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Objectives: Methotrexate (MTX) is one of the most commonly used medications to treat rheumatoid arthritis (RA). However, the effect of MTX treatment on cellular immune responses remains incompletely understood. This raises concerns about the vulnerability of these patients to emerging infections and following vaccination., Methods: In the current study, we investigated the impact of MTX treatment in patients with immune-mediated inflammatory disease on B and CD4 T cell SARS-CoV-2 vaccination responses. Eighteen patients with RA and two patients with psoriatic arthritis on MTX monotherapy were included, as well as 10 patients with RA without immunosuppressive treatment, and 29 healthy controls. CD4 T and B cell responses were analysed 7 days and 3-6 months after two SARS-CoV-2 messenger RNA vaccinations. High-dimensional flow cytometry analysis was used to analyse fresh whole blood, an activation-induced marker assay to measure antigen-specific CD4 T cells, and spike probes to study antigen-specific B cells., Results: Seven days following two SARS-CoV-2 vaccinations, total B and T cell counts were similar between MTX-treated patients and controls. In addition, spike-specific B cell frequencies were unaffected. Remarkably, the frequency of antigen-specific CD4 T cells was reduced in patients using MTX and correlated strongly with anti-RBD IgG antibodies. These results suggest that decreased CD4 T cell activity may result in slower vaccination antibody responses in MTX-treated patients., Conclusion: Taken together, MTX treatment reduces vaccine-induced CD4 T cell activation, which correlates with lower antibody responses., Trial Registration Number: NL8900., Competing Interests: Competing interests: FE, GW, SMvH and TWK report (governmental) grants from ZonMw to study immune response after SARS-Cov-2 vaccination in auto-immune diseases. FE also reports grants from Prinses Beatrix Spierfonds, CSL Behring, Kedrion, Terumo BCT, Grifols, Takeda Pharmaceutical and GBS-CIDP Foundation; consulting fees from UCB Pharma and CSl Behring; honoraria from Grifols. All other authors report no disclosures relevant to the manuscript., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Despite impaired humoral response in patients treated with immunosuppressants (ISPs), recent studies found similar severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection compared to controls. One potential explanation is the rapid generation of humoral response on infection, but evidence is lacking., Objectives: We investigated the longitudinal dynamics of the SARS-CoV-2 antibody repertoire after SARS-CoV-2 delta and omicron breakthrough infection in patients with immune-mediated inflammatory diseases (IMIDs) receiving ISP therapy and controls., Methods: As a prospective substudy of the national Target-to-B! (T2B!) consortium, we included IMID patients receiving ISPs therapy and controls who reported SARS-CoV-2 breakthrough infection between July 1, 2021, and April 1, 2022. To get an impression of the dynamics of the antibody repertoire, 3 antibody titers of wild-type RBD, wild-type S, and omicron RBD were measured at 4 time points after SARS-CoV-2 breakthrough infection., Results: We included 302 IMID patients receiving ISPs and 178 controls. Antibody titers increased up to 28 days after breakthrough infection in both groups. However, in IMID patients receiving therapy with anti-CD20 and sphingosine-1 phosphate receptor modulators, antibody titers were considerably lower compared to controls. In the anti-TNF group, we observed slightly lower antibody titers in the early stages and a faster decline of antibodies after infection compared to controls. Breakthrough infections were mostly mild, and hospitalization was required in less than 1% of cases., Conclusions: Most ISPs do not influence the dynamics of the SARS-CoV-2 antibody repertoire and exhibit a rapid recall response with cross-reactive antibody clones toward new virus variants. However, in patients treated with anti-CD20 therapy or sphingosine-1 phosphate receptor modulators, the dynamics were greatly impaired, and to a lesser extent in those who received anti-TNF. Nevertheless, only a few severe breakthrough cases were reported., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: There are concerns for safety regarding SARS-CoV-2 vaccines for patients with autoimmune neuromuscular disease. We compared daily functioning using disease-specific patient-reported outcome measures (PROMs) before and after SARS-CoV-2 vaccinations., Methods: In this substudy of a prospective observational cohort study (Target-to-B!), patients with myasthenia gravis (MG), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), and idiopathic inflammatory myopathy (IIM) vaccinated against SARS-CoV-2 were included. Surveys of daily functioning (Myasthenia Gravis Activities of Daily Living, Inflammatory Rasch-Built Overall Disability Scale, Multifocal Motor Neuropathy Rasch-Built Overall Disability Scale, and Health Assessment Questionnaire-Disability Index) were sent before first vaccination and every 60 days thereafter for up to 12 months. Regression models were constructed to assess differences in PROM scores related to vaccination, compared to scores unrelated to vaccination. We also assessed the proportion of patients with deterioration of at least the minimal clinically important difference (MCID) between before first vaccination and 60 days thereafter., Results: We included 325 patients (median age = 59 years, interquartile range = 47-67, 156 [48%] female sex), of whom 137 (42%) had MG, 79 (24%) had CIDP, 43 (13%) had MMN, and 66 (20%) had IIM. PROM scores related to vaccination did not differ from scores unrelated to vaccination. In paired PROMs, MCID for deterioration was observed in three of 49 (6%) MG patients, of whom none reported a treatment change. In CIDP, MCID for deterioration was observed in eight of 29 patients (28%), of whom two of eight (25%) reported a treatment change., Conclusions: SARS-CoV-2 vaccination had no effect on daily functioning in patients with autoimmune neuromuscular diseases, confirming its safety in these patients., (© 2024 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Background: Messenger RNA (mRNA) vaccines provide robust protection against SARS-CoV-2 in healthy individuals. However, immunity after vaccination of patients with multiple sclerosis (MS) treated with ocrelizumab (OCR), a B cell-depleting anti-CD20 monoclonal antibody, is not yet fully understood., Methods: In this study, deep immune profiling techniques were employed to investigate the immune response induced by SARS-CoV-2 mRNA vaccines in untreated patients with MS (n=21), OCR-treated patients with MS (n=57) and healthy individuals (n=30)., Results: Among OCR-treated patients with MS, 63% did not produce detectable levels of antibodies (non-seroconverted), and those who did have lower spike receptor-binding domain-specific IgG responses compared with healthy individuals and untreated patients with MS. Before vaccination, no discernible immunological differences were observed between non-seroconverted and seroconverted OCR-treated patients with MS. However, non-seroconverted patients received overall more OCR infusions, had shorter intervals since their last OCR infusion and displayed higher OCR serum concentrations at the time of their initial vaccination. Following two vaccinations, non-seroconverted patients displayed smaller B cell compartments but instead exhibited more robust activation of general CD4 + and CD8 + T cell compartments, as indicated by upregulation of CD38 and HLA-DR surface expression, when compared with seroconverted patients., Conclusion: These findings highlight the importance of optimising treatment regimens when scheduling SARS-CoV-2 vaccination for OCR-treated patients with MS to maximise their humoral and cellular immune responses. This study provides valuable insights for optimising vaccination strategies in OCR-treated patients with MS, including the identification of CD38 and HLA-DR as potential markers to explore vaccine efficacy in non-seroconverting OCR-treated patients with MS., Competing Interests: Competing interests: JK received research grants for multicentre investigator-initiated trials (DOT-MS trial, ClinicalTrials.gov Identifier: NCT04260711 (ZonMw) and BLOOMS trial (ZonMw and Treatmeds), ClinicalTrials.gov Identifier: NCT05296161); received consulting fees from F Hoffmann-La Roche, Biogen, Teva, Merck, Novartis and Sanofi/Genzyme (all payments to institution); reports speaker relationships with F Hoffmann-La Roche, Biogen, Immunic, Teva, Merck, Novartis and Sanofi/Genzyme (all payments to institution); and adjudication committee of MS clinical trial of Immunic (payments to institution only)., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: The noninflammatory immunoglobulin G4 (IgG4) is linked to tolerance and is unique to humans. Although poorly understood, prolonged antigenic stimulation and IL-4-signaling along the T helper 2-axis may be instrumental in IgG4 class switching. Recently, repeated SARS-CoV-2 mRNA vaccination has been linked to IgG4 skewing. Although widely used immunosuppressive drugs have been shown to only moderately affect humoral responses to SARS-CoV-2 mRNA vaccination, the effect on IgG4 switching has not been investigated., Methods: Here we study the impact of such immunosuppressive drugs, including the IL-4 receptor-blocking antibody dupilumab, on IgG4 skewing upon repeated SARS-CoV-2 mRNA vaccination. Receptor-binding domain (RBD) specific antibody responses were longitudinally measured in 600 individuals, including patients with immune-mediated inflammatory diseases treated with a TNF inhibitor (TNFi) and/or methotrexate (MTX), dupilumab, and healthy/untreated controls, after repeated mRNA vaccination., Results: We observed a substantial increase in the proportion of RBD-specific IgG4 antibodies (median 21%) in healthy/untreated controls after third vaccination. This IgG4 skewing was profoundly reduced in dupilumab-treated patients (<1%). Unexpectedly, an equally strong suppression of IgG4 skewing was observed in TNFi-treated patients (<1%), whereas MTX caused a modest reduction (7%). RBD-specific total IgG levels were hardly affected by these immunosuppressive drugs. Minimal skewing was observed, when primary vaccination was adenoviral vector-based., Conclusions: Our results imply a critical role for IL-4/IL-13 as well as TNF in vivo IgG4 class switching. These novel findings advance our understanding of IgG4 class switch dynamics, and may benefit humoral tolerance induction strategies, treatment of IgG4 pathologies and mRNA vaccine optimization., (© 2024 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

