Incidence, prevalence, and co-occurrence of autoimmune disorders over time and by age, sex, and socioeconomic status: a population-based cohort study of 22 million individuals in the UK.

BACKGROUND: A rise in the incidence of some autoimmune disorders has been described. However, contemporary estimates of the overall incidence of autoimmune diseases and trends over time are scarce and inconsistent. We aimed to investigate the incidence and prevalence of 19 of the most common autoimmune diseases in the UK, assess trends over time, and by sex, age, socioeconomic status, season, and region, and we examine rates of co-occurrence among autoimmune diseases. METHODS: In this UK population-based study, we used linked primary and secondary electronic health records from the Clinical Practice Research Datalink (CPRD), a cohort that is representative of the UK population in terms of age and sex and ethnicity. Eligible participants were men and women (no age restriction) with acceptable records, approved for Hospital Episodes Statistics and Office of National Statistics linkage, and registered with their general practice for at least 12 months during the study period. We calculated age and sex standardised incidence and prevalence of 19 autoimmune disorders from 2000 to 2019 and used negative binomial regression models to investigate temporal trends and variation by age, sex, socioeconomic status, season of onset, and geographical region in England. To characterise co-occurrence of autoimmune diseases, we calculated incidence rate ratios (IRRs), comparing incidence rates of comorbid autoimmune disease among individuals with a first (index) autoimmune disease with incidence rates in the general population, using negative binomial regression models, adjusted for age and sex. FINDINGS: Among the 22 009 375 individuals included in the study, 978 872 had a new diagnosis of at least one autoimmune disease between Jan 1, 2000, and June 30, 2019 (mean age 54·0 years [SD 21·4]). 625 879 (63·9%) of these diagnosed individuals were female and 352 993 (36·1%) were male. Over the study period, age and sex standardised incidence rates of any autoimmune diseases increased (IRR 2017-19 vs 2000-02 1·04 [95% CI 1·00-1·09]). The largest increases were seen in coeliac disease (2·19 [2·05-2·35]), Sjogren's syndrome (2·09 [1·84-2·37]), and Graves' disease (2·07 [1·92-2·22]); pernicious anaemia (0·79 [0·72-0·86]) and Hashimoto's thyroiditis (0·81 [0·75-0·86]) significantly decreased in incidence. Together, the 19 autoimmune disorders examined affected 10·2% of the population over the study period (1 912 200 [13·1%] women and 668 264 [7·4%] men). A socioeconomic gradient was evident across several diseases, including pernicious anaemia (most vs least deprived area IRR 1·72 [1·64-1·81]), rheumatoid arthritis (1·52 [1·45-1·59]), Graves' disease (1·36 [1·30-1·43]), and systemic lupus erythematosus (1·35 [1·25-1·46]). Seasonal variations were observed for childhood-onset type 1 diabetes (more commonly diagnosed in winter) and vitiligo (more commonly diagnosed in summer), and regional variations were observed for a range of conditions. Autoimmune disorders were commonly associated with each other, particularly Sjögren's syndrome, systemic lupus erythematosus, and systemic sclerosis. Individuals with childhood-onset type 1 diabetes also had significantly higher rates of Addison's disease (IRR 26·5 [95% CI 17·3-40·7]), coeliac disease (28·4 [25·2-32·0]), and thyroid disease (Hashimoto's thyroiditis 13·3 [11·8-14·9] and Graves' disease 6·7 [5·1-8·5]), and multiple sclerosis had a particularly low rate of co-occurrence with other autoimmune diseases. INTERPRETATION: Autoimmune diseases affect approximately one in ten individuals, and their burden continues to increase over time at varying rates across individual diseases. The socioeconomic, seasonal, and regional disparities observed among several autoimmune disorders in our study suggest environmental factors in disease pathogenesis. The inter-relations between autoimmune diseases are commensurate with shared pathogenetic mechanisms or predisposing factors, particularly among connective tissue diseases and among endocrine diseases. FUNDING: Research Foundation Flanders.

In vitro B cell experiments explore the role of CD24, CD38, and energy metabolism in ME/CFS.

