Impact of muscle biopsy on the clinical decision-making process in patients with suspected idiopathic inflammatory myopathy.

BACKGROUND: The significance of muscle biopsy as a diagnostic tool in idiopathic inflammatory myopathies (IIM) remains elusive. We aimed to determine the diagnostic weight that has been given to muscle biopsy in patients with suspected IIM, particularly in terms of clinical diagnosis and therapeutic decisions. MATERIAL AND METHODS: In this retrospective multicentric study, we analyzed muscle biopsy results of adult patients with suspected IIM referred to a tertiary center between January 1, 2007, and October 31, 2021. Information regarding referral department, suspected diagnosis, biopsy site, demographic, clinical, laboratory data, and imaging results were extracted. Statistical analyses included the level of agreement between suspected and histological diagnosis and calculation of diagnostic performance (positive and negative predictive values, positive and negative likelihood ratios, sensitivity, and specificity of muscle biopsy in relation to clinical diagnosis and/or treatment initiation). Performance was tested in different strata based on clinical pre-test probability. RESULTS: Among 758 muscle biopsies, IIM was histologically compatible in 357/758 (47.1%) cases. Proportion of IIM was higher if there was a solid clinical pre-test probability (64.3% vs. 42.4% vs. 48% for high, medium and low pre-test probability). Sensitivity and specificity of muscle biopsy were highest (82%) when the diagnosis by the clinician was used as outcome scenario. Negative predictive value was only moderate (between 63% and 80%) and lowest if autoantibodies were positive (35%). CONCLUSION: In patients with clinically suspected IIM, approximately 50% of biopsies revealed features indicative of IIM. Diagnostic performance of muscle biopsy was moderate to high depending on clinical pre-test probability.

Immunogenicity and safety of COVID-19 booster vaccination: A population-based clinical trial to identify the best vaccination strategy.

BACKGROUND: Various SARS-CoV-2 variants of concerns (VOCs) characterized by higher transmissibility and immune evasion have emerged. Despite reduced vaccine efficacy against VOCs, currently available vaccines provide protection. Population-based evidence on the humoral immune response after booster vaccination is crucial to guide future vaccination strategies and in preparation for imminent COVID-19 waves. METHODS: This multicenter, population-based cohort study included 4697 individuals ≥18 years of age who received a booster vaccination. Antibody levels against SARS-CoV-2 receptor binding domain (RBD) and neutralizing antibodies against wild-type (WT) virus and Omicron variants were assessed at baseline (day of booster vaccination) and after four weeks. Safety was evaluated daily within the first week using a participant-completed electronic diary. Antibody levels were compared across different vaccination strategies, taking into account individual host factors. RESULTS: Our main model including 3838 participants revealed that individuals who received a booster with mRNA-1273 compared to BNT162b2 vaccine had a significantly higher increase (95 %CI) in anti-RBD-antibody levels (37,707 BAU/mL [34,575-40,839] vs. 27,176 BAU/mL [26,265-28,087]), and of neutralization levels against WT (1,681 [1490-1872] vs. 1141 [1004-1278] and Omicron variant (422 [369-474] vs. 329 [284-374]). Neutralizing antibody titres highly correlated with anti-RBD antibodies, with neutralizing capacity 4.4 fold higher against WT compared to Omicron. No differences in safety were found between the two booster vaccines. CONCLUSION: Our study underlines the superiority of a booster vaccination with mRNA-1273, independent of the primary vaccination and therefore provides guidance on the vaccination strategy.

NLRP12 interacts with NLRP3 to block the activation of the human NLRP3 inflammasome.

