T cells are central drivers of inflammation across autoimmune and inflammatory diseases, yet current therapies inadequately target pathogenic T-cell pathways, limiting durable disease control. Here, we identified a novel, targetable transcriptional-metabolic axis that sustains inflammatory T-cell responses, characterized by NFATc1-regulated activation of MTHFD2-dependent one-carbon metabolism. We demonstrate that NFATc1 directly binds the MTHFD2 promoter region, driving metabolic reprogramming in activated T cells from rheumatoid arthritis (RA) patients as well as in experimental arthritis models. Pharmacological inhibition of MTHFD1/2 using the novel small molecule TH9619 suppresses proinflammatory cytokine production, expands Foxp3⁺ regulatory T cells and protects against cartilage and bone damage in vivo. Proteomic profiling reveals that TH9619 elicits a distinct molecular response in patients' T cells, divergent from the currently used anti-folate therapy, particularly in inadequate responders. These findings use RA as the proving ground to establish NFATc1-mediated MTHFD2 activation as a critical regulator of sustained T-cell-driven inflammation and support selective MTHFD1/2 inhibition as a novel, mechanism-based therapeutic strategy for RA.

Tumor-associated macrophages (TAMs) are major regulators of the tumor immune microenvironment and are frequently associated with poor clinical outcomes. Although TAMs often acquire immunosuppressive phenotypes in established tumors, they retain substantial plasticity and can be reprogrammed toward immunostimulatory states. The molecular mechanisms controlling these transitions remain incompletely defined. MNK1 and MNK2 both phosphorylate the translation initiation factor eIF4E, but their relative contributions to macrophage biology are unclear. Here we show that Mknk1 and Mknk2 are enriched in distinct TAM subsets and exert non-redundant control of eIF4E phosphorylation, establishing divergent translational and proteomic programs that shape macrophage function. Silencing of Mknk2 in TAMs enhanced adaptive immune responses, suppressed tumor growth and reprogrammed TAMs toward an angiostatic, anti-metastatic phenotype, whereas modulation of Mknk1 had minimal effects on tumor progression. These findings identify MNK2 as a dominant regulator of TAM-mediated immunosuppression and suggest MNK2 inhibition as a strategy to reprogram the tumor immune microenvironment toward anti-tumor immunity.

CRISPR screen data provides a valuable resource for understanding gene function and identifying potential drug targets. Here, we present Correlate, a freely accessible web application ( https://correlate.cmm.se ) that enables exploration of the Cancer Dependency Map (DepMap) CRISPR screen gene effects, hotspot mutations, and translocation/fusion data across more than 1,000 human cancer cell lines. The application supports two main use cases: (i) analysis of user-defined gene sets (e.g. CRISPR screen hits) to identify functionally linked genes based on correlations while providing an overview based on essentiality or user-provided screen statistics; and (ii) exploration of genes of interest in defined biological contexts, such as specific cancer types or mutational backgrounds, to generate hypotheses about gene function and dependencies. Additionally, Correlate supports experimental design by providing rapid overviews of gene essentiality and enabling the identification of cell lines with relevant mutational profiles. In contrast to knowledge-based approaches such as STRING and GSEA, which rely on prior biological annotations and curated interaction networks, Correlate identifies gene connections directly from functional CRISPR screen readouts, offering a complementary and data-driven perspective on gene network analysis. The application runs entirely in the browser, requires no installation or login, and integrates with the Green Listed v2.0 tool family for custom CRISPR screen design. HIGHLIGHTS ▪ Interactive web-based platform for bulk correlation analysis of user-defined gene sets using DepMap CRISPR screen data, requiring no installation or programming expertise. ▪ Identifies functional gene relationships from CRISPR screen readouts rather than curated annotations, offering a data-driven complement to tools such as GSEA and STRING. ▪ Enables contextual exploration of gene dependencies across cancer types and mutational backgrounds, supporting hypothesis generation about gene function and therapeutic targets. ▪ Supports experimental design through gene essentiality overviews, mutation and fusion analysis, and cell line identification, with optional integration of user-provided statistics from CRISPR screens, proteomics, or transcriptomics analyses.

