Primary immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by decreased platelet count due to enhanced platelet destruction and/or impaired platelet production. The estimated incidence of pediatric ITP is 2 to 5 per 100,000 children (1). Platelet production is a complex biological process that involves the generation and maturation of megakaryocytes, as well as the release of platelets. Platelets originate from megakaryocytes that express many immune receptors and cytokines in common with their “mother cells” (2). The classic pathogenesis of ITP is that antiplatelet antibodies mediate macrophage phagocytosis and the destruction of platelets. In recent years, the underlying mechanism of ITP has expanded to a more heterogeneous and complex immune pathophysiology including deficiency in T cell regulatory activity, altered megakaryocytic function (3–6). CD3⁺CD4⁺IFN-γ⁺ helper T lymphocyte (Th1) polarization is demonstrated by the increase in serum IFN-γ and IL-2 in ITP patients, and the Th1/Th2 ratio of T cells in the circulation and spleen is increased (7, 8). In addition, the percentage of CD3⁺CD4⁺IL-17⁺ helper T lymphocytes (Th17) to CD3⁺CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) is elevated at the newly diagnosed ITP stage (9). In our previous study, we used a range of machine learning algorithms in predicting the chronicity of pediatric ITP, and identified Th17, Th17-toTreg, Th1, and TCRαβ⁺CD4⁻CD8⁻ double negative T cells (DNT)/CD3⁺ T cells as the four most significant immune function indicators (10).

TCRαβ⁺DNT, a relatively small subpopulation comprising approximately 1-5% of all CD3⁺ T lymphocytes in peripheral blood. The most well-defined pathological condition characterized by the expansion of TCRαβ⁺DNT is autoimmune lymphoproliferative syndrome (ALPS), which is defined as exceeding 1.5% of total lymphocytes and/or >2.5% of CD3⁺ lymphocytes, according to current clinical guidelines (11, 12). In addition to serving as an important diagnostic marker for ALPS, TCRαβ⁺DNT has also been proven to play diverse functional roles in various diseases. First, TCRαβ⁺DNT can prevent the onset of and provide long-lasting protection against type 1 diabetes and graft versus host disease (GVHD) (13, 14). Achita et al. have confirmed that the infusion of allogeneic or xenogeneic TCRαβ⁺DNT alone does not cause GVHD due to the MHC-unrestricted characteristics (14). Second, multiple studies point to a critical anti-tumor activity exhibited by TCRαβ⁺DNT, producing IFN-γ, perforin and granzyme B, mediating the killing effect in hematologic malignancies and solid tumors (15–17). Lee and collaborators identified that TCRαβ⁺DNT demonstrated promising efficacy as an ex vivo therapeutic approach for the treatment of leukemia (17). Third, TCRαβ⁺DNT is capable of secreting proinflammatory cytokines, including IL-4, IL-17, and tumor necrosis factor-α (TNF-α), and plays a similar role to CD4⁺ Th cells. It contributes to several autoimmune diseases, such as systemic lupus erythematosus (SLE), psoriasis and Sjögren’s syndrome. TCRαβ⁺DNT can infiltrate the primary target organs, suggesting a direct and pathogenic role in tissue damage (18–20). Although the presence of DNT at sites of injury in autoimmunity strongly suggest their critical role in immune regulation, their potential immunomodulatory function in ITP remains poorly understood.

Glucocorticoids are the first-line treatment option for patients with ITP, especially dexamethasone, which is less toxic and more active against plasma cells. It has long been used in ITP for its global influence on the immune system by functionally suppressing both T- and B-cell reactions and restoring Tregs. High-dose dexamethasone (HD-DXM) treatment has been reported to restore the normal Th1/Th2 ratio, reduce Th17 cells, and increase Tregs (21–23). Moreover, evidence suggests that the intrinsic sensitivity of lymphocytes may be a major factor in determining their response to glucocorticoids (24, 25). However, some patients have a slow or no response to HD-DXM treatment, which implies the presence of other types of immune cells.

Here, we aimed to identify different immune function indicators that may contribute to the development and prognosis of newly diagnosed pediatric primary ITP, such as Th17, Th17-toTreg, Th1, and TCRαβ⁺DNT. In addition, we tried to further investigate the expression of blood TCRαβ⁺DNT in the pathogenesis of pediatric ITP, and analyze the association of the response to glucocorticoids with larger samples.

Imbalanced T-lymphocyte subpopulations have been shown to be involved in the autoimmune pathogenesis of pediatric ITP (3). In our study, to explore the contribution of various immune indicators to the pathophysiology of ITP, the percentages of Th17-to Treg, Th17, Th1, and TCRαβ⁺DNT were analyzed in the peripheral blood of patients with ITP and healthy controls. Similar to what Jingyao Ma et al. found (10), we observed Th17-to Treg, Th17, Th1, and TCRαβ⁺DNT might participate in the onset of newly diagnosed ITP. Furthermore, we compared these indicators at the initial diagnosis between OR and NR to HD-DXM. Univariate analysis revealed that only the TCRαβ⁺DNT proportion was a key factor for ITP patients with ITP. Taken together, these data suggest that TCRαβ⁺DNT could play a significant role in both the development of ITP and the treatment outcome of HD-DXM.

