Abatacept in individuals at high risk of rheumatoid arthritis (APIPPRA): a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial.

BACKGROUND: Individuals with serum antibodies to citrullinated protein antigens (ACPA), rheumatoid factor, and symptoms, such as inflammatory joint pain, are at high risk of developing rheumatoid arthritis. In the arthritis prevention in the pre-clinical phase of rheumatoid arthritis with abatacept (APIPPRA) trial, we aimed to evaluate the feasibility, efficacy, and acceptability of treating high risk individuals with the T-cell co-stimulation modulator abatacept. METHODS: The APIPPRA study was a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial done in 28 hospital-based early arthritis clinics in the UK and three in the Netherlands. Participants (aged ≥18 years) at risk of rheumatoid arthritis positive for ACPA and rheumatoid factor with inflammatory joint pain were recruited. Exclusion criteria included previous episodes of clinical synovitis and previous use of corticosteroids or disease-modifying antirheumatic drugs. Participants were randomly assigned (1:1) using a computer-generated permuted block randomisation (block sizes of 2 and 4) stratified by sex, smoking, and country, to 125 mg abatacept subcutaneous injections weekly or placebo for 12 months, and then followed up for 12 months. Masking was achieved by providing four kits (identical in appearance and packaging) with pre-filled syringes with coded labels of abatacept or placebo every 3 months. The primary endpoint was the time to development of clinical synovitis in three or more joints or rheumatoid arthritis according to American College of Rheumatology and European Alliance of Associations for Rheumatology 2010 criteria, whichever was met first. Synovitis was confirmed by ultrasonography. Follow-up was completed on Jan 13, 2021. All participants meeting the intention-to-treat principle were included in the analysis. This trial was registered with EudraCT (2013-003413-18). FINDINGS: Between Dec 22, 2014, and Jan 14, 2019, 280 individuals were evaluated for eligibility and, of 213 participants, 110 were randomly assigned to abatacept and 103 to placebo. During the treatment period, seven (6%) of 110 participants in the abatacept group and 30 (29%) of 103 participants in the placebo group met the primary endpoint. At 24 months, 27 (25%) of 110 participants in the abatacept group had progressed to rheumatoid arthritis, compared with 38 (37%) of 103 in the placebo group. The estimated proportion of participants remaining arthritis-free at 12 months was 92·8% (SE 2·6) in the abatacept group and 69·2% (4·7) in the placebo group. Kaplan-Meier arthritis-free survival plots over 24 months favoured abatacept (log-rank test p=0·044). The difference in restricted mean survival time between groups was 53 days (95% CI 28-78; p<0·0001) at 12 months and 99 days (95% CI 38-161; p=0·0016) at 24 months in favour of abatacept. During treatment, abatacept was associated with improvements in pain scores, functional wellbeing, and quality-of-life measurements, as well as low scores of subclinical synovitis by ultrasonography, compared with placebo. However, the effects were not sustained at 24 months. Seven serious adverse events occurred in the abatacept group and 11 in the placebo group, including one death in each group deemed unrelated to treatment. INTERPRETATION: Therapeutic intervention during the at-risk phase of rheumatoid arthritis is feasible, with acceptable safety profiles. T-cell co-stimulation modulation with abatacept for 12 months reduces progression to rheumatoid arthritis, with evidence of sustained efficacy beyond the treatment period, and with no new safety signals. FUNDING: Bristol Myers Squibb.

Oleic acid enhances proliferation and calcium mobilization of CD3/CD28 activated CD4+ T cells through incorporation into membrane lipids.

Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function.

Autoantibodies in rheumatoid arthritis - rheumatoid factor, anticitrullinated protein antibodies and beyond.