Inflammasomes are multiprotein complexes that drive inflammation and contribute to protective immunity against pathogens and immune pathology in autoinflammatory diseases. Inflammasomes assemble when an inflammasome scaffold protein senses an activating signal and forms a signaling platform with the inflammasome adaptor protein ASC. The NLRP subfamily of NOD-like receptors (NLRs) includes inflammasome nucleators (such as NLRP3) and also NLRP12, which is genetically linked to familial autoinflammatory disorders that resemble diseases caused by gain-of-function NLRP3 mutants that generate a hyperactive NLRP3 inflammasome. We performed a screen to identify ASC inflammasome-nucleating proteins among NLRs that have the canonical pyrin-NACHT-LRR domain structure. Only NLRP3 and NLRP6 could initiate ASC polymerization to form "specks," and NLRP12 failed to nucleate ASC polymerization. However, wild-type NLRP12 inhibited ASC inflammasome assembly induced by wild-type and gain-of-function mutant NLRP3, an effect not seen with disease-associated NLRP12 mutants. The capacity of NLRP12 to suppress NLRP3 inflammasome assembly was limited to human NLRP3 and was not observed for wild-type murine NLRP3. Furthermore, peripheral blood mononuclear cells from patients with an NLRP12 mutant-associated inflammatory disorder produced increased amounts of the inflammatory cytokine IL-1ß in response to NLRP3 stimulation. Thus, our findings provide insights into NLRP12 biology and suggest that NLRP3 inhibitors in clinical trials for NLRP3-driven diseases may also be effective in treating NLRP12-associated autoinflammatory diseases.

Selectivity, efficacy and safety of JAKinibs: new evidence for a still evolving story.

Fundamental insight gained over the last decades led to the discovery of cytokines as pivotal drivers of inflammatory diseases such as rheumatoid arthritis, psoriasis/psoriasis arthritis, inflammatory bowel diseases, atopic dermatitis and spondylarthritis. A deeper understanding of the pro-inflammatory and anti-inflammatory effects of various cytokines has prompted new cytokine-targeting therapies, which revolutionised the treatment options in the last years for patients with inflammatory disorders. Disease-associated immune responses typically involve a complex interplay of multiple cytokines. Therefore, blockade of one single cytokine does not necessarily lead to a persistent remission in all patients with inflammatory disorders and fostered new therapeutic strategies targeting intracellular pathways shared by multiple cytokines. By inhibiting JAK-STAT signalling pathways common to families of cytokines, JAK-inhibitors (JAKinibs) have created a new paradigm for the treatment of inflammatory diseases. Multiple agents have been approved for various disorders and more are being investigated for several new indications. Second-generation selective JAKinibs have been devised with the aim to achieve an increased selectivity and a possible reduced risk of side effects. In the current review, we will summarise the current body of evidence of pan versus selective JAKinibs and the most recent insights on new side effects and indications, including COVID-19.

A conformation-locking inhibitor of SLC15A4 with TASL proteostatic anti-inflammatory activity.

Dysregulation of pathogen-recognition pathways of the innate immune system is associated with multiple autoimmune disorders. Due to the intricacies of the molecular network involved, the identification of pathway- and disease-specific therapeutics has been challenging. Using a phenotypic assay monitoring the degradation of the immune adapter TASL, we identify feeblin, a chemical entity which inhibits the nucleic acid-sensing TLR7/8 pathway activating IRF5 by disrupting the SLC15A4-TASL adapter module. A high-resolution cryo-EM structure of feeblin with SLC15A4 reveals that the inhibitor binds a lysosomal outward-open conformation incompatible with TASL binding on the cytoplasmic side, leading to degradation of TASL. This mechanism of action exploits a conformational switch and converts a target-binding event into proteostatic regulation of the effector protein TASL, interrupting the TLR7/8-IRF5 signaling pathway and preventing downstream proinflammatory responses. Considering that all components involved have been genetically associated with systemic lupus erythematosus and that feeblin blocks responses in disease-relevant human immune cells from patients, the study represents a proof-of-concept for the development of therapeutics against this disease.

Cytokine-directed cellular cross-talk imprints synovial pathotypes in rheumatoid arthritis.

INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.

PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing.

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.

