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.

BACKGROUND:Systemic autoimmune rheumatic diseases (SARDs) are a heterogeneous group of autoimmune conditions characterized by immune system dysregulation leading to chronic inflammation and tissue damage. The overlapping clinical manifestations make differential diagnosis challenging, highlighting the need for novel biomarkers to facilitate early diagnosis, stratification, and personalized treatment. METHODS:Five SARDs including idiopathic inflammatory myopathies (n = 210), rheumatoid arthritis (n = 84), systemic sclerosis (n = 100), Sjögren disease (n = 99), and systemic lupus erythematosus (n = 99), as well as healthy controls (n = 400) and controls with acute infectious diseases (n = 218) were selected for plasma protein profiling using Olink Explore 1536. Differential abundance analysis and machine learning were used to identify proteins with both known and novel association to SARDs. RESULTS:The five SARDs share hundreds of proteins with consistently altered abundance compared to both healthy and infectious controls, reflecting common underlying molecular dysregulation. Despite the overlap, we identify multiple proteins with higher abundance specific to individual SARDs. Machine learning further enables accurate classification of the five SARDs, identifying a panel of 48 proteins with high discriminatory performance, several of which are also supported by differential abundance analysis. CONCLUSIONS:Altogether, this explorative cross-sectional study demonstrates the importance of a pan-disease approach, including also infectious and healthy controls, to identify robust and disease-informative protein panels for improved classification of SARDs. Protein levels from this study are available open access through the Human Protein Atlas, facilitating further plasma proteome research on autoimmune disease.

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.

OBJECTIVES:In SLE, expanded B cell subtypes like double-negative 2 (DN2) may harbour autoreactivity, which may be linked to impaired checkpoint regulation. We investigated checkpoint molecule CD72 on B cells, its clinical associations, and dynamic changes upon rituximab (RTX) treatment. METHODS:Thirty SLE patients (26 with active disease) were studied. Seven received RTX treatment with sampling at baseline, 3 months and 6 months. B cell phenotypes were analysed using spectral flow cytometry, and clinical associations were evaluated. B cell receptor (BCR) signalling was studied through downstream phosphorylation in vitro. RESULTS:Patients with active SLE showed increased frequencies of CD72-negative B cells in switched memory (SWM), activated naïve (aNAV), DN2 and plasmablast (PB) subsets compared with healthy controls (HCs). CD72-negative DN2 cells (CD27-IgD-CD21-CD11c+) were elevated in LN patients. These cells expressed higher CD95 and lower CD20 compared with HCs and canonical SLE DN2. Upon BCR stimulation, lupus CD72-negative SWM and DN2 B cells displayed increased pSYK phosphorylation compared with their CD72-positive counterparts and the overall cell population. CD72-negative DN2 frequencies associated positively with SLEDAI-2K and inversely with C3 levels and were increased in anti-dsDNA-positive patients. After RTX treatment, the remaining DN2 cells were primarily CD72-negative at 3 months. CONCLUSION:The DN2 B cell subset have been associated with autoimmunity. We showed that DN2 B cells in SLE have reduced CD72 expression, which is associated with anti-dsDNA positivity, complement consumption, and enhanced BCR downstream signalling. The relative resistance of CD72-negative B cells to RTX indicates that an impaired checkpoint programme in lupus B cells may potentially contribute to disease relapse. These findings require validation in larger cohorts.

OBJECTIVES:Autoantibodies targeting melanoma differentiation associated protein 5 (MDA5) are strongly associated with dermatomyositis (DM) and may contribute to its pathogenesis. Here we aimed to investigate MDA5+ B cells, their phenotype and generate MDA5 monoclonal antibodies to assess their epitope specificity. METHODS:MDA5-reactive B cells were captured from peripheral blood of patients with anti-MDA5+ DM (n = 3) using an MDA5-fluorescent probe. B cell receptor (BCR) sequences were analysed from single-sorted B cells (n = 240). Selected clones were re-expressed as IgG1 monoclonal antibodies (mAbs, n = 23). Reactivity was assessed using recombinant MDA5 protein constructs, peptide epitope mapping, ELISA, western blot and a commercial line blot assay. RESULTS:Of 240 anti-MDA5+ sorted B cells, 23 BCRs were re-expressed as mAbs, two of which showed high reactivity and specificity for MDA5. These antibody sequences originated from one CD19+IgD-CD27-CD38+ and one CD19+IgD-CD27+CD38+ IgG+ B cell with low somatic hypermutation (SHM). Both mAbs had nanomolar apparent affinity and bound to sites within the helicase domains of the MDA5 protein but with distinct epitope recognition. Serology screening confirmed targeting of a linear epitope identified in the mAb studies. CONCLUSION:Our results show that anti-MDA5+ B cells recognize the helicase domains, which are the enzymatically active domains of the protein. These results have implications for understanding the etiopathology of anti-MDA5+ DM and development of new antigen-specific therapies.

