After antigen encounter, long-lived antibody-secreting cells (ASC) secrete high-affinity circulating antibodies. In addition, memory B cells (MBC) are quickly reactivated upon antigen re-exposure and predominantly generate shorter-lived ASCs. Studies have suggested that MBC can differentiate into ASCs without recognizing their cognate antigen, a process known as “bystander activation”. This antigen-independent reactivation of MBC could help maintain circulating antibody levels, thereby protecting against future infections. To elucidate whether SARS-CoV-2 mRNA vaccination leads to bystander activation of B cells, the dynamics of antibody concentrations against six pathogen-specific antigens not encountered during the sampling period were analyzed over time. Deep profiling of antigen-specific B cell responses was simultaneously performed using multiparameter high-dimensional spectral flow cytometry. Antibody concentrations against tetanus toxoid (TT), respiratory syncytial virus (RSV), and influenza hemagglutinin (HA) unexpectedly increased 6 weeks after the first SARS-CoV-2 vaccination. Deep profiling of B cell differentiation stages demonstrated a short-term increase in influenza-specific IgG+ DN3 B cells, RSV-specific IgG+ CD11c+ activated B cells, and TT-specific IgG+ MBC following vaccination. In this study, we demonstrated at both the antibody and cellular levels that SARS-CoV-2 mRNA vaccination transiently activates distinct early activated B cell compartments directed against influenza HA, RSV, and TT.

Detection and characterization of antigen-specific T cells are important for studying immune responses upon infection, vaccination, or autoreactivity. The activation-induced marker (AIM) assay is a robust technique to identify and characterize antigen-specific CD4 and CD8 T cells. However, there is variability in the AIM assay, particularly in the type and number of activation markers used. In this study, we set out to define which marker combinations are most suited to optimally detect antigen-specific CD4 and CD8 T cells and if certain marker combinations preferentially detect specific CD4 T helper subsets. A multiparameter flow cytometry panel, including six common activation markers: CD40L, CD137, CD69, OX40, CD25, and PD-L1, was used for detecting antigen-specific T cells following infection (SARS-CoV-2 and CMV) or vaccination (mRNA-1273 SARS-CoV-2). We demonstrate that combining multiple activation markers increases the detection frequency of antigen-specific CD4 T cells compared to commonly used dual marker combinations. In addition, marker combinations including PD-L1 detected a higher frequency of antigen-specific CD4 T cells in SARS-CoV-2 and CMV infected but not in SARS-CoV-2-vaccinated individuals. Certain dual marker combinations preferentially detected specific CD4 T helper subsets. The majority of antigen-specific CD8 T cells were captured by the dual combination of CD69 plus CD25. In conclusion, combining CD137, CD69, OX40, CD25, and PD-L1 in an AIM assay results in robust and optimal detection of both specific CD4 T helper subsets and CD8 T cells in different antigenic contexts.

Patients with immune thrombocytopenia (ITP) exhibit substantial heterogeneity in treatment responses. Approximately 30%-50% of patients maintain remission after discontinuation of thrombopoietin receptor agonists (TPO-RA), suggesting immunomodulatory effects beyond enhanced thrombopoiesis. However, predictive or explanatory biomarkers for sustained remission after TPO-RA are currently lacking. In this study, we performed spectral flow cytometry on B cells of longitudinal samples from patients before, during and after treatment with the TPO-RA romiplostim. We observed that anergic B cells were the predominant B cell population within the peripheral blood in romiplostim-naïve patients (mean: 51.7%) and in healthy controls (mean: 47.8%). Paired longitudinal analyses showed a significant increase in anergic B cells during romiplostim treatment, with values exceeding those of healthy controls after 1 year of treatment (mean: 58.4%, p = 0.006). After tapering, the anergic B cells remained stably increased (mean: 59.7%), both in treatment-free patients as well as patients who restarted romiplostim. Within the switched- and antibody-secreting cell (ASC) compartment, a decrease of immunoglobulin G (IgG+) and immunoglobulin A (IgA+) ASC was observed during romiplostim (p = 0.04 and p = 0.08). These results reveal a previously unappreciated potential role for anergic B cells and ASCs in ITP and emphasize the need for further investigations of TPO-RA-induced B cell responses in ITP.

