Ultrahigh frequencies of peripherally matured LGI1- and CASPR2-reactive B cells characterize the cerebrospinal fluid in autoimmune encephalitis.

Intrathecal synthesis of central nervous system (CNS)-reactive autoantibodies is observed across patients with autoimmune encephalitis (AE), who show multiple residual neurobehavioral deficits and relapses despite immunotherapies. We leveraged two common forms of AE, mediated by leucine-rich glioma inactivated-1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies, as human models to comprehensively reconstruct and profile cerebrospinal fluid (CSF) B cell receptor (BCR) characteristics. We hypothesized that the resultant observations would both inform the observed therapeutic gap and determine the contribution of intrathecal maturation to pathogenic B cell lineages. From the CSF of three patients, 381 cognate-paired IgG BCRs were isolated by cell sorting and scRNA-seq, and 166 expressed as monoclonal antibodies (mAbs). Sixty-two percent of mAbs from singleton BCRs reacted with either LGI1 or CASPR2 and, strikingly, this rose to 100% of cells in clonal groups with ≥4 members. These autoantigen-reactivities were more concentrated within antibody-secreting cells (ASCs) versus B cells (P < 0.0001), and both these cell types were more differentiated than LGI1- and CASPR2-unreactive counterparts. Despite greater differentiation, autoantigen-reactive cells had acquired few mutations intrathecally and showed minimal variation in autoantigen affinities within clonal expansions. Also, limited CSF T cell receptor clonality was observed. In contrast, a comparison of germline-encoded BCRs versus the founder intrathecal clone revealed marked gains in both affinity and mutational distances (P = 0.004 and P < 0.0001, respectively). Taken together, in patients with LGI1 and CASPR2 antibody encephalitis, our results identify CSF as a compartment with a remarkably high frequency of clonally expanded autoantigen-reactive ASCs whose BCR maturity appears dominantly acquired outside the CNS.

Rituximab abrogates aquaporin-4-specific germinal center activity in patients with neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSDs) are caused by immunoglobulin G (IgG) autoantibodies directed against the water channel aquaporin-4 (AQP4). In NMOSDs, discrete clinical relapses lead to disability and are robustly prevented by the anti-CD20 therapeutic rituximab; however, its mechanism of action in autoantibody-mediated disorders remains poorly understood. We hypothesized that AQP4-IgG production in germinal centers (GCs) was a core feature of NMOSDs and could be terminated by rituximab. To investigate this directly, deep cervical lymph node (dCLN) aspirates (n = 36) and blood (n = 406) were studied in a total of 63 NMOSD patients. Clinical relapses were associated with AQP4-IgM generation or shifts in AQP4-IgG subclasses (odds ratio = 6.0; range of 3.3 to 10.8; P < 0.0001), features consistent with GC activity. From seven dCLN aspirates of patients not administered rituximab, AQP4-IgGs were detected alongside specific intranodal synthesis of AQP4-IgG. AQP4-reactive B cells were isolated from unmutated naive and mutated memory populations in both blood and dCLNs. After rituximab administration, fewer clinical relapses (annual relapse rate of 0.79 to 0; P < 0.001) were accompanied by marked reductions in both AQP4-IgG (fourfold; P = 0.004) and intranodal B cells (430-fold; P < 0.0001) from 11 dCLNs. Our findings implicate ongoing GC activity as a rituximab-sensitive driver of AQP4 antibody production. They may explain rituximab's clinical efficacy in several autoantibody-mediated diseases and highlight the potential value of direct GC measurements across autoimmune conditions.

Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis.