OBJECTIVES:Around 30% of patients with rheumatoid arthritis (RA) lack rheumatoid factor and anti-citrullinated protein antibodies (ACPA) complicating diagnosis and potentially delaying treatment. We hypothesised that innate immune mechanisms might be more prominent in ACPA- RA. METHODS:We performed single-cell RNA sequencing of mononuclear cells from peripheral blood (PBMC) and synovial fluid (SFMC) of patients with ACPA- and ACPA+ RA (n = 4 per group: discovery cohort; n = 8 per group: validation cohort). Dendritic cells and proinflammatory cytokine production were analysed by flow cytometry on SFMC from patients with ACPA- RA, ACPA+ RA, and psoriatic arthritis. Interferon (IFN) levels in synovial fluid (SF) and serum were measured in these groups. RESULTS:Several macrophage subsets and cDC2 were enriched in ACPA- RA SF whereas the frequency of Tph and B cells was increased in ACPA+ RA SF. Type I IFN-stimulated genes were detected in SFMC, but not PBMC, of patients with ACPA- RA. A type I IFN signature was also observed in synovial tissue from two patients with ACPA- RA in an independent dataset. IFN levels were higher in SF than serum but IFN-α/β production did not differ between ACPA+ and ACPA- RA. CONCLUSIONS:This study identifies a distinct innate cell composition and type I IFN gene response in synovial joints, but not in peripheral blood, of patients with ACPA- RA. Similar IFN levels across groups suggest the IFN signature may have been primed before the cells entered the joints. These findings provide a foundation for future research on type I IFN responses in ACPA- RA.

OBJECTIVES:Anti-Jo1+ antisynthetase syndrome (ASyS) is characterised by autoantibodies targeting histidyl t-RNA synthetase (HisRS), association with HLA-DRB1*03:01 and a distinct clinical phenotype including interstitial lung disease, myositis, arthritis, and mechanic's hands. Previous studies of autoreactive HisRS-specific CD4+T cells point to yet undiscovered T cell epitopes. We aimed to identify new epitopes on HisRS to investigate the presence of autoreactive T cells and their corresponding T-cell receptor (TCR) repertoire from patients with ASyS. METHODS:Peptides from HisRS N-terminal region with appropriate major histocompatibility complex (MHC) anchor residues were selected for in vitro binding assays. The peptide (HisRS41-55) with the highest HLA-DRB1*03:01 binding affinity was selected for studies with HLA-class II tetramers. Peripheral blood mononuclear cells (PBMCs) from patients with ASyS with HLA-DRB1*03:01 (n = 12) were stimulated in vitro with peptide and peptide-HLA-DRB1*03:01 tetramers were used to detect HisRS+CD4+T cells. Single TCR sequencing of captured T cells allowed analyses of the underlying TCR repertoire. RESULTS:We identified a new T cell epitope on HisRS with high affinity for HLA-DRB1*03:01. Autoreactive HisRS+CD4+T cells were detected in PBMCs of patients (n = 6/12). TCR repertoire analysis of HisRS+CD4+T cells revealed shared gene V-alpha and beta usages. Moreover, HisRS+CD4+T cells persisted after treatment in 2 patients (P2 and P4) and 2 identical T cell clones were detected between the initial and follow-up time points in 1 patient (P2). CONCLUSIONS:Autoreactive T-cells targeting a new HisRS epitope were identified indicating T cell reactivity to diverse epitopes of the HisRS protein in patients with anti-Jo1 autoantibodies. Furthermore, we demonstrated the TCR repertoire of autoreactive HisRS+CD4+T cells in patients. Persistence of these T-cells and specific clones may be contributing to disease.