Autoreactive T cells recognizing citrullinated antigens, although rare and difficult to study, are implicated in rheumatoid arthritis (RA). To allow functional studies and manipulation of such T cells, we have generated and characterized transgenic mice expressing a TCR cloned from an RA patient and reactive with a prominent target, citrullinated tenascin C (citTNC). A humanized TCR transgenic (hTCR-tg) mouse recognizing the citrullinated TNC22 (citTNC22) antigen in an HLA-DRB1*04:01 (HLA-DR4) restricted manner was developed by genetically engineering a chimeric TCR expressing murine constant domains and human V(D)J sequences. hTCR-tg mice were immune phenotyped in murine H-2 b and humanized HLA-DR4 backgrounds using full spectrum flow cytometry, cytokine ELISAs and fluorospot assays at steady-state and after antigen challenge. Additionally, we investigated the presence of antibodies to citTNC and arthritis following citTNC protein immunization. Thymic selection of hTCR T cells differed between the two MHC-II alleles, with a normal CD4+ T cell development observed solely under HLA-DR4 restriction. The chimeric hTCR maintained its citTNC22 specificity in vitro and ex vivo, without cross-reactivity to native TNC22 or other citrullinated autoantigens. hTCR-tg CD4+ T cells responded to antigen challenge in vivo and provided support to IgG class-switch and antigen-specific antibody production. Moreover, protein immunized hTCR-tg mice developed arthritis after periarticular challenge with citTNC. hTCR-tg mice expressing an RA patient-derived autoreactive TCR develop functional antigen-specific and HLA-restricted CD4+ T cells. The combination of humanized HLA-DR4 and TNC22 mice will be a valuable tool in the development of antigen-specific therapies translatable to human disease.

Dysregulation of adipocyte function is an important component of metabolic and cardiovascular diseases. It is increasingly evident that interorgan neuroimmune crosstalk in adipose tissue maintains adipose tissue functionality, yet the molecular mechanisms are incompletely understood. The vagus nerve regulates numerous physiological functions, including inflammation and weight control, even in tissues lacking direct cholinergic innervation. In this study, we examined whether vagal signaling is involved in maintaining baseline homeostasis in epididymal white adipose tissue (eWAT) by investigating how disrupting vagus signaling affects adipose tissue physiology. As expected, vagotomized male animals had lower body weight and epididymal white adipose tissue mass as compared with controls. Vagotomized animals showed increased Ly6G+ cell infiltration in eWAT. Interestingly, vagotomy-associated weight loss was significantly attenuated in neutrophil-deficient Ly6GcreMcl1fl/fl mice and in mice treated with repeated injections of anti-Ly6G antibodies. Together, these observations indicate that there is a Ly6G-mediated vagotomy-associated reduction in weight and reveal that Ly6G+ cells participate in the regulation of eWAT energy homeostasis.

OBJECTIVE:Somatic DNMT3A mutations are the most common drivers of clonal hematopoiesis in patients with rheumatoid arthritis (RA) and have been associated with seropositive disease and increased markers of inflammation. These mutations are predominantly hypomorphic or dominant-negative, reducing DNMT3A function. We tested whether Dnmt3a mutations enhance the pathogenicity of autoreactive CD4+ T cells. METHODS:Using the KRN+ TCR transgenic transfer model of autoimmune arthritis, in which autoreactive CD4+ T follicular helper (Tfh) cells drive disease, we assessed the impact of Dnmt3a mutations introduced by CRISPR-Cas9 or Cd4cre-mediated recombination. Competitive bone marrow chimeras, immunization with sheep red blood cells, and autoreactive T cell transfer were used to evaluate Tfh responses, antibody production, and joint inflammation. RESULTS:Loss of DNMT3A function in CD4+ T cells consistently reduced Tfh responses and IgG production during both immunization-driven and autoreactive activation. In the KRN+ transfer model, mice receiving Dnmt3a-mutant autoreactive CD4+ T cells developed significantly attenuated joint inflammation, accompanied by reduced systemic interleukin-6 and autoantibody levels. CONCLUSION:Wild-type Dnmt3a supports Tfh responses under both immunization-driven and autoreactive conditions. Dnmt3a loss-of-function in CD4+ T cells limits autoreactive inflammation, suggesting that clinical associations in RA may reflect effects in other hematopoietic lineages or T cell functions beyond Tfh-dependent responses.