The accelerated waning of immunity and reduced effect of booster in patients treated with bDMARD and tsDMARD after SARS-CoV-2 mRNA vaccination.

Objectives: This study aimed to assess the duration of humoral responses after two doses of SARS-CoV-2 mRNA vaccines in patients with inflammatory joint diseases and IBD and booster vaccination compared with healthy controls. It also aimed to analyze factors influencing the quantity and quality of the immune response. Methods: We enrolled 41 patients with rheumatoid arthritis (RA), 35 with seronegative spondyloarthritis (SpA), and 41 suffering from inflammatory bowel disease (IBD), excluding those receiving B-cell-depleting therapies. We assessed total anti-SARS-CoV-2 spike antibodies (Abs) and neutralizing Ab titers 6 months after two and then after three doses of mRNA vaccines compared with healthy controls. We analyzed the influence of therapies on the humoral response. Results: Patients receiving biological or targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) showed reduced anti-SARS-CoV-2 S Abs and neutralizing Ab titers compared with HC or patients receiving conventional synthetic (cs)DMARDs 6 months after the first two vaccination doses. Anti-SARS-CoV-2 S titers of patients with b/tsDMARDs declined more rapidly, leading to a significant reduction in the duration of vaccination-induced immunity after two doses of SARS-CoV-2 mRNA vaccines. While 23% of HC and 19% of patients receiving csDMARDs were without detectable neutralizing Abs 6 months after the first two vaccination doses, this number was 62% in patients receiving b/tsDMARDs and 52% in patients receiving a combination of csDMARDs and b/tsDMARDs. Booster vaccination led to increased anti-SARS-CoV-2 S Abs in all HC and patients. However, anti-SARS-CoV-2 S Abs after booster vaccination was diminished in patients receiving b/tsDMARDs, either alone or in combination with csDMARDs compared to HC. Conclusion: Patients receiving b/tsDMARDs have significantly reduced Abs and neutralizing Ab titers 6 months after mRNA vaccination against SARS-CoV-2. This was due to a faster decline in Ab levels, indicating a significantly reduced duration of vaccination-induced immunity compared with HC or patients receiving csDMARDs. In addition, they display a reduced response to a booster vaccination, warranting earlier booster vaccination strategies in patients under b/tsDMARD therapy, according to their specific Ab levels.

A genome-wide CRISPR functional survey of the human phagocytosis molecular machinery.

Phagocytosis, the process by which cells engulf large particles, plays a vital role in driving tissue clearance and host defense. Its dysregulation is connected to autoimmunity, toxic accumulation of proteins, and increased risks for infections. Despite its importance, we lack full understanding of all molecular components involved in the process. To create a functional map in human cells, we performed a genome-wide CRISPRko FACS screen that identified 716 genes. Mapping those hits to a comprehensive protein-protein interaction network annotated for functional cellular processes allowed retrieval of protein complexes identified multiple times and detection of missing phagocytosis regulators. In addition to known components, such as the Arp2/3 complex, the vacuolar-ATPase-Rag machinery, and the Wave-2 complex, we identified and validated new phagocytosis-relevant functions, including the oligosaccharyltransferase complex (MAGT1/SLC58A1, DDOST, STT3B, and RPN2) and the hypusine pathway (eIF5A, DHPS, and DOHH). Overall, our phagocytosis network comprises elements of cargo uptake, shuffling, and biotransformation through the cell, providing a resource for the identification of potential novel drivers for diseases of the endo-lysosomal system. Our approach of integrating protein-protein interaction offers a broadly applicable way to functionally interpret genome-wide screens.

Accelerated waning of immune responses to a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases.