Introduction: Disturbances of energy metabolism contribute to the clinical manifestations of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Previously, we found that B cells from ME/CFS patients have an increased expression of CD24, a modulator of many cellular functions including those of cell stress. The relative ability of B cells from ME/CFS patients and healthy controls (HC) to respond to rapid changes in energy demand was compared. Methods: CD24, the ectonucleotidases CD39 and CD73, the NAD-degrading enzyme CD38, and mitochondrial mass (MM) were measured following cross-linking of the B cell receptor and costimulation with either T-cell-dependent or Toll-like-receptor-9-dependent agonists. The levels of metabolites consumed/produced were measured using 1H-NMR spectroscopy and analyzed in relation to cell growth and immunophenotype. Results: Proliferating B cells from patients with ME/CFS showed a lower mitochondrial mass and a significantly increased usage of essential amino acids compared with those from HC, with a significantly delayed loss of CD24 and an increased expression of CD38 following stimulation. Discussion: The immunophenotype results suggested the triggering of a stress response in ME/CFS B cells associated with the increased usage of additional substrates to maintain necessary ATP levels. Disturbances in energy metabolism in ME/CFS B cells were thus confirmed in a dynamic in vitro model, providing the basis for further mechanistic investigations.

Autoimmune diseases and cardiovascular risk: a population-based study on 19 autoimmune diseases and 12 cardiovascular diseases in 22 million individuals in the UK.

BACKGROUND: Some autoimmune diseases are associated with an increased risk of cardiovascular disease. We aimed to determine whether or not this is true, and to what extent, for a broad range of autoimmune conditions. METHODS: In this population-based study, we used linked primary and secondary care records from the Clinical Practice Research Datalink (CPRD), GOLD and Aurum datasets, to assemble a cohort of individuals across the UK who were newly diagnosed with any of 19 autoimmune diseases between Jan 1, 2000, and Dec 31, 2017, younger than 80 years at diagnosis, and free of cardiovascular diseases up to 12 months after diagnosis. We also assembled a matched cohort with up to five individuals matched on age, sex, socioeconomic status, region, and calendar year, who were free of autoimmune disease and free of cardiovascular diseases up to 12 months after study entry. Both cohorts were followed up until June 30, 2019. We investigated the incidence of 12 cardiovascular outcomes and used Cox proportional hazards models to examine differences in patients with and without autoimmune diseases. FINDINGS: Of 22 009 375 individuals identified from the CPRD databases, we identified 446 449 eligible individuals with autoimmune diseases and 2 102 830 matched controls. In the autoimmune cohort, mean age at diagnosis was 46·2 years (SD 19·8), and 271 410 (60·8%) were women and 175 039 (39·2%) were men. 68 413 (15·3%) people with and 231 410 (11·0%) without autoimmune diseases developed incident cardiovascular disease during a median of 6·2 years (IQR 2·7-10·8) of follow-up. The incidence rate of cardiovascular disease was 23·3 events per 1000 patient-years among patients with autoimmune disease and 15·0 events per 1000 patient-years among those without an autoimmune disease (hazard ratio [HR] 1·56 [95% CI 1·52-1·59]). An increased risk of cardiovascular disease with autoimmune disease was seen for every individual cardiovascular disease and increased progressively with the number of autoimmune diseases present (one disease: HR 1·41 [95% CI 1·37-1·45]; two diseases: 2·63 [2·49-2·78]); three or more diseases: 3·79 [3·36-4·27]), and in younger age groups (age <45 years: 2·33 [2·16-2·51]; 55-64 years: 1·76 [1·67-1·85]; ≥75 years: 1·30 [1·24-1·36]). Among autoimmune diseases, systemic sclerosis (3·59 [2·81-4·59]), Addison's disease (2·83 [1·96-4·09]), systemic lupus erythematosus (2·82 [2·38-3·33]), and type 1 diabetes (2·36 [2·21-2·52]) had the highest overall cardiovascular risk. INTERPRETATION: These findings warrant targeted cardiovascular prevention measures, in particular in younger patients with autoimmune diseases, and further research into pathophysiological mechanisms underlying these complications. FUNDING: Horizon 2020 Marie Sklodowska-Curie Actions and European Society of Cardiology.

Alterations in B- and circulating T-follicular helper cell subsets in immune thrombotic thrombocytopenic purpura.