SARS-CoV-2-specific CD8 + T cells recognize conserved viral peptides and in the absence of cross-reactive antibodies form an important line of protection against emerging viral variants as they ameliorate disease severity. SARS-CoV-2 mRNA vaccines induce robust spike-specific antibody and T cell responses in healthy individuals, but their effectiveness in patients with chronic immune-mediated inflammatory disorders (IMIDs) is less well defined. These patients are often treated with systemic immunosuppressants, which may negatively affect vaccine-induced immunity. Indeed, TNF inhibitor (TNFi)-treated inflammatory bowel disease (IBD) patients display reduced ability to maintain SARS-CoV-2 antibody responses post-vaccination, yet the effects on CD8 + T cells remain unclear. Here, we analyzed the impact of IBD and TNFi treatment on mRNA-1273 vaccine-induced CD8 + T cell responses compared to healthy controls in SARS-CoV-2 experienced and inexperienced patients. CD8 + T cells were analyzed for their ability to recognize 32 SARS-CoV-2-specific epitopes, restricted by 10 common HLA class I allotypes using heterotetramer combinatorial coding. This strategy allowed in-depth ex vivo profiling of the vaccine-induced CD8 + T cell responses using phenotypic and activation markers. mRNA vaccination of TNFi-treated and untreated IBD patients induced robust spike-specific CD8 + T cell responses with a predominant central memory and activated phenotype, comparable to those in healthy controls. Prominent non-spike-specific CD8 + T cell responses were observed in SARS-CoV-2 experienced donors prior to vaccination. Non-spike-specific CD8 + T cells persisted and spike-specific CD8 + T cells notably expanded after vaccination in these patient cohorts. Our data demonstrate that regardless of TNFi treatment or prior SARS-CoV-2 infection, IBD patients benefit from vaccination by inducing a robust spike-specific CD8 + T cell response., Competing Interests: Declaration of competing interest No conflict of interest to report., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Humoral responses after SARS-CoV-2 vaccination are greatly impaired in multiple sclerosis (MS) patients on fingolimod. Effects of repeated vaccination and infections on long-term responses are unclear., Methods: Prospective study in 60 MS patients on fingolimod measuring humoral responses after up to four vaccinations and 8 months after fourth vaccination., Results: Anti-WH1 antibody titers increased with each additional vaccination. At long-term follow-up titers increased further and most patients developed new humoral responses against the BA.1 omicron variant., Conclusion: Repeated SARS-CoV-2 vaccinations boost humoral immunity and, probably together with SARS-CoV-2 infections, induce humoral responses on the long-term in almost all patients., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: FE and TK report (governmental) grants from ZonMw to study immune response after SARS-CoV-2 vaccination in auto-immune diseases. FE also reports grants from Prinses Beatrix Spierfonds, CSL Behring, Kedrion, Terumo BCT, Grifols, Takeda Pharmaceutical Company, and GBS-CIDP Foundation; consulting fees from UCB Pharma and CSL Behring; honoraria from Grifols. JK has speaking relationships with Merck Serono, Biogen Idec, TEVA, Sanofi, Genzyme, Roche, and Novartis; Amsterdam UMC, location VUmc, MS Center Amsterdam has received financial support for research activities from Merck Serono, Bayer Schering Pharma, Biogen Idec, GlaxoSmithKline, Roche, Teva, Sanofi, Genzyme, GlaxoSmithKline, and Novartis. No other disclosures were reported.

Background: Rheumatoid factors (RFs) are autoantibodies that target the Fc region of IgG, and are found in patients with rheumatic diseases as well as in the healthy population. Many studies suggest that an immune trigger may (transiently) elicit RF responses. However, discrepancies between different studies make it difficult to determine if and to which degree RF reactivity can be triggered by vaccination or infection., Objective: We quantitatively explored longitudinal RF responses after SARS-CoV-2 vaccination and infection in a well-defined, large cohort using a dual ELISA method that differentiates between true RF reactivity and background IgM reactivity. In addition, we reviewed existing literature on RF responses after vaccination and infection., Methods: 151 healthy participants and 30 RA patients were included to measure IgM-RF reactivity before and after SARS-CoV-2 vaccinations by ELISA. Additionally, IgM-RF responses after a SARS-CoV-2 breakthrough infection were studied in 51 healthy participants., Results: Published prevalence studies in subjects after infection report up to 85% IgM-RF seropositivity. However, seroconversion studies (both infection and vaccination) report much lower incidences of 2-33%, with a trend of lower percentages observed in larger studies. In the current study, SARS-CoV-2 vaccination triggered low-level IgM-RF responses in 5.5% (8/151) of cases, of which 1.5% (2/151) with a level above 10 AU/mL. Breakthrough infection was accompanied by development of an IgM-RF response in 2% (1/51) of cases., Conclusion: Our study indicates that de novo RF induction following vaccination or infection is an uncommon event, which does not lead to RF epitope spreading., Competing Interests: TR and GW are inventors on a patent application based on the use of bioengineered IgG targets for the characterization of rheumatoid factor reactivity patterns. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Keijzer, Oskam, Ooijevaar-de Heer, Steenhuis, Keijser, Wieske, van Dam, Stalman, Kummer, Boekel, Kuijpers, ten Brinke, van Ham, Eftimov, Tas, Wolbink and Rispens.)

γδ T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are γδ T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than γδ T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of Vδ2 + T cells, while Vδ1 + T cells likewise have anti-tumor potential. Here, we investigated whether γδ T cells directly isolated from blood could be efficiently expanded while maintaining function. γδ T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure γδ T cells. Isolated Vδ1 + and Vδ2 + T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. MACS/FACS isolated and PHA stimulated γδ T cells expanded as good as immobilized antibody mediated stimulated cells in PBMCs, but delivered purer cells. After expansion, potential effector functions of γδ T cells were demonstrated by IFN-γ, TNF-α and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure γδ T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, γδ T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Verkerk, Pappot, Jorritsma, King, Duurland, Spaapen and van Ham.)

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences.

