Elevated Peripheral CD161 ⁺ Treg Cells and Their Clinical Significance in Behçet Syndrome

Abstract

Objective

Behçet syndrome (BS) is a systemic autoimmune vasculitis characterized by immune dysregulation involving multiple immune cell subsets. CD161⁺ Treg cells exhibit proinflammatory properties and impair immune regulation during inflammation. This study aimed to investigate the alterations in CD161⁺ Treg cells in BS and their clinical relevance, particularly in disease pathogenesis and neurologic involvement.

Methods

This prospective multicenter study included 182 patients diagnosed with BS at the three hospitals between 2018 and 2024, 166 patients with systemic lupus erythematosus (SLE) and 149 patients with rheumatoid arthritis (RA) and 114 patients with healthy controls (HCs). Demographic and clinical data were recorded. CD4⁺CD25^highCD127^lowCD161⁺ T cells (CD161⁺ Treg cells) in peripheral blood were analyzed via flow cytometry. Statistical analysis included the Wilcoxon rank‐sum test, Fisher's exact test, and logistic regression, with P < 0.05 considered significant.

Results

Patients with BS had a significantly higher proportion among total Treg cells and CD4⁺ T cells, as well as higher absolute number of CD161⁺ Treg cells compared to HCs. CD161⁺ Treg cell levels negatively correlated with Foxp3⁺ Treg cells and positively correlated with Teff cells. Patients with BS had higher absolute number of CD161⁺ Treg cells than that in patients with SLE and in patients with RA. Moreover, patients with BS with higher erythrocyte sedimentation rate or C‐reactive protein or with neurologic involvement exhibited higher CD161⁺ Treg cells, which were identified as a risk factor for neurologic involvement. Among 41 patients with BS observed after treatment, CD161⁺ Treg cells significantly decreased, correlating with reduced disease activity.

Conclusion

Patients with BS exhibit an increased CD161⁺ Treg cells in peripheral blood, which may contribute to immune dysregulation and neurologic involvement. The reduction of CD161⁺ Treg cells following treatment suggests their potential role as a biomarker for disease activity in BS.

INTRODUCTION

Behçet syndrome (BS) is a chronic, multisystem inflammatory disorder characterized by autoimmunity‐driven inflammation. Although BS can occur in individuals of all age groups, young males are disproportionately affected. Clinically, BS is marked by prolonged disease activity and episodic exacerbations. ¹ , ² As a systemic vasculitis, BS involves multiple organ systems, with mucocutaneous lesions being the most common clinical manifestation. Articular involvement is frequently observed, while ocular complications, such as uveitis, are considered hallmark characteristics of the disease. Additionally, the involvement of the gastrointestinal, cardiovascular, and nervous systems also highlights the systemic nature of BS. ¹ This clinical heterogeneity complicates diagnosis, emphasizing the importance of early recognition to mitigate disease progression and prevent severe complications.

The pathogenesis of BS is multifactorial, involving a complex interplay between genetic predisposition, environmental triggers, and immune dysregulation. ² , ³ , ⁴ , ⁵ , ⁶ , ⁷ Central to this immune dysregulation are T cell subsets, which play a pivotal role in propagating the inflammatory cascade. Notably, Th1 and Th17 cells have been implicated in mediating the inflammatory response through the secretion of a spectrum of proinflammatory cytokines. ⁸ , ⁹ Th1 cells, in particular, are significantly enriched in the peripheral blood, intestinal tissues, and skin lesions of patients with BS, where they contribute to tissue damage by producing cytokines such as interferon‐γ (IFNγ). Similarly, Th17 cells exhibit heightened activity in BS, promoting neutrophil and macrophage recruitment and amplifying inflammation under the influence of interleukin‐23 (IL‐23). ³ , ⁸ , ⁹ Treg cells, a subset of immune cells with immunosuppressive and immunomodulatory functions, are dysregulated in BS. Studies have demonstrated that functional impairments in Treg cells contribute to an imbalance in the Th17/Treg cell ratio, exacerbating immune dysregulation in BS. ¹⁰ , ¹¹ , ¹² Specifically, cutaneous Ly‐positive Treg cells are reduced in the peripheral blood of patients with BS, further supporting their role in disease pathogenesis. ¹³ Consequently, Treg cells have emerged as a focal point in understanding the immunopathogenesis of BS.