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint damage. Despite available treatments, many patients fail to achieve adequate disease control, prompting interest in complementary approaches. In traditional Chinese medicine, multi-herbal formulations such as Formulation C (Qushi-Juanbi Granules) are used clinically to treat RA. Among its component plants, S. costus (S. costus) has long been recognized for its anti-inflammatory properties that have largely been attributed to reactive α-methylene-γ-lactone-containing sesquiterpene lactones. However, comparatively little is known about the activities of non-reactive compounds.ObjectiveThis study aimed to investigate the anti-inflammatory and anti-arthritic potential of S. costus and to determine the contribution of reactive sesquiterpene lactones to this effect.Materials and methodsExtracts from Formulation C plants were screened in cell-based assays targeting RA-relevant pathways (NF-κB, NFAT, STAT3/STAT5), prostanoid formation in a synovial fibroblast cell line, and cytokine production in primary B cells. The aqueous extract of S. costus and an electrophile-depleted version were prepared by elution through thiol-bonded silica and tested in vitro and in the KRN T cell transfer arthritis model in vivo. Seven compounds were isolated from S. costus and their activity characterized in vitro.ResultsScreening identified the aqueous extract of S. costus with potent in vitro activities, mainly affecting the NF-κB and NFAT pathways, and the B cell assay. This activity was lost upon electrophilic-compound depletion, and the sesquiterpene lactones costunolide and dehydrocostus lactone were identified as major active constituents. In vivo, the aqueous extract modestly reduced paw swelling during early disease phase, whereas the electrophile-depleted extract showed minimal activity.ConclusionThis study applies a straightforward thiol-reactive compound depletion method to explore the bioactive constituents of S. costus, a strategy that can be broadly applied to other natural product extracts. Our findings indicate that sesquiterpene lactones are major contributors to the overall anti-inflammatory activity of S. costus, while other, non-thiol-reactive compounds may also play a role for in vivo activity. Overall, these results provide a more detailed characterization of the bioactive profile of S. costus and support its future mechanistic and preclinical investigation for RA research.

Custom CRISPR screens are powerful tools for rapid, hypothesis-driven discovery, but their design is often complex and time-consuming. Green Listed v2.0 simplifies this process with an intuitive workflow for designing custom CRISPR spacer libraries and supports downstream analysis for all users, irrespective of their computational experience. The web application features a user-friendly graphical interface freely accessible at https://greenlisted.cmm.se. Version 2.0 includes significant upgrades to the original 2016 version that were implemented based on user feedback. This includes a new gene synonym tool, expanded library options, optimized output lists, performance improvements, and linked scripts for the rational design of custom CRISPR screen gene sets.

Supplementary Data from CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies

Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that poses a significant socio-economic burden. Traditional Chinese medicine (TCM) herbal formulations, with their long history of clinical use, represent a promising source for novel anti-rheumatic drugs. However, the complexity of RA pathogenesis and the chemical diversity of TCM formulations present challenges in understanding compound-target interactions. Purpose This study aimed to identify the anti-inflammatory constituents from the individual plant species included in the clinically used TCM formulation (“Formulation A”), and to characterize their effects on key inflammatory pathways. Formulation A is currently undergoing a double-blind, randomized clinical trial. A key innovation of this work is the integration of a phenotypic screening strategy, bridging the gap between typical single-species natural product studies and formulation-level decoction studies. Methods A custom panel of phenotypic in vitro assays reflecting key inflammatory pathways relevant to RA (NF-ĸB, NFAT, STAT3, and STAT5) was developed. A suite of plant extracts and fractions was generated, then screened in the above assays. Effects on cytokine production in primary human B cells (IL-6, TNF-α, and GM-CSF) and prostaglandin production (PGD2, PGE2, PGF2α and TxB2) in a fibroblast cell line were also evaluated. Results Plant species within Formulation A displayed a spectrum of biological activities, from minor to broad and more targeted effects. The strongest anti-inflammatory activities were observed in nonpolar extracts and fractions, reflecting the utility of organic solvent extraction for accessing pharmacologically potent constituents that may be underrepresented in traditional water-based decoctions. Two new compounds from Atractylodes lancea were identified, alongside 37 known compounds from other species exhibiting both new and previously known anti-inflammatory activities. These findings also highlight specific molecular effects of the formulation’s components on inflammatory signaling pathways and mediators. Conclusion This study provides mechanistic insights into the anti-inflammatory effects of plant constituents present in Formulation A, based on the systematic analysis of each individual plant species. Moreover, it establishes a robust phenotypic screening platform for the systematic discovery of new anti-rheumatic agents from natural sources.