Background: CD11c + Tbet + B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c + Tbet + B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity., Objectives: We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c + B-cell subsets-age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells-and compared them to their canonical CD11c - counterparts., Methods: Dynamics of the response of the 3 CD11c + B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c + B-cell subsets were functionally characterized by optimized in vitro cultures., Results: In contrast to a durable expansion of antigen-specific CD11c - memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c + Tbet + B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c - counterparts., Conclusion: Overall, CD11c + Tbet + B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre-antibody-secreting cell phenotype., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressants (ISPs) may have impaired long-term humoral immune responses and increased disease activity after SARS-CoV-2 infection. We aimed to investigate long-term humoral immune responses against SARS-CoV-2 and increased disease activity after a primary SARS-CoV-2 infection in unvaccinated IMID patients on ISPs., Methods: IMID patients on active treatment with ISPs and controls (i.e. IMID patients not on ISP and healthy controls) with a confirmed SARS-CoV-2 infection before first vaccination were included from an ongoing prospective cohort study (T2B! study). Clinical data on infections and increased disease activity were registered using electronic surveys and health records. A serum sample was collected before first vaccination to measure SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies., Results: In total, 193 IMID patients on ISP and 113 controls were included. Serum samples from 185 participants were available, with a median time of 173 days between infection and sample collection. The rate of seropositive IMID patients on ISPs was 78% compared to 100% in controls (p < 0.001). Seropositivity rates were lowest in patients on anti-CD20 (40.0%) and anti-tumor necrosis factor (TNF) agents (60.5%), as compared to other ISPs (p < 0.001 and p < 0.001, respectively). Increased disease activity after infection was reported by 68 of 260 patients (26.2%; 95% CI 21.2-31.8%), leading to ISP intensification in 6 out of these 68 patients (8.8%)., Conclusion: IMID patients using ISPs showed reduced long-term humoral immune responses after primary SARS-CoV-2 infection, which was mainly attributed to treatment with anti-CD20 and anti-TNF agents. Increased disease activity after SARS-CoV-2 infection was reported commonly, but was mostly mild., Trial Registration: NL74974.018.20, Trial ID: NL8900. Registered on 9 September 2020., (© 2023. The Author(s).)

Background: It is unclear which patients with multiple sclerosis (MS) are most susceptible for omicron breakthrough infections., Methods: We assessed omicron breakthrough infections in vaccinated patients with MS with and without disease-modifying therapies enrolled in an ongoing large prospective study. We longitudinally studied humoral responses after primary and booster vaccinations and breakthrough infections., Results: Omicron breakthrough infections were reported in 110/312 (36%) patients with MS, and in 105/110 (96%) infections were mild. Omicron breakthrough infections occurred more frequently in patients treated with anti-CD20 therapies and sphingosine-1 phosphate receptor (S1PR) modulators, patients with impaired humoral responses after primary immunisation (regardless of treatment) and patients without prior SARS-CoV-2 infections. After infection, antibody titres increased in patients on S1PR modulator treatment while anti-CD20 treated patients did not show an increase., Conclusions: SARS-COV-2 omicron breakthrough infections are more prevalent in patients with MS on anti-CD20 therapies and S1PR modulators compared with other patients with MS, which correlated with decreased humoral responses after vaccination. Humoral responses after infection were higher in S1PR modulator-treated patients in comparison to patients on anti-CD20 therapies, suggesting that immunological protection from contracting infection or repeated exposures may differ between these therapies., Competing Interests: Competing interests: JK reported speaking and consulting relationships with Biogen, Genzyme, Merck, Novartis, Roche, Sanofi and TEVA. Amsterdam UMC, location VUmc, MS Center Amsterdam has received financial support for research activities from Biogen, Celgene, Genzyme, Merck, Novartis, Roche, Sanofi and TEVA. TR reports consulting fees from Novartis. No other disclosures were reported., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions., Competing Interests: NV, SP, PU, CM, GE, TJ, MS, KB, KH, TR, JS, At, MB, Sv No competing interests declared, (© 2023, Verstegen, Pollastro et al.)

Ocrelizumab, an anti-CD20 monoclonal antibody, counteracts induction of humoral immune responses after severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccinations in patients with multiple sclerosis (MS). We aimed to assess if serum ocrelizumab concentration measured at the time of vaccination could predict the humoral response after SARS-CoV-2 vaccination. In 52 patients with MS, we found ocrelizumab concentration at the time of vaccination to be a good predictor for SARS-CoV-2 IgG anti-RBD titers after vaccination (comparable to B-cell count). As the course of ocrelizumab concentration may be predicted using pharmacokinetic models, this may be a superior biomarker to guide optimal timing for vaccinations in B-cell depleted patients with MS. ANN NEUROL 2023;93:103-108., (© 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

The generation of high-affinity antibodies requires an efficient germinal center (GC) response. As differentiating B cells cycle between GC dark and light zones they encounter different oxygen pressures ( p O 2 ). However, it is essentially unknown if and how variations in p O 2 affect B cell differentiation, in particular for humans. Using optimized in vitro cultures together with in-depth assessment of B cell phenotype and signaling pathways, we show that oxygen is a critical regulator of human naive B cell differentiation and class switch recombination. Normoxia promotes differentiation into functional antibody secreting cells, while a population of CD27 ++ B cells was uniquely generated under hypoxia. Moreover, time-dependent transitions between hypoxic and normoxic p O 2 during culture - reminiscent of in vivo GC cyclic re-entry - steer different human B cell differentiation trajectories and IgG class switch recombination. Taken together, we identified multiple mechanisms trough which oxygen pressure governs human B cell differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Koers, Marsman, Steuten, Tol, Derksen, ten Brinke, van Ham and Rispens.)