CD161, a lectin‐like receptor encoded by the killer cell lectin‐like receptor B1 (KLRB1) gene, is primarily associated with natural killer cells. ¹⁴ , ¹⁵ Its ligand, CLEC2D, is expressed by myeloid and malignant cells, and the CD161‐CLEC2D pathway has been implicated in tumor proliferation and immune escape. ¹⁵ CD161 has been found to be expressed on Th17 cells and defines a distinct subset of CD4⁺CD25⁺Foxp3⁺ Treg cells that secrete inflammatory cytokines, such as IL‐17 and IFNγ, under inflammatory conditions. ¹⁴ , ¹⁶ , ¹⁷ This suggests that CD161⁺ Treg cells may play a significant role in immune homeostasis disruption and the pathogenesis of autoimmune diseases. The involvement of CD161⁺ Treg cells has been validated in multiple autoimmune disorders, ¹⁸ , ¹⁹ , ²⁰ highlighting their broader relevance in autoimmunity. Given this context, we conducted this study to elucidate the role of CD161⁺ Treg cells in BS pathogenesis and explore their potential association with the clinical manifestations of the disease.

METHODS

Human participants

This prospective multicenter study was conducted in China. A total of 182 adult patients diagnosed with BS in the Rheumatology and Immunology Departments of Peking University People's Hospital, Qingdao Women and Children's Hospital and Shijiazhuang People's Hospital between 2018 and 2024 were enrolled. All patients met the International Criteria for Behçet's Disease. ²¹ Patients were excluded if they had overlapping autoimmune diseases, such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE), as well as any active infection or malignant tumor. Disease activity of BS was assessed using the Behçet's Disease Current Activity Form (BDCAF), ²² with all patients in the active phase at enrollment (BDCAF ≥ 1). In addition, 114 healthy volunteers were recruited as healthy controls (HCs). SLE and RA are two relatively common systemic autoimmune diseases; therefore, we also enrolled 166 patients with SLE and 149 patients with RA as disease controls. Disease activity in patients with SLE and RA was assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI‐2000) ²³ and the Disease Activity Score in 28 joints (DAS28), ²⁴ respectively. All enrolled patients had active disease, defined as a SLEDAI‐2000 score ≥6 for SLE and a DAS28 score ≥3.2 for RA.

Clinical features

Demographic and clinical characteristics, including sex, age, disease duration, systemic involvements (mucocutaneous, articular, ocular, cardiovascular, gastrointestinal, and neurologic), laboratory parameters (erythrocyte sedimentation rate [ESR] and C‐reactive protein [CRP]), and previous medications (glucocorticoids [GCs], immunosuppressors [IS], immunomodulators [IMs], and biologic agents) of patients with BS were documented during their initial clinical visit. Elevated ESR was defined as >15 mm/h in males and >20 mm/h in females, while elevated CRP was defined as >8 mg/L, and the severity of the disease was assessed using the Krause score. ²⁵

Flow cytometry analysis

A single‐platform, flow cytometry‐based absolute count technique was used to quantify the percentage and absolute numbers of lymphocytes in each subset. Briefly, 50 μL of EDTA‐anticoagulated venous blood was added to BD Trucount tubes (BD, #340334) using reverse pipetting. Cells were stained for 15 minutes with 10 μL of anti‐CD3‐FITC/CD8‐PE/CD45‐PerCP/CD4‐APC and anti‐CD3‐FITC/CD16+CD56‐PE/CD45‐PerCP/CD19‐APC antibodies from the BD Multitest IMK kit (BD, #662965), ensuring no direct contact with blood. Subsequently, 450 μL of 1X BD Multitest IMK kit lysing solution (BD, #349202) was added, followed by a 15‐minute incubation. Samples were analyzed within 24 hours using MultiSET software on a BD FACSCanto flow cytometer (BD Biosciences).

