthmatic mice were treated with PLGA-levamisole nanoparticles. Then, AHR, BALF, and blood cell counts, levels of the IgG1 subclass, total and OVA-specific IgE, IL2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-25, IL-33, INF-γ, and TNF-α, gene expression of FoxP3, T-bet, RORγt, PU.1, GATA3, FcεRII, CysLT1R, eotaxin, and ADAM10, and lung histopathology were evaluated. PLGA-LMHCl with considered characteristics could control airway hyper-responsiveness, eosinophils in the BALF, levels of immunoglobulins, Th2-, Th9-, and Th17-derived cytokines and pivotal genes, eosinophilic inflammation, hyperplasia of the goblet cell, and hyperproduction of mucus and could increase Th1- and Treg-derived cytokines and also pivotal genes. It could also modulate the ADAM10 activity and had no effect on the number of neutrophils in the bloodstream. The novel safe nanodrug had no side effect on the bone marrow to produce neutrophils and could control the allegro-immuno-inflammatory response of asthma.

Factors influencing the immune response over 15 months after SARS-CoV-2 infection: A longitudinal population-wide study in the Faroe Islands.

BackgroundThe durability of SARS‐CoV‐2 antibody response and the resulting immunity to COVID‐19 is unclear.ObjectivesTo investigate long‐term humoral immunity to SARS‐CoV‐2.MethodsIn this nationwide, longitudinal study, we determined antibody response in 411 patients aged 0–93 years from two waves of infections (March to December 2020) contributing 1063 blood samples. Each individual had blood drawn on 4–5 occasions 1–15 months after disease onset. We measured total anti‐SARS‐CoV‐2 receptor‐binding domain (RBD) antibody using a qualitative RBD sandwich ELISA, IgM, IgG and IgA levels using an quantitative in‐house ELISA‐based assay and neutralizing antibodies (NAbs) using an in‐house ELISA‐based pseudoneutralizing assay. IgG subclasses were analyzed in a subset of samples by ELISA‐based assay. We used nonlinear models to study the durability of SARS‐CoV‐2 antibody responses and its influence over time.ResultsAfter 15 months, 94% still had detectable circulating antibodies, mainly the IgG isotype, and 92% had detectable NAbs. The distribution of IgG antibodies varied significantly over time, characterized by a biphasic pattern with an initial decline followed by a plateau after approximately 7 months. However, the NAbs remained relatively stable throughout the period. The strength of the antibody response was influenced by smoking and hospitalization, with lower IgG levels in smokers and higher levels in hospitalized individuals. Antibody stability over time was mainly associated with male sex and older age with higher initial levels but more marked decrease.ConclusionsThe humoral immune response to SARS‐CoV‐2 infection varies depending on behavioral factors and disease severity, and antibody stability over 15 months was associated with sex and age.

315.11: Tacrolimus Withdrawal After Mesenchymal Stromal Cell Therapy Is Associated With Donor-Specific Antibody Formation in Kidney Transplant Recipients

Introduction: Recently, the clinical phase II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy is a safe method to facilitate tacrolimus withdrawal in kidney transplant recipients (KTRs). Here, we analyzed de novo donor-specific antibody (dnDSA) formation and HLA molecular mismatch in MSC-treated KTRs. Methods: All patients underwent a first, living-donor kidney transplantation and received alemtuzumab induction, steroids, tacrolimus and everolimus. Patients in the MSC arm of the study received two infusions of autologous MSC at week 6 and 7 post-transplantation. Subsequently, tacrolimus was tapered and completely withdrawn at week 8. Patient sera were tested by luminex single antigen bead (SAB) assays (Immucor) for dnDSA assignment; positivity was defined as MFI > 1000. DSA were further characterized using a modified SAB assay to detect IgG1, IgG2, IgG3 and IgG4 subclasses. Complement binding was assessed with a C3d assay (Immucor). Patients and donors were HLA typed on 11 loci by next-generation sequencing. Eplet mismatches were calculated using HLAMatchmaker 2.0. Results: In total, 3 out of 27 patients (11%) in the control group and 11 out of 29 patients (38%) in the MSC group developed dnDSA, of which the majority was directed against HLA-DQ. The majority of DSA were complement binding and had different IgG subclasses. Although patients in the MSC group had an increased rate of dnDSA formation, the degree of antigen mismatch was similar to the control group (Figure 1). Cox regression showed that both MSC treatment group (HR=4.247, P=0.032) and HLA class II antibody-verified eplet mismatch (HR=1.176 per single eplet mismatch, P=0.001) were independently associated with dnDSA development. Despite the increased dnDSA formation in MSC-treated patients, incidence of rejection or graft loss was not increased and eGFR was stable at 2 years follow-up (Figure 2). Conclusion: Tacrolimus withdrawal after MSC therapy is associated with an increased rate of dnDSA formation in KTRs. However, no increased risk of rejection or inferior graft function was found, raising the question on the pathogenicity of these DSA, despite the presence of complement fixing IgG subclasses. We found that patients with a high HLA class II eplet mismatch load were less likely to tolerate tacrolimus withdrawal without developing dnDSA, which is in line with previous findings that HLA eplet mismatch load modulates tacrolimus through levels required to prevent dnDSA formation. Further research is warranted to explore HLA molecular mismatch load as a biomarker to guide personalized immunosuppression in transplantation. Jon J van Rood Transplantatiefonds.