Germinal centers (GCs) are specialized sites within secondary lymphoid organs where B cells expand, are selected, and mature to produce high-quality antibodies. Their structural complexity makes them difficult to model in vitro. Here, we developed a human 3D lymphoid culture system combining lymphoid and stromal cells to better mimic GC environments than conventional 2D cultures. Tonsil cells were cultured with or without fibroblastic reticular cells (FRCs) in 2D or 3D hydrogels and stimulated with viral antigens or vaccines. FRC-supported 3D cultures significantly improved B and T cell survival and promoted reaggregation into follicle-like structures with. 3D FRC-supported co-cultures higher levels of antigen-specific antibodies, increased frequencies of S- or HA-specific B cells, and enhanced differentiation into antibody-secreting cells. Importantly, these cultures also showed reduced cell death and lower bystander activation and CXCR4 and CXCR5 expression on CD27+CD38+ B cells indicated GC-like polarization. Autologous and allogeneic FRCs performed comparably, supporting the scalability of the model for high-throughput applications. This 3D platform offers a more physiologically relevant system for studying human GC-associated immune responses and may facilitate mechanistic research and screening of vaccine immunogens and adjuvants in a controlled laboratory setting.

In humans, the stages and dynamics of B cell development after antigen encounter remain unclear. Identifying early B cell differentiation stages could reveal biomarkers for humoral immunity and potential targets to prevent unwanted antibody responses. We characterized antigen-specific B cell responses longitudinally after SARS-CoV-2 mRNA vaccination using multiparameter spectral flow cytometry. Spike-specific IgG+ CD27+ CD71+ activated B cells (ActBCs), presumed to be germinal center-derived and IgG+ DN2 extrafollicular B cells, dominated the early antigen-specific B cell response, while memory B cells were the main population 6 months after vaccination. Within the IgG+ ActBC compartment, we delineated six novel clusters with specific contraction dynamics. Following the second vaccination, certain ActBC clusters displayed sustained expansion over time, being phenotypically similar to memory B cells, while others strongly expanded and subsequently contracted. Several of the rapidly contracting ActBC clusters expressed CD11c, a defining marker for atypical B cells, suggesting a possible extrafollicular origin of these clusters. The transient presence of heterogeneous ActBC clusters was also observed for total B cells when gated in an antigen-independent manner. Characterization of novel ActBC clusters early after antigen encounter helps delineate and dissect the complexity of B cell differentiation, which is vital for understanding unwanted B cell responses.

Solid organ transplantation remains the only curative treatment for end-stage organ diseases. A critical aspect of enhancing long-term graft survival is to prevent antibody-mediated rejection caused by donor-specific antibodies (DSAs). DSAs are formed when donor alloantigen-specific B cells differentiate into antibody-secreting cells. In this review, we explore what is known about the relationship between treatment with extracorporeal photopheresis (ECP) and its effects on undesired B-cell activation and DSA formation. Current preliminary evidence suggests that ECP, when used as an adjuvant therapy, displays significant benefits, including allograft survival, decreased circulating DSAs, and downregulated activation of the allogeneic immune response, possibly through expansion of regulatory B cells. Despite these promising findings, the precise mechanisms through which ECP affects B-cell fate remain incompletely understood. Further research into specific B-cell subpopulations is necessary to fully elucidate the role of ECP in modulating pathways involved in DSA formation, which might allow more effective management of antibody-mediated rejection.

B cells regulate immune responses via antibody production and antigen (Ag) presentation. Although B cell depletion is used therapeutically, it may be associated with side effects, highlighting the need for alternative B cell-targeted approaches. While tolerogenic dendritic cells (tolDC) are known to modulate T cell responses, their impact on B cells is poorly defined. We show that IL-10-producing tolDC (DCIL-10) regulate B cell responses through direct and indirect mechanisms. DCIL-10 enhances T cell-independent human B cell proliferation and differentiation while suppressing Ag-specific memory B cells via T cell inhibition in vitro and dampen Ag-specific IgG production in preclinical humanized and murine models. These findings reveal a dual mechanism by which DCIL-10 regulates B cell responses, broadening their application as a cell-based approach to treating immune-mediated diseases by targeting both B and T cells.

