CD40 is a member of the TNF-α receptor superfamily and is found in several types of cells, including B cells and myeloid cells, where it is expressed constitutively. CD40L is expressed on activated T cells and is usually not detected in resting T cells. The CD40L expression is detectable in activated Th1, Th2, Th17, follicular helper T cells (Tfh), and Tregs. However, the expression of CD40L in Tregs is significantly reduced compared to Th. Moreover, CD40L induces CD40 trimerization and mediates downstream signaling pathways. The CD40/CD40L pathway plays a vital role in adaptive immunity. CD40 on B cells binding to CD40L facilitates B cells proliferation, activation, and antibody production. In the presence of other T cell activation signals (MHC-II and B7), CD40 can promote the proliferation and differentiation of T cell into specific types with the assistance of cytokines (68).

In patients with TED, the expression of CD40 on OFs is reported to be higher compared to healthy individuals. Additionally, under the stimulation of IFN-γ secreted by T cells, CD40 on OFs of TED patients gets significantly upregulated (69). CD40 activation exacerbates orbital inflammation and extraocular muscle fibrosis. It has been shown in vitro that CD40 activation leads to an increased synthesis of IL-6, IL-8, prostaglandin E2 (PGE2), MCP-1, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin, and also results in increased secretion of hyaluronic acid (56, 70–72). Several variables are involved in the pathogenesis of TED via the CD40 pathway. Adipsin, a serine protease homolog, has been implicated in the CD40-mediated stimulation of OFs in individuals with TED. Adipocytes mainly release Adipsin, but macrophages and monocytes can also generate it. Adipsin is a vital adipokine and one of the components of complement factor D, participating in the pathogenesis of various autoimmune diseases. Activation of TED-OFs by CD40L or IGF-1 causes fibroblasts to differentiate into adipocytes, leading to increased synthesis and secretion of Adipsin. This further increases the expression of downstream cytokines, including IL-6, IL-8, PGE2, ICAM-1, and CCL2 (73). In addition, there is a notable elevation in the expression of sphingosine-1-phosphate (S1P) in the orbital tissues of patients with TED. S1P has been demonstrated to stimulate fibrosis, lipid synthesis, and the release of inflammatory cytokines in OFs of individuals with TED. Research has demonstrated that CD40 enhances the production of S1P in TED-OFs and facilitates T cell migration (74). This study proposed that lipid metabolism may regulatethe migration of T cells to orbital tissues and that controlling the activity of enzymes associated with S1P metabolism might be an effective strategy for managing inflammation in the orbit. Furthermore, the TSHR and CD40 expression levels on CD34⁺ fibrocytes in the peripheral blood of individuals with TED are markedly elevated. TSH and CD40L can upregulate IL-6, IL-8, and IL-12 expression of CD34⁺ fibrocytes obtained from the peripheral blood of patients with TED (75–77). The genetic polymorphism of CD40 also indicates its important role in the pathogenesis of TED. A meta-analysis revealed that the increased frequency of the T allele at the CD40 rs1883832 locus was associated with a reduced risk of developing TED (78). The mutation at the CD40 rs1883832 locus may reduce the transcription of CD40 mRNA and thus inhibit the activation of OFs (79).