T follicular helper (Tfh) cells regulate development of antigen-specific B-cell immunity. We prospectively investigated B-cell and circulating Tfh (cTfh) cell subsets in 45 patients with immune thrombotic thrombocytopenic purpura (iTTP) at presentation and longitudinally after rituximab (RTX). B-cell phenotype was altered at acute iTTP presentation with decreased transitional cells and post-germinal center (post-GC) memory B cells and increased plasmablasts compared with healthy controls. A higher percentage of plasmablasts was associated with higher anti-ADAMTS13 IgG and lower ADAMTS13 antigen levels. In asymptomatic patients with ADAMTS13 relapse, there were increased naïve B cells and a global decrease in memory subsets, with a trend to increased plasmablasts. Total circulating Tfh (CD4+CXCR5+) and PD1+ Tfh cells were decreased at iTTP presentation. CD80 expression was decreased on IgD+ memory cells and double-negative memory cells in acute iTTP. At repopulation after B-cell depletion in de novo iTTP, post-GC and double-negative memory B cells were reduced compared with pre-RTX. RTX did not cause alteration in cTfh cell frequency. The subsequent kinetics of naïve, transitional, memory B cells and plasmablasts did not differ significantly between patients who went on to relapse vs those who remained in remission. In summary, acute iTTP is characterized by dysregulation of B- and cTfh cell homeostasis with depletion of post-GC memory cells and cTfh cells and increased plasmablasts. Changes in CD80 expression on B cells further suggest altered interactions with T cells.

Long-term persistence of rituximab in patients with rheumatoid arthritis: an evaluation of the UCL cohort from 1998 to 2020.

OBJECTIVES: B cell depletion therapy based on rituximab in patients with RA was pioneered at University College London Hospitals/University College London in 1998. The objective of this study was to evaluate long-term persistence of rituximab and identify factors associated with discontinuation of treatment. METHODS: Retrospective review of medical records from all rituximab-treated RA patients followed up in a dedicated clinic (1998-2020). Data collected included gender, disease duration, previous DMARDs, autoantibody status, age and concomitant therapy at first cycle, length of follow-up, and number of cycles. Drug survival and factors associated with drug discontinuation were analysed using Kaplan-Meier survival curves, log-rank test and Cox regression analysis. RESULTS: A total of 404 patients were included. Median disease duration and age at time of first rituximab cycle were 10 and 57 years, respectively. Median total follow-up was 55 months and median number of cycles five. 93.1% of patients were seropositive. Overall, 31.2% of patients stopped rituximab, with the largest reason for discontinuing being primary inefficacy (42.1%). Comparison of Kaplan-Meier curves showed that rituximab drug survival was lower in seronegative patients and in patients who had previously failed at least one biologic DMARD (bDMARD). Cox regression analysis revealed that rituximab discontinuation was associated with a greater number of previous bDMARDs. CONCLUSION: Many patients with RA achieve good control of their disease with repeated cycles of rituximab treatment. The most common reasons for treatment discontinuation were either primary or secondary inefficacy. Patients who were seronegative and who had previously failed other bDMARDs were more at risk of drug discontinuation.

Disparity in peripheral and renal B-cell depletion with rituximab in systemic lupus erythematosus: an opportunity for obinutuzumab?

OBJECTIVES: To investigate key factors that may contribute to the variability of rituximab-mediated peripheral and renal B cell depletion (BCD) in SLE. METHODS: We analysed: (i) CD19+ B cell counts in patients with SLE before and 1, 2, 3 and 6 months after treatment with rituximab, comparing them with RA patients; (ii) the presence of B cells in renal biopsies after rituximab therapy; (iii) whether the duration of BCD correlated with patient demographics and B cell expression of CD20 and FcγRIIb; and (iv) the effect of B cell activation factor (BAFF) on the efficiency of rituximab and obinutuzumab at inducing BCD in whole blood assays, in vitro. RESULTS: In SLE (n = 71), the duration of BCD was shorter compared with RA (n = 27). B cells were detectable in renal biopsy samples (n = 6) after treatment with rituximab in all patients with poor response while peripheral blood B cells remained low or undetectable in the same patients. There were no significant relationships between peripheral BCD and patient age, disease duration, serum C3 levels or the level of expression of B cell surface proteins CD20 and FcγRIIb. Obinutuzumab was more efficient than rituximab at inducing BCD in whole blood assays, regardless of excess BAFF. CONCLUSIONS: BCD in SLE is less efficient than in RA. Renal B cell presence following rituximab treatment was associated with poor outcomes. No significant relationships between any measured B cell related, clinical or laboratory parameters and the efficiency of BCD by rituximab was found. Obinutuzumab was superior to rituximab at inducing BCD.

Serum markers of B-cell activation in pregnant women with atopic asthma.