Background Repeated antigen exposure can result in a shifting antibody repertoire. The mechanisms by which this occurs and consequences for cross-variant protection against evolving pathogens remain incompletely understood, particularly in the context of immunosuppressive treatments used in patients with immune-mediated inflammatory diseases (IMID). Methods To investigate this, we characterised longitudinal changes in the anti-SARS-CoV-2 antibody repertoire over the course of three SARS-CoV-2 mRNA vaccinations in patients with IMIDs treated with methotrexate (MTX) and/or tumour necrosis factor-inhibitors (TNFi), anti-CD20 monoclonal antibodies, no systemic therapy, and healthy controls (total N = 878). We determined serum antibody titres against the receptor-binding domain (RBD) of Wuhan-Hu-1 (WH1) and Omicron BA.1 spike proteins, and assessed ratios thereof between groups as a proxy for cross-reactivity. Findings We observe emerging anti-BA.1 RBD reactivity over time, notably following a third vaccination. This may be partly explained by affinity maturation, as evaluated by inhibition of ACE2-RBD interactions. Similar trends were seen in patients treated with MTX and/or TNFi, but not in patients on anti-CD20 therapy. SARS-CoV-2 infection prior to vaccination accelerated these effects initially while leading to comparable results after three vaccinations. Interpretation MTX and TNFi do not qualitatively alter the evolution of the antibody repertoire in response to repeated antigen exposure, whereas anti-CD20 does. These insights may help to optimise vaccination strategies for patients with immune-mediated inflammatory diseases. Funding This study was supported by ZonMw (The Netherlands Organization for Health Research and Development) and SGF (Collaborating Health Funds). Copyright (c) 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

During the COVID-19 pandemic, the daily life of many patients with dermatological immune-mediated inflammatory diseases (DIMIDs), such as atopic dermatitis (AD), psoriasis, and vitiligo, was impacted by social restrictions caused by (fear of) morbidity, mortality associated with COVID-19, and vaccine hesitancy. This prospective observational, multicenter, multidisciplinary cohort study explored the impact of COVID-19 disease and vaccination on DIMIDs, specifically AD, psoriasis, and vitiligo. Data from patients with DIMIDs were collected as part of the Target2B! study (between February 2021 and October 2022). We analyzed the differences in baseline characteristics, risk of developing COVID-19, proportion of DIMIDs in patients reaching seroconversion upon vaccination per DIMID, and self-reported increase in DIMID activity by multivariable logistic regression and sensitivity analyses. A total of 424 patients with DIMID were included. COVID-19 disease commonly occurred in patients with vitiligo (51.1%), AD (42.0%), and psoriasis (34.3%) (p = 0.038). COVID-19 was not associated with the use of immunosuppressive therapy. Three patients (two with AD and one with vitiligo) were hospitalized due to COVID-19. Nearly all patients with DIMIDs exhibited effective seroconversion after regular vaccination regimens (vitiligo 100%, psoriasis 97.9%, AD 96.5%). Increased DIMID activity after COVID-19 (6.6%) or severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) vaccination (12.26%) was reported in a minority of patients, with baseline progressive disease (disease activity 3 months preceding baseline survey) being the only associated risk factor (COVID-19: odds ratio [OR], 4.27 [p = 0.02]; vaccination OR, 3.45 [p = 0.002]). In conclusion, no alarming signs were shown in this study regarding (severe) COVID-19 in patients with AD, psoriasis, or vitiligo. Vaccination against COVID-19 is advised in patients with DIMIDs. Moreover, patients with DIMIDs can safely continue their immunosuppressant therapy, since this does not increase the risk of COVID-19, while vaccination-induced humoral responses are adequate. In only a minority of patients, increased DIMID activity after COVID-19 or SARS-CoV-2 vaccination occurred.

OBJECTIVES:Tumour necrosis factor inhibitors (TNFi) are widely used and effective as treatment for immune-mediated inflammatory diseases (IMIDs). However, TNFi therapy causes a faster waning of antibody responses following vaccination. The underlying cause by which TNFi affect humoral immunity remains to be elucidated. The formation of long-lasting, high-affinity antibodies after vaccination results from germinal centre (GC)-derived, T cell-dependent B-cell responses. Therefore, this study investigated how TNFi affect the formation and maintenance of antigen-specific B- and CD4+ T-cell responses following SARS-CoV-2 mRNA vaccination. METHODS:SARS-CoV-2 spike-specific B-cell responses were characterised using spectral flow cytometry. Spike-specific CD4+ T cells were measured using an activation-induced marker assay. 15 patients with inflammatory bowel disease (IBD) treated with TNFi were compared with 9 IBD patients without systemic immunosuppression and 10 healthy controls. RESULTS:Spike-specific CD4+T-cell frequency and phenotype, including T follicular helper cells, were not affected by TNFi. Total spike-specific B-cell frequencies were reduced in TNFi-treated patients. Deep phenotyping revealed lower IgG+memory B-cell frequencies in TNFi-treated patients 3-6 months after vaccination. These data were confirmed in TNFi-treated rheumatoid arthritis patients. Interestingly, already at day 7 after the second vaccination, TNFi therapy reduced the induction of class-switched CD11c- CD71+activated B cells, which are believed to be GC-derived. Conversely, CD11c+B cells, associated with extrafollicular B-cell responses, were not affected by TNFi therapy. CONCLUSIONS:These data suggest that TNFi therapy affects the differentiation of GC-derived B cells, which may explain its effect on humoral immune responses.

