Detection and phenotypic characterisation of acetylcholine receptor (AChR)-reactive CD4⁺ T cells in myasthenia gravis using antigen-reactive T cell enrichment (ARTE) and multiparameter flow cytometry

Generalized myasthenia gravis (MG) is a chronic, relapse-prone autoimmune disease driven by pathogenic autoantibodies, most often directed against the acetylcholine receptor (AChR). Although established treatments such as thymectomy can lead to clinical improvement, a substantial proportion of patients experience incomplete remission or disease relapse, and no validated immunological biomarkers exist to predict long-term outcomes after surgery. In parallel, new targeted therapies, including efgartigimod alfa (Vyvgart®), a neonatal Fc receptor antagonist, have recently entered clinical practice; however, biomarkers that reflect underlying immune mechanisms or predict long-term therapeutic response are likewise lacking. Recent studies in other chronic antibody-mediated autoimmune diseases have identified a population of autoreactive CD4⁺ T cells with an exhaustion-like phenotype (ThEx) that persists despite treatment while retaining B cell helper capacity, potentially explaining chronicity and relapse in antibody-mediated autoimmunity. Whether such ThEx-like autoreactive CD4⁺ T cells are present in MG, and how they are affected by thymectomy or emerging therapies, remains unknown. To address this, we applied antigen-reactive T cell enrichment (ARTE) combined with multiparametric flow cytometry to directly detect and phenotype rare AChR-specific CD4⁺ T cells ex vivo. ARTE was used to track the frequency and phenotype of AChR-reactive CD4⁺ T cells before and after efgartigimod therapy and thymectomy. In parallel, comprehensive peripheral T and B cell immunophenotyping was performed, and in thymectomy cases, T and B cell subsets were additionally analysed in thymic tissue. Initial data show that AChR-reactive CD4⁺ T cells were present at higher frequencies in MG patients than in healthy controls and exhibited increased FOXP3 expression together with the inhibitory receptors PD-1 and TIGIT, while retaining a conventional T helper lineage, consistent with an exhaustion-like (ThEx) phenotype. Therapy-associated changes in the frequency and phenotype of AChR-reactive CD4⁺ T cells were observed, accompanied by alterations in peripheral T and B cell compartments following both efgartigimod therapy and thymectomy. Further in-depth phenotypic characterisation of peripheral and thymic immune cell subsets is ongoing. Overall, these preliminary findings demonstrate the feasibility of integrating autoreactive CD4⁺ T cell analysis with broader T and B cell immunophenotyping to explore immune signatures relevant to treatment response and disease course in MG.