Autoantibodies against the extracellular domain of the N-methyl-d-aspartate receptor (NMDAR) NR1 subunit cause a severe and common form of encephalitis. To better understand their generation, we aimed to characterize and identify human germinal centres actively participating in NMDAR-specific autoimmunization by sampling patient blood, CSF, ovarian teratoma tissue and, directly from the putative site of human CNS lymphatic drainage, cervical lymph nodes. From serum, both NR1-IgA and NR1-IgM were detected more frequently in NMDAR-antibody encephalitis patients versus controls (both P < 0.0001). Within patients, ovarian teratoma status was associated with a higher frequency of NR1-IgA positivity in serum (OR = 3.1; P < 0.0001) and CSF (OR = 3.8, P = 0.047), particularly early in disease and before ovarian teratoma resection. Consistent with this immunoglobulin class bias, ovarian teratoma samples showed intratumoral production of both NR1-IgG and NR1-IgA and, by single cell RNA sequencing, contained expanded highly-mutated IgA clones with an ovarian teratoma-restricted B cell population. Multiplex histology suggested tertiary lymphoid architectures in ovarian teratomas with dense B cell foci expressing the germinal centre marker BCL6, CD21+ follicular dendritic cells, and the NR1 subunit, alongside lymphatic vessels and high endothelial vasculature. Cultured teratoma explants and dissociated intratumoral B cells secreted NR1-IgGs in culture. Hence, ovarian teratomas showed structural and functional evidence of NR1-specific germinal centres. On exploring classical secondary lymphoid organs, B cells cultured from cervical lymph nodes of patients with NMDAR-antibody encephalitis produced NR1-IgG in 3/7 cultures, from patients with the highest serum NR1-IgG levels (P < 0.05). By contrast, NR1-IgG secretion was observed neither from cervical lymph nodes in disease controls nor in patients with adequately resected ovarian teratomas. Our multimodal evaluations provide convergent anatomical and functional evidence of NMDAR-autoantibody production from active germinal centres within both intratumoral tertiary lymphoid structures and traditional secondary lymphoid organs, the cervical lymph nodes. Furthermore, we develop a cervical lymph node sampling protocol that can be used to directly explore immune activity in health and disease at this emerging neuroimmune interface.

Divergent chemokine receptor expression and the consequence for human IgG4 B cell responses.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of TFH /TH2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20+ B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.

Activated T-Follicular Helper 2 Cells Are Associated With Disease Activity in IgG4-Related Sclerosing Cholangitis and Pancreatitis.

OBJECTIVES: Immunoglobulin G4-related sclerosing cholangitis (IgG4-SC) and autoimmune pancreatitis (AIP) are characterized by an abundance of circulating and tissue IgG4-positive plasma cells. T-follicular helper (Tfh) cells are necessary for B-cell differentiation into plasma cells. We aimed at elucidating the presence and phenotype of Tfh cells and their relationship with disease activity in IgG4-SC/AIP. METHODS: Circulating Tfh-cell subsets were characterized by multiparametric flow cytometry in IgG4-SC/AIP (n = 18), disease controls with primary sclerosing cholangitis (n = 8), and healthy controls (HCs, n = 9). Tissue Tfh cells were characterized in IgG4-SC/AIP (n = 12) and disease control (n = 10) specimens. Activated PD1+ Tfh cells were cocultured with CD27+ memory B cells to assess their capacity to support B-cell differentiation. Disease activity was assessed using the IgG4-responder index and clinical parameters. RESULTS: Activated circulating PD-1+CXCR5+ Tfh cells were expanded in active vs inactive IgG4-SC/AIP, primary sclerosing cholangitis, and HC (P < 0.01), with enhanced PD-1 expression on all Tfh-cell subsets (Tfh1, P = 0.003; Tfh2, P = 0.0006; Th17, P = 0.003). Expansion of CD27+CD38+CD19lo plasmablasts in active disease vs HC (P = 0.01) correlated with the PD-1+ Tfh2 subset (r = 0.69, P = 0.03). Increased IL-4 and IL-21 cytokine production from stimulated cells of IgG4-SC/AIP, important in IgG4 class switch and proliferation, correlated with PD-1+ Tfh2 (r = 0.89, P = 0.02) and PD-1+ Tfh17 (r = 0.83, P = 0.03) subsets. Coculture of PD1+ Tfh with CD27+ B cells induced higher IgG4 expression than with PD1- Tfh (P = 0.008). PD-1+ Tfh2 cells were strongly associated with clinical markers of disease activity: sIgG4 (r = 0.70, P = 0.002), sIgE (r = 0.66, P = 0.006), and IgG4-responder index (r = 0.60, P = 0.006). Activated CXCR5+ Tfh cells homed to lymphoid follicles in IgG4-SC/AIP tissues. CONCLUSIONS: Circulating and tissue-activated Tfh cells are expanded in IgG4-SC/AIP, correlate with disease activity, and can drive class switch and proliferation of IgG4-committed B cells. PD1+ Tfh2 cells may be a biomarker of active disease and a potential target for immunotherapy.