The CD40/CD40L pathway mediates the interaction between activated T cells and OFs in TED. Feldon et al. demonstrated that the expression of CD40L on T cells is essential for the activation of OFs in individuals with TED (80). The binding of CD40 on OFs and CD40L (CD154) on T cells in TED patients leads to OFs proliferation, and blocking CD40 or CD40L significantly reduces OFs proliferation. Th17 cells in the peripheral blood of patients with TED can interact with OFs through the CD40-CD40L pathway. Additionally, they can stimulate the secretion of IL-17 and bind to the IL-17RA receptor on the cell membrane of OFs. This interaction activates the MAPK pathway, promoting RANTES secretion by OFs (28) and further promoting the migration of Th17 and Th1 cells to the orbit. In an in vitro model, The concurrent administration of IL-17 and CD40L resulted in a significant augmentation in the production and release of RANTES in OFs. However, the IL-17 alone did not demonstrate any noteworthy secretion of RANTES. This suggests that the activation of CD40 upstream is accountable for the response to IL-17. In addition to the activation of OFs by T cells, the CD40/CD40L pathway also plays a key role in the activation of T cells by OFs in TED patients, evidenced by TED-OFs promoting the proliferation of autologous T cells (81). The proportion of CD4⁺ CD28^- T cells in the peripheral blood of TED is reported to be significantly increased in comparison to normal individuals and is positively correlated with disease severity (82). CD40L may be crucial in activating T cells in TED or other autoimmune diseases when CD28, the second T cell activation signal, is absent. The activation of CD4⁺ CD28^- T cells is potentially reliant on the activation of CD40L. This is evident as inhibitory antibodies against CD40L considerably impede the activation of CD4⁺ CD28^- T cells (82). The precise mechanism of action of CD4⁺ CD28^- T cells, which contribute to orbital inflammation in TED, remains unclear. However, in the thyroid tissue of GD patients, activated CD4⁺ CD28^- T cells can secrete various inflammatory factors, promoting local tissue inflammation and the secretion of TRAb by B cellsleading to thyroid cell proliferation and tissue hyperplasia. Compared with CD4⁺ CD28⁺ T cells, CD4⁺ CD28^- T cells are insensitive to signals for cell apoptosis (83), suggesting that T cell apoptosis deficiency may be involved in the orbital inflammation of TED.

Ultimately, CD40 shows potential as a viable focus for addressing TED therapeutically. RNA aptamers specifically targeted CD40(CD40Apt) in TED mice, thereby significantly inhibiting the CD40-CD40L pathway. Moreover, CD40Apt reduced the expression of CD40, collagen I, TGF-β, and α-SMA in mice’s extraocular muscles and orbital fat tissue. Additionally, the aptamers effectively suppressed the Erk, p38, JNK, and NF-κB signaling pathways (84). The therapeutic effect of CFZ533 (CD40 monoclonal antibody) on TED still needs further research. However, CD40 is widely expressed in various cells throughout the body, including B cells, macrophages, dendritic cells (DC), platelets, endothelial cells, and epithelial cells (68). Close monitoring of systemic side effects is necessary when using therapies that target CD40.

CTLA-4, also referred to as CD152, is mainly found in Tregs and activated T cells. Its principal function is to control the activation of T cells during the early stage. This procedure mostly takes place within the lymph nodes. In naïve T cells, CTLA-4 is present in intracellular vesicles. The binding of CD28 and CD80/CD86 causes CTLA-4 to migrate to the membrane of T cells. In comparison to CD28, CTLA-4 shows a higher affinity for B7. Therefore, CTLA-4 competitively binds to the ligands CD80 and CD86 on APCs, transmitting inhibitory signals for T cell activation and preventing their further proliferation (85). CTLA-4 inhibitors hinder the transmission of inhibitory signals from CTLA-4 to cells, stimulating the proliferation of T cells that target tumor antigens and strengthen the immune system’s ability to eliminate tumor cells (7).

Currently, ipilimumab and tremelimumab are FDA-approved CTLA-4 inhibitors (86). There have been reports of cancer patients developing Graves’ disease following therapy with ipilimumab or tremelimumab (87–89). Administration of CTLA-4 inhibitors, either alone or in combination with PD-1/PD-L1 inhibitors, can lead to acute exacerbation of pre-existing Graves’ disease in advanced melanoma patients (90, 91). An initial case of TED development after four doses of ipilimumab treatment was reported by Min et al., with the patient presenting severe eye pain, proptosis, and periorbital edema. The CT and MRI scans revealed significant thickening of the extraocular muscles, but laboratory testing indicated a normal concentration of TRAb (17). Subsequently, McElnea, Borodic, Sheldon, and Sagiv independently documented instances of tumor patients who had no prior record of Graves’ disease developing TED after receiving ipilimumab therapy (18–21). These patients presented varying thyroid function and TRAb ( Table 1 ). Rhea reported a case of a patient with Graves’ disease who had a sudden and rapid worsening of TED symptoms after receiving ipilimumab treatment. The patient’s periorbital edema improved significantly after discontinuing ipilimumab and undergoing glucocorticoid pulse therapy. However, there was a subsequent recurrence of TED while receiving pembrolizumab treatment (22). Sagiv also documented a case of a patient experiencing TED after receiving treatment with tremelimumab, a recently approved monoclonal antibody that targets CTLA-4. The patient exhibited notable thickening of the four rectus muscles, and abnormalities in thyroid function were reported (21). These case reports suggest that CTLA-4 may underlie the pathogenesis of TED.