Objectives: To compare the cumulative incidence and disease severity of reported SARS-CoV-2 omicron breakthrough infections between patients with immune-mediated inflammatory diseases (IMID) on immunosuppressants and controls, and to investigate determinants for breakthrough infections., Methods: Data were used from an ongoing national prospective multicentre cohort study on SARS-CoV-2 vaccination responses in patients with IMID in the Netherlands (Target-to-B! (T2B!) study). Patients wih IMID on immunosuppressants and controls (patients with IMID not on immunosuppressants and healthy controls) who completed primary immunisation were included. The observation period was between 1 January 2022 and 1 April 2022, during which the SARS-CoV-2 omicron (BA.1 and BA.2 subvariant) was dominant. A SARS-CoV-2 breakthrough infection was defined as a reported positive PCR and/or antigen test at least 14 days after primary immunisation. A multivariate logistic regression model was used to investigate determinants., Results: 1593 patients with IMID on immunosuppressants and 579 controls were included. The cumulative incidence of breakthrough infections was 472/1593 (29.6%; 95% CI 27% to 32%) in patients with IMID on immunosuppressants and 181/579 (31.3%; 95% CI 28% to 35%) in controls (p=0.42). Three (0.5%) participants had severe disease. Seroconversion after primary immunisation (relative risk, RR 0.71; 95% CI 0.52 to 0.96), additional vaccinations (RR 0.61; 95% CI 0.49 to 0.76) and a prior SARS-CoV-2 infection (RR 0.60; 95% CI 0.48 to 0.75) were associated with decreased risk of breakthrough infection., Conclusions: The cumulative incidence of reported SARS-CoV-2 omicron breakthrough infections was high, but similar between patients with IMID on immunosuppressants and controls, and disease severity was mostly mild. Additional vaccinations and prior SARS-CoV-2 infections may reduce the incidence of breakthrough infections., Competing Interests: Competing interests: FE and TWK report (governmental) grants from ZonMw to studyimmune response after SARS-Cov-2 vaccination in autoimmune diseases.FE also reports grants from Prinses Beatrix Spierfonds, CSL Behring,Kedrion, Terumo BCT, Grifols, Takeda Pharmaceutical Company, andGBS-CIDP Foundation; consulting fees from UCB Pharma and CSlBehring; and honoraria from Grifols. AJvdK reports grants from CSLBehring and participation on an advisory board for Argen-X. ML reports agrant from Galapagos not related to this study, and honoraria from BristolMyers Squibb, Pfizer, Takeda, and Tillotts. PIS is involved in clinical trialswith many pharmaceutical industries that manufacture drugs used for thetreatment of, for example, psoriasis and atopic dermatitis, for whichfinancial compensation is paid to the department or hospital, and is achief investigator of the TREAT NL registry taskforce and SECURE-ADregistry. MWB is a secretary for the Dutch Experimental DermatologyBoard; head of the pigmentary disorders group within the DutchDermatology Board; and reports honoraria from Pfizer, Sanofi, Novartis,and Fondation René Touraine. JK has speaking relationships with MerckSerono, Biogen Idec, TEVA, Sanofi, Genzyme, Roche, and Novartis;received financial support to his institution for researchactivities from Merck Serono, Bayer Shcering Pharma, Biogen Idec,GlaxoSmithKline (GSK), Roche, Teva, Sanofi, Genzyme, and Novartis. BHreports unpaid positions as a medical adviser for several patient groups, aboard position for ERN-SKIN, and associate editor for The British Journalof Dermatology; reports grants from AbbVIe, Akari Therapeutics, Celgene, and Novartis; consulting fees from UCB Pharma, Novartis, and Janssen; and honoraria from AbbVie. JJGMV reports consulting fees from Argenx,Alexion, and NMD Pharma, and is a co-inventor on patent applicationsbased on MuSK-related research. DJH reportsgrants from AbbVie, AstraZeneca, Janssen, LEO Pharma, and UCB;honoraria from AbbVie, Galderma, Janssen, Lilly, Pfizer, Sanofi, and UCB;and a paid position on an advisory board for BIOMAP IMI. PAvDparticipated on an advisory board for Octapharma. PvP reports grantsfrom Alexion Pharma and GSK, and participation on advisory boards forGSK and Vifor Pharma. GRAMD’H reports consulting fees from AbbVie,Agomab, AstraZeneca, AM Pharma, AMT, Arena Pharmaceuticals, BristolMyers Squibb, Boehringer Ingelheim, Celltrion, Eli Lilly, ExeliomBiosciences, Exo Biologics, Galapagos, Index Pharmaceuticals, Kaleido,Roche, Gilead, GSK, Gossamerbio, Pfizer, Immunic, Johnson andJohnson, Origo, Polpharma, Procise Diagnostics, PrometheusLaboratories, Prometheus Biosciences, Progenity, and Protagonist;honoraria from AbbVie, Arena, Galapagos, Gilead, Pfizer, Bristol MyersSquibb, and Takeda; and participation on advisory boards for AbbVie,Seres Health, Galapagos, and AstraZeneca. RBT reports honoraria fromSobi and Norgine, and participation on an advisory board for Norgine.SHG is a board member of the Dutch Society of Clinical Neurophysiology(unpaid), reports grants from Prinses Beatrix Spierfonds, and receivedspeaker fees from Shire/Takeda. KAHZ reports paid data safetymonitoring board positions for Torrent and Foresee. All other authorsdeclare no competing interests., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: During the COVID-19 pandemic, certain disease modifying therapies (DMTs) used in multiple sclerosis (MS), such as anti-CD20 therapies, have been associated with decreased humoral responses after SARS-CoV-2 vaccination. Hybrid immunity, referring to immunity after both vaccination and SARS-CoV-2 infection might increase humoral responses., Methods: This was a substudy of two prospective cohort studies on SARS-CoV-2 antibodies after SARS-CoV-2 infection and vaccination. RBD-specific IgG titers of patients with MS and healthy controls who had experienced SARS-CoV-2 infection prior to the first vaccination were compared with those patients and healthy controls without prior infection. Humoral responses were measured at various time points after SARS-CoV-2 infection in convalescent patients and all patients prior to the first vaccination, 28 days after the first vaccination, and 28 days after the second vaccination., Results: One hundred and two individuals [of which 34 patients with MS and DMTs (natalizumab or ocrelizumab), 30 patients without DMTs, and 38 healthy controls] were included. Fifty one of these individuals were convalescent. Median SARS-CoV-2 antibody titers were higher after the first vaccination in convalescent individuals compared with individuals without infection prior to vaccination. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers were comparable after the second vaccination in patients with MS with and without prior infection. However, in the convalescent ocrelizumab-treated patients, SARS-CoV-2 antibody titers did not increase after vaccinations., Conclusion: In patients with MS without anti-CD20 therapies, SARS-CoV-2 infection before vaccination increases humoral responses after the first vaccination, similar to the healthy controls. In patients with MS treated with ocrelizumab (convalescent and non-convalescent), humoral responses remained low., Competing Interests: FE reports consulting fees from UCB Pharma and CSl Behring; honoraria from Grifols. TR reports consulting fees from Novartis. Jki reported speaking and consulting relationships with Biogen, Genzyme, Merck, Novartis, Roche, Sanofi, and TEVA. Amsterdam UMC, location VUmc, MS Center Amsterdam has received financial support for research activities from Biogen, Celgene, Genzyme, Merck, Novartis, Roche, Sanofi, and TEVA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van Dam, Hogenboom, Stalman, Kummer, Steenhuis, Keijser, Brinke, van Ham, Kuijpers, Rispens, Wieske, Eftimov, Strijbis, Killestein and van Kempen.)

Objectives: High-magnitude CD8 + T cell responses are associated with mild COVID-19 disease; however, the underlying characteristics that define CD8 + T cell-mediated protection are not well understood. The antigenic breadth and the immunodominance hierarchies of epitope-specific CD8 + T cells remain largely unexplored and are essential for the development of next-generation broad-protective vaccines. This study identified a broad spectrum of conserved SARS-CoV-2 CD8 + T cell epitopes and defined their respective immunodominance and phenotypic profiles following SARS-CoV-2 infection., Methods: CD8 + T cells from 51 convalescent COVID-19 donors were analysed for their ability to recognise 133 predicted and previously described SARS-CoV-2-derived peptides restricted by 11 common HLA class I allotypes using heterotetramer combinatorial coding, which combined with phenotypic markers allowed in-depth ex vivo profiling of CD8 + T cell responses at quantitative and phenotypic levels., Results: A comprehensive panel of 49 mostly conserved SARS-CoV-2-specific CD8 + T cell epitopes, including five newly identified low-magnitude epitopes, was established. We confirmed the immunodominance of HLA-A*01:01/ORF1ab 1637-1646 and B*07:02/N 105-113 and identified B*35:01/N 325-333 as a third epitope with immunodominant features. The magnitude of subdominant epitope responses, including A*03:01/N 361-369 and A*02:01/S 269-277 , depended on the donors' HLA-I context. All epitopes expressed prevalent memory phenotypes, with the highest memory frequencies in severe COVID-19 donors., Conclusion: SARS-CoV-2 infection induces a predominant CD8 + T memory response directed against a broad spectrum of conserved SARS-CoV-2 epitopes, which likely contributes to long-term protection against severe disease. The observed immunodominance hierarchy emphasises the importance of T cell epitopes derived from nonspike proteins to the overall protective and cross-reactive immune response, which could aid future vaccine strategies., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Human naïve B cells are notoriously difficult to differentiate into antibody-secreting cells (ASCs) in vitro while maintaining sufficient cell numbers to evaluate the differentiation process. B cells require T follicular helper (T FH ) cell-derived signals like CD40L and IL-21 during germinal center (GC) responses to undergo differentiation into ASCs. Cognate interactions between B and T FH cells are transient; after T FH contact, B cells cycle between GC light and dark zones where T FH contact is present and absent, respectively. Here, we elucidated that the efficacy of naïve B cells in ACS differentiation is dramatically enhanced by the release of CD40L stimulation. Multiparameter phospho-flow and transcription factor (TF)-flow cytometry revealed that termination of CD40L stimulation downmodulates NF-κB and STAT3 signaling. Furthermore, the termination of CD40 signaling downmodulates C-MYC, while promoting ASC TFs BLIMP1 and XBP-1s. Reduced levels of C-MYC in the differentiating B cells are later associated with crucial downmodulation of the B cell signature TF PAX5 specifically upon the termination of CD40 signaling, resulting in the differentiation of BLIMP1 high expressing cells into ASCs. The data presented here are the first steps to provide further insights how the transient nature of CD40 signaling is in fact needed for efficient human naïve B cell differentiation to ASCs., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy.

