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.