Freshly collected whole‐blood samples were processed within 24 hours. For T cell subset analysis, immunophenotyping was performed using conjugated antihuman murine monoclonal antibodies, including anti‐CD3‐APC‐H7, anti‐CD4‐FITC, anti‐CD25‐PE, anti‐CD127 (IL‐7Rα)‐BV605, anti‐CD45RA‐BV510, anti‐CD197 (CCR7)‐BV421, anti‐PD‐1‐PE‐CY7, anti‐human CD185 (CXCR5) Alexa Fluor 647, anti‐CD161‐BV421, and anti‐CD3‐PerCP (all from BD Pharmingen). For surface staining, 100 μL of whole blood was incubated with antibodies for 15 minutes at room temperature in the dark. Erythrocytes were lysed by adding 2 mL of 1X BD Multitest IMK kit lysing solution for 10 minutes, followed by centrifugation at 600g for 5 minutes and removal of the supernatant. The stained cells were washed with phosphate‐buffered saline and stored in a cool, dark environment until flow cytometry analysis. Labeled T cell subsets were acquired on a FACSAria II flow cytometer (BD Biosciences) and analyzed using FlowJo v10 (TreeStar). CD161⁺ Treg cells were defined as CD3⁺CD4⁺CD25^highCD127^lowCD161⁺ T cells, with the representative gating strategy depicted in Figure S1.

Follow‐up

Among the 182 patients with BS, 41 individuals were longitudinally observed, and data on therapy, changes in peripheral CD161⁺ Treg cells levels, clinical manifestations, and disease activity were recorded during the follow‐up period. The median follow‐up duration was 4.0 (3.0, 9.5) months.

Statistical analysis

All statistical analyses were performed using SPSS software (version 23; SPSS Inc.) and the GraphPad Prism software (version 10.1.2; GraphPad Software, Inc.). Student's t‐test was used to compare continuous variables with a normal distribution between groups, while the Wilcoxon rank‐sum test was applied to continuous variables that did not follow a normal distribution. Fisher's exact tests were used to analyze categorical variables. Quantitative data are presented as means (SD) or medians (interquartile range), depending on the data distribution. Spearman's correlation analysis was used to assess associations between CD161⁺ Treg cells, Foxp3⁺ Treg cells, and Teff cells. Risk factors were evaluated using logistic regression analysis. Statistical significance was set at P < 0.05.

Ethics approval

This study was conducted in compliance with the Declaration of Helsinki and received ethical approval from the Ethics Committee of Peking University People's Hospital (approval no. 2023PHB163‐001). Written informed consent was obtained from all participants before their inclusion in the study.

Data availability

The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

RESULTS

Patient characteristics

A total of 94 male and 88 female patients with BS were included in the study, with a median age of 37.0 (30.0, 50.3) years and a median disease duration of 84.0 (24.0, 180.0) months (median [IQR]). The sex distribution and age of the 114 HCs (50 males, 64 females; median age, 43.5 [32.0, 55.0] years) did not significantly differ from those of patients with BS. Patient characteristics at enrollment, including clinical features and treatment, are shown in Table S1. The median BDCAF score at enrollment was 4.0 (3.0, 5.0) (median [IQR]). At enrollment, oral ulcers were the most prevalent manifestation, occurring in 173 patients (95.1%). Additionally, 117 (64.3%) patients presented with genital ulcers. Skin involvement was observed in 128 patients (70.3%), including pseudofolliculitis (n = 87, 47.8%), erythema nodosum (n = 50, 27.5%), and pustules (n = 15, 8.2%). Ocular involvement was noted in 42 patients (23.1%), including uveitis (19.8%) and retinal vasculitis (6.6%). Gastrointestinal involvement was present in 45 patients (24.7%), comprising intestinal ulcer (19.8%), hemorrhage (7.1%), esophageal lesion (3.8%). Articular involvement was identified in 56 patients (30.8%), comprising arthralgia (n = 56, 30.8%) and arthritis (n = 38, 20.9%). Cardiovascular involvement was identified in 61 patients (33.5%) and included venous embolism (n = 32, 17.7%), arterial aneurysm (n = 20, 11.0%), arterial thrombosis (n = 9, 4.9%), arterial stenosis (n = 33, 18.1%), and valvular disease (n = 8, 4.4%). Neurologic involvement was present in 27 patients (14.8%), including central nervous system (CNS) parenchymal involvement (n = 20, 11.0%), intracranial venous sinus thrombosis (IVST) (n = 8, 4.4%), and peripheral neuropathy (n = 5, 2.7%). In addition, 33 patients (18.1%) presented with fever (Table S1).

