Discussion
OX40, a member of the TNFR superfamily, is mainly expressed on activated CD4+ T cells. Its cognate ligand OX40L, a member of the TNF superfamily, is predominantly expressed on activated ADCs and on some endothelial cells, mast T cells and activated T cells. Accumulating evidence has shown that the OX40-OX40L pathway plays a crucial role in the pathogenesis of multiple autoimmune diseases[19-22]. Furthermore, a correlation between the expression of OX40 on CD4+ T cells and disease severity has been observed in individuals with autoimmune diseases such as SLE[13, 23]. The OX40-OX40L interaction contributes to promoting the activation, proliferation and long-term survival of effector T cells, preserving cellular memory, facilitating the production of effector cytokines, and suppressing regulatory function by activating the PI3K-PKB/NF-κB/NFAT pathways[7]. Here, we detected the expression of membrane-bound and soluble OX40 and OX40L and explored the functions, and potential mechanisms, as well as the clinical significance of OX40/OX40L signaling in the occurrence and development of MG.
In this study, we found that the expression of the positive costimulatory molecule OX40 on CD4+ T cells from patients with MG was significantly higher than that on cells from the HC group, and OX40 expression on CD4+ T cells was positively correlated with the concentration of AchR-Ab, consistent with previous research results[24]. In contrast previous studies or new findings, CD4+ OX40 expression on CD4+ T cells from patients with MG was not related to the age at onset in our study[24], which may be associated with the sample size and disease activity. In addition, our study showed that OX40L expression on CD19+ B cells and CD14+ mononuclear cells in the MG group was significantly upregulated compared with that in the HC group. The subgroup analysis revealed significantly higher expression of OX40 on CD4+ T cells from the GMG group than on cells from the the OMG group and patients with thymoma or thymic hyperplasia than in those without thymoma or thymic hyperplasia, and OX40L expression was significantly increased on CD14+mononuclear cells from the GMG group compared with that in the OMG group. The correlation analysis showed that OX40 expression on CD4+ T cells was positively correlated with QMGs. Based on these results, the expression of OX40 on CD4+ T cells may be related to the severity of MG, and the OX40/OX40L signal participates in the immunopathological process of MG. To the best of our knowledge, the present study is the first to discover increased expression of OX40/OX40L on the peripheral blood lymphocytes of patients with MG. Therefore, we speculate that the OX40-OX40L interactions may contribute to the activation, proliferation and long-term survival of effector T cells, promote B cell differentiation to secrete autoantibodies and inhibit cell apoptosis in patients with MG, which may generate immune disorders and immune injury.
Because MG is an intricate, heterogeneous disease, the expression of these molecules may dynamically change across the disease course. In the present study, significantly higher OX40 expression was detected on CD4+ T cells from the USMG, RSMG and PSMG groups than on cells from the HC group. OX40L expression on CD19+B cells was significantly increased in the PSMG group compared with the HC group. OX40L was expressed at higher levels on CD14+mononuclear cells from the RSMG group than on cells from the HC group. These results indicated that the OX40/OX40L pathways in MG mainly play regulatory roles in the later stage of the disease. Furthermore, compared with the USMG and PSMG groups, the expression of OX40 on CD4+ T cells from the RSMG group was significantly increased. The results of the correlation analysis indicate that OX40 expression on CD4+ T cells was positively correlated with the AchR-Ab concentration, while OX40L expression on CD19+B cells and CD14+mononuclear cells was negatively correlated with the disease duration in the RSMG group. These results suggested that the expression of OX40 on CD4+ T cells may be closely related to the recurrence of MG. These phenomena may be related to the mechanism of OX40/OX40L signaling in T cells. On the one hand, from the perspective of T cell survival, Song, Salek-Ardakani [25] et al. found that OX40-deficient T cells normally differentiated and proliferated into effector T cells 2–3 days after the activation of TCR signaling. Nevertheless, the survival rate was significantly decreased after 12–13 days of activation, suggesting that the OX40/OX40L pathways might not affect the early stage of T cell proliferation but promote the activation of T cells and prolong their survival in the later stage. On the other hand, from the perspective of T cell function, OX40-OX40L interactions promote the generation of memory T cells and maintain their survival[26-29], and the long-term survival of memory CD4+ T cells after antigen restimulation promotes their rapid differentiation into effector T cells, which may be one of the causes of disease recurrence [30].