PROBLEM: As maternal atopy represents a risk factor for the development of atopy in offspring, we aimed to assess how pregnancy affects B-cell activation markers in women with atopic asthma and whether they correlate with risk manifestations for allergy in newborns from mothers with atopic asthma. METHOD OF STUDY: Pregnant women with atopic asthma (AP) in the third trimester of gestation and nonpregnant women with atopic asthma (ANP) were prospectively recruited and compared to respective healthy counterparts (HP and HNP). All pregnant women were also assessed during the postpartum period until 6 weeks after delivery (HP/PP and AP/PP). Newborns were clinically evaluated at the age of 6 months. Peripheral blood samples were taken from each woman at each time point. Soluble CD23 (sCD23), B-cell activating factor (BAFF), IgA, IgG, IgM, kappa (κ), and lambda (λ) free light chains (FLC) were quantified in serum samples. RESULTS: The AP group presented increased sCD23 (p < 0.05) and BAFF (p < 0.001) levels compared to the ANP group and even higher levels of sCD23 during the postpartum period (p < 0.001). Moreover, the cutoffs of 6.74 g/L for IgG (sensitivity 90.9%, specificity 77.8%) and of 11.30 mg/L for λ FLC (sensitivity 81.8%, specificity 88.9%) in the AP group were predictive factors for the manifestation of allergy in their offspring. CONCLUSIONS: After delivery, the dynamics of sCD23 and BAFF changed significantly in the AP group. Furthermore, we found novel predictive factors for allergy manifestations in the children of these women, with potential clinical application.

Adverse infusion reactions to rituximab in systemic lupus erythematosus: a retrospective analysis.

BACKGROUND: To undertake a retrospective review of patients with SLE who had received Rituximab in order to determine the rates and associated patient characteristics of clinically significant adverse infusion reactions. METHODS: A descriptive analysis was undertaken of each infusion reaction, which was then assessed using the clinical information available to hypothesise on the possible underlying mechanism(s). RESULTS: Records of 136 SLE patients previously treated with 481 individual infusions of Rituximab were reviewed. A total of 22 patients (17.6%) had 28 (5.8% of total infusions) documented clinically significant adverse infusion reactions. Average age at first Rituximab infusion in patients without a reaction was 37 years (range 16-73) compared with 30 years (range 18-56) in those with a reaction. A high proportion of men (18.2%) experienced an infusion reaction. Severity and type of reaction varied. 6.4% of those who had a reaction were not retreated. CONCLUSIONS: While Rituximab remains an important tool in the treatment of SLE it is important to be aware that rates of infusion reactions may be more significant in SLE than in other diseases. A prospective study is required to better characterise the reactions.

Serum markers of B-cell activation in pregnancy during late gestation, delivery, and the postpartum period.

PROBLEM: B cells are vital for the normal evolution of pregnancy due to their humoral and possible regulatory activities. Our group and others have documented that circulating B-cell subsets undergo changes from normal late pregnancy to the postpartum period. However, the underlying mechanisms are poorly understood. Therefore, this study examined the degree of B-cell activation in normal pregnancy by analyzing the levels of serum markers in healthy pregnant women during the third trimester of pregnancy, the day of delivery, and the postpartum period. METHOD OF STUDY: A prospective study including pregnant and non-pregnant women attending routine care was undertaken at a hospital clinic. Sociodemographic and clinical data were collected, along with peripheral blood samples. The serum levels of soluble CD23 (sCD23), B-cell-activating factor (BAFF), kappa (κ) and lambda (λ) free light chains (FLC), IgA, IgG, and IgM were quantified. RESULTS: Our study included 43 third trimester pregnant and 35 non-pregnant women. In the pregnant women, the median levels of sCD23, BAFF, IgG, and κ FLC were significantly higher during the postpartum period than during the third trimester of pregnancy. Compared to the non-pregnant women, the third trimester pregnant women had higher median BAFF levels and lower sCD23, IgA, IgG, and FLC levels. CONCLUSION: Changes in serum markers of B-cell kinetics that occur during pregnancy often persist into the postpartum period and affect the secretion of immunoglobulins from different classes. Further studies are needed to clarify the biological significance of our observations.