The previous treatments of the patients are shown in Table S1. Based on their prior treatment regimens, the patients were divided into five groups: treatment‐naïve (n = 80; 44.0%), GC monotherapy (n = 17; 9.3%), IS/IM therapy (n = 31; 17.0%), GC + IS/IM therapy (n = 39; 21.4%), and biologic agent therapy (n = 15; 8.2%). The Mann–Whitney U test revealed no significant differences in the proportion of CD161⁺ Treg cells within the Treg cell population and the absolute number of CD161⁺ Treg cells between the treatment‐naïve group and the other four treatment groups (Figure S2).

Increased CD161 ⁺ Treg cells in the peripheral blood of patients with BS

We employed the Mann–Whitney U test to compare the levels of peripheral blood CD161⁺ Treg cells between patients with BS and HCs. Compared to HCs, the proportion of CD161⁺ Treg cells among the total Treg cells (12.9 [10.0, 16.7] vs 10.5 [8.6, 13.1], P < 0.001) (median [IQR]) and among CD4⁺ T cells (1.10 [0.75, 1.53] vs 0.83 [0.64, 1.09], P < 0.001) (median [IQR]) in peripheral blood was significantly higher. The absolute number (/μL) of CD161⁺ Treg cells in peripheral blood was also significantly higher than that in HCs (7.82 [4.71, 11.59] vs 6.38 [4.63, 9.42], P = 0.015) (median [IQR]) (Figure 1A–C). We assessed the correlation between CD161⁺ Treg cells and other immune cells using linear regression analysis. In patients with BS, the proportion of CD161⁺ Treg cells among total Treg cells in peripheral blood was negatively correlated with the proportion of Foxp3⁺ Treg cells among CD4⁺ T cells (R = −0.353, P < 0.001) and positively correlated with the proportion of Teff cells among CD4⁺ T cells (R = 0.343, P < 0.001) (Figure 1C and D).

Figure 1.

Comparison of CD161⁺ Treg cells in peripheral blood between patients with BS and HCs and linear regression of CD161⁺ Treg cells, Foxp3⁺ Treg cells, and Teff cells in patients with BS. (A) The proportion of CD161⁺ Treg cells among total Treg cells in patients with BS was significantly higher than that in HCs. (B) The proportion of CD161⁺ Treg cells among CD4⁺ T cell cells in patients with BS was significantly higher than that in HCs. (C) The absolute number of CD161⁺ Treg cells in patients with BS was significantly higher than that in HCs. Error bars represent the median with interquartile range and min/max values. (D) The proportion of CD161⁺ Treg cells among total Treg cells was negatively correlated with the proportion of Foxp3⁺ Treg cells among CD4⁺ T cells. (E) The proportion of CD161⁺ Treg cells among total Treg cells was positively correlated with the proportion of Teff cells. BS, Behçet syndrome; HC, healthy control.

Comparisons of CD161 ⁺ Treg cells in different autoimmune disorders

We compared the level of CD161⁺ Treg cells between patients with BS and patients with SLE and patients with RA by Mann–Whitney U test. The results demonstrated that the proportion of CD161⁺ Treg cells within the total Treg cell population (12.9 [10.0, 16.7] vs 11.5 [7.7, 16.1], P = 0.012) (median [IQR]) and the absolute number of CD161⁺ Treg cells (7.82 [4.71, 11.59] vs 4.54 [2.47, 7.03], P < 0.001) (median [IQR]) were significantly higher in patients with BS compared to patients with SLE. Furthermore, patients with BS exhibited a markedly increased proportion of CD161⁺ Treg cells among CD4⁺ T cells (1.10 [0.75, 1.53] vs 0.81 [0.55, 1.25], P < 0.001) (median [IQR]) and increased absolute number of CD161⁺ Treg cells (7.82 [4.71, 11.59] vs 5.10 [3.57, 8.03], P < 0.001) (median [IQR]) relative to patients with RA (Figure 2).

Figure 2.