Genetic association links disordered autoimmunity 1 (DIORA1) to numerous autoimmune rheumatic diseases, including systemic lupus erythematosus, Sjögren's disease, rheumatoid arthritis, polymyositis, and systemic sclerosis. However, its cellular function has remained unknown. Here, we identify the Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinases (MRCK kinases) family of serine/threonine kinases-key regulators of actomyosin contractility and cell motility-as direct interactors of DIORA1. Through interaction mapping, we show that DIORA1 binds three distinct modules of MRCK kinases, including the conserved kinase inhibitory motif, C1-PH, and citron homology domains. DIORA1 knockdown in human cells altered cellular phosphorylation patterns and reduced phosphorylation of known MRCK targets. RNA-sequencing and proteomic analyses revealed upregulation of epithelial-mesenchymal transition genes and proteins, and functional analyses confirmed increased cell invasion, following knockdown of DIORA1. Together, these findings identify the autoimmunity-associated DIORA1 protein as an interactor of MRCK kinases and a regulator of cell motility.

Colorectal cancer (CRC) is a leading cause of cancer mortality and is characterized by a high tumor mutational burden, making it responsive to immunotherapy. We developed a bioinformatic and manufacturing pipeline for personalized cancer vaccines and evaluated it in the MC38 colon adenocarcinoma mouse model. Thirty-six high-scoring neoantigens (NAGs) were identified by exome and transcriptome analysis, produced as six purified polypeptides, and coupled to paramagnetic beads. Intralymphatic vaccination of C57BL/6 mice with NAG beads induced robust NAG-specific T cell and antibody responses, resulting in significant inhibition of MC38 tumor growth. Treated tumors displayed increased necrosis and CD8+ T cell infiltration. Compared with soluble peptides, bead-coupled antigens elicited superior protection. Studies in T cell-deficient and antibody-depleted mice confirmed that both CD4+ and CD8+ T cells mediated the antitumor effect. These findings highlight the potential of NAG bead vaccination as an effective immunotherapy for CRC. ### Competing Interest Statement The authors have declared no competing interest. Cancer- och allergifonden European Innovation Council Eurostars Swedish Research Council, https://ror.org/03zttf063 Swedish Cancer Society, https://ror.org/0527jb766 Swedish Brain Foundation European Union’s Horizon 2020 research and innovation program

Immunotherapy has revolutionized cancer treatment but its efficacy depends on a robust immune response in the tumor. Silencing of the tumor suppressor p53 is common in tumors, and can affect the recruitment and activation of different immune cells, leading to immune evasion and poor therapy response. We found that the p53 activating stapled peptide MDM2/MDMX inhibitor Sulanemadlin (ALRN-6924) inhibited p53 wild-type cancer cell growth in vitro and in vivo. In mice carrying p53 wild-type CT26.WT tumors, monotherapy with the PD-1 inhibitor DX400 or Sulanemadlin delayed tumor doubling time by 50% and 37% respectively, while combination therapy decreased tumor doubling time by 93% leading to an increased median survival time. Sulanemadlin treatment led to increased immunogenicity and combination treatment with PD-1 inhibition resulted in an increased tumor infiltration of lymphocytes. This combination treatment strategy could potentially turn partial responders into responders of immunotherapy, expanding the patient target group for PD-1-targeting immunotherapy.

The differentiation of hematopoietic stem and progenitor cells into the various cell lineages is regulated by cell-intrinsic factors and the progenitors' microenvironment. Experiments in mice have allowed the identification of transcriptional modulators that control mast cell differentiation. However, a comprehensive approach that allows efficient disruption of individual transcriptional modulators in primary human hematopoietic progenitors coupled with a mast cell formation readout has not been described. Here, we report a simple electroporation- and ribonucleoprotein-based knockout system that allows the identification of genes that are required and dispensable for human mast cell differentiation. We show that the transcription factor MITF is upregulated in human mast cell progenitors and reveal that the loss of MITF results in the suppressed formation of mast cells. By contrast, CITED2, another transcriptional modulator that is upregulated along the mast cell differentiation trajectory, was dispensable for human mast cell differentiation. Taken together, we report a CRISPR/Cas9-based framework that serves to identify genes involved in regulating the formation of human mast cells, and the results uncover the role of two transcriptional modulators in controlling human mast cell differentiation.

