Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent and progressive inflammation of the synovial joints, leading to debilitating arthritis and heterogeneous extra-articular manifestations (1). The high incidence, teratogenicity, disability, and poor prognosis associated with RA present significant challenges and demand effective management strategies (2, 3). Currently, the clinical treatment of RA relies heavily on glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs), and disease-modifying anti-rheumatic drugs (DMARDs) such as synthetic or biologic agents (4, 5). While these medications play a crucial role in alleviating joint symptoms and halting disease progression, they often come with potential side effects including nausea, anorexia, and bone marrow suppression due to individual variations (4, 5). As the need for new anti-RA therapies becomes increasingly urgent, both clinical and basic research efforts are continuously being conducted to better understand the pathogenesis of this complex disease.

The etiology of RA remains incompletely understood, with genetic, autoimmune, and environmental factors being considered as the primary causes (6). Individuals with genetic predisposition may experience abnormal immune responses in synovium triggered by environmental factors, resulting in synovitis characterized by joint swelling, pain, and irreversible damage (7). T lymphocyte subgroups and their released pro-inflammatory or anti-inflammatory cytokines exist widely in synovitis and play an indispensable role in perpetuating the inflammatory response (8, 9). Good progress has been made in the development and application of biological agents targeting proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6, and IL-17 (10). IL-6, a pleiotropic pro-inflammatory cytokine with various biological activities, participates in a variety of biological activities such as immune regulation, hematopoiesis, and inflammatory response (11). IL-6 amplifies the effects of cytokines such as TNF-α and IL-17, indirectly influencing the activation of tyrosine protein kinases 1 and 2 via binding with IL-6R. This ultimately leads to the release of TNF-α, IL-17, and other cytokines, contributing to the development of arthritis and extra-articular lesions (12, 13). In addition, IL-6 promotes the proliferation, differentiation, and maturation of B cells, and subsequently causes serious antibody deposition in the synovium and exacerbation of joint injury (14). Up-regulation of IL-6 has even been observed prior to the onset of RA (15). As expected, the humanized anti-interleukin-6 receptor (IL-6R) antibody, tocilizumab (TCZ), has shown high efficacy in treating patients with RA. However, limitations still exist (16–18).

Evidence from numerous studies indicates that the imbalance of CD4+T cell subsets, especially the significant reduction in T regulatory cells (Tregs) responsible for maintaining immune homeostasis and suppressing inflammatory response, contributes to joint damage and disease deterioration in RA (19). Tregs have emerged as potential therapeutic targets for many autoimmune diseases such as RA (20, 21). Initial data indicate that the number of circulating Tregs in RA patients decreases with increasing disease activity, which can be reversed by exogenous supplementation with low doses of IL-2 (22). IL-2, a cytokine with multiple immunomodulatory functions, has been widely recognized for its ability to selectively expand Tregs and maintain or restore the balance of CD4+T subgroup balance and immune tolerance (23, 24). Encouraging progress has also been made in combining exogenous IL-2 with DMARDs such as methotrexate and TNF inhibitors, but not with TCZ (25, 26).

Previous studies have confirmed the efficacy and safety of low-dose IL-2 in the treatment of RA. In the current study, we analyzed the effects of low-dose IL-2 combined with TCZ on clinical symptoms, immune-related indicators, and CD4+T lymphocyte subsets in RA patients, and evaluated its safety.

We have previously reported that Tregs are significantly reduced with the aggravation of disease in RA patients, and combined with low-dose IL-2 therapy can maintain or even rebuild autoimmune homeostasis by promoting the proliferation of Treg cells, thus delaying the deterioration of disease (22, 30). Great heterogeneity exists in the clinical presentation and immune mechanisms of RA, as a consequence, not all patients respond similarly to the same drug. This study aims to observe the effects of low-dose IL-2 combined with TCZ on arthritis symptoms and immune cells in active RA patients, based on the good results of low-dose IL-2 combined with bDMARDs, excluding TCZ, reported in previous studies (25, 26). The results of this study showed that low-dose IL-2 combined with TCZ treatment could safely and effectively reduce arthritis symptoms in RA patients, promote the more stable proliferation of Tregs, and reduce the proliferation of Th2, Th17, and Th17/Treg caused by IL-2 treatment alone. This is of great value for patients with immune disorders mediated by high levels of Th2 and Th17.

The dysregulation of the immune response, mainly including the imbalance of immune cells and cytokines, has been widely proven to contribute to the pathogenesis and progression of RA (31). More notably, the disorders of immune modulate cells not only exacerbate synovitis, progressive bone injury, and progressive destruction of articular cartilage but are also closely associated with complications such as pulmonary interstitial fibrosis and coronary artery disease that increase mortality in RA patients (32, 33). Therefore, targeting cytokines or immunomodulatory drugs are gradually entering clinical therapy.