PURPOSE OF REVIEW: RA is characterized by the presence of autoantibodies among which rheumatoid factors (RFs) and antimodified protein antibodies (AMPA) are serological hallmarks of the disease. In recent years, several novel insights into the biology, immunogenetics and clinical relevance of these autoantibodies have been obtained, which deserve to be discussed in more detail. RECENT FINDINGS: RFs from RA patients seem to target distinct epitopes which appear to be quite specific for RA. Determination of immunoglobulin A (IgA) isotypes of RF and anticitrullinated protein antibodies (ACPA) may provide prognostic information because their presence is associated with reduced therapeutic responses to TNF inhibitors. Furthermore, IgA levels are increased in RA patients and IgA immune complexes are more potent than immunoglobulin G (IgG) complexes in inducing NET formation. Concerning AMPAs, investigations on variable domain glycosylation (VDG) revealed effects on antigen binding and activation of autoreactive B cells. Studies on pathogenetic involvement of ACPA suggest Janus-faced roles: on the one hand, ACPA may be involved in joint destruction and pain perception while on the other hand protective anti-inflammatory effects may be attributed to a subset of ACPAs. SUMMARY: The autoimmune response in RA is extremely complex and still far from being fully understood. Antibodies are not only valuable diagnostic biomarkers but also seem to play pivotal roles in the pathophysiology of RA.

Immune response to post-translationally modified proteins in rheumatoid arthritis: what makes it special?

Rheumatoid arthritis (RA) exhibits common characteristics with numerous other autoimmune diseases, including the presence of susceptibility genes and the presence of disease-specific autoantibodies. Anti-citrullinated protein antibodies (ACPA) are the hallmarking autoantibodies in RA and the anti-citrullinated protein immune response has been implicated in disease pathogenesis. Insight into the immunological pathways leading to anti-citrullinated protein immunity will not only aid understanding of RA pathogenesis, but may also contribute to elucidation of similar mechanisms in other autoantibody-positive autoimmune diseases. Similarly, lessons learnt in other human autoimmune diseases might be relevant to understand potential drivers of RA. In this review, we will summarise several novel insights into the biology of the anti-citrullinated protein response and their clinical associations that have been obtained in recent years. These insights include the identification of glycans in the variable domain of ACPA, the realisation that ACPA are polyreactive towards other post-translational modifications on proteins, as well as new awareness of the contributing role of mucosal sites to the development of the ACPA response. These findings will be mirrored to emerging concepts obtained in other human (autoimmune) disease characterised by disease-specific autoantibodies. Together with an updated understanding of genetic and environmental risk factors and fresh perspectives on how the microbiome could contribute to antibody formation, these advancements coalesce to a progressively clearer picture of the B cell reaction to modified antigens in the progression of RA.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

A whole blood-based functional assay to characterize immunoglobulin A effector functions.

Most investigations on the immune cell-activating potency of IgA used purified total IgA and/or specific isolated cell populations. As IgA2 has been reported to be more pro-inflammatory than IgA1, we aimed to employ a fast and convenient whole blood-based assay to individually probe the capacity of the two IgA subclasses to activate immune cells in close physiological conditions. To this end, whole blood from healthy donors (n = 10) was stimulated with immobilized IgA1, IgA2m1 or IgA2m2 (the two main allotypic variants of IgA2). Activation of major leukocyte subsets was measured using a 10-color flow cytometry panel providing access to the expression of 5 activation markers on 6 different immune cell subsets. While capturing some heterogeneity of responses among donors, IgA2m1 and IgA2m2 systematically showed a stronger activation profile compared to IgA1 in a variety of dimensions. For example, both IgA2 allotypes led to stronger modulations of CD54, CD11b, CD62L, CD66b or CD69, on both or either monocytes or neutrophils, indicating a more pronounced pro-inflammatory effect for this subclass than IgA1. By taking into account donor-specific soluble and cellular components this whole blood-based functional approach provides new perspectives to further investigate IgA effector functions in mechanistic studies and/or translational research.

Antigen-specific Fab profiling achieves molecular-resolution analysis of human autoantibody repertoires in rheumatoid arthritis.