Differential effects of oestrogen on class-switch recombination of B cells dependent on sex chromosome complement: a cross-sectional study

<h2>Abstract</h2><h3>Background</h3> Cisgender women have stronger humoral immune responses than do cisgender men, which partly explains their enhanced responses to infection and vaccination, and the female sex bias in many B-cell driven autoimmune disorders. Oestrogen has been shown to augment class-switch recombination defined as the process whereby B cells switch to IgG, IgA, and IgE isotypes. Murine work suggests that this process occurs via upregulation of the enzyme activation-induced cytidine deaminase, encoded by the <i>AICDA</i> gene, responsible for DNA mutations necessary for class-switch recombination. However, the effect of sex determinants on class-switch recombination remains understudied in humans. We aimed to investigate the effect of sex-chromosomal complement and hormonal milieu on B-cell class-switch recombination. <h3>Methods</h3> This cross-sectional study was done at University College London in the UK. We used an in vivo human model with peripheral blood samples from self-defined healthy cisgender men and women (aged 14–31 years) and healthy transgender men and women (aged 15–19 years) who received treatment with gonadotropin-releasing hormone analogue with or without additional testosterone (for transgender men) or oestradiol (for transgender women). Tanner stage during puberty was determined by a self-reported questionnaire for cisgender participants and by a physician-determined assessment before starting treatment for transgender participants. All participants were defined as Tanner stages 4–5 (after puberty) and had no history of autoimmune disorders, serious infections, or other medical conditions. None received vaccines or immune-modifying treatment 3 weeks before the donation. Peripheral blood mononuclear cell phenotyping was performed using flow cytometry and serum phenotyping was done using a LEGENDplex immunoassay (BioLegend, San Diego, USA). RNA sequencing analysis was done for gene expression. An unpaired t test, Mann-Whitney <i>U</i> tests, or a one-way ANOVA were used for statistical analysis, as appropriate. <h3>Findings</h3> Between July 10, 2015, and July 30, 2021, blood samples were obtained from 153 participants (43 cisgender men, 62 cisgender women, 25 transgender men, and 23 transgender women). RNA sequencing was carried out on samples from 22 additional participants (four cisgender males, six cisgender females, five transgender males, and seven transgender females). None were excluded. The median age of participants was 18·0 years (IQR 17·2–19·3). Cisgender men had significantly lower percentages of class-switched (IgD^– CD27⁺) B cells than did cisgender women (unpaired <i>t</i> test mean 12·00 <i>vs</i> 16·07, 95% CI –6·65 to –1·47; p=0·0024), specifically pertaining to decreased IgG⁺ B cells (mean 10·50 <i>vs</i> 17·16, –10·99 to –2·34; p=0·0094). IgG subclasses 1–3 are implicated in response to infection, whereas IgG4 appears to have immunoregulatory effects. Cisgender men showed a higher IgG4 to IgG1 serum ratio compared with cisgender women (mean 0·15 <i>vs</i> 0·06, 0·03 to 0·15; p=0·0039). Oestrogen blockade on a XX background (gonadotropin-releasing hormone analogue) in transgender men resulted in a reduced proportion of class-switched B cells compared with cisgender women (ANOVA mean 10·13 <i>vs</i> 16·07, 1·45 to 10·42; p=0·0062). The ratio of IgG1 to IgG4 subclasses was unaffected. Oestrogen (oestradiol) treatment on a XY background in transgender women showed no overall effect on class-switching (ANOVA mean 9·55 <i>vs</i> 12·00 in cisgender men, –1·25 to 6·15; p=0·26), but IgG4 to IgG1 ratios decreased significantly (Mann-Whitney <i>U</i> median 0·05 [IQR 0·03 to 0·12] <i>vs</i> 0·15 [0·08 to 0·20]; p=0·015). Preliminary analysis showed that <i>AICDA</i> expression was lower in cisgender men (median 1·66 [1·62 to 1·72] <i>vs</i> 1·85 [1·75 to 1·90] in cisgender women; p=0·038), and oestrogen blockade in transgender men resulted in a non-statistically significant reduction compared with cisgender women (median 1·91 [1·86 to 1·97] <i>vs</i> 2·05 [1·94 to 2·10]; p=0·13). No differences were observed in <i>AICDA</i> expression between transgender women treated with oestrogen and cisgender men (median 1·12 [1·09 to 1·20] <i>vs</i> 1·07 [1·04 to 1·15]; p=0·23). <h3>Interpretation</h3> Oestrogen differentially affects B-cell class-switch recombination depending on the sex chromosome complement. On the XX background, oestrogen was associated with increased class-switch recombination, whereas on the XY background this effect was not seen. Further work is needed to elucidate the molecular mechanisms underlying these observations. <h3>Funding</h3> NIHR UCLH Biomedical Research Centre and Lupus UK.