Distinct HLA associations of LGI1 and CASPR2-antibody diseases.

The recent biochemical distinction between antibodies against leucine-rich, glioma-inactivated-1 (LGI1), contactin-associated protein-2 (CASPR2) and intracellular epitopes of voltage-gated potassium-channels (VGKCs) demands aetiological explanations. Given established associations between human leucocyte antigen (HLA) alleles and adverse drug reactions, and our clinical observation of frequent adverse drugs reactions in patients with LGI1 antibodies, we compared HLA alleles between healthy controls (n = 5553) and 111 Caucasian patients with VGKC-complex autoantibodies. In patients with LGI1 antibodies (n = 68), HLA-DRB1*07:01 was strongly represented [odds ratio = 27.6 (95% confidence interval 12.9-72.2), P = 4.1 × 10-26]. In contrast, patients with CASPR2 antibodies (n = 31) showed over-representation of HLA-DRB1*11:01 [odds ratio = 9.4 (95% confidence interval 4.6-19.3), P = 5.7 × 10-6]. Other allelic associations for patients with LGI1 antibodies reflected linkage, and significant haplotypic associations included HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02, by comparison to DRB1*11:01-DQA1*05:01-DQB1*03:01 in CASPR2-antibody patients. Conditional analysis in LGI1-antibody patients resolved further independent class I and II associations. By comparison, patients with both LGI1 and CASPR2 antibodies (n = 3) carried yet another complement of HLA variants, and patients with intracellular VGKC antibodies (n = 9) lacked significant HLA associations. Within LGI1- or CASPR2-antibody patients, HLA associations did not correlate with clinical features. In silico predictions identified unique CASPR2- and LGI1-derived peptides potentially presented by the respective over-represented HLA molecules. These highly significant HLA associations dichotomize the underlying immunology in patients with LGI1 or CASPR2 antibodies, and inform T cell specificities and cellular interactions at disease initiation.10.1093/brain/awy109_video1awy109media15796480660001.

Condition-dependent generation of aquaporin-4 antibodies from circulating B cells in neuromyelitis optica.

Autoantibodies to aquaporin-4 (AQP4) are pathogenic in neuromyelitis optica spectrum disorder (NMOSD). However, it is not known which B cells are the major contributors to circulating AQP4 antibodies nor which conditions promote their generation. Our experiments showed CD19+CD27++CD38++ circulating ex vivo antibody-secreting cells did not produce AQP4 antibodies under several culture conditions. To question whether other cells in circulation were capable of AQP4 antibody production, B cells were differentiated into antibody-secreting cells in vitro. Unfractionated peripheral blood mononuclear cells, isolated from 12 patients with NMOSD and a wide range of serum AQP4 antibody levels (91-26 610 units), were cultured with factors that mimicked established associations of NMOSD including T cell help, concurrent infections and cytokines reported to be elevated in NMOSD. Overall, the in vitro generation of CD19+CD27++CD38++ cells across several culture conditions correlated closely with the total IgG secreted (P < 0.0001, r = 0.71), but not the amount of AQP4 antibody. AQP4 antibody production was enhanced by CD40-ligand (P = 0.005), and by interleukin-2 plus toll-like receptor stimulation versus interleukin-21-predominant conditions (P < 0.0001), and did not require antigen. Across NMOSD patients, this in vitro generation of AQP4 antibodies correlated well with serum AQP4 antibody levels (P = 0.0023, r = 0.81). To understand how early within B cell lineages this AQP4 specificity was generated, purified B cell subsets were activated under these optimized conditions. Naïve pre-germinal centre B cells (CD19+CD27-IgD+) differentiated to secrete AQP4 antibodies as frequently as post-germinal centre cells (CD19+CD27+). Taken together, these human cell-culture experiments demonstrate that preformed B cells, rather than ex vivo circulating antibody-secreting cells, possess AQP4 reactivity. Their differentiation and AQP4 antibody secretion is preferentially driven by select cytokines and these cells may make the dominant contribution to serum AQP4 antibodies. Furthermore, as AQP4-specific B cells can derive from likely autoreactive naïve populations an early, pre-germinal centre loss of immunological tolerance appears present in some patients with NMOSD. This study has implications for understanding mechanisms of disease perpetuation and for rational choice of immunotherapies in NMOSD. Furthermore, the in vitro model presents an opportunity to apply condition-specific approaches to patients with NMOSD and may be a paradigm to study other antibody-mediated diseases.awy010media15732448284001.