Background: Patients affected by different types of autoimmune diseases, including common conditions such as multiple sclerosis (MS) and rheumatoid arthritis (RA), are often treated with immunosuppressants to suppress disease activity. It is not fully understood how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and cellular immunity induced by infection and/or upon vaccination is affected by immunosuppressants., Methods: The dynamics of cellular immune reactivation upon vaccination of SARS-CoV-2 experienced MS patients treated with the humanized anti-CD20 monoclonal antibody ocrelizumab (OCR) and RA patients treated with methotrexate (MTX) monotherapy were analyzed at great depth via high-dimensional flow cytometry of whole blood samples upon vaccination with the SARS-CoV-2 mRNA-1273 (Moderna) vaccine. Longitudinal B and T cell immune responses were compared to SARS-CoV-2 experienced healthy controls (HCs) before and 7 days after the first and second vaccination., Results: OCR-treated MS patients exhibit a preserved recall response of CD8 + T central memory cells following first vaccination compared to HCs and a similar CD4 + circulating T follicular helper 1 and T helper 1 dynamics, whereas humoral and B cell responses were strongly impaired resulting in absence of SARS-CoV-2-specific humoral immunity. MTX treatment significantly delayed antibody levels and B reactivation following the first vaccination, including sustained inhibition of overall reactivation marker dynamics of the responding CD4 + and CD8 + T cells., Conclusions: Together, these findings indicate that SARS-CoV-2 experienced MS-OCR patients may still benefit from vaccination by inducing a broad CD8 + T cell response which has been associated with milder disease outcome. The delayed vaccine-induced IgG kinetics in RA-MTX patients indicate an increased risk after the first vaccination, which might require additional shielding or alternative strategies such as treatment interruptions in vulnerable patients., Funding: This research project was supported by ZonMw (The Netherlands Organization for Health Research and Development, #10430072010007), the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#792532 and #860003), the European Commission (SUPPORT-E, #101015756) and by PPOC (#20&#95;21 L2506), the NHMRC Leadership Investigator Grant (#1173871)., Competing Interests: NV, RH, Jv, LK, CK, TA, LK, MS, MD, Rd, Nd, Cv, AB, EM, KK, Kv, ES, LB, GW, ST, Zv, LW, TK, FE, TR, Sv, At, Cv No competing interests declared, JK has speaking relationships with Merck, Biogen, TEVA, Sanofi, Genzyme, Roche and Novartis. AmsterdamUMC, location VUmc, MS Center Amsterdam has received financial support for research activities from Merck, Celgene, Biogen, GlaxoSmithKline, Immunic, Roche, Teva, Sanofi, Genzyme, and Novartis, (© 2022, Verstegen et al.)

Background/methods: For mechanistic studies, in-vitro human B-cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T-cell-dependent (TD) and T-cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols make the interpretation of results challenging. The aim of the present study was to achieve the most optimal B-cell differentiation conditions using isolated CD19 + B cells and peripheral blood mononuclear cell (PBMC) cultures. We addressed multiple seeding densities, different durations of culturing, and various combinations of TD and TI stimuli including B-cell receptor (BCR) triggering. B-cell expansion, proliferation, and differentiation were analyzed after 6 and 9 days by measuring B-cell proliferation and expansion, plasmablast and plasma cell formation, and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared., Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B-cells and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2,500 and 25,000 B-cells per culture well for the TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B-cell cultures, which allows dismissal of additional B-cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed a little effect on phenotypic B-cell differentiation; however, it interferes with Ig secretion measurements. The addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B-cell differentiation and Ig secretion in B-cell but not in PBMC cultures. With this approach, efficient B-cell differentiation and Ig secretion were accomplished when starting from fresh or cryopreserved samples., Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more in-depth analysis of B-cell differentiation in primary human B-cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B-cell differentiation of patient samples from different cohorts of B-cell-mediated diseases., Competing Interests: The authors declare that this research was conducted in the absence of commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor JG declared a shared affiliation with the authors DV and TK., (Copyright © 2022 Marsman, Verhoeven, Koers, Rispens, ten Brinke, van Ham and Kuijpers.)

