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• Leflunomide could be a therapeutic candidate for MG treatment. • The germinal center response in EAMG rats was reduced by leflunomide. • Leflunomide rebuilds the balance of CD4+ Th cells by suppressing Th1 cells and promoting Treg cells. • Leflunomide inhibits Tfh cells through the IL-21/STAT3 pathway. Leflunomide, an immunosuppressive disease-modifying anti-rheumatic drug (DMARD), is widely used in the treatment of rheumatoid arthritis (RA), psoriatic arthritis (PA) as well as multiple sclerosis. However, its role in myasthenia gravis (MG) has not yet been clearly explored. Here, we investigated the effect of leflunomide on experimental autoimmune myasthenia gravis (EAMG) in vivo and in vitro. The results demonstrated that leflunomide alleviated the severity of EAMG associated with reduced serum total anti-acetylcholine receptor (AChR) IgG levels. During the development of EAMG, the increase of follicular helper T cells (Tfh) 1, Tfh 17 cells and decrease of follicular regulatory T cells (Tfr) were reversely altered after leflunomide administration. Our work further found that leflunomide might inhibit Tfh cells through the IL-21/STAT3 pathway to reduce the secretion of antibodies by B cells. In addition, leflunomide rebuilt the balance of Th1/Th2/Th17/Treg subsets. These results suggested that leflunomide ameliorated EAMG severity by regulating humoral immune responses and Th cell profiles thereby providing a novel effective treatment strategy for MG. [ABSTRACT FROM AUTHOR]

• JAK2 inhibitors could be a therapeutic candidate for MG treatment. • JAK2 inhibitors rebuilds the balance of CD4+ Th cells by suppressing Th17 cells and promoting Treg cells. • JAK2 inhibitors balances Th17/Treg cells via regulating the JAK2/STAT3-AKT/mTOR signaling pathway. An imbalance in Th17/regulatory T (Treg) cells is the major pathogenic mechanism underlying myasthenia gravis (MG). JAK2 inhibitors selectively inhibit JAK2 and reduce inflammatory responses. However, there have been no studies examining the therapeutic effects of JAK2 inhibitors in the context of MG. Here, an experimental autoimmune MG (EAMG) rat model was established to explore the therapeutic effect of JAK2 inhibitors on EAMG rats immunized with the AChR α-subunit (97–116 peptide). A JAK2 inhibitor was administered to EAMG rats both in vivo and in vitro. The following experimental methods were used to evaluate the effects of JAK2 inhibitors. The behavioral scores and body weights of the rats were assessed on alternate days. Serum anti-AChR (97–116) IgG and cytokine levels were detected using ELISA. CD4+ T cell subsets and related transcription factors in mononuclear cells were detected using flow cytometry and qPCR, respectively. The expression levels of protein molecules in the signaling pathway were detected by western blotting, and the neuromuscular junctions were observed using immunofluorescence. The results revealed that JAK2 inhibitors could regulate Th17/Treg balance in vivo and in vitro. JAK2 inhibitors reduced the immune response in EAMG rats (including reducing pro-inflammatory cytokines and postsynaptic membrane complement deposition), improved clinical symptoms, and increased AChR aggregation in the postsynaptic membrane. Meanwhile, this study demonstrated that JAK2 inhibitor treatment suppressed the phosphorylation of JAK2/STAT3 and AKT/mTOR pathways and decreased the expression level of the IL-23 receptor. This study reveals that there is crosstalk between the JAK2/STAT3 and AKT/mTOR pathways in EAMG rats. JAK2 inhibitors can ameliorate EAMG by regulating Th17/Treg balance by inhibiting both signaling pathways. Our study provides new potential therapeutic targets for MG immunotherapy. [ABSTRACT FROM AUTHOR]