Introduction

Acute ischemic stroke (AIS) ranks among the primary contributors to mortality and long-term disability worldwide, exerting considerable economic strain on both society and families.¹ Cerebral collateral circulation serves a pivotal function in safeguarding the brain from ischemic and reperfusion injury.² In the event of stenosis or occlusion of cerebral blood vessels, collateral circulation permits the delivery of blood to the infarct-adjacent region, commonly referred to as the penumbra, thus offering protection to the ischemic brain tissue in that area.³ Good collateral status (GCS) has been linked to improved clinical outcomes in AIS patients undergoing intravenous thrombolysis or endovascular therapy.⁴

Clinically, the assessment of collateral circulation is predominantly performed using imaging techniques, with digital subtraction angiography (DSA) widely acknowledged as the premier approach for detecting collateral circulation status.⁵ Despite its accuracy, the invasive nature of DSA limits its widespread use. Computed tomography angiography (CTA), valued for its non-invasive approach, has emerged as a preferable method for evaluating collateral circulation in AIS patients.⁶ However, various neuroimaging techniques are often confined to major stroke centers and are inaccessible in numerous other regions, hindering the ability to monitor dynamic changes in collateral circulation during long-term follow-up of ischemic stroke.⁷ At present, effective and sensitive biomarkers for predicting collateral circulation remain unavailable. Thus, identifying reliable biomarkers is of great significance for predicting collateral circulation and determining the clinical prognosis.

Inflammation is recognized as a key factor in the pathophysiology of ischemic stroke, encompassing a complex interplay of various cell types and effector molecules, impacting tissues beyond the central nervous system.⁸ Uncontrolled and excessive inflammatory responses exacerbate ischemic brain damage and impede post-stroke cerebral recovery. Regulatory T cells (Tregs), a vital subgroup of anti-inflammatory T cells, serve a function in mediating immune tolerance and ensuring tissue homeostasis.^9,10 Tregs are involved in post-ischemic stroke inflammatory and immune responses via multiple mechanisms, significantly contributing to the limitation of brain damage and the promotion of recovery after stroke. In addition to stroke, research indicates that Tregs play a critical role in cardiovascular diseases, diabetes, and other conditions. In cardiovascular diseases, Tregs can limit the inflammatory response in atherosclerotic plaques, thereby reducing plaque rupture and subsequent cardiovascular events.¹¹ Similarly, in diabetes, Tregs are involved in maintaining immune tolerance, and their dysfunction is closely associated with the onset and progression of type 2 diabetes.¹²

Interleukin-2 (IL-2), commonly called T cell growth factor, is produced by T lymphocytes in response to antigenic stimulation and is crucial for regulatory T cells expansion. Previous studies have demonstrated that defects in the IL-2 receptor and its downstream signaling pathways are closely linked to the pathogenesis of type 1 diabetes, and enhancing IL-2 signaling can prevent and reverse the disease by restoring Tregs cell function.¹³ Additionally, research has shown that a single intravenous injection of recombinant human interleukin-2 (rhIL-2) improves cardiac function after myocardial infarction by promoting angiogenesis and activating natural killer (NK) cells.¹⁴ Following ischemic stroke, IL-2 exerts neuroprotective effects by promoting Tregs expansion and inhibiting inflammation.

Nevertheless, it remains unclear whether Tregs and IL-2 have additional roles following ischemic stroke, particularly concerning their potential impact on cerebral collateral circulation post-ischemia. This study aims to investigate the association between two systemic immune-inflammatory markers, Tregs and IL-2, and the collateral circulation status in AIS patients through flow cytometry and enzyme-linked immunosorbent assay (ELISA), highlighting their potential as biomarkers for predicting and improving treatment outcomes.

Materials and Methods

Study Design and Subjects

The study design flowchart is presented in Figure 1. This investigation is a retrospective, single-center, observational case-control study. Between September 2023 and May 2024, AIS patients who received treatment at the Department of Neurology, The First Hospital of Anhui University of Science & Technology, were prospectively enrolled.

Figure 1 Flow chart of the study design.