N-methyl-D-aspartate receptor antibody production from germinal center reactions: Therapeutic implications.

INTRODUCTION: N-methyl-D-aspartate receptor (NMDAR) antibody encephalitis is mediated by immunoglobulin G (IgG) autoantibodies directed against the NR1 subunit of the NMDAR. Around 20% of patients have an underlying ovarian teratoma, and the condition responds to early immunotherapies and ovarian teratoma removal. However, despite clear therapeutic relevance, mechanisms of NR1-IgG production and the contribution of germinal center B cells to NR1-IgG levels are unknown. METHODS: Clinical data and longitudinal paired serum NR1-reactive IgM and IgG levels from 10 patients with NMDAR-antibody encephalitis were determined. Peripheral blood mononuclear cells from these 10 patients, and two available ovarian teratomas, were stimulated with combinations of immune factors and tested for secretion of total IgG and NR1-specific antibodies. RESULTS: In addition to disease-defining NR1-IgG, serum NR1-IgM was found in 6 of 10 patients. NR1-IgM levels were typically highest around disease onset and detected for several months into the disease course. Moreover, circulating patient B cells were differentiated into CD19+ CD27++ CD38++ antibody-secreting cells in vitro and, from 90% of patients, secreted NR1-IgM and NR1-IgG. Secreted levels of NR1-IgG correlated with serum NR1-IgG (p < 0.0001), and this was observed across the varying disease durations, suggestive of an ongoing process. Furthermore, ovarian teratoma tissue contained infiltrating lymphocytes which produced NR1-IgG in culture. INTERPRETATION: Serum NR1-IgM and NR1-IgG, alongside the consistent production of NR1-IgG from circulating B cells and from ovarian teratomas suggest that ongoing germinal center reactions may account for the peripheral cell populations which secrete NR1-IgG. Cells participating in germinal center reactions might be a therapeutic target for the treatment of NMDAR-antibody encephalitis. Ann Neurol 2018;83:553-561.

The importance of early immunotherapy in patients with faciobrachial dystonic seizures.

Faciobrachial dystonic seizures and limbic encephalitis closely associate with antibodies to leucine-rich glioma-inactivated 1 (LGI1). Here, we describe 103 consecutive patients with faciobrachial dystonic seizures and LGI1 antibodies to understand clinical, therapeutic and serological differences between those with and without cognitive impairment, and to determine whether cessation of faciobrachial dystonic seizures can prevent cognitive impairment. The 22/103 patients without cognitive impairment typically had normal brain MRI, EEGs and serum sodium levels (P < 0.0001). Overall, cessation of faciobrachial dystonic seizures with antiepileptic drugs alone occurred in only 9/89 (10%) patients. By contrast, 51% showed cessation of faciobrachial dystonic seizures 30 days after addition of immunotherapy (P < 0.0001), with earlier cessation in cognitively normal patients (P = 0.038). Indeed, expedited immunotherapy (P = 0.031) and normal cognition (P = 0.0014) also predicted reduced disability at 24 months. Furthermore, of 80 patients with faciobrachial dystonic seizures as their initial feature, 56% developed cognitive impairment after 90 days of active faciobrachial dystonic seizures. Whereas only one patient developed cognitive impairment after cessation of faciobrachial dystonic seizures (P < 0.0001). All patients had IgG4-LGI1 antibodies, but those with cognitive impairment had higher proportions of complement-fixing IgG1 antibodies (P = 0.03). Both subclasses caused LGI1-ADAM22 complex internalization, a potential non-inflammatory epileptogenic mechanism. In summary, faciobrachial dystonic seizures show striking time-sensitive responses to immunotherapy, and their cessation can prevent the development of cognitive impairment.awx323media15681705685001.