The presence of autoantibodies is a defining feature of many autoimmune diseases. The number of unique autoantibody clones is conceivably limited by immune tolerance mechanisms, but unknown due to limitations of the currently applied technologies. Here, we introduce an autoantigen-specific liquid chromatography-mass spectrometry-based IgG1 Fab profiling approach using the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA) as an example. We show that each patient harbors a unique and diverse ACPA IgG1 repertoire dominated by only a few antibody clones. In contrast to the total plasma IgG1 antibody repertoire, the ACPA IgG1 sub-repertoire is characterised by an expansion of antibodies that harbor one, two or even more Fab glycans, and different glycovariants of the same clone can be detected. Together, our data indicate that the autoantibody response in a prominent human autoimmune disease is complex, unique to each patient and dominated by a relatively low number of clones.

N-linked Fc glycosylation is not required for IgG-B-cell receptor function in a GC-derived B-cell line.

IgG secreted by B cells carry asparagine N(297)-linked glycans in the fragment crystallizable (Fc) region. Changes in Fc glycosylation are related to health or disease and are functionally relevant, as IgG without Fc glycans cannot bind to Fcɣ receptors or complement factors. However, it is currently unknown whether ɣ-heavy chain (ɣHC) glycans also influence the function of membrane-bound IgG-B-cell receptors (BCR) and thus the outcome of the B-cell immune response. Here, we show in a germinal center (GC)-derived human B-cell line that ɣHC glycans do not affect membrane expression of IgG-BCRs. Furthermore, antigen binding or other BCR-facilitated mechanisms appear unaffected, including BCR downmodulation or BCR-mediated signaling. As expected, secreted IgG lacking Fc glycosylation is unable to carry out effector functions. Together, these observations indicate that IgG-Fc glycosylation serves as a mechanism to control the effector functions of antibodies, but does not regulate the activation of IgG-switched B cells, as its absence had no apparent impact on BCR function.

Acetylated bacterial proteins as potent antigens inducing an anti-modified protein antibody response.

OBJECTIVE: Gut-residing bacteria, such as Escherichia coli, can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses cross-reactive to modified self-proteins and be recognised by human AMPA (hAMPA). METHODS: E. coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E. coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA. RESULTS: Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins. CONCLUSIONS: Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing cross-reactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.

Type 17-specific immune pathways are active in early spondyloarthritis.

OBJECTIVE: Undifferentiated, early inflammatory arthritis (EIA) can differentiate into seropositive or seronegative rheumatoid arthritis (RA), peripheral spondyloarthritis (SpA) or remain as seronegative undifferentiated inflammatory arthritis (UIA). Little is known about immune pathways active in the early stages of SpA and seronegative UIA, in contrast to detailed knowledge of seropositive RA. The aim of this study was to examine if specific immune pathways were active in synovial CD4+ and CD8+ T cells in EIA. METHODS: Synovial fluid (SF) samples from 30 patients with EIA were analysed for expression of IL-17A, IFNγ and TNFα in CD8+ or CD4+ T cells. Final clinical diagnoses were made at least 12 months after sample collection, by two independent clinicians blind to the study data. RESULTS: Flow cytometry analysis of all EIA samples indicated considerable variation in synovial IL-17A+CD8+ T cells (Tc17) cell frequencies between patients. The group with a final diagnosis of SpA (psoriatic arthritis or peripheral SpA, n=14) showed a significant enrichment in the percentage of synovial Tc17 cells compared with the group later diagnosed with seronegative UIA (n=10). The small number of patients later diagnosed with seropositive RA (n=6) patients had few Tc17 cells, similar to our previous findings in established disease. In contrast, RA SF contained a significantly higher percentage of CD8+IFNγ+ T cells compared with SpA or seronegative UIA. CONCLUSION: These results suggest that adaptive T cell cytokine pathways differ not only between RA and SpA but also seronegative UIA early in the disease process, with a particular activation of Tc17 pathways in early SpA.