Background: Concerns have been raised regarding the risks of SARS-CoV-2 breakthrough infections in vaccinated patients with immune-mediated inflammatory diseases treated with immunosuppressants, but clinical data on breakthrough infections are still scarce. The primary objective of this study was to compare the incidence and severity of SARS-CoV-2 breakthrough infections between patients with immune-mediated inflammatory diseases using immunosuppressants, and controls (patients with immune-mediated inflammatory diseases not taking immunosuppressants and healthy controls) who had received full COVID-19 vaccinations. The secondary objective was to explore determinants of breakthrough infections of the delta (B.1.617.2) variant of SARS-CoV-2, including humoral immune responses after vaccination., Methods: In this substudy, we pooled data collected in two large ongoing prospective multicentre cohort studies conducted in the Netherlands (Target to-B! [T2B!] study and Amsterdam Rheumatology Center COVID [ARC-COVID] study). Both studies recruited adult patients (age ≥18 years) with immune-mediated inflammatory diseases and healthy controls. We sourced clinical data from standardised electronic case record forms, digital questionnaires, and medical files. We only included individuals who were vaccinated against SARS-CoV-2. For T2B!, participants were recruited between Feb 2 and Aug 1, 2021, and for ARC-COVID, participants were recruited between April 26, 2020, and March 1, 2021. In this study we assessed data on breakthrough infections collected between July 1 and Dec 15, 2021, a period in which the delta SARS-CoV-2 variant was the dominant variant in the Netherlands. We defined a SARS-CoV-2 breakthrough infection as a PCR-confirmed or antigen test-confirmed SARS-CoV-2 infection that occurred at least 14 days after vaccination. All breakthrough infections during this period were assumed to be due to the delta variant due to its dominance during the study period. We analysed post-vaccination serum samples for anti-receptor binding domain (RBD) antibodies to assess the humoral vaccination response (T2B! study only) and anti-nucleocapsid antibodies to identify asymptomatic breakthrough infections (ARC-COVID study only). We used multivariable logistic regression analyses to explore potential clinical and humoral determinants associated with the odds of breakthrough infections. The T2B! study is registered with the Dutch Trial Register, Trial ID NL8900, and the ARC-COVID study is registered with Dutch Trial Register, trial ID NL8513., Findings: We included 3207 patients with immune-mediated inflammatory diseases who receive immunosuppressants, and 1807 controls (985 patients with immune-mediated inflammatory disease not on immunosuppressants and 822 healthy controls). Among patients receiving immunosuppressants, mean age was 53 years (SD 14), 2042 (64%) of 3207 were female and 1165 (36%) were male; among patients not receiving immunosuppressants, mean age was 54 years (SD 14), 598 (61%) of 985 were female and 387 (39%) were male; and among healthy controls, mean age was 57 years (SD 13), 549 (67%) of 822 were female and 273 (33%) were male. The cumulative incidence of PCR-test or antigen-test confirmed SARS-CoV-2 breakthrough infections was similar in patients on immunosuppressants (148 of 3207; 4·6% [95% CI 3·9-5·4]), patients not on immunosuppressants (52 of 985; 5·3% [95% CI 4·0-6·9]), and healthy controls (33 of 822; 4·0% [95% CI 2·8-5·6]). There was no difference in the odds of breakthrough infection for patients with immune-mediate inflammatory disease on immunosuppressants versus combined controls (ie, patients not on immunosuppressants and healthy controls; adjusted odds ratio 0·88 [95% CI 0·66-1·18]). Seroconversion after vaccination (odds ratio 0·58 [95% CI 0·34-0·98]; T2B! cohort only) and SARS-CoV-2 infection before vaccination (0·34 [0·18-0·56]) were associated with a lower odds of breakthrough infections., Interpretation: The incidence and severity of SARS-CoV-2 breakthrough infections in patients with immune-mediated inflammatory diseases on immunosuppressants was similar to that in controls. However, caution might still be warranted for those on anti-CD20 therapy and those with traditional risk factors., Funding: ZonMw (the Netherlands Organization for Health Research and Development) and Reade foundation., Competing Interests: FE and TWK report (governmental) grants from ZonMw (the Netherlands Organization for Health Research and Development) to study immune responses after SARS-CoV-2 vaccination in autoimmune diseases. FE also reports grants from Prinses Beatrix Spierfonds, CSL Behring, Kedrion, Terumo BCT, Grifols, Takeda Pharmaceutical Company, and Guillain-Barré Syndrome-Chronic Inflammatory Demyelinating Polyneuropathy (GBS-CIDP) Foundation; consulting fees from UCB Pharma and CSl Behring; and honoraria from Grifols. AJvdK reports grants from CSL Behring and participation on an advisory board for Argen-X. MLö reports a grant from Galapagos NV not related to this study, and honoraria from Bristol Myers Squibb, Pfizer, Takeda, and Tillotts. PIS is involved in clinical trials with Regeneron, Sanofi, Leopharma, Lilly, AbbVie, Boerhinger, Celgene, Janssen, and UCB, which manufacture drugs used for the treatment of conditions, including psoriasis and atopic dermatitis, for which financial compensation is paid to their department or hospital, and is a chief investigator of the TREAT NL registry taskforce and SECURE-AD registry. MWB is a secretary for the Dutch Experimental Dermatology Board; head of the pigmentary disorders group within the Dutch Dermatology Board; and reports honoraria from Pfizer, Sanofi, Novartis, and Fondation René Touraine. JKi has consulting relationships with Merck Serono, Biogen Idec, Teva, Genzyme, Sanofi, Roche, and Novartis; Amsterdam UMC, location VUmc, MS Center Amsterdam has received financial support for research activities from Merck, Celgene, Biogen, GlaxoSmithKline, Immunic, Roche, Teva, Sanofi, Genzyme, and Novartis. BH reports unpaid positions as a medical adviser for several patient groups, a board position for European Reference Network for rare skin diseases (ERN-Skin), and associate editor for The British Journal of Dermatology; reports grants from AbbVIe, Akari Therapeutics, Celgene, and Novartis; consulting fees from UCB Pharma, Novartis, and Janssen; and honoraria from AbbVie. JJGMV reports consulting fees from Argen-X, Alexion, and NMD Pharma, and is a co-inventor on a patent applications based on MuSK-related research (patent number 9574015). DJH reports grants from AbbVie, AstraZeneca, Janssen, LEO Pharma, and UCB; honoraria from AbbVie, Galderma, Janssen, Lilly, Pfizer, Sanofi, and UCB; and a paid position on an advisory board for Biomarkers in Atopic Dermatitis and Psoriasis (BIOMAP IMI). PAvD has participated on an advisory board for Octapharma. PvP reports grants from Alexion Pharma and GSK, and participation on advisory boards for GSK and Vifor Pharma. GRAMD reports consulting fees from AbbVie, Agomab, AstraZeneca, AM Pharma, AMT, Arena Pharmaceuticals, Bristol Myers Squibb, Boehringer Ingelheim, Celltrion, Eli Lilly, Exeliom Biosciences, Exo Biologics, Galapagos, Index Pharmaceuticals, Kaleido, Roche, Gilead, GSK, Gossamerbio, Pfizer, Immunic, Johnson & Johnson, Origo, Polpharma, Procise Diagnostics, Prometheus Laboratories, Prometheus Biosciences, Progenity, and Protagonist; honoraria from AbbVie, Arena, Galapagos NV, Gilead, Pfizer, Bristol Myers Squibb, and Takeda; and participation on advisory boards for AbbVie, Seres Health, Galapagos NV, and AstraZeneca. RBT reports honoraria from Sobi and Norgine and participation on an advisory board for Norgine. HSG is a board member of the Dutch Society of Clinical Neurophysiology (unpaid), reports grants from Prinses Beatrix Spierfonds, and has received speaker fees from Shire/Takeda. KAHZ reports paid data safety monitoring board positions for Torrent and Foresee. All other authors declare no competing interests., (© 2022 Published by Elsevier Ltd.)