The inclusion criteria were as follows: (1) A clinical diagnosis of AIS verified through cranial magnetic resonance imaging and diffusion-weighted imaging; (2) Symptom onset within 3 days; (3) Patients aged 18 years or older; (4) The presence of intracranial atherosclerotic stenosis (≥50%) or occlusion in the anterior circulation, confirmed by CTA within 7 days after symptom onset.

The exclusion criteria included the following: (1) Intracranial hemorrhage identified through neuroimaging; (2) A confirmed life expectancy of less than 6 months, attributable to cancer or severe systemic diseases; (3) Presence of chronic inflammatory conditions, pregnancy, or being a kidney transplant recipient; (4) A history of trauma, surgical procedures, or administration of steroids, immunosuppressants, immunomodulators, or antibiotics within 30 days prior to hospitalization; (5) Evidence of active infection (body temperature >37.5 °C, increased white blood cell [WBC] count), symptoms such as productive cough, urinary dysfunction, or diarrhea; (6) Thrombolysis or thrombectomy following stroke onset, as well as a baseline modified Rankin Scale (mRS) score of ≥2.

Collateral circulation status was not utilized as an inclusion criterion in this study. It was assessed by independent, blinded observers through the previously established TAN scoring system.¹⁵ AIS patients were categorized into two groups: individuals with GCS (TAN 2–3) and those with poor collateral circulation (PCS, TAN 0–1). Healthy volunteers, free of any stroke history, were selected as the control group and matched with AIS patients based on age and gender, minimizing the confounding effects of other risk factors.

Clinical Examination

Demographic information, including age, epidemiological details, and comorbidities, was extracted from the participant’s medical records. Laboratory findings such as WBC count, renal function, lipid profile, cystatin C, fasting blood glucose, lipoprotein (a), and homocysteine levels were recorded. Upon admission, professionally trained neurologists evaluated the severity of neurological deficits in all AIS patients utilizing the National Institutes of Health Stroke Scale (NIHSS). At 90 days following the onset of stroke, the mRS was employed to score the patients.

Measurement of Peripheral Blood Tregs and IL-2

Peripheral blood samples (2 mL) were drawn from AIS patients on the second-day post-admission, with control group samples obtained during routine physical examinations, all under fasting conditions. Flow cytometry was employed to examine the expression of specific surface antigens on circulating Tregs. Specifically, 100 μL of peripheral blood underwent incubation with the following monoclonal antibodies for labeling: 20 μL anti-CD3, 20 μL anti-CD4, 20 μL anti-CD25, and 20 μL anti-CD127 (BD, Franklin Lakes, NJ, USA). The cells were evaluated utilizing a FACSCanto II flow cytometer (BD, Franklin Lakes, NJ, USA) and processed via Kaluza analysis software (Beckman Coulter, Brea, CA, USA). Tregs were defined as CD4+/CD25+/CD127 low-stained cells within the lymphocyte gate. For all analyses, Tregs% was represented as the proportion of Tregs relative to the total CD4+ lymphocyte population.

Additionally, 4 mL of peripheral blood was collected under fasting conditions on the second day (for controls, this occurred during the physical examination). Plasma IL-2 levels were quantified using an ELISA kit (IL-2 ELISA kit, double antibody sandwich method, catalog number 11321001; Hunan Xieguang Biotechnology Co., Ltd., Hunan, China) per the supplier’s protocols.

Statistical Analysis

All statistical analyses were conducted utilizing SPSS 27.0 software (IBM, Chicago, IL, USA). Quantitative data with normal distribution were denoted as mean ± standard deviation and compared between groups through t-tests. Data with non-normal distribution were denoted as median (interquartile range) [M(IQR)] and examined utilizing Mann–Whitney U-tests. Categorical variables were displayed as frequency (percentage) [n(%)] and examined utilizing χ² tests. Bivariate correlations were assessed with Pearson coefficients for normally distributed variables and Spearman coefficients for non-normally distributed variables. Logistic regression analysis was applied to explore the association between Tregs and IL-2 with varying collateral circulation states. A receiver operating characteristic (ROC) curve evaluation was executed to assess Tregs’ and IL-2’s predictive ability to regulate different collateral circulation statuses. A P-value < 0.05 was deemed statistically significant.