Rheumatoid arthritis (RA) is the most common inflammatory joint disease, and early diagnosis is key for effective disease management. CD69 is one of the earliest cell surface markers seen at the surface of activated immune cells, and CD69 is upregulated in synovial tissue in patients with active RA. In this study, we evaluated the performance of a CD69-targeting PET agent, [68Ga]Ga-DOTA-ZCAM241, for early disease detection in a model of inflammatory arthritis. Methods: A model of inflammatory arthritis was induced by transferring splenocytes from KRN T -cell receptor transgenic B6 mice into T -cell- deficient I-Ag7 major histocompatibility complex class II-expressing recipient mice. The mice were examined longitudinally by [68Ga]Ga- DOTA-ZCAM241 PET/CT before and 3, 7, and 12 d after induction of arthritis. Disease progression was monitored by clinical parameters, including measuring body weight and scoring the swelling of the paws. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the paws was analyzed and expressed as SUVmean. Tissue biopsy samples were analyzed for CD69 expression by flow cytometry or immunostaining for a histologic correlate. A second group of mice was examined by a nonbinding, size -matched Affibody molecule as the control. Results: Clinical symptoms appeared 5-7 d after induction of arthritis. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the joints was negligible at baseline but increased gradually after disease induction. An elevated PET signal was found on day 3, before the appearance of clinical symptoms. The uptake of [68Ga]Ga-DOTA-ZCAM241 correlated with the clinical score and disease severity. The presence of CD69-positive cells in the joints and lymph nodes was confirmed by flow cytometry and immunostaining. The uptake of the nonbinding tracer that was the negative control also increased gradually with disease progression, although to a lesser extent than with [68Ga]Ga-DOTA-ZCAM241. Conclusion: The uptake of [68Ga]Ga-DOTA-ZCAM241 in the inflamed joints preceded the clinical symptoms in the KRN T -cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [68Ga]Ga-DOTA-ZCAM241 is thus a promising PET imaging marker of activated immune cells in tissue during RA onset.

Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease with an unpredictable course towards progressive disability. Treating progressive MS is challenging due to limited insights into the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. In PPMS, we found hypermethylation of the 1q21.1 locus, controlled by PPMS-specific genetic variations and influencing the expression of proximal genes (CHD1L, PRKAB2) in the brain. Evidence from reporter assay and CRISPR/dCas9 experiments supports a causal link between methylation and expression and correlation network analysis further implicates these genes in PPMS brain processes. Knock-down of CHD1L in human iPSC-derived neurons and knock-out of chd1l in zebrafish led to developmental and functional deficits of neurons. Thus, several lines of evidence suggest a distinct genetic-epigenetic-transcriptional interplay in the 1q21.1 locus potentially contributing to PPMS pathogenesis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation, strongly associated with the activity of autoreactive CD4+ T cells. DNMT3A mutations are the most common somatic mutations found in the hematopoietic system of patients with rheumatoid arthritis. However, the role of DNMT3A in CD4+ T cells and CD4+ T follicular helper (Tfh) cells is poorly understood. Since somatic mutations are not identified in standard genome-wide association studies, somatic mutations’ impact on the etiology of diseases could be underestimated. Here, we thoroughly characterized and used the KRN+ splenocyte transfer model of autoimmune joint inflammation and inactivated Dnmt3a using CRISPR-Cas9 and standard Cre/loxP approaches. Experiments with competitive bone marrow (BM) chimeras identified a positive role for Dnmt3a in Tfh differentiation, which was validated by comparing mice with Dnmt3a mutations in CD4+ cells to animals with WT Dnmt3a . In conclusion, We identify that Dnmt3a mutations limit normal and autoreactive Tfh differentiation.Key findings ### Competing Interest StatementFW has received consulting fees from SmartCella Solutions, which is outside the scope of the submitted study.

High‐throughput drug screening enables the discovery of new anticancer drugs. Although monolayer cell cultures are commonly used for screening, their limited complexity and translational efficiency require alternative models. Three‐dimensional cell cultures, such as multicellular tumor spheroids (MCTS), mimic tumor architecture and offer promising opportunities for drug discovery. In this study, we developed a neuroblastoma MCTS model for high‐content drug screening. We also aimed to decipher the mechanisms underlying synergistic drug combinations in this disease model. Several agents from different therapeutic categories and with different mechanisms of action were tested alone or in combination with selective inhibition of prostaglandin E 2 by pharmacological inhibition of microsomal prostaglandin E synthase‐1 (mPGES‐1). After a systematic investigation of the sensitivity of individual agents and the effects of pairwise combinations, GFP‐transfected MCTS were used in a confirmatory screen to validate the hits. Finally, inhibitory effects on multidrug resistance proteins were examined. In summary, we demonstrate how MCTS‐based high‐throughput drug screening has the potential to uncover effective drug combinations and provide insights into the mechanism of synergy between an mPGES‐1 inhibitor and chemotherapeutic agents.

