Karine Chemin

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. 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. 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. 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.

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.

GPR183 is a chemotactic GPCR involved in immune cell migration. Using AI-driven virtual screening and biophysical assays, we identify inverse agonists. From 70 compounds and a subsequent hit expansion, compound 78 emerges as a potent inhibitor of constitutive and agonist-induced Gi signaling as well as β-arrestin2 recruitment. Binding within the receptor core is confirmed by a conformational biosensor, molecular dynamics simulations, and mutagenesis. The compound also blocks agonist-driven migration of peripheral blood mononuclear cells ex vivo with very high potency. Additionally, our analyses reveal key features of GPR183 activation, highlighting tyrosine 260 (Y260 ^6.51 ) in transmembrane helix 6 as critical. Mutation of this residue alters compound 78 efficacy as well as induces receptor signaling bias, indicating a switch mechanism. Overall, this study provides tools to probe GPR183 function, identifies a chemical scaffold, and advances understanding of receptor activation, supporting therapeutic targeting in inflammatory, autoimmune, and cancer-related diseases.

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.

GPR183 is a chemotactic G protein-coupled receptor implicated in immune cell migration and various diseases. Here, we report the discovery of novel GPR183 inverse agonists using an artificial intelligence-driven virtual screening approach combined with biophysical sensors including a first-in-class conformational biosensor. From an initial screen of 70 compounds and a hit expansion, a selected candidate - compound 78 - demonstrated potent inhibition of GPR183’s constitutive and agonist-stimulated Gi activation as well β-arrestin2 recruitment. The binding of the compound to the receptor core was confirmed by studies using a new conformational sensor based on intramolecular BRET, molecular dynamics, and site-directed mutagenesis experiments. Finally, the compound 78 was able to block agonist-induced migration of peripheral blood mononuclear cells ex vivo . This work provides a novel chemical scaffold for GPR183 modulation. It introduces a new tool for dissecting receptor function, paving the way for therapeutic exploration in inflammatory, autoimmune, and neoplastic disorders.

Objectives: The aim of this study was to combine deep T cell phenotyping with assessment of citrulline-reactive CD4+T cells in the pre-rheumatoid arthritis (RA) phase. Methods: 20 anti-CCP2 positive individuals (HLA-DRB1*04:01) presenting musculoskeletal complaints without clinical or ultrasound signs of synovitis; 10 arthritis progressors and 10 matched non-arthritis progressors were included. Longitudinal samples (1–3 time points) of peripheral blood mononuclear cells were assessed using HLA-class II tetramers with 12 different citrullinated candidate autoantigens combined in a >20-colour spectral flow cytometry panel. Results: The baseline CD4+T cell phenotype was similar between individuals who progressed to arthritis (ie, in the pre-RA phase) and the non-progressors, when studying markers associated with Th1, Th17, T-peripheral and T-regulatory cells as well as with T-cell activation. Citrulline-reactive CD4+T cells were present in both groups but at significantly lower frequency in the progressor group. CD4+T cells specific for citrullinated tenascin-C were the most frequently observed among the progressors, and their frequencies diminished during follow-up that is, closer to arthritis onset. Notably, PD-1 and CD95 expression on the memory cit-tenascin-C-specific T cells in this group indicated repeated antigen exposure. Conclusions: Our data lend support to citrullinated tenascin-C as an interesting T cell antigen in RA. Moreover, lower frequency of circulating citrulline-specific cells in arthritis progressing individuals suggest an initiated homing of these cells to the joints and/or their associated lymph nodes in the pre-RA phase and a possible window of opportunity for therapeutic preventive interventions.

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. 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. 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. 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.

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 (T _RM ) 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 T _RM 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.