CD24 Expression and B Cell Maturation Shows a Novel Link With Energy Metabolism: Potential Implications for Patients With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

CD24 expression on pro-B cells plays a role in B cell selection and development in the bone marrow. We previously detected higher CD24 expression and frequency within IgD+ naïve and memory B cells in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) compared with age-matched healthy controls (HC). Here, we investigated the relationship between CD24 expression and B cell maturation. In vitro stimulation of isolated B cells in response to conventional agonists were used to follow the dynamics of CD24 positivity during proliferation and differentiation (or maturation). The relationship between CD24 expression to cycles of proliferation and metabolism in purified B cells from HC was also investigated using phospho-flow (phosphorylation of AMPK-pAMPK), 1proton nuclear magnetic resonance and Mitotracker Far-red (Mitochondrial mass-MM). In vitro, in the absence of stimulation, there was an increased percentage of CD24+ viable B cells in ME/CFS patients compared to HC (p < 0.05) following 5 days culture. Following stimulation with B cell agonists, percentage of CD24+B cells in both naïve and memory B cell populations decreased. P < 0.01). There was a negative relationship between percentage of CD24+B cells with MM (R2 = 0.76; p < 0.01), which was subsequently lost over sequential cycles of proliferation. There was a significant correlation between CD24 expression on B cells and the usage of glucose and secretion of lactate in vitro. Short term ligation of the B cell receptor with anti-IgM antibody significantly reduced the viability of CD24+ memory B cells compared to those cross-linked by anti-IgD or anti-IgG antibody. A clear difference was found between naïve and memory B cells with respect to CD24 expression and pAMPK, most notably a strong positive association in IgD+IgM+ memory B cells. In vitro findings confirmed dysregulation of CD24-expressing B cells from ME/CFS patients previously suggested by immunophenotype studies of B cells from peripheral blood. CD24-negative B cells underwent productive proliferation whereas CD24+ B cells were either unresponsive or susceptible to cell death upon BCR-engagement alone. We suggest that CD24 expression may reflect variations in energy metabolism on different B cell subsets.

Detection of Experimental and Clinical Immune Complexes by Measuring SHIP-1 Recruitment to the Inhibitory FcγRIIB.

Fc γ receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcγRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain-containing inositol phosphatase 1 to the ITIM of this receptor. Such recruitment required prior cross-linking of an ITAM-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signaling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, rituximab-anti-idiotype complexes, and anti-trinitrophenol-trinitrophenol complexes in a sensitive manner (≤1 µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus and rheumatoid arthritis were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

Obinutuzumab induces superior B-cell cytotoxicity to rituximab in rheumatoid arthritis and systemic lupus erythematosus patient samples.

Objective: A proportion of RA and SLE patients treated with standard doses of rituximab (RTX) display inefficient B cell deletion and poor clinical responses that can be augmented by delivering higher doses, indicating that standard-dose RTX is a sub-optimal therapy in these patients. This study aimed to investigate whether better responses could be achieved with mechanistically different anti-CD20 mAbs. Methods: We compared RTX with obinutuzumab (OBZ), a new-generation, glycoengineered type II anti-CD20 mAb, in a series of in vitro assays measuring B cell cytotoxicity in RA and SLE patient samples. Results: We found that OBZ was at least 2-fold more efficient than RTX at inducing B-cell cytotoxicity in in vitro whole blood assays. Dissecting this difference, we found that RTX elicited more potent complement-dependent cellular cytotoxicity than OBZ. In contrast, OBZ was more effective at evoking Fc gamma receptor-mediated effector mechanisms, including activation of NK cells and neutrophils, probably due to stronger interaction with Fc gamma receptors and the ability of OBZ to remain at the cell surface following CD20 engagement, whereas RTX became internalized. OBZ was also more efficient at inducing direct cell death. This was true for all CD19 + B cells as a whole and in naïve (IgD + CD27 - ) and switched (IgD - CD27 + ) memory B cells specifically, a higher frequency of which is associated with poor clinical response after RTX. Conclusion: Taken together, these data provide a mechanistic basis for resistance to rituximab-induced B-cell depletion, and for considering obinutuzumab as an alternative B-cell depleting agent in RA and SLE.

Chronic fatigue syndrome and the immune system: Where are we now?