TCRαβ⁺DNT, as a minor portion of αβ T cells that lack CD4 and CD8 markers, has been considered to contribute to many autoimmune diseases, such as autoimmune lymphoproliferative syndrome (ALPS), systemic Lupus Erythematosus (SLE), and psoriasis, along with their ability to help B cells produce autoantibodies and various pro-inflammatory cytokines including IL-17, IFN-γ, and IL-4 (11, 18, 19). However, in some studies, TCRαβ⁺DNT has been found to potentially exert an immunosuppressive function in specific diseases, such as graft versus host disease (GVHD) and type 1 diabetes (T1D), by suppressing B-cell proliferation and producing IL-10 (28, 29). Therefore, TCRαβ⁺DNT may play a pro-inflammatory or immunosuppressive role under different conditions. Based on the above findings, we conducted a comprehensive analysis of the alterations in TCRαβ⁺DNT in pediatric ITP and evaluated the impact of HD-DXM on this specific subset.

In the present study, we found that the level of TCRαβ⁺DNT was increased in pediatric patients with newly diagnosed ITP. This is consistent with prior studies that found that an abnormal proportion of total lymphocytes or CD3⁺ T lymphocytes probably contributed to the development of other autoimmune disorders (30, 31). It suggested that an elevated TCRαβ⁺DNT proportion may be involved in the immune imbalance of ITP.

HD-DXM, which is generally accepted as the most common first-line treatment for pediatric ITP, is thought to have an extensive effect on the immune system. According to previous studies, lymphocyte subsets can influence or predict their response to corticosteroids in patients with ITP, including abnormal CD4:CD8 ratios, higher Th17 levels, and lower Treg levels (21–23, 32). Lu et al. described a subtype of adenosine receptor CD39⁺Tregs that was decreased in patients with ITP, but after HD-DXM therapy, the responding patients showed an increase in Tregs and their ability to immunosuppress was improved (33). Additionally, previous studies have indicated that changes in platelet mass play a significant role in modulating Tregs (34, 35). One potential explanation for the reversal of Treg defects by increased platelet count is their ability to secrete substantial quantities of TGF-β1, a critical molecular responsible for inducing Tregs (36). Generally speaking, aberrant T cell differentiation and proliferation can be rectified by dexamethasone. However, no study has previously described the significance of TCRαβ⁺DNT in dexamethasone therapy. Our data showed that a higher TCRαβ⁺DNT level at initial diagnosis was associated with a poor response to HD-DXM. Patients with lower levels of TCR αβ⁺DNT had a sensitive response to glucocorticoids. This implies that TCRαβ⁺DNT might play a predictive role in HD-DXM efficacy.

Furthermore, the recovery of TCRαβ⁺DNT levels was more obvious after HD-DXM in the OR group than in the NR group. Yan’s et al. found that conventional methylprednisolone effectively reduced DNT cells, but not IL-17-producing DNT cells, in patients with autoantibody-associated vasculitis (AAV) (37). A study of primary Sjogren’s syndrome showed that expanded-disease-associated IL-17⁺ DNT cells may be resistant to dexamethasone treatment (38). These results suggest that there is a special subpopulation of DNT cells resistant to glucocorticoids, which may account for the unchanged level of TCRαβ⁺DNT after HD-DXM therapy in the NR group. Therefore, it might be helpful to predict the outcomes of HD-DXM by detecting the level of TCRαβ⁺DNT initially and after treatment.

Although this study utilized a larger sample size to explore the role of TCRαβ⁺DNT in pediatric ITP, some limitations still exist. First, the observation period was short, and the predictive role of TCRαβ⁺DNT in the long-term efficacy or prognosis of HD-DXM was not investigated. Second, this study design did not implement the recommended control-to-case ratio of 1:1. Despite these limitations, TCRαβ⁺DNT is involved in the development of pediatric ITP and affects the response to glucocorticoids.

In conclusion, our findings suggest that TCRαβ⁺DNT in the peripheral blood might contribute to T cell-mediated immune dysregulation in pediatric ITP and may be involved in immune responses to HD-DXM. We systematically analyzed the alterations in TCRαβ⁺DNT in pediatric patients with ITP. This demonstrates that the proportion of TCRαβ⁺DNT is higher in ITP patients than in healthy controls. The percentage of TCRαβ⁺DNT was significantly higher in NR patients at initial diagnosis. Interestingly, elevated TCRαβ⁺DNT can be effectively corrected by glucocorticoids in the OR group, rather than in the NR group. Overall, TCRαβ⁺DNT may play a significant role in the pathogenesis of pediatric ITP and influence the efficacy of HD-DXM therapy. Moreover, further studies are needed to explore the underlying mechanism of TCRαβ⁺DNT in pediatric patients with ITP.