Results

Participant Characteristics

This investigation encompassed 46 healthy controls, 60 patients with AIS and GCS, and 57 patients with AIS and PCS (Table 1). In comparison to the healthy control group, AIS patients demonstrated lower levels of lymphocytes, Tregs and IL-2 (Figure 2A and B), along with elevated systolic and diastolic blood pressure, a greater incidence of hypertension and diabetes, and increased levels of WBCs, neutrophil percentage (NEUT%), neutrophils, fasting blood glucose, homocysteine (HCY), creatinine (Cr), Cystatin-C, and low-density lipoprotein (LDL). No notable disparities in age, gender, other medical histories, or laboratory data were identified between the groups (Table 1).

Table 1 Baseline Characteristics of Control and Patients with AIS

Figure 2 (A) Plasma IL-2 levels were lower in the AIS group compared to the control group. (B) Plasma Treg levels were lower in the AIS group compared to the control group. (C) Plasma IL-2 levels were lower in the PCS group compared to the GCS group. (D) Plasma Treg levels were lower in the PSC group compared to the GCS group. Indicators (**): express p<0.01, Indicators (***): express p<0.001.

In addition, when compared to the PCS group, patients in the GCS group showed higher Treg and IL-2 levels (Figure 2C and D), lower NIHSS scores upon admission, lower mRS scores 90 days post-stroke, lower blood urea nitrogen (BUN) levels, and higher triglyceride (TG) levels. No notable disparities in age, gender, blood pressure, or vascular risk factors were noted between the GCS and PCS groups. Furthermore, no notable differences in other laboratory data were found between these two groups (Table 1).

Correlation Between Peripheral Blood Tregs and IL-2 Levels and Clinical Indicators

Correlation analyses, utilizing Pearson for normally distributed variables and Spearman for non-normally distributed variables, were performed to assess the relationships between Tregs, IL-2, and clinical indicators in AIS patients (Figure 3A–E). A significant positive association was detected between Tregs and IL-2 levels (r = 0.3849; p < 0.0001). Negative correlations were noted between Tregs levels and NIHSS scores (r = −0.2701; p = 0.0032), 90-day mRS scores (r = −0.4763; p < 0.0001), and SBP (r = −0.2472; p = 0.0072) (Supplementary Figure 1A). Similarly, IL-2 levels displayed negative associations with NIHSS scores (r = −0.1958; p = 0.0319), 90-day mRS scores (r = −0.2742; p = 0.0028), and systolic blood pressure (r = −0.2765; p = 0.0025) (Supplementary Figure 1B), while exhibiting a positive link to glucose (GLU) levels (r = 0.1886; p = 0.0417) (Supplementary Figure 1C).

Figure 3 Correlation between peripheral blood Tregs and IL-2 levels with clinical indicators. (A) Treg levels show a significant positive correlation with IL-2 levels. (B) Treg levels are negatively correlated with NIHSS scores. (C) IL-2 levels are negatively correlated with NIHSS scores. (D) Treg levels are negatively correlated with 90-day mRS scores. (E) IL-2 levels are negatively correlated with 90-day mRS scores.

Predictive Potential of Tregs and IL-2

To assess the predictive potential of Tregs and IL-2, logistic regression analysis, a nomogram, and ROC curve analysis were employed. Logistic regression analysis demonstrated that Tregs and IL-2 levels were linked to collateral circulation in both unadjusted and adjusted models. After adjusting for age, gender, blood pressure, vascular risk factors, and variables with p < 0.05 in the univariate analysis (laboratory data, NIHSS score at admission), low levels of Tregs and IL-2 remained independent predictors of PCS (compared to the GCS group) (Table 2). Moreover, a nomogram model was constructed to predict collateral circulation risk factors (Figures 4, 5, Supplementary Figure 2). These findings support the potential of plasma Tregs and IL-2 levels as biomarkers for predicting collateral circulation in AIS patients. Subsequently, ROC curve analysis was executed to assess the diagnostic efficiency of Tregs and IL-2. The area under the ROC curve (AUC) for Tregs in distinguishing GCS patients from PCS patients was 0.741 (0.652–0.830), while the AUC for IL-2 was 0.710 (0.618–0.803) (Figure 6A, Table 2). Furthermore, the AUC for the combination of Tregs and IL-2 reached 0.779 (Figure 6B).