PV106 / #522 Poster Topic:AS12 - Genetics, Epigenetics, Transcriptomics Using their autoantibody profile, patients with systemic lupus erythematosus (SLE) can be grouped into 4 less heterogeneous subgroups [1] Subgroup 1 was dominated by anti-SSA/SSB, Subgroup 2 by anti-nucleosome/Sm/RNP/dsDNA, Subgroup 3 by aPL, and Subgroup 4 was negative for 13 antibodies tested, but ANA ever positive. Those subgroups differed in cytokine levels, clinical manifestations, andHLA-DRB1gene associations.[1] Therefore, we hypothesize that the pathogenic mechanisms are different among these subgroups of patients. We aimed to evaluate whether there are differences in known SLE genetic risk factors and protein levels among antibody-defined SLE subgroups. We analyzed 448 patients from our previous study[1] to address differences in genetic risk factors. We compared the SLE polygenic risk scores (PRS) previously described by Reid et al 2020.[2] Using a double-sided Mann-Whitney U test, we tested differences in the distribution of 2 PRS: 1 including 4 Single Nucleotide Polymorphisms (SNPs) in theHLAregion or 57 non-HLASNPs across the subgroups. Furthermore, untargeted liquid chromatography-mass spectrometry (LC-MS) was implemented using plasma samples from 100 SLE patients, 25 representing each subgroup. Differential expression analysis was performed using linear modeling with the limma package,[3] and pairwise comparisons were conducted among the subgroups. P-values for both approaches were corrected using a False Discovery Rate (FDR) multiple-testing correction. Adjusted values (P-adj) lower than 0.05 were considered significant. Subgroup 1 displayed significantly higherHLA-PRS compared to subgroup 2 (P-adj = 9.27e-06), subgroup 3 (P-adj =1.65e-10), and subgroup 4 (P-adj =3.64e-04) (Figure 1). Similarly, subgroup 2 had a significantly higher i-PRS than subgroup 3 (P-adj = 7.3e-03). Conversely, for PRS calculated using SNPs outside the HLA region, scores of subgroup 1 were significantly lower than scores from subgroups 2 (P-adj = 3.89e-02) and 3 (P-adj = 3.89e-02), suggesting a higher contribution of SNPs in the HLA region to the overall higher genetic risk of subgroup 1. Differential expression analysis of proteins (n=2625) between the subgroups revealed that an isoform of Complement C4-B (C4B) was significantly overexpressed (P-adj 3.72e0-5) in subgroup 1 compared to subgroup 3, followed by C2, which indicates involvement of the complement system in this subgroup. Likewise, an isoform of Immunoglobulin heavy constant gamma 3 (IGHG3) was significantly overexpressed (P-adj 2.3e-03) in subgroup 2 compared to subgroup 4 (Figure 2). These preliminary results support the concept that unanalyzed or unknown autoantibodies and B cell involvement may be present in patients of subgroup 4. Figure 1. Figure 2. Subgroup 1 exhibited higherHLA-PRS than the other subgroups and elevated C4b levels compared to Subgroup 3. This is consistent with the high linkage disequilibrium betweenC4gene andHLArisk variants. These preliminary findings support the hypothesis of subgroup-specific pathogenic mechanisms among antibody-defined SLE subgroups of patients. We will further analyze this dataset to incorporate PRS relevant to immune cell phenotypes and calculate PRS targeted to SLE subgroups. We will also validate these findings in independent populations.References:[1.] Diaz-Gallo LM. ACR Open Rheumatol 2022;4(1):27-39. [2.] Reid S. Ann Rheum Dis 2020;79(3):363-9. [3.] Ritchie ME. Nucleic Acids Res 2015;43(7):e47.

OBJECTIVES Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation-associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein. METHODS We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n = 8) and serum (n = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using Western Blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains. RESULTS All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein. CONCLUSIONS Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade.

Supplementary Figure 2 from TLR3 as a Biomarker for the Therapeutic Efficacy of Double-stranded RNA in Breast Cancer

Supplementary Table 1 from TLR3 as a Biomarker for the Therapeutic Efficacy of Double-stranded RNA in Breast Cancer

The involvement of citrulline-specific CD4+ T cells in anti-citrulline protein antibody-positive rheumatoid arthritis (RA) is well described, whereas less attention has been given to CD8+ T cells. New data suggest that CD8+ T cells also contribute to citrulline-specific immune responses in RA.