BACKGROUND: A 3rd COVID-19 vaccination is currently recommended for patients under immunosuppression. However, a fast decline of antibodies against the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein has been observed. Currently it remains unclear whether immunosuppressive therapy affects kinetics of humoral and cellular immune responses. METHODS: 50 patients under immunosuppression and 42 healthy controls (HCs) received a 3rd dose of an mRNA-based vaccine and were monitored over a 12-weeks period. Humoral immune response was assessed 4 and 12 weeks after 3rd dose. Antibodies were quantified using the Elecsys Anti-SARS-CoV-2 Spike immunoassay against the receptor-binding domain (RBD) of the spike protein. SARS-CoV-2-specific T cell responses were quantified by IFN-γ ELISpot assays. Adverse events, including SARS-CoV-2 infections, were monitored over a 12-week period. RESULTS: At week 12, reduced anti-RBD antibody levels were observed in IMID patients as compared to HCs (median antibody level 5345 BAU/ml [1781-10,208] versus 9650 BAU/ml [6633-16,050], p < 0.001). Reduction in relative antibody levels was significantly higher in IMID patients as compared to HCs at week 12 (p < 0.001). Lowest anti-RBD antibody levels were detected in IMID patients who received biological disease-modifying anti-rheumatic drugs (DMARDs) or a combination therapy with conventional synthetic and biological DMARDs. Number of SARS-CoV-2-specific T cells against wildtype and Omicron variants remained stable over 12 weeks in IMID patients. No serious adverse events were reported. CONCLUSION: Due to a fast decline in anti-RBD antibodies in IMID patients an early 4th vaccination should be considered in this vulnerable group of patients.

Reduced immunogenicity of BNT162b2 booster vaccination in combination with a tetravalent influenza vaccination: results of a prospective cohort study in 838 health workers.

OBJECTIVE: To investigate the immunogenicity and safety of BNT162b2 booster vaccination with and without a tetravalent influenza vaccine. METHODS: A prospective, open-label cohort study on immunogenicity and safety of COVID-19 booster vaccination with or without a tetravalent influenza vaccine was performed. Eight hundred thirty-eight health care workers were included in the following study arms: BNT162b2 booster-only, influenza-vaccine-only or combination of both. Levels of antibodies against SARS-CoV-2 spike receptor binding domain, and haemagglutinin inhibition tested for four different influenza strains (A(H1N1)pdm09, A(H3N2), B/Victoria, B/Yamagata) were measured at the time of vaccination and 4 weeks later. RESULTS: After 4 weeks, median (interquartile range) levels of antibodies against the receptor binding domain of the viral spike (S) protein and relative change from baseline were high in individuals who received BNTb162b2 booster vaccination only (absolute: 16 600 [10 980-24 360] vs. 12 630 [8198-18 750] BAU/mL [p < 0.0001]; relative increase: 49% [23.6-95.3] vs. 40% [21.9-80.6] [p 0.048]; booster-only n = 521 vs. combination-arm n = 229 respectively). Results were confirmed after matching for sex, age, body mass index, baseline antibody levels and vaccine compound received for primary immunization (absolute: 13 930 [10 610-22 760] vs. 12 520 [8710-17 940]; [p 0.031]; relative increase: 55.7% [27.8-98.5] vs. 42.2% [22.9-74.5]; p 0.045). Adverse events were almost identical in the booster-only and the combination-arm, but numerically low in the influenza arm (525/536 [97.9%] vs. 235/240 [97.9%] vs. 26/33 [78.8 %]). DISCUSSION: Although no safety concerns occurred, our study provides evidence on reduced immunogenicity of a BNT162b2 booster vaccination in combination with a tetravalent influenza vaccine. Further studies investigating new influenza variants as well as potential differences vaccine effectiveness are needed.

Safety and immunogenicity of a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases compared with healthy controls.