Objectives: To evaluate whether a third vaccination shows an added effect on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T-cell responses in patients with multiple sclerosis treated with ocrelizumab or fingolimod., Methods: This is a substudy of a prospective multicenter study on SARS-CoV-2 vaccination in patients with immune-mediated diseases. Patients with MS treated with ocrelizumab, fingolimod, and no disease-modifying therapies and healthy controls were included. The number of interferon (IFN)-γ secreting SARS-CoV-2-specific T cells at multiple time points before and after 3 SARS-CoV-2 vaccinations were evaluated., Results: In ocrelizumab-treated patients (N = 24), IFN-γ-producing SARS-CoV-2-specific T-cell responses were induced after 2 vaccinations with median levels comparable to healthy controls (N = 12) and patients with MS without disease-modifying therapies (N = 10). A third vaccination in ocrelizumab-treated patients (N = 8) boosted T-cell responses that had declined after the second vaccination, but did not lead to higher overall T-cell responses as compared to immediately after a second vaccination. In fingolimod-treated patients, no SARS-CoV-2-specific T cells were detected after second (N = 12) and third (N = 9) vaccinations., Discussion: In ocrelizumab-treated patients with MS, a third SARS-CoV-2 vaccination had no additive effect on the maximal T-cell response but did induce a boost response. In fingolimod-treated patients, no T-cell responses could be detected following both a second and third SARS-CoV-2 vaccination., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: Disease-specific studies have reported impaired humoral responses after SARS-CoV-2 vaccination in patients with immune-mediated inflammatory disorders treated with specific immunosuppressants. Disease-overarching studies, and data on recall responses and third vaccinations are scarce. Our primary objective was to investigate the effects of immunosuppressive monotherapies on the humoral immune response after SARS-CoV-2 vaccination in patients with prevalent immune-mediated inflammatory disorders., Methods: We did a cohort study in participants treated in outpatient clinics in seven university hospitals and one rheumatology treatment centre in the Netherlands as well as participants included in two national cohort studies on COVID-19-related disease severity. We included patients aged older than 18 years, diagnosed with any of the prespecified immune-mediated inflammatory disorders, who were able to understand and complete questionnaires in Dutch. Participants with immune-mediated inflammatory disorders who were not on systemic immunosuppressants and healthy participants were included as controls. Anti-receptor binding domain IgG responses and neutralisation capacity were monitored following standard vaccination regimens and a three-vaccination regimen in subgroups. Hybrid immune responses-ie, vaccination after previous SARS-CoV-2 infection-were studied as a proxy for recall responses., Findings: Between Feb 2 and Aug 1, 2021, we included 3222 participants in our cohort. Sera from 2339 participants, 1869 without and 470 participants with previous SARS-CoV-2 infection were analysed (mean age 49·9 years [SD 13·7]; 1470 [62·8%] females and 869 [37·2%] males). Humoral responses did not differ between disorders. Anti-CD20 therapy, sphingosine 1-phosphate receptor (S1P) modulators, and mycophenolate mofetil combined with corticosteroids were associated with lower relative risks for reaching seroconversion following standard vaccination (0·32 [95% CI 0·19-0·49] for anti-CD20 therapy, 0·35 [0·21-0·55] for S1P modulators, and 0·61 [0·40-0·90] for mycophenolate mofetil combined with corticosteroids). A third vaccination increased seroconversion for mycophenolate mofetil combination treatments (from 52·6% after the second vaccination to 89·5% after the third) but not significantly for anti-CD20 therapies (from 36·8% to 45·6%) and S1P modulators (from 35·5% to 48·4%). Most other immunosuppressant groups showed moderately reduced antibody titres after standard vaccination that did not increase after a third vaccination, although seroconversion rates and neutralisation capacity were unaffected. In participants with previous SARS-CoV-2 infection, SARS-CoV-2 antibodies were boosted after vaccination, regardless of immunosuppressive treatment., Interpretation: Humoral responses following vaccination are impaired by specific immunosuppressants. After standard vaccination regimens, patients with immune-mediated inflammatory disorders taking most immunosuppressants show similar seroconversion to controls, although antibody titres might be moderately reduced. As neutralisation capacity and recall responses are also preserved in these patients, this is not likely to translate to loss of (short-term) protection. In patients on immunosuppressants showing poor humoral responses after standard vaccination regimens, a third vaccination resulted in additional seroconversion in patients taking mycophenolate mofetil combination treatments, whereas the effect of a third vaccination in patients on anti-CD20 therapy and S1P modulators was limited., Funding: ZonMw (The Netherlands Organization for Health Research and Development)., Competing Interests: FE and TWK report (governmental) grants from ZonMw to study immune response after SARS-Cov-2 vaccination in autoimmune diseases. FE also reports grants from Prinses Beatrix Spierfonds, CSL Behring, Kedrion, Terumo BCT, Grifols, Takeda Pharmaceutical Company, and GBS-CIDP Foundation; consulting fees from UCB Pharma and CSL Behring; and honoraria from Grifols. AJvdK reports grants from CSL Behring and participation on an advisory board for Argen-X. ML reports a grant from Galapagos not related to this study, and honoraria from Bristol Myers Squibb, Pfizer, Takeda, and Tillotts. PIS is involved in clinical trials with many pharmaceutical industries that manufacture drugs used for the treatment of, for example, psoriasis and atopic dermatitis, for which financial compensation is paid to the department or hospital, and is a chief investigator of the TREAT NL registry taskforce and SECURE-AD registry. MWB is a secretary for the Dutch Experimental Dermatology Board; head of the pigmentary disorders group within the Dutch Dermatology Board; and reports honoraria from Pfizer, Sanofi, Novartis, and Fondation René Touraine. JK has speaking relationships with Merck Serono, Biogen Idec, TEVA, Sanofi, Genzyme, Roche, and Novartis; received financial support to his institution for research activities from Merck Serono, Bayer Shcering Pharma, Biogen Idec, GlaxoSmithKline (GSK), Roche, Teva, Sanofi, Genzyme, and Novartis. BH reports unpaid positions as a medical adviser for several patient groups, a board position for ERN-SKIN, and associate editor for The British Journal of Dermatology; reports grants from AbbVie, Akari Therapeutics, Celgene, and Novartis; consulting fees from UCB Pharma, Novartis, and Janssen; and honoraria from AbbVie. JJGMV reports consulting fees from Argenx, Alexion, and NMD Pharma, and is a co-inventor on patent applications based on MuSK protein-related research. DJH reports grants from AbbVie, AstraZeneca, Janssen, LEO Pharma, and UCB; honoraria from AbbVie, Galderma, Janssen, Lilly, Pfizer, Sanofi, and UCB; and a paid position on an advisory board for BIOMAP IMI. PAvD participated on an advisory board for Octapharma. PvP reports grants from Alexion Pharma and GSK, and participation on advisory boards for GSK and Vifor Pharma. GRAMD'H reports consulting fees from AbbVie, Agomab, AstraZeneca, AM Pharma, AMT, Arena Pharmaceuticals, Bristol Myers Squibb, Boehringer Ingelheim, Celltrion, Eli Lilly, Exeliom Biosciences, Exo Biologics, Galapagos, Index Pharmaceuticals, Kaleido, Roche, Gilead, GSK, Gossamerbio, Pfizer, Immunic, Johnson and Johnson, Origo, Polpharma, Procise Diagnostics, Prometheus Laboratories, Prometheus Biosciences, Progenity, and Protagonist; honoraria from AbbVie, Arena, Galapagos, Gilead, Pfizer, Bristol Myers Squibb, and Takeda; and participation on advisory boards for AbbVie, Seres Health, Galapagos, and AstraZeneca. RBT reports honoraria from Sobi and Norgine, and participation on an advisory board for Norgine. HSG is a board member of the Dutch Society of Clinical Neurophysiology (unpaid), reports grants from Prinses Beatrix Spierfonds, and received speaker fees from Shire/Takeda. KAHZ reports paid data safety monitoring board positions for Torrent and Foresee. All other authors declare no competing interests., (© 2022 Elsevier Ltd. All rights reserved.)

Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Koers, Pollastro, Tol, Pico-Knijnenburg, Derksen, van Schouwenburg, van der Burg, van Ham and Rispens.)

Background: Research on the disease severity of COVID-19 in patients with rheumatic immune-mediated inflammatory diseases (IMIDs) has been inconclusive, and long-term prospective data on the development of SARS-CoV-2 antibodies in these patients are lacking., Methods: Adult patients with rheumatic IMIDs from the Amsterdam Rheumatology and Immunology Center, Amsterdam were invited to participate. All patients were asked to recruit their own sex-matched and age-matched control subject. Clinical data were collected via online questionnaires (at baseline, and after 1-4 and 5-9 months of follow-up). Serum samples were collected twice and analysed for the presence of SARS-CoV-2-specific antibodies. Subsequently, IgG titres were quantified in samples with a positive test result., Findings: In total, 3080 consecutive patients and 1102 controls with comparable age and sex distribution were included for analyses. Patients were more frequently hospitalised compared with controls when infected with SARS-CoV-2; 7% vs 0.7% (adjusted OR: 7.33, 95% CI: 0.96 to 55.77). Only treatment with B-cell targeting therapy was independently associated with an increased risk of COVID-19-related hospitalisation (adjusted OR: 14.62, 95% CI: 2.31 to 92.39). IgG antibody titres were higher in hospitalised compared with non-hospitalised patients, and slowly declined with time in similar patterns for patients in all treatment subgroups and controls., Interpretation: We observed that patients with rheumatic IMIDs, especially those treated with B-cell targeting therapy, were more likely to be hospitalised when infected with SARS-CoV-2. Treatment with conventional synthetic disease-modifying antirheumatic drugs (DMARDs) and biological DMARDs other than B-cell targeting agents is unlikely to have negative effects on the development of long-lasting humoral immunity against SARS-CoV-2., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Studies have suggested incremental short-term adverse events (AE) after repeated vaccination. In this report, we assessed occurrence and risk factors for short-term AEs following repeated SARS-CoV-2 vaccination in patients with various immune-mediated inflammatory diseases (IMIDs)., Methods: Self-reported daily questionnaires on AEs during the first 7 days after vaccination were obtained of 2259 individuals (2081 patients and 178 controls) participating in an ongoing prospective multicenter cohort study on SARS-CoV-2 vaccination in patients with various IMIDs in the Netherlands (T2B-COVID). Relative risks were calculated for potential risk factors associated with clinically relevant AE (rAE), defined as AE lasting longer than 2 days or impacting daily life., Results: In total, 5454 vaccinations were recorded (1737 first, 1992 second and 1478 third vaccinations). Multiple sclerosis, Crohn's disease and rheumatoid arthritis were the largest disease groups. rAEs were reported by 57.3% (95% CI 54.8-59.8) of patients after the first vaccination, 61.5% (95% CI 59.2-63.7) after the second vaccination and 58% (95% CI 55.3-60.6) after the third vaccination. At day 7 after the first, second and third vaccination, respectively, 7.6% (95% CI 6.3-9.1), 7.4% (95% CI 6.2-8.7) and 6.8% (95% CI 5.4-8.3) of patients still reported AEs impacting daily life. Hospital admissions and allergic reactions were uncommon (<0.7%). Female sex (aRR 1.43, 95% CI 1.32-1.56), age below 50 (aRR 1.14, 95% CI 1.06-1.23), a preceding SARS-CoV-2 infection (aRR 1.14, 95% CI 1.01-1.29) and having an IMID (aRR 1.16, 95% CI 1.01-1.34) were associated with increased risk of rAEs following a vaccination. Compared to the second vaccination, the first vaccination was associated with a lower risk of rAEs (aRR 0.92, 95% CI 0.84-0.99) while a third vaccination was not associated with increased risk on rAEs (aRR 0.93, 95% CI 0.84-1.02). BNT162b2 vaccines were associated with lower risk on rAEs compared to CX-024414 (aRR 0.86, 95% CI 0.80-0.93)., Conclusions: A third SARS-CoV-2 vaccination was not associated with increased risk of rAEs in IMID patients compared to the second vaccination. Patients with an IMID have a modestly increased risk of rAEs after vaccination when compared to controls. Most AEs are resolved within 7 days; hospital admissions and allergic reactions were uncommon., Trial Registration: NL74974.018.20 , Trial ID: NL8900. Registered on 9 September 2020., (© 2022. The Author(s).)