Sex-specific risk of anti-topoisomerase antibodies on mortality and disease severity in systemic sclerosis: 10-year analysis of the Leiden CCISS and EUSTAR cohorts.

BACKGROUND: We aimed to evaluate sex-specific risk of anti-topoisomerase I antibodies (ATA) on mortality, diffuse cutaneous systemic sclerosis, interstitial lung disease, and pulmonary hypertension in two cohorts of people with systemic sclerosis. METHODS: This study was a 10-year analysis of the prospective Leiden Combined Care in Systemic Sclerosis (CCISS) cohort in the Netherlands and the international European Scleroderma Trials and Research (EUSTAR) cohort. We included participants with systemic sclerosis according to the 2013 American College of Rheumatology-European League Against Rheumatism (ACR-EULAR) classification criteria; available autoantibody status; available skin subtyping; at least one available radiographic assessment of interstitial lung disease; and with a known date of disease onset. People with systemic sclerosis were categorised in six risk groups by sex and autoantibody status (anti-centromere antibody [ACA]-positive female, ACA-positive male, ACA and ATA-negative female, ACA and ATA-negative male, ATA-positive female, and ATA-positive male). We constructed Kaplan-Meier curves and Cox proportional hazard models, accounting for left-truncated survival to prevent bias because the date of disease onset (first non-Raynaud's symptom) preceded the date of cohort entry for all patients. The primary outcome was all-cause mortality and the secondary outcomes were diffuse cutaneous systemic sclerosis, interstitial lung disease, and pulmonary hypertension. FINDINGS: 445 (63%) of 708 participants between April 1, 2009, and Jan 1, 2022, in CCISS (101 [23%] male and 344 [77%] female) and 4263 (50%) of 8590 between June 1, 2004, and March 28, 2018, in EUSTAR (783 [18%] male and 3480 [82%] female) were eligible for this study. In both cohorts, ATA expression occurred significantly more often in males than in females (39 [39%] of 101 males vs 67 [19%] of 344 females in CCISS; p<0·0001 and 381 [49%] of 783 males vs 1323 [38%] of 3480 females in EUSTAR; p<0·0001). According to estimated survival rates, 30% of ATA-positive males versus 12% of ATA-positive females died in the CCISS cohort and 33% versus 15% died in the EUSTAR cohort within 10 years. After adjustment for age, race, and autoantibody status, male sex remained the most important risk factor for all-cause mortality (HR 2·9 [95% CI 1·5-5·5] in CCISS, p=0·0018; and HR 2·6 [2·0-3·4] in EUSTAR, p<0·0001). INTERPRETATION: We show that the association between male sex and increased mortality in systemic sclerosis cannot be explained by higher ATA prevalence. However, additional research on the effect of sex-specific characteristics on people with systemic sclerosis is required. FUNDING: None.

The autoimmune response as a potential target for tolerance induction before the development of rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease that affects the synovial joints. Although treatment options and efficacy have increased substantially in the past two decades, the disease cannot be cured or prevented. Therefore, rheumatoid arthritis still has a considerable effect on the quality of life of patients, not only because life-long medication is often required, but also because residual disease activity leads to progressive loss of function in the musculoskeletal system and extra-articular morbidity. Key future goals in the management of rheumatoid arthritis are the ability to induce long-lasting drug-free remission in patients with the disease (ie, to achieve a cure), and to prevent disease before it emerges. To reach these goals, it is pivotal to understand the autoimmune response underlying rheumatoid arthritis pathogenesis and to develop ways to permanently silence it (ie, to induce tolerance). For preventive studies, the identification of markers (clinical, immunological, and biological) predictive of future disease is crucial, as prevention of disease will not be feasible without the ability to identify relevant at-risk target populations. In this Series paper, we review the autoimmune response underlying rheumatoid arthritis, how rheumatoid arthritis-specific autoimmunity develops and evolves during the transition from health to disease, and how tolerance studies could be designed to achieve prevention or cure of the disease.