OBJECTIVES: A third COVID-19 vaccination is recommended for immunosuppressed patients. However, data on immunogenicity and safety of a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMIDs) are sparse and therefore addressed within this clinical trial. METHODS: 60 immunosuppressed patients and 48 healthy controls (HCs) received a third vaccination with an mRNA vaccine. The primary endpoint was defined as the presence of antibody levels against the receptor-binding domain (RBD)>1500 BAU/mL in patients with IMIDs versus HCs. Further endpoints included differences in neutralising antibodies and cellular immune responses after the third vaccination. Reactogenicity was recorded for 7 days, and safety was evaluated until week 4. RESULTS: Rate of individuals with anti-RBD antibodies>1500 BAU/mL was not significantly different after the third vaccination between patients with IMIDs and HCs (91% vs 100% p=0.101). Anti-RBD and neutralising antibody levels were significantly lower in patients with IMIDs after the third vaccination than in HCs (p=0.002 and p=0.016, respectively). In contrast, fold increase in antibody levels between week 0 and 4 was higher in patients with IMIDs. Treatment with biological (b) disease-modifying anti-rheumatic drugs (DMARD) or combination of bDMARDs and conventional synthetic DMARDs was associated with reduced antibody levels. Enhanced cellular immune response to wild type and Omicron peptide stimulation was observed after the third vaccination. No serious adverse event was attributed to the third vaccination. CONCLUSION: Our clinical trial data support the immunogenicity and safety of a third COVID-19 vaccination in patients with IMIDs. However, effects of DMARD therapy on immunogenicity should be considered. TRIAL REGISTRATION NUMBER: EudraCT No: 2021-002693-10.

Multiparametric Prediction Models for Coronavirus Disease 2019 Vaccine Selection: Results of a Comparative Population-Based Cohort Study.

BACKGROUND: An understanding vaccine-dependent effects on protective and sustained humoral immune response is crucial to planning future vaccination strategies against coronavirus disease 2019 (COVID-19). METHODS: In this multicenter, population-based, cohort study including 4601 individuals after primary vaccination against COVID-19 ≥ 4 months earlier we compared factors associated with residual antibody levels against severe acute respiratory syndrome coronavirus-2 receptor-binding domain (RBD) across different vaccination strategies (BNT162b2, mRNA-1273, or ChAdOx1). RESULTS: Our main model including 3787 individuals (2 × BNT162b2, n = 2271; 2 × mRNA-1273, n = 251; 2 × ChAdOx1, n = 1265), predicted significantly lower levels of anti-RBD antibodies after 6 months in individuals vaccinated with ChAdOx1 (392.7 binding antibody units per milliliter [BAU/mL]) compared with those vaccinated with BNT162b2 (1179.5 BAU/mL) or mRNA-1273 (2098.2 BAU/mL). Vaccine-dependent association of antibody levels was found for age with a significant predicted difference in BAU/ml per year for BNT162b2 (-21.5; 95% confidence interval [CI], -24.7 to -18.3) and no significant association for mRNA-1273 (-4.0; 95% CI, -20.0 to 12.1) or ChAdOx1 (1.7; 95% CI, .2 to 3.1). The predicted decrease over time since full immunization was highest in mRNA-1273 (-23.4; 95% CI, -31.4 to -15.4) compared with BNT162b2 (-5.9; 95% CI, -7 to -4.8). CONCLUSIONS: Our study revealed population-based evidence of vaccine-dependent effects of age and time since full immunization on humoral immune response. Findings underline the importance of individualized vaccine selection, especially in elderly individuals.

Heterologous vector versus homologous mRNA COVID-19 booster vaccination in non-seroconverted immunosuppressed patients: a randomized controlled trial.

Impaired response to COVID-19 vaccination is of particular concern in immunosuppressed patients. To determine the best vaccination strategy for this vulnerable group we performed a single center, 1:1 randomized blinded clinical trial. Patients who failed to seroconvert upon two mRNA vaccinations (BNT162b2 or mRNA-1273) are randomized to receive either a third dose of the same mRNA or the vector vaccine ChAdOx1 nCoV-19. Primary endpoint is the difference in SARS-CoV-2 spike antibody seroconversion rate between vector and mRNA vaccinated patients four weeks after the third dose. Secondary outcomes include cellular immune responses. Seroconversion rates at week four are significantly higher in the mRNA (homologous vaccination, 15/24, 63%) as compared to the vector vaccine group (heterologous vaccination, 4/22, 18%). SARS-CoV-2-specific T-cell responses are reduced but could be increased after a third dose of either vector or mRNA vaccine. In a multivariable logistic regression analysis, patient age and vaccine type are associated with seroconversion. No serious adverse event is attributed to COVID-19 booster vaccination. Efficacy and safety data underline the importance of a booster vaccination and support the use of a homologous mRNA booster vaccination in immunosuppressed patients.Trial registration: EudraCT No.: 2021-002693-10.