No evidence to support a role for Helicobacter pylori infection and plasminogen binding protein in autoimmune pancreatitis and IgG4-related disease in a UK cohort.

BACKGROUND AND OBJECTIVES: Helicobacter pylori (H.pylori) plasminogen binding protein (PBP) has been proposed as an antigen triggering autoimmune pancreatitis (AIP), the pancreatic manifestation of IgG4-related disease (IgG4-RD). We investigated exposure to H. pylori infection, cytokine response and immunological memory to H. pylori PBP in a prospective IgG4-RD cohort in the UK. METHODS: Clinical and endoscopic evidence of peptic ulceration, serological H. pylori exposure and serum IgG4 levels were obtained in 55 IgG4-RD patients and 52 disease controls (DC) with autoimmune or inflammatory conditions with an elevated serum IgG4. Gastric and duodenal tissues were assessed for H. pylori and immunostained for IgG4. B and T cell ELISpot and cytokine luminex assays were used to detect immune responses to H. pylori PBP. RESULTS: 85% of IgG4-RD patients had pancreatic and/or biliary disease, 89% had extra-pancreatic manifestations, and 84% had an increased serum IgG4. Clinical dyspepsia (35.2%), gastritis (58%), peptic ulceration (7.4%) and H. pylori colonisation (24%) in IgG4-RD was similar to DC. In IgG4-RD, gastric tissue contained a chronic inflammatory infiltrate with a low IgG4+ plasma-cell count (<10/HPF; range 1-4/HPF), and duodenal specimens had an increased IgG4 count (>10/HPF; range 7-54) compared with DC (p < 0.01). Th1 and Th2 cytokine response and immunological B-cell memory to H. pylori PBP did not differ between IgG4-RD and DC. CONCLUSIONS: In a prospective UK cohort, the prevalence of gastric ulceration, exposure to H. pylori, cytokine response and immunological memory to H. pylori PBP did not differ in IgG4-RD patients compared with DC. This study does not support a role for H. pylori PBP as a microbial antigen in IgG4-RD. KEYWORDS FOR ABSTRACT: Peptic ulceration, Antigens, B cells, T cells, Interleukins, Helicobacter pylori.

Increases in IgE, Eosinophils, and Mast Cells Can be Used in Diagnosis and to Predict Relapse of IgG4-Related Disease.