In addition to the membrane-bound forms, costimulatory molecules also exist in soluble forms. Soluble costimulatory molecules are generated through the proteolytic cleavage[31] or mRNA splicing[32] of membrane-bound molecules. sOX40 and sOX40L may be cleaved from membrane molecules, but the specific mechanism remains unclear[12, 33]. We detected the plasma levels of sOX40 and sOX40L in patients with MG for the first time to explore the roles of the soluble molecules. The plasma sOX40 levels in the MG group, especially in the USMG group, were significantly decreased compared with those in the HC group, consistent with the findings of unbalanced expression of the membrane and soluble form of OX40 in individuals with type 1 diabetes[12]. Moreover, plasma sOX40 levels were positively correlated with QMGs and disease duration in the MG group. Therefore, sOX40 may be associated with disease severity in patients with MG. Studies have confirmed that sOX40 binds to mOX40L on ADCs to interfere with the positive signals transduced by the OX40/OX40L pathway and suppress T cell activation [34-36]. Therefore, we speculated that activated T cells may be inhibited by sOX40-induced blockade of OX40/OX40L, and then inactivated T cells would generate less sOX40 in the early stage of MG. In the dynamic process of disease progression, similar to mOX40, the levels of sOX40 in the RSMG group were significantly higher than those in the USMG and PSMG groups, and the level of sOX40 in 13 patients with recurrent MG was significantly higher than that in patients in remission. Based on the findings, we hypothesized that the increase in sOX40 levels in the RSMG group may be due to increased shedding from mOX40.
No significant difference in the plasma sOX40 levels was observed between the MG group and HC group. The dynamic observation of the molecules showed that sOX40L levels in the PSMG group were significantly decreased compared with those in the HC, USMG and RSMG groups. Moreover, the levels of sOX40L in 13 patients with MG in the remission stage were significantly lower than those in the recurrence stage. The correlation analysis showed that sOX40L levels were negatively correlated with the disease duration. Experiments have shown that sOX40L promotes late proliferation and activation states of T cells but does not influence early proliferation and activation[37, 38]. Researchers have speculated that the binding of sOX40L in the peripheral blood to OX40 on activated T cells would allow T cells to receive continuous persistent and positive stimulation signals[39, 40], leading to relative immune system hyperactivity in the later stages of MG. Excessive consumption of sOX40L may result in decreased sOX40L levels in later disease stages.
In our study, the concentration of sOX40L was dozens of times higher than that of sOX40. We hypothesized that sOX40L may be a functional molecule and may enhance the positive signals transduced by the OX40/OX40L pathway in the later stage of MG, which may be an important mechanism for reinitiating an immune response, but this hypothesis requires further exploration in the future. ROC curves showed the moderate predictive value of the expression levels of OX40 and sOX40L in CD4+ T cells for monitoring MG recurrence. However, the sensitivity was low, and further large-scale cohort studies should be conducted in the future.
In summary, this study suggested that abnormal activation of the OX40/OX40L pathway participates in the immunopathological process of MG. sOX40L may regulate the positive signals transduced by the OX40/OX40L pathway in the later stage of the disease, leading to the activation and proliferation of effector T cells and the subsequent progression of MG. Membrane-bound and soluble OX40 expression levels are correlated with MG disease activity and severity, and OX40 expression on CD4+ T cells may be related to the recurrence of MG. Further studies targeting the specific mechanisms underlying the involvement of the OX40/OX40L pathway in MG-related immune disorders will provide new targets and directions for the treatment of MG.