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterised by multiple symptoms including fatigue, headaches and cognitive impairment, which have a significantly adverse effect on the normal functioning and well-being of the individual. These symptoms are often triggered or worsened following physical or mental exertion. ME/CFS has long been thought of as having a significant immunological component, but reports describing changes in immune function are often inconsistent between study groups. Although the wide range of physical, neurocognitive and autonomic symptoms reported have seriously hampered attempts to understand pathophysiological pathways, investment in biomedical research in ME/CFS is finally increasing with a number of novel and promising investigations being published. The onset of ME/CFS may often be linked to (viral) infections which would be consistent with a variety of alterations in natural killer (NK) cell function as described by a number of different groups. Consistency in cytokine data has been lacking so far, although recently more sophisticated approaches have led to more robust data from large patient cohorts. New hope has also been given to sufferers with the possibility that therapies that deplete B cells can result in clinical improvement. To understand the pathogenic mechanism in this complex condition, it is important to consider repeated analysis in different cohorts. In this review, we will discuss the potential of different components of the immune system to be involved in the pathogenesis of ME/CFS.

Antibodies to Cyclic Citrullinated Peptides in Patients With Juvenile Idiopathic Arthritis and Patients With Rheumatoid Arthritis: Shared Expression of the Inherently Autoreactive 9G4 Idiotype.

OBJECTIVE: Antibodies to cyclic citrullinated peptides (anti-CCP) in rheumatoid arthritis (RA) can express the inherently autoreactive gene VH 4-34, detected using the rat monoclonal antibody 9G4. Patients with the polyarticular subtype of juvenile idiopathic arthritis (JIA) share some but not all of the features of adult patients with RA. This study was undertaken to compare serologic findings for rheumatoid factor (RF), anti-CCP, and 9G4-expressing anti-CCP in a large JIA cohort with a cohort of adult RA patients. METHODS: Serum from 88 patients with polyarticular JIA, 29 patients with enthesitis-related arthritis, 38 patients with extended oligoarthritis, 31 adolescent controls, 35 patients with RA, and 30 adult controls were tested for RF, for IgG, IgA, and IgM anti-CCP, and for 9G4-expressing anti-CCP by enzyme-linked immunosorbent assay. Total serum 9G4-positive IgM was also measured. RESULTS: Of 65 patients with RF-negative polyarticular JIA, 4 (6.2%) were IgG anti-CCP positive. Sera from 20 of 23 patients with RF-positive polyarticular JIA (87.0%), 24 of 35 patients with RA (68.6%), and 1 patient with extended oligoarthritis contained IgG anti-CCP. IgA and IgM anti-CCP levels were lower in the adolescent group (P < 0.01). Levels of 9G4-expressing anti-CCP were higher in patients with RF-positive polyarticular JIA than in those with RF-negative polyarticular JIA (P < 0.0001). Median levels of 9G4-expressing anti-CCP in patients with RF-positive polyarticular JIA and those with RA did not differ. Expression of 9G4 on serum total IgM was greater in patients with RF-positive polyarticular JIA than other adolescent groups (P < 0.01), but similar to adult RF-positive RA. CONCLUSION: In healthy individuals, 9G4-positive B cells comprise 5-10% of the peripheral blood pool but serum immunoglobulins utilizing VH 4-34 are disproportionately low. The idiotope recognized by 9G4 was detected on anti-CCP antibodies in >80% of patients with RF-positive polyarticular JIA. VH 4-34 usage by anti-CCP in both JIA and RA patients suggest elicitation of these autoantibodies through shared pathogenic B cell selection processes.

Pragmatic Treatment of Patients With Systemic Lupus Erythematosus With Rituximab: Long-Term Effects on Serum Immunoglobulins.

OBJECTIVE: B cell-depletion therapy based on rituximab is a therapeutic option for refractory disease in patients with systemic lupus erythematosus (SLE). The aim of this observational study was to document long-term effects on B cell function by following serum immunoglobulin levels in patients with SLE treated with rituximab in routine clinical practice. METHODS: We included 57 consecutive patients with SLE treated with rituximab and concomitant/sequential immunosuppressants and measured serum total IgG, IgM, and IgA and IgG anti-dsDNA antibodies, over a median of 48 months most recent followup. Flow cytometry was used prospectively to assess B cell phenotypes in 17 of 57 patients. RESULTS: Twelve patients (21%) had persistent IgM hypogammaglobulinemia (<0.4 gm/liter), and 4 of 57 (5%) had low IgG (<7 gm/liter) at the most recent followup (range 12-144 months). This was not associated with serious adverse events or high anti-double-stranded DNA (anti-dsDNA) antibodies (>1,000 IU/ml; normal <50 IU/ml). Factors predictive of low serum IgM included baseline serum IgM ≤0.8 gm/liter (receiver operator curve analysis) and subsequent therapy with mycophenolate mofetil (MMF; odds ratio 6.8, compared with other immunosuppressants). In patients maintaining normal IgM levels (9 of 17), the frequency of circulating IgD+CD27+ B cells was significantly higher (P = 0.05). At 12 months after rituximab, 7 of 30 SLE patients with baseline anti-dsDNA ≤1,000 IU/ml had lost seropositivity. CONCLUSION: Lower baseline serum IgM levels and sequential therapy with MMF were predictive of IgM hypogammaglobulinemia after rituximab in SLE, but this was not associated with higher levels of anti-dsDNA antibodies or an increased risk of infections. This provides useful directions for clinicians regarding rituximab and sequential immunosuppressive treatment for patients with SLE.