The structural complexity of secondary lymphoid organs (SLOs) and their role in shaping antigen-specific B cell responses, pose significant challenges in modeling human germinal center (GC) response in vitro . A human 3D lymphoid model incorporating lymphoid and stromal cell types recapitulates key immune and structural features, enabling the study of antigen-specific B and T cell interactions beyond current 2D culture limitations. In this study, human tonsil cells were cultured with and without tonsil-derived fibroblastic reticular cells (FRCs) either in 2D or within a 3D PEG-4MAL hydrogel culture. Antigen-specific B cell responses in co-cultures were studied by comparing unstimulated cultures to stimulation with antigen (SARS-CoV-2 spike (S) or Influenza hemagglutinin (HA), both with or without adjuvant R848), S-nanoparticles and influenza vaccines. Combination of FRCs with the 3D matrix significantly improved B and T cell survival and facilitated reaggregation into follicle-like structures. Antigen-specific responses were most pronounced in 3D FRC-supported co-cultures, with increasing S- or HA-specific B cell frequencies, antibody-secreting cell differentiation, and secretion of antigen-specific antibodies. Importantly, cell death and unspecific bystander activation was lowest in 3D FRC-supported cultures. Additionally, GC-associated chemokine receptors CXCR4 and CXCR5 showed distinct expression patterns on CD27⁺CD38⁺ B cells, reflecting GC-like dark and light zone organization typically observed in SLOs in vivo . Autologous and allogeneic FRC-supported cultures yielded comparable results, demonstrating the platform’s potential for high-throughput applications. The 3D FRC-supported lymphoid cultures offer a physiologically relevant platform for studying human GC responses in vitro , supporting mechanistic research into adaptive immunity and enabling the screening of vaccine immunogens and adjuvants in a controlled setting. ### Competing Interest Statement The authors have declared no competing interest.

Immunosenescence, age-related immune dysregulation, reduces immunity upon vaccinations and infections. Cytomegalovirus (CMV) infection results in declining naïve (Tnaïve) and increasing terminally differentiated (Temra) T cell populations, further aggravating immune aging. Both immunosenescence and CMV have been speculated to hamper the formation of protective T-cell immunity against novel or emerging pathogens. The SARS-CoV-2 pandemic presented a unique opportunity to examine the impact of age and/or CMV on the generation of de novo SARS-CoV-2-specific CD8+ T cell responses in 40 younger (22-40 years) and 37 older (50-66 years) convalescent individuals. Heterotetramer combinatorial coding combined with phenotypic markers were used to study 35 SARS-CoV-2 epitope-specific CD8+ T cell populations directly ex vivo. Neither age nor CMV affected SARS-CoV-2-specific CD8+ T cell frequencies, despite reduced total CD8+ Tnaïve cells in older CMV- and CMV+ individuals. Robust SARS-CoV-2-specific central memory CD8+ T (Tcm) responses were detected in younger and older adults regardless of CMV status. Our data demonstrate that immune aging and CMV status did not impact the SARS-CoV-2-specific CD8+ T cell response. However, SARS-CoV-2-specific CD8+ T cells of older CMV- individuals displayed the lowest stem cell memory (Tscm), highest Temra and PD1+ populations, suggesting that age, not CMV, may impact long-term SARS-CoV-2 immunity.