Immunogenicity and safety of a fourth COVID-19 vaccination in rituximab-treated patients: an open-label extension study.

OBJECTIVES: Patients under rituximab therapy are at high risk for a severe COVID-19 disease course. Humoral immune responses to SARS-CoV-2 vaccination are vastly diminished in B-cell-depleted patients, even after a third vaccine dose. However, it remains unclear whether these patients benefit from a fourth vaccination and whether continued rituximab therapy affects antibody development. METHODS: In this open-label extension trial, 37 rituximab-treated patients who received a third dose with either a vector or mRNA-based vaccine were vaccinated a fourth time with an mRNA-based vaccine (mRNA-1273 or BNT162b2). Key endpoints included the humoral and cellular immune response as well as safety after a fourth vaccination. RESULTS: The number of patients who seroconverted increased from 12/36 (33%) to 21/36 (58%) following the fourth COVID-19 vaccination. In patients with detectable antibodies to the spike protein's receptor-binding domain (median: 8.0 binding antibody units (BAU)/mL (quartiles: 0.4; 13.8)), elevated levels were observed after the fourth vaccination (134.0 BAU/mL (quartiles: 25.5; 1026.0)). Seroconversion and antibody increase were strongly diminished in patients who received rituximab treatment between the third and the fourth vaccination. The cellular immune response declined 12 weeks after the third vaccination, but could only be slightly enhanced by a fourth vaccination. No unexpected safety signals were detected, one serious adverse event not related to vaccination occurred. CONCLUSIONS: A fourth vaccine dose is immunogenic in a fraction of rituximab-treated patients. Continuation of rituximab treatment reduced humoral immune response, suggesting that rituximab affects a second booster vaccination. It might therefore be considered to postpone rituximab treatment in clinically stable patients. TRIAL REGISTRATION NUMBER: 2021-002348-57.

TNFR2 is critical for TNF-induced rheumatoid arthritis fibroblast-like synoviocyte inflammation.

OBJECTIVES: TNF-induced activation of fibroblast-like synoviocytes (FLS) is a critical determinant for synovial inflammation and joint destruction in RA. The detrimental role of TNF-receptor 1 (TNFR1) has thoroughly been characterized. The contributions of TNFR2, however, are largely unknown. This study was performed to delineate the role of TNFR2 in human FLS activation. METHODS: TNFR2 expression in synovial tissue samples was determined by immunohistochemistry. Expression of TNFR2 was silenced using RNAi or CRISPR/Cas9 technologies. Global transcriptional changes were determined by RNA-seq. QPCR, ELISA and immunoblotting were used to validate RNA-seq results and to uncover pathways operating downstream of TNFR2 in FLS. RESULTS: TNFR2 expression was increased in RA when compared with OA synovial tissues. In particular, RA-FLS demonstrated higher levels of TNFR2 when compared with OA-FLS. TNFR2 expression in RA-FLS correlated with RA disease activity, synovial T- and B-cell infiltration. TNF and IL1ß were identified as inflammatory mediators that upregulate TNFR2 in RA-FLS. Silencing of TNFR2 in RA-FLS markedly diminished the TNF-induced expression of inflammatory cytokines and chemokines, including CXCR3-binding chemokines and the B-cell activating factor TNFSF13B. Immunobiochemical analyses revealed that TNFR2-mediated expression of inflammatory mediators critically depends on STAT1. CONCLUSION: Our results define a critical role for TNFR2 in FLS-driven inflammation and unfold its participation in the unresolved course of synovial inflammation in RA.