BACKGROUND & AIMS: IgG subclass 4-related disease (IgG4-RD) is characterized by increased serum levels of IgG4 and infiltration of biliary, pancreatic, and other tissues by IgG4-positive plasma cells. We assessed the prevalence of allergy and/or atopy, serum, and tissue IgE antibodies, and blood and tissue eosinophils in patients with IgG4-RD. We investigated the association between serum IgE and diagnosis and relapse of this disease. METHODS: We performed a prospective study of 48 patients with IgG4-RD, 42 patients with an increased serum level of IgG4 with other inflammatory and autoimmune conditions (disease control subjects), and 51 healthy individuals (healthy control subjects) recruited from Oxford, United Kingdom from March 2010 through March 2014, and followed for a median of 41 months (range, 3-73 months). Serum levels of immunoglobulin were measured at diagnosis, during steroid treatment, and at disease relapse for patients with IgG4-RD; levels at diagnosis were compared with baseline levels of control subjects. Allergen-specific IgEs were measured using the IgE ImmunoCAP. Levels and distribution of IgG4 and IgE antibodies in lymphoid, biliary, and pancreatic tissues from patients with IgG4-RD and disease control subjects were measured by immunohistochemistry. We analyzed data using the Spearman rank correlation and receiver operating characteristic curves. RESULTS: Serum levels of IgG4 increased to 1.4 g/L or more, and IgE increased to 125 kIU/L or more, in 81% and 54% of patients with IgG4-RD, respectively, compared with 6% and 16% of healthy control subjects (P < .0001). Peripheral blood eosinophilia was detected in 38% of patients with IgG4-RD versus 9% of healthy control subjects (P = .004). Of patients with IgG4-RD, 63% had a history of allergy and 40% had a history of atopy with an IgE-specific response; these values were 60% and 53% in patients with increased serum levels of IgE (P < .05). Level of IgE at diagnosis >480 kIU/L distinguished patients with IgG4-RD from disease control subjects with 86% specificity, 36% sensitivity, and a likelihood ratio of 3.2. Level of IgE at diagnosis >380 kIU/L identified patients with disease relapse with 88% specificity, 64% sensitivity, and a likelihood ratio of 5.4. IgE-positive mast cells and eosinophilia were observed in lymphoid, biliary, and pancreatic tissue samples from 50% and 86% of patients with IgG4-RD, respectively. CONCLUSIONS: In a prospective study, we associated IgG4-RD with allergy, atopy, eosinophilia, increased serum levels of IgE, and IgE-positive mast cells in lymphoid, biliary, and pancreatic tissue. An IgE-mediated allergic response therefore seems to develop in most patients with IgG4-RD; levels of IgE might be used in diagnosis and predicting relapse.

Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies.

OBJECTIVES: Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined. METHODS: Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin (125I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of 125I-αDTX and 125I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2. RESULTS: VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated 125I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical-serological correlations and a limited immunotherapy response. CONCLUSIONS: Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against non-mammalian αDTX. These antibodies should no longer be classified as neuronal-surface antibodies. They consequently lack pathogenic potential and do not in themselves support the use of immunotherapies.

Elevated Serum IgG4 Levels in Diagnosis, Treatment Response, Organ Involvement, and Relapse in a Prospective IgG4-Related Disease UK Cohort.

OBJECTIVES: Elevated serum immunoglobulin G4 (IgG4) levels have been associated with autoimmune pancreatitis and IgG4-related disease (IgG4-RD) for over a decade. However, an elevated serum IgG4 is not specific for the disease. There have been inconsistent reports of its use in diagnosis, as a marker of disease relapse, and its relationship to organ involvement in retrospective cohorts. The aims of this study were to ascertain conditions that are associated with an elevated serum IgG4 and to investigate the role of IgG4 in diagnosis, relapse, and organ involvement in a prospective cohort of patients with IgG4-RD. METHODS: We evaluated serum IgG4 measurements in the Oxford Immunology Laboratory over 6 years. Patients in whom serum IgG4 was requested to differentiate IgG4-RD from other diseases were recruited into a longitudinal follow-up study to determine final diagnosis. In a prospective cohort of IgG4-RD patients, organ involvement, response to therapy, and disease relapse were determined. RESULTS: Two thousand and sixty-seven samples from 1,510 patients had serum IgG4 measured. Of these, IgG4 was elevated (≥1.4 g l(-1)) in 243 (16.1%) patients. The main indication (85.6%) was to distinguish between IgG4-RD and non-IgG4-RD conditions. Only 5.1% of patients who had serum IgG4 measured for this purpose had a final diagnosis of IgG4-RD. Of those with an elevated serum IgG4, 22.4% met IgG4-RD diagnostic criteria. Serum IgG4 was elevated in 48 (82.8%) of IgG4-RD patients. An IgG4 cutoff of 1.4 g l(-1) gave a sensitivity of 82.8% and specificity of 84.7% to diagnose IgG4-RD. Increasing this to 2.8 g l(-1) increased specificity to 96.2% and negative predictive value to 97.7%, with a lower sensitivity of 56.9% and positive predictive value of 44.5%. Serum IgG4 levels fell with corticosteroid therapy, but this was not disease-specific. A serum IgG4 of ≥2.8 g l(-1) at diagnosis was associated with multi-organ involvement and risk of relapse. CONCLUSIONS: Serum IgG4 levels are elevated in multiple non-IgG4-RD inflammatory and malignant conditions, with less than one-quarter of those with an elevated IgG4 meeting IgG4-RD diagnostic criteria. A serum IgG4 of ≥2.8 g l(-1) is useful in distinguishing between IgG4-RD and non-IgG4-RD diagnoses, predicting multiple-organ involvement and risk of relapse in IgG4-RD.