B cell depletion with rituximab in patients with rheumatoid arthritis: Multiplex bead array reveals the kinetics of IgG and IgA antibodies to citrullinated antigens.

The serology of patients with Rheumatoid arthritis (RA) is characterized by persistently raised levels of autoantibodies: Rheumatoid Factors (RhF) against Fc of IgG, and to citrullinated (Cit) protein/peptide sequences: ACPA, recognizing multiple Cit-sequences. B cell depletion therapy based on rituximab delivers good clinical responses in RA patients, particularly in the seropositive group, with responses sometimes lasting beyond the phase of B cell reconstitution. In general, ACPA levels fall following rituximab, but fluctuations with respect to predicting relapse have proved disappointing. In order to identify possible immunodominant specificities within either IgG- or IgA-ACPA we used a Multiplex bead-based array consisting of 30 Cit-peptides/proteins and 22 corresponding native sequences. The kinetics of the serum ACPA response to individual specificities was measured at key points (Baseline, B cell depletion phase, Relapse) within an initial cycle of rituximab therapy in 16 consecutive patients with severe, active RA. All had achieved significant decreases in Disease Activity Scores-28 and maintained B cell depletion in the peripheral blood (<5 CD19+cells/µl) for at least 3 months. At Baseline, mean fluorescence intensity shown by individual IgG- and IgA-ACPA were strongly correlated (R(2) = 0.75; p < 0.0001) but IgA-ACPA were approximately 10-fold lower. Data were Z-normalised in order to compare serial results and antibody classes. At Baseline, a total of 68 IgG- and 51 IgA-ACPA had Z-scores ≥ 1 (above population mean) were identified, with at least one Cit-antigen identified in each serum. ACPA to individual specificities subsequently fluctuated with 3 different patterns. Most 51/68 (75%) IgG- and 48/51 IgA-ACPA (94%) fell between Baseline and Depletion, of which 57% IgG- and 65% IgA-ACPA rebounded pre-Relapse. Interestingly, 17/68 IgG-ACPA (25%) and some IgA-ACPA (3/51; 6%) transiently increased from Baseline, subsequently falling pre-Relapse. Individual responses to particular Cit-epitopes were not linked to particular patterns of fluctuation, but IgG- and IgA-ACPA to individual Cit-antigens often followed similar courses. Some new IgG- and IgA-ACPA, generally to different Cit-antigens however, arose at Relapse in 4 patients. The complexities of the ACPA response after rituximab may therefore reflect its ability to deplete or modify the function of parent B cell clones, which varies between patients. Although relapse following rituximab invariably follows naïve B cell exit from the bone marrow, these studies show that interactions between both 'new' and residual autoreactive memory B cells may be key to resumption of symptoms. The lack of identification of any immunodominant specificity suggests that the process of citrullination, rather than any particular Cit-antigen drives the autoimmune response in RA patients.

Internalization of rituximab and the efficiency of B Cell depletion in rheumatoid arthritis and systemic lupus erythematosus.