According to the production of cytokines and the expression of main transcription factors, CD4+T cells are roughly divided into CD4+T helper (Th) cells such as Th1, Th2, and Th17 cells, which can regulate the inflammatory environment, promote antibody production, control innate immunity and stimulate immune memory, and anti-inflammatory CD4+T groups, namely Treg, which inhibit inflammation, dominant self-tolerance, maintain immune homeostasis and control immune responses to prevent autoimmune diseases (34). Inflammation is usually accompanied by dysregulation of CD4+T cell subsets and cytokine networks, which play a dominant role in immune regulation, characterized by excessive proliferation of inflammatory cells or depletion or dysfunction of regulatory cells, and ultimately the inflammation gets aggravated (19, 21). As observed in this study, the absolute count of peripheral Tregs and the ratio of Th17 to Treg were strongly associated with the disease activities of RA. Specifically, the absolute counts of Treg cells were significantly negatively correlated with the DAS28, ESR, CRP, TJC, and SJC levels, while Th17/Treg was significantly positively correlated with DAS28, ESR, TJC, and SJC, which demonstrated the regulation of Treg cells levels and Th17/Treg ratio were beneficial to the recovery of RA.

IL-2, produced by T-cell activation, is an essential cytokine for lymphocyte growth. T cell proliferation in mice with knockout of the IL-2 receptor and IL-2 expression gene was uncontrolled and combined with progressive fatal systemic autoimmune disease, which could be prevented by adoptive transfer of Tregs, highlighting the importance of effective IL-2 signaling in immune function (35, 36). As demonstrated in this study, low doses of IL-2 can improve the condition by promoting Tregs proliferation in RA patients. Consistent with the findings, a prospective study performed by Rosenzwajg M. demonstrated the value of low-dose IL-2 in 11 autoimmune diseases, proposing that all patients had relatively specific amplification and activation of Tregs, and were well tolerated to low-dose IL-2 regardless of disease and concomitant therapy (24). However, IL-2 is regarded as the central factor responsible for the proliferation, differentiation, and function of CD4+T cell subsets, and can also promote or inhibit the expression of specific Th cell gene programs, including Th2, and Th17 cells (37). This action of IL-2 signaling is extremely detrimental in patients with immune disorders characterized by high levels of Th cells such as Th2. Therefore, it may be feasible to co-regulate immune homeostasis with other DMARD drugs.

Given the complexity of the immune network in RA, biological agents targeting immune cells and cytokines have emerged in an endless stream in recent years, among which infliximab targeting TNF-α and TCZ targeting IL-6R are the most widely used in clinical practice. Circulating IL-6 levels are elevated in RA patients, and there is a correlation between IL-6 levels in the affected joints and inflammatory markers. IL-6 is required for the development of collagen-induced arthritis (CIA), and blocking IL-6 receptors improved joint disease in CIA mice (38, 39). IL-6R-mediated JAK-STAT signaling pathway induces or aggravates RA lesions by promoting B cell activation and T cell proliferation (12, 13). The binding of IL-6 to its receptor induces the expression of RORγt by phosphorylation of JAK-mediated STAT3, thereby promoting the release of pro-inflammatory cytokines IL-17 and IL-22, recruiting granulocytes, and promoting the development of an amplifying loop for inflammation (40).

Tocilizumab, the first humanized monoclonal antibody against IL-6R, could inhibit all IL-6R-mediated signaling by intravenous or subcutaneous injection, alone or in combination with methotrexate, for the treatment of RA in adults who have either responded inadequately to or were intolerant of previous therapy with ≥ 1 csDMARDs or TNF inhibitor (41). Our study observed that in RA patients with high levels of Th2, Th17, and Th17/Treg on the basis of the original treatment, exogenous TCZ and low dose of IL-2 can rapidly and safely reduce the DAS28 score and alleviate the disease in a short period, effectively promote the moderate proliferation of Tregs and reduce the level of Th cells, which is consistent with the previous results of TCZ combined with other DMARDs (42). Meanwhile, more female patients were recruited in this study, but relevant literature showed that gender did not affect the distribution ratio of peripheral blood immune cells and the therapeutic effect of tocilizumab, so it was believed that gender did not affect the judgment of results in this study (43).

Our study has a few limitations, such as a single-center study with a small sample size and short observation time. Additionally, the lack of cytokine data was considered a limitation of this study. Since this was a retrospective study, data from all patients were not available for several variables.

In conclusion, this study further confirms that reduced Tregs are an integral factor in the deterioration of RA, which can be improved or even reversed by exogenous low doses of IL-2. However, combined TCZ can safely and effectively reduce Th cells while appropriately Treg for RA patients with high Th cell levels, thus regulating immune homeostasis and contributing to preventing disease deterioration.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by This study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (2016 KY-007). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

SZ: Investigation, Methodology, Resources, Writing – original draft. HC: Data curation, Software, Writing – original draft. JW: Investigation, Methodology, Resources, Writing – original draft. HS: Formal analysis, Methodology, Supervision, Writing – review & editing. TC: Data curation, Investigation, Writing – original draft. RP: Data curation, Methodology, Validation, Writing – review & editing. QS: Methodology, Validation, Writing – original draft. HZ: Investigation, Methodology, Software, Writing – review & editing. XL: Conceptualization, Resources, Writing – review & editing.