Researchers have been intrigued by the association between the risk of TED development and the polymorphism in the CTLA-4 gene for many years. Vaidya et al. first discovered that the G-carrying genotype in the CTLA-4 A/G polymorphism is associated with an increased risk of TED and the frequency of the G allele is positively correlated with the severity of TED (92). There has been considerable research interest in the SNP49 in exon 1 of the CTLA-4 gene. However, the findings from different studies have yielded inconsistent conclusions (93). Studies on gene polymorphisms in various ethnic groups have shown that the G allele at SNP49 is associated with an increased risk of developing TED (94). The findings of a systematic review indicated that the A/G polymorphism at the SNP49 in exon 1 of the CTLA-4 gene has been linked to the development of TED in Europeans. However, this association does not appear to be statistically significant in Asians. Among Europeans, the GG genotype has been observed to significantly elevate the likelihood of developing TED. Additionally, individuals carrying the G allele face a notably higher risk than those with the A allele (95). The mechanism may rely on the fact that the G allele at the SNP49 in exon 1 of CTLA4 leads to the substitution of threonine (Thr) for alanine (Ala), which results in incorrect processing of the CTLA-4 in the endoplasmic reticulum. This causes a reduction in glycosylation efficiency and decreases the expression of CTLA-4 on the cell membrane (96). Subsequent studies have shown a negative correlation between the “TT” genotype frequency at the CTLA-4 rs733618 and TED. Haplotype analysis indicates that Crs733618Crs16840252 may potentially affect the risk of development of TED among patients with GD (14). The T allele at position -318 in the CTLA-4 promoter region is also speculated to increase the risk of TED in GD patients (94, 97).

The exact function of CTLA-4 in the development of TED still needs to be fully understood. The expression of Foxp3 (a vital transcription factor in Tregs differentiation and maturation) and CD40 is significantly increased in the orbital fat tissue of TED patients. In contrast, the expression level of CTLA-4 decreases significantly. According to the study, a positive correlation was found between the severity of the disease and the expression levels of Foxp3 and CD40. On the other hand, there is a negative correlation between the severity of the disease and the expression levels of CTLA-4, CD28, and CD40L (58). This indicates that Tregs infiltrate the orbital tissue of individuals with TED, but their ability to suppress inflammatory responses is impaired. The compromised function of Tregs may be associated with the reduced production of CTLA-4, which reduces the suppressive signal for T cell activation.

Unlike CTLA-4, the inhibitory effect of PD-1 on T cells usually occurs in the peripheral tissues (98). Typically, following the TCR and MHC-antigen peptide complex become linked, the proximal signaling molecules of TCR undergo phosphorylation and trigger the activation of downstream PI3K-Akt-mTOR, Ras-MEK-ERK signaling pathways to relay signals that activate T cells. Tumor cells have aberrantly increased expression of PD-L1. Following the interaction between TCR and the MHC-antigen peptide complex, PD-L1 specifically binds to PD-1 on the surface of T cells. This binding triggers the phosphorylation of tyrosine in the intracellular domain of PD-1, which then recruits SHP-2 to the ITIM motif. As a result, the phosphorylation of proximal signaling molecules of TCR, such as Lck and ZAP-70, is blocked. This disruption leads to abnormal downstream activation signal transduction of T cells, ultimately causing abnormalities in the T cell cycle, gene transcription, cell metabolism, and epigenetics. Consequently, the recognition and killing of tumor cells by T cells are compromised (99).