Increased IgG4 responses to multiple food and animal antigens indicate a polyclonal expansion and differentiation of pre-existing B cells in IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition, characterised by an elevated serum IgG4 concentration and abundant IgG4-positive plasma cells in the involved organs. An important question is whether the elevated IgG4 response is causal or a reflection of immune-regulatory mechanisms of the disease. OBJECTIVES: To investigate if the IgG4 response in IgG4-RD represents a generalised polyclonal amplification by examining the response to common environmental antigens. METHODS: Serum from 24 patients with IgG4-RD (14 treatment-naive, 10 treatment-experienced), 9 patients with primary sclerosing cholangitis and an elevated serum IgG4 (PSC-high IgG4), and 18 healthy controls were tested against egg white and yolk, milk, banana, cat, peanut, rice and wheat antigens by radioimmunoassay. RESULTS: We demonstrated an elevated polyclonal IgG4 response to multiple antigens in patients with IgG4-RD and in PSC-high IgG4, compared with healthy controls. There was a strong correlation between serum IgG4 and antigen-specific responses. Responses to antigens were higher in treatment-naive compared with treatment-experienced patients with IgG4-RD. Serum electrophoresis and immunofixation demonstrated polyclonality. CONCLUSIONS: This is the first study to show enhanced levels of polyclonal IgG4 to multiple antigens in IgG4-RD. This supports that elevated IgG4 levels reflect an aberrant immunological regulation of the overall IgG4 response, but does not exclude that causality of disease could be antigen-driven.

Human B cells promote T-cell plasticity to optimize antibody response by inducing coexpression of T(H)1/T(FH) signatures.

BACKGROUND: B cells mediate humoral immunity against pathogens but also direct CD4(+) T-cell responses. Recent plasticity studies in mice have challenged the concept of strict fate commitment during CD4(+) T-cell differentiation into distinct subsets. OBJECTIVE: We sought to elucidate the contribution of human antigen-primed B cells in CD4(+) T-cell responses that support humoral immunity. METHODS: CD4(+) T-cell differentiation by primary human B cells was investigated in in vitro cocultures by using tetanus toxoid and Salmonella species as antigen models. T-cell differentiation was assessed by using intracellular cytokines and subset-specific transcription factors and markers. IgM and IgG formation was analyzed by means of ELISA. RESULTS: Human B cells, but not dendritic cells, induce prominent and stable coexpression of TH1 and follicular helper T (TFH) cell characteristics during priming and on antigen recall. TH1/TFH cells coexpress the TH1 and TFH effector cytokines IFN-γ and IL-21 and the TFH marker CXCR5, demonstrating that the coexpressed TH1 and TFH subset-specifying transcription factors T-box transcription factor (T-bet) and B cell lymphoma 6 are both functionally active. B cell-derived IL-6 and IL-12 controlled respective expression of IL-21 and IFN-γ, with IL-21 being key for humoral immunity. CONCLUSION: Human B cells exploit CD4(+) T-cell plasticity to create flexibility in the effector T-cell response. Induction of a T-cell subset coexpressing IL-21 and IFN-γ might combine IL-21-mediated T-cell aid for antibody production while maintaining TH1 cytokine expression to support other cellular immune defenses.