Table 2 Correlation Analysis of Biomarkers with Different Collaterals

Figure 4 Nomogram for collateral circulation risk factors.

Figure 5 Decision curve analysis for the collateral circulation risk nomogram.

Figure 6 Diagnostic efficiency of Tregs and IL-2. (A) ROC curves of plasma Tregs and IL-2 distinguish between GCS and PCS groups. (B) ROC curve for the combination of Tregs and IL-2.

Subgroup Analysis

To further investigate the relationship between Tregs, IL-2, and collateral circulation status, we performed a series of subgroup analyses. As illustrated in Figure 7, none of the subgroups, including age, sex, the presence of hypertension or diabetes, as well as smoking and alcohol consumption, significantly modified the association between Tregs, IL-2, and collateral circulation status (all P values for interaction > 0.05).

Figure 7 Subgroup analyses of the association between the Tregs, IL-2, and collateral circulation status.

Discussion

In this study, plasma Tregs and IL-2 levels were compared between AIS patients and control groups, as well as between GCS and PCS patients. The key findings are as follows: (1) Plasma Tregs and IL-2 levels effectively distinguished GCS from PCS patients, with PCS patients displaying significantly lower levels of both markers in comparison to GCS patients and healthy controls. (2) A notable negative association was observed between Tregs and IL-2 levels and NIHSS scores at admission, as well as 90-day mRS scores. (3) Plasma Tregs and IL-2 levels were strongly linked to collateral circulation status in individuals with AIS, with lower levels of these markers identified as independent predictors of PCS. In conclusion, these results underscore the predictive value of plasma Tregs and IL-2 for assessing collateral circulation status in AIS patients, suggesting their potential as biomarkers and therapeutic targets for this patient population.

An increasing body of research indicates that collateral circulation is a key factor in determining clinical outcomes in AIS patients, regardless of whether they undergo acute reperfusion therapy.^16,17 In individuals with adequate cerebral collateral blood supply, brain tissue within the affected vascular region may remain viable for an extended duration before evolving into an ischemic core.¹⁵ In this investigation, GCS patients were observed to exhibit milder neurological impairments. Moreover, 90-day functional outcomes, as measured by mRS scores, revealed that GCS patients had better daily living abilities. These observations are consistent with the findings of earlier research.

The development of collateral circulation in patients with AIS is influenced by several factors, including genetics, aging, sex, neurological pathways, and vascular risk conditions such as diabetes and hypertension.¹⁸ It has been suggested that heightened inflammatory responses following ischemic stroke can negatively influence collateral circulation formation.¹⁹ Inflammation negatively impacts collateral vessel formation through various mechanisms, one of which may involve a reduction in the number of endothelial progenitor cells, thereby hindering the development of collateral vessels.²⁰ Early brain edema is partially mediated by inflammatory pathways, which may increase the resistance in collateral arterioles, ultimately leading to collateral circulation failure.^21,22 Tregs play a crucial role in this process. Prior studies have highlighted the critical role of Tregs in maintaining immune tolerance and mitigating inflammatory collateral damage by regulating self-reactive immune responses.^23,24 One investigation revealed that Tregs are capable of inhibiting the expression of neutrophil-derived matrix metalloproteinase-9 (MMP-9) and safeguarding the blood-brain barrier to mitigate cerebral damage after AIS. This mechanism requires cell-to-cell interaction and is mediated through PD-1/PD-L1 signaling.²⁵ Furthermore, Tregs modulate the function of effector T cells by secreting inhibitory cytokines such as TGF-β, IL-10, and IL-35, thereby suppressing excessive inflammatory responses and reducing the potential damage to collateral vessels. Through this immune regulatory function, Tregs help alleviate inflammatory damage in collateral circulation and promote vascular stability and functional recovery. Moreover, another study observed a correlation between low MMP-9 levels and high monocyte chemoattractant protein-1 levels with GCS status prior to treatment.²⁶ These studies suggest that Tregs, by regulating immune responses and particularly through the secretion of inhibitory cytokines to control inflammation, may play a significant role in the formation of collateral circulation following ischemic stroke. This study found that early peripheral blood Tregs in ischemic stroke patients were reduced when compared with a healthy control group, aligning with findings from Ruhnau et al.²⁷ Furthermore, a negative correlation was identified between Tregs and 90-day mRS scores (r = −0.2742; p = 0.0028), indicating that elevated levels of Tregs are predictive of improved clinical outcomes. It was also demonstrated that GCS patients exhibited higher plasma Tregs levels than PCS patients, suggesting that elevated Tregs levels may enhance the development of collateral circulation.