Table 1 shows that some cancer patients experienced TED due to the administration of PD-1 or PD-L1 inhibitors. Park et al. reported the first case of bilateral TED following the treatment with pembrolizumab (PD-1 inhibitor) (23). After treatment with pembrolizumab, the patient developed asymmetric exophthalmos and diplopia in both eyes; however, the thyroid function remained normal. For patients with pre-existing Graves’ disease, there are reports of the occurrence of TED after treatment with nivolumab (PD-1 inhibitor) (21). Research has furthermore discovered that the occurrence of hyperthyroidism and hypothyroidism in individuals with cancer who use PD-1 inhibitors is significantly higher compared to those who receive CTLA-4 inhibitors or PD-L1 inhibitors alone (100), suggesting that immune imbalance caused by PD-1 abnormalities may play an essential role in TED. Further investigation revealed a significant increase in the presence of PD-1 on the outer layer of CD4⁺ T cells and CD8⁺ T cells in the peripheral blood of individuals with TED. Adding PD-L1 to the co-culture system of T cells and OFs significantly inhibited the secretion of IFN-γ, IL-1β, TNF-α, and IL-2. Moreover, it significantly downregulated the CD40L of T cells. The downregulation of CD40L may have a therapeutic impact on TED by downregulating the activation of CD40 on the surface of OFs (31). This work elucidated the mechanism of regulation of the CD40/CD40L pathway in patients with TED and proposed that PD-L1 might serve as a promising therapeutic agent for the treatment of TED. Furthermore, there was a significant increase in the proportion of PD-1⁺ cells in CD4⁺ T, CD8⁺ T, and CD19⁺ B cells in the peripheral blood of patients with GD (101). However, the involvement of PD-1 on the surface of B cells, in developing TED is still understudied. The role of CD19⁺ PD-1⁺ B cells in the pathogenesis of TED and the disease diagnosis may have potential implications against the production of autoantibodies.

Recent research has indicated that patients with TED exhibited a significant increase in MHC II, B7, and PD-L1 expression on CD34⁺ fibrocytes. In clinical trials, moderate to severe TED patients treated with teprotumumab showed a substantial reduction in MHC II, B7, and PD-L1 expression. However, this effect was not observed in patients receiving a placebo (36). Since PD-L1 plays an inhibitory role in T cell activation, and MHCII and B7 are two important co-stimulatory molecules, this study suggests that MHC II and B7 may mask the immunosuppressive effect of PD-L1 on CD34⁺ fibrocytes in TED patients. The upregulation of PD-L1 may be an adaptive response to excessive immune activation. The study also found that the secretion of Slit2 by OFs significantly downregulated the expression of PD-L1 on CD34⁺ fibrocytes. Suppressing the expression of Slit2 in patients with TED resulted in a significant increase in the expression of PD-L1. These findings indicate that in the orbital microenvironment, Slit2 may intensify local inflammation by modifying the expression of immune checkpoints.

The major histocompatibility complex (MHC), also referred to as human leukocyte antigen (HLA), has a role in the development of TED due to its gene polymorphism. HLA is divided into type I (HLA-A, B, C) and type II (DP, DQ, DR). Type II HLA, or MHC II, is usually expressed on the surface of APCs, providing the first signal for CD4⁺ T cells activation, and can enhance the inflammatory response. In particular, MHC II is also expressed in the OFs and is essential for stimulating T cells in individuals with TED (80). The presence of TED increases MHC II in CD34⁺ fibrocytes. However, when IGF-IR inhibitors (teprotumumab, lisitinib, 1H7) or IGF-IR knockdownare used, the expression levels of MHC II decrease. This decrease is accompanied by a reduction in the number of CD4⁺ T cells adhering to CD34⁺ fibrocytes and a decrease in the secretion of IL-17A, which is mainly secreted by Th17 cells. These findings suggest that IGF-IR inhibitors may restore immune tolerance in TED patients by regulating the expression of immune checkpoints associated with T cell activation in CD34⁺ fibrocytes and influencing the proliferation and differentiation of CD4⁺ T cells (36). The presence of MHC II on OFs and fibrocytes, rather than MHC I, suggests their close direct interaction with CD4⁺ T cells in the pathogenesis of TED. Similarly, numerous studies have reported that the variation in type II HLA genes is linked to a higher risk of developing TED (102). However, there is less research on the differences in type I HLA variation between TED patients and healthy individuals, indicating that the CD4⁺ T cell subset may significantly impact the progression of TED.