OBJECTIVE: Rituximab, a type I anti-CD20 monoclonal antibody (mAb), induces incomplete B cell depletion in some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), thus contributing to a poor clinical response. The mechanisms of this resistance remain elusive. The purpose of this study was to determine whether type II mAb are more efficient than type I mAb at depleting B cells from RA and SLE patients, whether internalization influences the efficiency of depletion, and whether Fcγ receptor type IIb (FcγRIIb) and the B cell receptor regulate this internalization process. METHODS: We used an in vitro whole blood B cell-depletion assay to assess the efficiency of depletion, flow cytometry to study cell surface protein expression, and surface fluorescence-quenching assays to assess rituximab internalization, in samples from patients with RA and patients with SLE. Paired t-test or Mann-Whitney U test was used to compare groups, and Spearman's rank correlation test was used to assess correlation. RESULTS: We found that type II mAb internalized significantly less rituximab than type I mAb and depleted B cells from patients with RA and SLE at least 2-fold more efficiently than type I mAb. Internalization of rituximab was highly variable between patients, was regulated by FcγRIIb, and inversely correlated with cytotoxicity in whole blood B cell-depletion assays. The lowest levels of internalization were seen in IgD- B cells, including postswitched (IgD-CD27+) memory cells. Internalization of type I anti-CD20 mAb was also partially inhibited by anti-IgM stimulation. CONCLUSION: Variability in internalization of rituximab was observed and was correlated with impaired B cell depletion. Therefore, slower-internalizing type II mAb should be considered as alternative B cell-depleting agents for the treatment of RA and SLE.

Response to rituximab: has the original hypothesis been confirmed?

Before the use of rituximab, the strongest accepted evidence for an association between B-cells and rheumatoid arthritis (RA) was that clinical disease was associated with serum autoantibodies. The ability to remove B-cells with rituximab has also revealed the relative importance of the different immunological parameters that underlie the clinical symptoms of RA. First, seropositive patients have a significantly more predictable and favorable clinical response to rituximab than seronegative patients. Second, the kinetics of the clinical response, with a delay of weeks or months after depletion, suggest that it is a B-cell product (autoantibody) and not B-cells per se that need to be reduced for remission to occur. Third, removal of B-cells from joints may not be closely associated with clinical improvement, although maintenance of plasma cell counts in joints has been associated with poorer responses. The requirement of 'new' B-cells generated from the bone marrow for relapse to occur suggests that selection of autoreactive B-cell clones in the periphery may also be necessary for their survival and differentiation into autoantibody-producing cells. The initial hypothesis suggested that the autoimmune response underlying the pathogenesis of RA was self-sustaining. This would seem to be confirmed, as relapse inevitably follows a variable period of reduced clinical symptoms induced by rituximab. In addition, a dominant role for autoantibodies seems to have strong support from clinical practice. In addition to their possible role in the pathogenesis of RA in the form of immune complexes, further investigation is necessary to determine whether autoantibodies contribute to perpetuation of changes in central B-cell tolerance in these patients.

Expression of the inherently autoreactive idiotope 9G4 on autoantibodies to citrullinated peptides and on rheumatoid factors in patients with early and established rheumatoid arthritis.

BACKGROUND: The pre-symptomatic stage of Rheumatoid arthritis (RA) is associated with pro-inflammatory cytokines and autoantibodies. High levels and epitope spread by Rheumatoid factors (RhF) and autoantibodies to citrullinated proteins signify progression towards disease expression. In established RA, the persistence of high autoantibody levels reflects production by both long-lived plasma cells and short-lived plasmablasts. Neither the relative contributions to pathogenesis by autoantibodies from either source, nor the factors responsible for deciding the fate of autoantigen specific 'parent' B-cells, is understood. Phenotypic markers identifying subsets of autoreactive B-cells are therefore of interest in understanding the origin and perpetuation of the autoimmune response in RA. One such phenotypic marker is the rat monoclonal antibody, 9G4, which recognises an idiotope on immunoglobuins derived from the inherently autoreactive VH-gene, VH4-34. We therefore investigated whether the 9G4 idiotope was expressed on autoantibodies in patients with RA. METHODOLOGY/PRINCIPAL FINDINGS: Sera from 19 patients with established RA and those with <1year history of untreated polyarthritis either resolving into RA (n = 42) or non-RA diagnosis (n = 31) were included. Autoantibodies to cyclic citrullinated peptides (CCP), RhF and co-expression of the 9G4 idiotope were measured by ELISA. 9G4 recognised a population of anti-CCP antibodies in the majority of sera from patients with established disease and also in samples from patients with early disaese. 9G4+RhF levels were generally lower and not associated with positivity for, or levels of 9G4+CCP. CONCLUSIONS/SIGNIFICANCE: The persistence of 9G4+ immunoglobulins, of any isotype, in serum is rare. We describe here the novel finding of 9G4 expression on anti-CCP antibodies in patients from the earliest symptoms of RA through to established disease. Our results suggest that 9G4 expression on anti-CCP autoantibodies was not due to polyclonal expansion of VH4-34-encoded immunoglobulins. These studies may therefore provide a new focus for investigation into the evolution of the autoimmune response in RA patients.