Beyond their immunoregulatory role, emerging evidence underscores the significant involvement of Tregs in angiogenesis.²⁸ The involvement of Tregs in angiogenesis has been investigated across a range of disease conditions, particularly in the field of oncology, due to their considerable therapeutic potential. The relationship between angiogenesis and Tregs can be classified into two mechanisms: one involves the VEGF signaling pathway, while the other is mediated through the regulation of other immune cells and the secretion of pro-angiogenic cytokines and growth factors. Studies providing direct evidence of Tregs regulating VEGF signaling are relatively scarce. Li et al²⁹ demonstrated that Helios+ Tregs facilitate angiogenesis in the bone marrow of acute lymphoblastic leukemia mice via the VEGF-A/VEGFR-2 pathway. Similarly, Facciabene et al³⁰ discovered that tumor hypoxia triggers an upregulation of chemokine CC-chemokine ligand 28, facilitating increased Tregs levels in tumors, which in turn promote angiogenesis by elevating VEGFA levels in the tumor microenvironment. In addition to their VEGF-mediated effects, Tregs can also indirectly influence angiogenesis by regulating the function of other immune cells. D’Alessio et al³¹ found that Tregs promote pulmonary angiogenesis by regulating macrophages and their cytokines, and their depletion leads to reduced angiogenesis and a decrease in macrophage numbers. Another study showed that hyperforin enhances Tregs infiltration in the ischemic hemisphere, increasing Treg-derived cytokines such as IL-10 and TGF-β in an astrocyte IL-6-dependent manner, thereby promoting neurovascular regeneration post-stroke.³² In conclusion, these findings support the hypothesis that Tregs may offer a protective effect on collateral circulation. However, the precise mechanisms require further elucidation.

IL-2, recognized as a growth factor essential for the proliferation of human T cells, plays a pivotal role in facilitating the differentiation and conversion of naive CD4+ T cells.³³ Numerous studies have demonstrated a close association between IL-2 and Tregs. Post-stroke treatment with IL-2:IL-2 antibody complexes has been shown to elevate Tregs populations.³⁴ Moreover, additional research indicates that IL-2, beyond its function in the generation of splenic and peripherally induced Tregs and maintaining the balance of peripherally induced Tregs, is crucial through its activation of signal transducer and activator of transcription 5 to enhance the suppressive function of Tregs.³⁵ Although IL-2 plays a critical role in immune regulation by promoting the proliferation and functional enhancement of Tregs, its effects following ischemic stroke extend beyond immune tolerance. In fact, IL-2 may indirectly enhance the formation of collateral circulation and angiogenesis by increasing the number and suppressive activity of Tregs, thereby contributing positively to tissue repair and recovery.