The expression of Tim-3 is increased on the activated CD4⁺ T cells. Activation of Tim-3 suppresses the proliferation of T cells and the secretion of cytokines, leading to a decrease in the body’s inflammatory response and the preservation of peripheral immune tolerance. Research has demonstrated that individuals with TED exhibit significant reductions in the expression of Tim-3 on Th1 and Th17 cells in their peripheral blood in comparison to patients with GD. Furthermore, there is an inverse relationship between the number of Th1 and Th17 cells in the peripheral blood of TED patients and the proportion of Tim-3⁺ cells (30). This suggests that the downregulation of Tim-3 may disrupt immune tolerance in the body, increasing the immune activity of Th1 and Th17 cells and causing excessive local inflammation in the orbit region. Furthermore, research has shown that the presence of Gal-9 (the ligand for Tim-3) on DC cells in the peripheral blood of patients with TED is significantly reduced, and its expression is inversely associated with CAS. Adding Gal-9 to a co-culture system of peripheral blood DCs and lymphocytes significantly inhibits the secretion of Th1, Th2, and Th17-related cytokines. These studies reveal that dysfunction of the Gal-9/Tim-3 pathway may be associated with the pathogenesis of TED (103).

B7.1 (CD80) and B7.2 (CD86) molecules are co-stimulatory molecules on the surface of APCs. These molecules competitively interact with CD28 against CTLA-4. The interaction between B7 and CD28 serves as the second signal in the T cell activation pathway, resulting in the proliferation, and differentiation of T cells, along with the release of downstream inflammatory cytokines (104). In patients with GD, the polymorphism of CD80 and CD86 genes is related to the risk of TED. Multifactorial dimensionality reduction analysis revealed that the interaction between CD80-rs9289131 and CD86-rs9872483 has a protective effect on the onset of TED (104). A study revealed that B7 molecules were consistently expressed on CD34⁺ fibrocytes, and the expression of B7 molecules may be significantly increased by thyrotropin or M22 (an antibody that stimulates the TSHR). Conversely, Slit2 decreases the expression of B7 molecules. These findings indicate that OFs regulate the expression of B7 molecules on CD34⁺ fibrocytes,which significantly impact T cell activation.

The mRNA levels of CD28 in the orbital tissues of severe TED patients are significantly lower than those in mild TED patients. Nevertheless, the degree of downregulation is less pronounced compared to CTLA-4 (58), indicating that the equilibrium between CD28 and CTLA-4 plays a role in preserving the immune homeostasis of the orbit.

CD52 is widely expressed on mature B and T cells, with relatively low expression on NK cells and other leukocytes. CD52 is not expressed in hematopoietic stem cells (105). Alemtuzumab is a monoclonal antibody that specifically inhibits the activity of CD52. The FDA has approved it for treating multiple sclerosis and B-cell chronic lymphocytic leukemia. Alemtuzumab primarily works by quickly reducing the number of mature lymphocytes in the bloodstream and then using the regeneration ability of hematopoietic stem cells to restore a balanced immune system in the body. A subset of multiple sclerosis patients developed TED as a consequence of taking alemtuzumab therapy (105–108). Presently, there is a lack of research on the involvement of CD52 in the development of TED. During immune system reconstitution, lymphocytes undergo rapid proliferation and sustained activation. This can lead to an imbalance in the ratio of Tregs to Th1 and Th2 cells, thereby exacerbating the body’s inflammatory response and stimulating the production of autoantibodies (109). Furthermore, following the administration of alemtuzumab, there was a significant increase in the levels of IL-21 in the peripheral blood of multiple sclerosis patients who had recently developed autoimmune diseases. The activation of the IL-21 receptor on effector T cells can stimulate the production of autoreactive T cells, potentially facilitated by IL-21 (110).