Supplementary Figure Legends 1-2, Table Legend and Methods from TLR3 as a Biomarker for the Therapeutic Efficacy of Double-stranded RNA in Breast Cancer

Idiopathic inflammatory myopathies (IIM) are rare autoimmune systemic diseases characterized by muscle weakness and the presence of muscle-infiltrating T cells. IIM represent a clinical challenge due to heterogeneity of symptoms and variability of response to immunosuppressive treatment. Here, we performed in-depth single-cell sequencing on muscle-infiltrating T cells and peripheral blood memory T cells in six patients with recently diagnosed IIM. We identified tissue resident memory T-cell (TRM ) signatures including the expression of HOBIT, XCL1 and CXCR6 in the muscle biopsies of all patients with IIM. Clonally expanded T-cell clones were mainly found among cytotoxic and TRM implying their role in the disease pathogenesis. Finally, identical expanded T-cell clones persisting at follow-up in the muscle tissue of two patients suggest their involvement in disease chronicity. Our study reveals a muscle tissue resident memory T-cell signature in patients with IIM and a transcriptomic map to identify novel therapeutic targets in IIM.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease involving autoreactivity to proteinase 3 (PR3) as demonstrated by presence of ANCAs. While autoantibodies are screened for diagnosis, autoreactive T cells and their features are less well-studied. Here, we investigated PR3-specific CD4+T cell responses and features of autoreactive T cells in patients with PR3-AAV, using a cohort of 72 patients with either active or inactive disease. Autoreactive PR3-specific CD4+T cells producing interferon g in response to protein stimulation were found to express the G-protein coupled receptor 56 (GPR56), a cell surface marker that distinguishes T cells with cytotoxic capacity. GPR56+CD4+T cells were significantly more prominent in the blood of patients with inactive as compared to active disease, suggesting that these cells were affected by immunosuppression and/or that they migrated from the circulation to sites of organ involvement. Indeed, GPR56+CD4+T cells were identified in T-cell infiltrates of affected kidneys and an association with immunosuppressive therapy was found. Moreover, distinct TCR gene segment usage and shared (public) T cell clones were found for the PR3-reactive TCRs. Shared T cell clones were found in different patients with AAV carrying the disease-associated HLA- DP allele, demonstrating convergence of the autoreactive T cell repertoire. Thus, we identified a CD4+T cell signature in blood and in affected kidneys that display PR3 autoreactivity and associates with T cell cytotoxicity. Our data provide a basis for novel rationales for both immune monitoring and future therapeutic intervention in PR3-AAV.

Objective CD4+ T cells are implicated in rheumatoid arthritis (RA) pathology from the strong association between RA and certain HLA class II gene variants. This study was undertaken to examine the synovial T cell receptor (TCR) repertoire, T cell phenotypes, and T cell specificities in small joints of RA patients at time of diagnosis before therapeutic intervention. Methods Sixteen patients, of whom 11 patients were anti–citrullinated protein antibody (ACPA)–positive and 5 patients were ACPA–, underwent ultrasound‐guided synovial biopsy of a small joint (n = 13) or arthroscopic synovial biopsy of a large joint (n = 3), followed by direct sorting of single T cells for paired sequencing of the αβ TCR together with flow cytometry analysis. TCRs from expanded CD4+ T cell clones of 4 patients carrying an HLA–DRB1*04:01 allele were artificially reexpressed to study antigen specificity. Results T cell analysis demonstrated CD4+ dominance and the presence of peripheral helper T–like cells in both patient groups. We identified >4,000 unique TCR sequences, as well as 225 clonal expansions. Additionally, T cells with double α‐chains were a recurring feature. We identified a biased gene usage of the V β chain segment TRBV20‐1 in CD4+ cells from ACPA+ patients. In vitro stimulation of T cell lines expressing selected TCRs with an extensive panel of citrullinated and viral peptides identified several different virus‐specific TCRs (e.g., human cytomegalovirus and human herpesvirus 2). Still, the majority of clones remained orphans with unknown specificity. Conclusion Minimally invasive biopsies of the RA synovium allow for single‐cell TCR sequencing and phenotyping. Clonally expanded, viral‐reactive T cells account for part of the diverse CD4+ T cell repertoire. TRBV20‐1 bias in ACPA+ patients suggests recognition of common antigens. image