Improving our understanding of B cell transition to memory B cells (MBCs) and antibody-secreting cells (ASCs) is crucial for clinical monitoring and vaccine strategies. To explore these dynamics, we compared prepandemic antigen responses (influenza hemagglutinin, respiratory syncytial virus fusion glycoprotein, and tetanus toxoid) with recently encountered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen responses in convalescent COVID-19 patients using spectral flow cytometry. Our analysis revealed the CD43+CD71+IgG+ activated B cell subset, highly enriched for SARS-CoV-2 specificities, as a juncture for ASC and MBC differentiation, with CD86+ phenotypically similar to ASCs and CD86- to IgG+ MBCs. Moreover, subpopulations within IgG+ MBCs were further identified based on CD73 and CD24 expression. Activated MBCs (CD73-/CD24lo) were predominantly SARS-CoV-2-specific, while resting MBCs (CD73+/CD24hi) recognized prepandemic antigens. A CD95- subcluster within resting MBCs accounted for over 40% of prepandemic-specific cells, indicating long-lasting memory. These findings advance our understanding of IgG+ MBC and ASC development stages, shedding light on the decision-making process guiding their differentiation.

Treatment efficacy of patients receiving anti-TNF antibodies is limited by the formation of anti-drug antibodies. These are observed in most adalimumab-treated rheumatoid arthritis patients, despite the adjuvant-free and human sequence-derived nature of the antibody. The class switched phenotype and high affinity of these antibodies suggest CD4 T-cell involvement in their formation. In this study, we investigated the potential epitopes in the functional domain of adalimumab and assessed their actual HLA II presentation and induction of CD4 T-cell responses in exposed patients. The binding strength of overlapping adalimumab-derived peptides to 27 DR and 14 DQ HLA alleles was predicted in silico. 10 strong and 44 medium-binding 10-mer peptides were identified within the variable regions of the heavy and light chain of adalimumab. HLA-DR-mediated antigen presentation of selected peptides by monocyte-derived dendritic cells was determined by mass spectrometry of the peptide pool eluted from isolated HLA-DR complexes. Binding of the variable region peptides of heavy (H41-62) and light chains (L18-39) was demonstrated. The presence of adalimumab-specific CD4 T-cells in adalimumab-experienced patients was investigated via peptide stimulation of peripheral blood mononuclear cells and assessment of T-cell proliferation. Anti-adalimumab CD4 T-cell responses were observed against four variable region peptides in a group of adalimumab-experienced RA patients. Some of these responses were also present in healthy control donors. This study identifies immunologically relevant CD4 T-cell epitopes in the variable region of the human therapeutic antibody adalimumab based on RA patients' reactivity. Modification of these epitopes or concomitant therapy that targets or prevents adalimumab-specific T cell responses could be beneficial for patients with significant anti-drug responses.

The development of an effective antitumor response relies on the synergistic actions of various immune cells that recognize tumor cells via distinct receptors. Tumors, however, often manipulate receptor-ligand interactions to evade recognition by the immune system. Recently, we highlighted the role of neolacto-series glycosphingolipids (nsGSLs), produced by the enzyme β1,3-N-acetylglucosaminyltransferase 5 (B3GNT5), in tumor immune escape. We previously demonstrated that loss of signal peptide peptidase like 3 (SPPL3), an inhibitor of B3GNT5, results in elevated levels of nsGSLs and impairs CD8 T cell activation. The impact of loss of SPPL3 and an elevated nsGSL profile in tumor cells on innate immune recognition remains to be elucidated. This study investigates the antitumor efficacy of neutrophils, NK cells, and γδ T cells on tumor cells lacking SPPL3. Our findings demonstrate that SPPL3-deficient target cells are less susceptible to trogocytosis by neutrophils and killing by NK cells and γδ T cells. Mechanistically, SPPL3 influences trogocytosis and γδ T cell-instigated killing through modulation of nsGSL expression, whereas SPPL3-mediated reduced killing by NK cells is nsGSL-independent. The nsGSL-dependent SPPL3 sensitivity depends on the proximity of surface receptor domains to the cell membrane and the affinity of receptor-ligand interactions as shown with various sets of defined antibodies. Thus, SPPL3 expression by tumor cells alters crosstalk with immune cells through the receptor-ligand interactome thereby driving escape not only from adaptive but also from innate immunity. These data underline the importance of investigating a potential synergism of GSL synthesis inhibitors with current immune cell-activating immunotherapies.

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.

BackgroundMessenger 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.MethodsIn 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).ResultsAmong 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.ConclusionThese 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.

BackgroundRheumatoid 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.ObjectiveWe 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.Methods151 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.ResultsPublished 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.ConclusionOur study indicates that de novo RF induction following vaccination or infection is an uncommon event, which does not lead to RF epitope spreading.