Comparison of CD161⁺ Treg cells between patients with BS and patients with SLE, patients with RA and HCs. (A) The proportion of CD161⁺ Treg cells among total Treg cells in patients with BS was significantly higher than that in patents with SLE. (B) The proportion of CD161⁺ Treg cells among CD4⁺ T cells in patients with BS was significantly higher than that in patients with RA. Error bars represent the median with interquartile range and min/max values. (C) The absolute number of CD161⁺ Treg cells in patients with BS was significantly higher than that in patents with SLE and patients with RA. BS, Behçet syndrome; HC, healthy control; ns, xxx; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.

Association between CD161 ⁺ Treg cells and neurologic involvement in BS

We compared CD161⁺ Treg cell levels among patients with different symptoms by Mann–Whitney U test (Table 1). Patients with neurologic involvement showed a significantly higher proportion of CD161⁺ Treg cells within the total Treg cell population (16.6 [12.8, 20.6] vs 12.1 [9.6, 15.8], P < 0.001) (median [IQR]) and a greater absolute number of CD161⁺ Treg cells (11.97 [8.28, 15.99] vs 7.53 [4.42, 10.84], P = 0.001) (median [IQR]) in peripheral blood. Regarding specific neurologic manifestations, patients with CNS parenchymal lesions exhibited both a higher proportion of CD161⁺ Treg cells among total Treg cells (15.6 [12.6, 20.5] vs 12.8 [9.6, 15.9], P = 0.002) (median [IQR]) and a higher absolute number (12.09 [8.41, 15.36] vs 7.61 [4.46, 10.90], P = 0.001) (median [IQR]) compared to those without such lesions. Similarly, patients with IVST had significantly elevated values in both the ratio of CD161⁺ Treg cells/Treg cells (17.3 [13.2, 21.1] vs 12.8 [9.9, 16.6], P = 0.022) (median [IQR]) and absolute number (12.41 [9.48, 14.68] vs 7.64 [4.62, 11.27], P = 0.009) (median [IQR]). Patients with peripheral neuropathy also showed a higher proportion of CD161⁺ Treg cells among total Treg cells (17.7 [15.4, 20.6] vs 12.9 [9.9, 16.6], P = 0.041) (median [IQR]), but the absolute count did not differ significantly between groups. Complete clinical characteristics and their associations with CD161⁺ Treg cells are provided in Table S2, while the specific CD161⁺ Treg cell levels across different clinical conditions are illustrated in Figures S3 and S4. We assessed disease activity using the Krause score and stratified patients into two groups for comparison: those with a score ≤5 (n = 100) and those with a score ≥6 (n = 82). However, no significant difference in CD161⁺ Treg cell levels was observed between the two groups (Figures S3 and S4). In addition to clinical features, elevated markers of inflammation were associated with higher CD161⁺ Treg cell levels. Patients with increased ESR or CRP had both a higher proportion of CD161⁺ Treg cells among total Treg cells and a greater absolute number of CD161⁺ Treg cells (P < 0.05; Figure 3).

Table 1.

Association of different CD161⁺ Treg cells and different clinical manifestations of patients with BS^*

	CD161+ Treg cells/Treg cells, %		P	CD161+ Treg cells, /μL		P
	Present	Absent		Present	Absent
Oral ulcer	12.9 (10.0, 16.7)	13.1 (10.7, 14.4)	0.871	7.81 (4.71, 11.41)	9.65 (6.15, 14.55)	0.376
Genital ulcer	12.9 (10.0, 16.6)	12.6 (10.0, 17.1)	0.978	8.04 (5.21, 11.59)	7.53 (4.46, 11.41)	0.429
Skin manifestation	12.9 (9.9, 16.6)	13.1 (10.0, 17.3)	0.667	8.08 (4.83, 12.07)	7.74 (4.46, 10.06)	0.195
Ocular involvement	13.2 (9.3, 16.7)	12.8 (10.0, 16.7)	0.850	8.40 (5.91, 13.67)	7.72 (4.54, 11.18)	0.244
Gastrointestinal involvement	11.8 (9.6, 16.7)	13.2 (10.3, 16.6)	0.186	6.06 (4.24, 10.04)	8.27 (5.42, 11.93)	0.079
Articular involvement	13.5 (10.1, 19.0)	12.8 (9.6, 15.5)	0.108	8.01 (4.72, 12.89)	7.78 (4.71, 11.27)	0.558
Cardiovascular involvement	12.5 (9.9, 15.8)	13.0 (10.0, 16.7)	0.916	8.04 (4.71, 12.65)	7.81 (4.91, 11.10)	0.817
Neurologic involvement	16.6 (12.8, 20.6)	12.1 (9.6, 15.8)	<0.001 a	11.97 (8.28, 15.99)	7.53 (4.42, 10.84)	0.001 a
CNS parenchymal lesions	15.6 (12.6, 20.5)	12.8 (9.6, 15.9)	0.002 a	12.09 (8.41, 15.36)	7.61 (4.46, 10.90)	0.001 a
Intracranial venous sinus thrombosis	17.3 (13.2, 21.1)	12.8 (9.9, 16.6)	0.022 a	12.41 (9.48, 14.68)	7.64 (4.62, 11.27)	0.009 a
Peripheral neuropathy	17.7 (15.4, 20.6)	12.9 (9.9, 16.6)	0.041 a	8.28 (4.51, 20.64)	7.81 (4.78, 11.52)	0.753