Additional heterologous versus homologous booster vaccination in immunosuppressed patients without SARS-CoV-2 antibody seroconversion after primary mRNA vaccination: a randomised controlled trial.

OBJECTIVES: SARS-CoV-2-induced COVID-19 has led to exponentially rising mortality, particularly in immunosuppressed patients, who inadequately respond to conventional COVID-19 vaccination. METHODS: In this blinded randomised clinical trial, we compare the efficacy and safety of an additional booster vaccination with a vector versus mRNA vaccine in non-seroconverted patients. We assigned 60 patients under rituximab treatment, who did not seroconvert after their primary mRNA vaccination with either BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna), to receive a third dose, either using the same mRNA or the vector vaccine ChAdOx1 nCoV-19 (Oxford-AstraZeneca). Patients were stratified according to the presence of peripheral B cells. The primary efficacy endpoint was the difference in the SARS-CoV-2 antibody seroconversion rate between vector (heterologous) and mRNA (homologous) vaccinated patients by week 4. Key secondary endpoints included the overall seroconversion and cellular immune response; safety was assessed at week 1 and week 4. RESULTS: Seroconversion rates at week 4 were comparable between vector (6/27 patients, 22%) and mRNA (9/28, 32%) vaccines (p=0.6). Overall, 27% of patients seroconverted; specific T cell responses were observed in 20/20 (100%) vector versus 13/16 (81%) mRNA vaccinated patients. Newly induced humoral and/or cellular responses occurred in 9/11 (82%) patients. 3/37 (8%) of patients without and 12/18 (67%) of the patients with detectable peripheral B cells seroconverted. No serious adverse events, related to immunisation, were observed. CONCLUSIONS: This enhanced humoral and/or cellular immune response supports an additional booster vaccination in non-seroconverted patients irrespective of a heterologous or homologous vaccination regimen.

SARS-CoV-2 vaccination in rituximab-treated patients: B cells promote humoral immune responses in the presence of T-cell-mediated immunity.

OBJECTIVES: Evidence suggests that B cell-depleting therapy with rituximab (RTX) affects humoral immune response after vaccination. It remains unclear whether RTX-treated patients can develop a humoral and T-cell-mediated immune response against SARS-CoV-2 after immunisation. METHODS: Patients under RTX treatment (n=74) were vaccinated twice with either mRNA-1273 or BNT162b2. Antibodies were quantified using the Elecsys Anti-SARS-CoV-2 S immunoassay against the receptor-binding domain (RBD) of the spike protein and neutralisation tests. SARS-CoV-2-specific T-cell responses were quantified by IFN-γ enzyme-linked immunosorbent spot assays. Prepandemic healthy individuals (n=5), as well as healthy individuals (n=10) vaccinated with BNT162b2, served as controls. RESULTS: All healthy controls developed antibodies against the SARS-CoV-2 RBD of the spike protein, but only 39% of the patients under RTX treatment seroconverted. Antibodies against SARS-CoV-2 RBD significantly correlated with neutralising antibodies (τ=0.74, p<0.001). Patients without detectable CD19+ peripheral B cells (n=36) did not develop specific antibodies, except for one patient. Circulating B cells correlated with the levels of antibodies (τ=0.4, p<0.001). However, even patients with a low number of B cells (<1%) mounted detectable SARS-CoV-2-specific antibody responses. SARS-CoV-2-specific T cells were detected in 58% of the patients, independent of a humoral immune response. CONCLUSIONS: The data suggest that vaccination can induce SARS-CoV-2-specific antibodies in RTX-treated patients, once peripheral B cells at least partially repopulate. Moreover, SARS-CoV-2-specific T cells that evolved in more than half of the vaccinated patients may exert protective effects independent of humoral immune responses.

TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus12-14.