Certainly, existing studies also suggest that IL-2 can promote angiogenesis by activating other immune cells. Previous research has demonstrated that IL-2-activated NK cells interact with TNF-α-stimulated endothelial cells to stimulate their proliferation and promote angiogenesis through mechanisms involving α4β7 integrin and killer cell lectin-like receptor 1.³⁶ Additionally, IL-2 promotes angiogenesis by activating ROS and Akt signaling pathways, which enhance the expression of angiogenic factors such as VEGF and PDGF, stimulate tube formation in human umbilical vein endothelial cells, and its angiogenic effects have been confirmed in animal models.³⁷ In this investigation, a notable positive association between Tregs and IL-2 levels (r = 0.3849; p < 0.0001) was observed, corroborating the previously established link between IL-2 and Tregs. Additionally, IL-2 levels were inversely linked to NIHSS scores (r = −0.1958; p = 0.0319) and 90-day mRS scores (r = −0.2742; p = 0.0028), implying that IL-2 could potentially serve as a biomarker for predicting functional outcomes and disease severity. Furthermore, IL-2 levels in the peripheral blood of AIS patients were noted to be diminished in the early stages relative to those in the healthy control group, with PCS patients exhibiting lower IL-2 levels than GCS patients. Supporting these findings, Nayak et al³⁸ also reported that IL-2 may act as a biomarker for forecasting collateral circulation. However, it remains to be determined whether IL-2 influences collateral circulation through Tregs regulation or other pathways, warranting further investigation in future research.

Although Tregs represent only a small fraction of peripheral blood T cells and are present at low levels in ischemic brain tissue, effective strategies for stroke treatment have been developed through the ex vivo expansion of naturally derived thymus-originated autologous Tregs or by using biological and pharmacological agents to induce or expand Tregs in vivo. In recent years, novel Tregs genetic engineering techniques³⁹ have been developed to enhance the functionality, specificity, and delivery efficiency of Tregs. These techniques have shown initial progress in clinical trials related to transplantation and are expected to provide further support for the treatment of stroke and other neurological diseases. Moreover, several studies have demonstrated that IL-2 primarily exerts its therapeutic effects by increasing the number of Tregs in stroke models. Systemic injection of IL-2/IL-2Ab complexes has been shown to effectively increase Tregs numbers in blood and lymphoid tissues, reduce infarct volume, alleviate neuroinflammation, and improve sensory and motor function.⁴⁰ Additionally, IL-2 gene delivery targeting astrocytes has been shown to specifically increase brain Tregs, thereby providing effective protection against inflammatory damage in the brain.⁴¹ This discovery offers a potential delivery platform for clinical applications. Overall, these therapeutic strategies offer diverse treatment options for ischemic stroke and demonstrate significant potential. However, the efficacy and safety of these approaches require further validation through additional clinical trials, particularly for their application in stroke treatment. As research progresses and clinical validation continues, Tregs cell therapies and IL-2 are expected to become key treatment modalities for stroke and other neurological diseases.

Certain limitations of this investigation must be recognized. First, the single-phase CTA employed here may not be the most suitable method for the evaluation of collateral circulation. More comprehensive approaches, such as multi-phase CTA or DSA, could provide more precise assessments. Furthermore, the study was restricted to analyzing peripheral blood samples due to the difficulty of obtaining Tregs and IL-2 data from the brains of AIS patients. Additionally, since the collection of blood samples occurred outside the acute reperfusion therapy window for cerebral infarction, it remains uncertain whether Tregs and IL-2 levels can accurately reflect the status of collateral circulation during this critical therapeutic period, necessitating further investigation in future research. Lastly, the sample size was limited, further research with larger sample sizes is needed to explore the relationship between Tregs, IL-2, and collateral circulation. Additionally, more clinical trials or multi-center studies are required to validate the generalizability of these results.

Conclusion

The results of this investigation demonstrated that reduced levels of Tregs and IL-2 were linked to PCS status before treatment, indicating a potential relationship between the status of collateral circulation and immune inflammation. Peripheral blood Tregs and IL-2 concentrations might function as useful prognostic indicators for evaluating collateral circulation status. In the future, the regulation of Tregs and IL-2 levels could offer promising therapeutic approaches for enhancing cerebral collateral circulation, and providing new therapeutic targets for acute ischemic stroke.