Background Anti-citrullinated protein autoantibodies (ACPA) are known to associate with strong risk factors for rheumatoid arthritis (RA), as well as worse clinical prognosis[1]. Due to their high specificity in RA, ACPA are part of the classification criteria and regarded as being essential in the identification of individuals at risk of developing RA. However, not every ACPA-positive at-risk individual progresses to a clinical diagnosis[2], suggesting that the presence of ACPA per se does not dictate the clinical outcome. We have recently shown that ACPA are in fact predominantly anti-inflammatory and present different biological effects in a murine model of arthritis[3]. Having a wide diversity of ACPA clones isolated from different tissues of RA patients at our disposal, we have continued to analyze different monoclonal as well as polyclonal ACPA in their capacity to affect inflammatory processes in vivo. Objectives To determine whether ACPA with pathogenic features, i.e. capable of increasing or sustaining joint inflammation, can be found among different isolated ACPA clones from patients with established RA. Methods Immunoglobulin from single B cells from RA patients were sequenced and recombinantly expressed as monoclonal human (h)IgG1 in Expi293F cells, followed by validation of citrulline reactivity. Here, we tested 5 new monoclonal ACPA isolated based on anti-citrullinated tetramer staining of B cells[4]. Polyclonal ACPA IgG from 34 RA patients were enriched via a CCP2 affinity column. The unbound IgG fraction was used as the flow-through control. Arthritis was induced in BALB/c mice by i.v. transfer of 1.5mg of an arthritogenic cocktail of anti-type II collagen (CII) antibodies (Chondrex, USA) followed by administration of 25ug of LPS (E. coli strain O55:B5) 3 days later. Individually, 1mg of purified ACPA monomers, 2mg of ACPA-pool or 2mg flow-through IgG were transferred i.v. together with the arthritogenic antibody cocktail. An isotype control antibody and a previously identified anti-inflammatory ACPA clone (hE02 and hC03, respectively)[3] were used as experimental reference conditions. Arthritis development was monitored daily by a quantitative scoring system of inflamed joints – toes, knuckles, and wrist/ankle of front/hind paws. Statistical analysis was performed with repetitive-measure one-way ANOVA with Geisser-Greenhouse correction and Holm-Šidák’s multiple comparisons test. Results Adding to our previous data with 8 monoclonal ACPA[3], where 3 of them did not alter the disease course, and 4 displayed strong anti-inflammatory properties, we observed an additional 4 newly tested ACPA clones that did not influence arthritis development. Furthermore, we identified one novel monoclonal ACPA (hF2C05) that displays a pro-arthritogenic effect (p<0.01). When assessing a polyclonal ACPA sample, we observed a dominant anti-inflammatory effect of the ACPA-pool in comparison to its non-ACPA flow-through IgG fraction, displayed by a significantly reduced disease prevalence (p<0.001). Conclusion Our preliminary data focusing on distinct monoclonal ACPA, as well as a new set of patient-derived polyclonal ACPA-pool, strengthens the notion that ACPA clones differ significantly between them, and within the same individual. Although we were able to identify an ACPA clone with arthritogenic potential, the majority of ACPA display no aptitude to affect joint inflammation or are anti-inflammatory. Together, this diversity of inflammatory effects calls for a re-evaluation of the proposed role of ACPA in RA, where their heterogeneity must be considered. References [1] Hair, M. J. H. et al. Arthritis Rheumatol 66, 513–522 (2014) [2] Chirivi, R. G. S. et al. Cell Mol Immunol 18, 1528–1544 (2021) [3] Raposo, B. et al. Ann Rheum Dis ard-2022-223417 (2023) doi:10.1136/ard-2022-223417 [4] Titcombe, P. J. et al. Arthritis Rheumatol 70, 1933–1945 (2018) Acknowledgements We would like to acknowledge the following agencies for financial support of the current work: IMI project RTCure (777357), ERC consolidation grant (2017-7722209_PREVENT RA), the Swedish Research Council (VR; 2019-01664) and Ulla and Gustaf af Uggla Foundation (2020-0009). Disclosure of Interests None Declared.