Objective: The objective of this study was to measure humoral responses after SARS-CoV-2 vaccination in MS patients treated with ocrelizumab (OCR) compared to MS patients without disease modifying therapies (DMTs) in relation to timing of vaccination and B-cell count., Methods: OCR treated patients were divided into an early and a late group (cut-off time 12 weeks between infusion and first vaccination). Patients were vaccinated with mRNA-1273 (Moderna). B-cells were measured at baseline (time of first vaccination) and SARS-CoV-2 antibodies were measured at baseline, day 28, 42, 52 and 70., Results: 87 patients were included (62 OCR patients, 29 patients without DMTs). At day 70, seroconversion occurred in 39.3% of OCR patients compared to 100% of MS patients without DMTs. In OCR patients, seroconversion varied between 26% (early group) to 50% (late group) and between 27% (low B-cells) to 56% (at least 1 detectable B-cell/µL)., Conclusions: Low B-cell counts prior to vaccination and shorter time between OCR infusion and vaccination may negatively influence humoral response but does not preclude seroconversion. We advise OCR treated patients to get their first vaccination as soon as possible. In case of an additional booster vaccination, timing of vaccination based on B-cell count and time after last infusion may be considered., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Data are scarce on immunogenicity of COVID-19 vaccines in patients with autoimmune diseases, who are often treated with immunosuppressive drugs. We aimed to investigate the effect of different immunosuppressive drugs on antibody development after COVID-19 vaccination in patients with autoimmune diseases., Methods: In this study, we used serum samples collected from patients with autoimmune diseases and healthy controls who were included in two ongoing prospective cohort studies in the Netherlands. Participants were eligible for inclusion in this substudy if they had been vaccinated with any COVID-19 vaccine via the Dutch national vaccine programme, which at the time was prioritising vaccination of older individuals. Samples were collected after the first or second COVID-19 vaccination. No serial samples were collected. Seroconversion rates and IgG antibody titres against the receptor-binding domain of the SARS-CoV-2 spike protein were measured. Logistic and linear regression analyses were used to investigate the association between medication use at the time of vaccination and at least until sampling, seroconversion rates, and IgG antibody titres. The studies from which data were collected are registered on the Netherlands Trial Register, Trial ID NL8513, and ClinicalTrials.org, NCT04498286., Findings: Between April 26, 2020, and March 1, 2021, 3682 patients with rheumatic diseases, 546 patients with multiple sclerosis, and 1147 healthy controls were recruited to participate in the two prospective cohort studies. Samples were collected from patients with autoimmune diseases (n=632) and healthy controls (n=289) after their first (507 patients and 239 controls) or second (125 patients and 50 controls) COVID-19 vaccination. The mean age of both patients and controls was 63 years (SD 11), and 423 (67%) of 632 patients with autoimmune diseases and 195 (67%) of 289 controls were female. Among participants without previous SARS-CoV-2 infection, seroconversion after first vaccination were significantly lower in patients than in controls (210 [49%] of 432 patients vs 154 [73%] of 210 controls; adjusted odds ratio 0·33 [95% CI 0·23-0·48]; p<0·0001), mainly due to lower seroconversion in patients treated with methotrexate or anti-CD20 therapies. After the second vaccination, seroconversion exceeded 80% in all patient treatment subgroups, except among those treated with anti-CD20 therapies (three [43%] of seven patients). We observed no difference in seroconversion and IgG antibody titres between patients with a previous SARS-CoV-2 infection who had received a single vaccine dose (72 [96%] of 75 patients, median IgG titre 127 AU/mL [IQR 27-300]) and patients without a previous SARS-CoV-2 infection who had received two vaccine doses (97 [92%] of 106 patients, median IgG titre 49 AU/mL [17-134])., Interpretation: Our data suggest that seroconversion after a first COVID-19 vaccination is delayed in older patients on specific immunosuppressive drugs, but that second or repeated exposure to SARS-CoV-2, either via infection or vaccination, improves humoral immunity in patients treated with immunosuppressive drugs. Therefore, delayed second dosing of COVID-19 vaccines should be avoided in patients receiving immunosuppressive drugs. Future studies that include younger patients need to be done to confirm the generalisability of our results., Funding: ZonMw, Reade Foundation, and MS Center Amsterdam., Competing Interests: JK reports grants from Biogen Idec, Novatis, TEVA, Bayer Schering Pharma, GlaxoSmithKline, Merck Serono, Genzyme, and Roche outside of the submitted work. All other authors declare no competing interests., (© 2021 Published by Elsevier Ltd.)

Germinal center (GC) reactions are vital to the correct functioning of the adaptive immune system, through formation of high affinity, class switched antibodies. GCs are transient anatomical structures in secondary lymphoid organs where specific B cells, after recognition of antigen and with T cell help, undergo class switching. Subsequently, B cells cycle between zones of proliferation and somatic hypermutation and zones where renewed antigen acquisition and T cell help allows for selection of high affinity B cells (affinity maturation). Eventually GC B cells first differentiate into long-lived memory B cells (MBC) and finally into plasma cells (PC) that partially migrate to the bone marrow to encapsulate into long-lived survival niches. The regulation of GC reactions is a highly dynamically coordinated process that occurs between various cells and molecules that change in their signals. Here, we present a system-level perspective of T cell-mediated GC B cell differentiation, presenting and discussing the experimental and computational efforts on the regulation of the GCs. We aim to integrate Systems Biology with B cell biology, to advance elucidation of the regulation of high-affinity, class switched antibody formation, thus to shed light on the delicate functioning of the adaptive immune system. Specifically, we: i) review experimental findings of internal and external factors driving various GC dynamics, such as GC initiation, maturation and GCBC fate determination; ii) draw comparisons between experimental observations and mathematical modeling investigations; and iii) discuss and reflect on current strategies of modeling efforts, to elucidate B cell behavior during the GC tract. Finally, perspectives are specifically given on to the areas where a Systems Biology approach may be useful to predict novel GCBC-T cell interaction dynamics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Verstegen, Ubels, Westerhoff, van Ham and Barberis.)

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Objectives: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms., Methods: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling., Results: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay., Conclusion: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)