^*BS, Behçet syndrome; CNS, central nervous system.

^a P < 0.05.

Figure 3.

(A and B) CD161⁺ Treg cells/Treg cells and the absolute number of CD161⁺ Treg cells in patients with BS with elevated ESR were significantly higher than that in patients with normal ESR. (C and D) CD161⁺ Treg cells/Treg cells and the absolute number of CD161⁺ Treg cells in patients with BS with elevated CRP were significantly higher than that in patients with normal CRP. BS, Behçet syndrome; CRP, C‐reactive protein; ESR, erythrocyte sedimentation rate.

To investigate the risk of neurologic involvement in BS, we performed a logistic regression analysis using the proportion of CD161⁺ Treg cells within the Treg cell population and absolute number of CD161⁺ Treg cells as variables. Binary logistic regression analysis revealed that an increased proportion of CD161⁺ Treg cells among total Treg cells was a risk factor for the development of neurologic involvement in patients with BS (P = 0.014, odds ratio [OR] 1.099, 95% confidence interval [CI] 1.020–1.186). However, an increased proportion of CD161⁺ Treg cells absolute number was not associated with neurologic involvement (P = 0.826, OR 1.032, 95% CI 0.964–1.106) (Table S3).

The proportion of CD161 ⁺ Treg cells among Treg cells after treatment

Forty‐one patients with BS were followed up after treatment and the information of patients followed up is listed in Table S4. By Mann–Whitney U test, the median proportion of CD161⁺ Treg cells among total Treg cells (13.2 [9.0, 18.6] vs 9.7 [6.9, 12.8], P = 0.006) (median [IQR]) and the absolute number of CD161⁺ Treg cells (8.30 [5.03, 14.12] vs 6.19 [2.87, 10.61], P = 0.035) (median [IQR]) in these patients significantly decreased after treatment compared to baseline (Figure 4A–D) The median BDCAF score also significantly decreased compared to baseline after treatment (3.0 [3.0, 4.0] vs 2.0 [1.0, 3.0], P < 0.001) (median [IQR]) (Figure 4E). In 30 of these patients, the proportion of CD161⁺ Treg cells decreased, and in 28/30 of these patients, BDCAF scores were lower after treatment. In contrast, only 5/11 patients with increased CD161⁺ Treg cells had lower BDCAF scores after treatment. Fisher's exact test analysis demonstrated that decreased CD161⁺ Treg cells among total Treg cells after treatment was associated with lower disease activity (P = 0.002) (Figure 4F and G). Comparison of CD161⁺ Treg cell decline across treatments, performed using Fisher's exact tests, revealed a significantly more pronounced effect in the mycophenolate mofetil group (P = 0.039) (Table S5).

Figure 4.

CD161⁺ Treg cells in patients and BDCAF of patients before and after treatment. (A) This panel illustrates the changes in the proportion of CD161⁺ Treg cells among total Treg cells in 41 individuals with BS before and after treatment. (B) The proportion of CD161⁺ Treg cells among total Treg cells in 41 patients with BS after treatment was significantly lower than that before. Error bars represent the median with interquartile range. (C) This panel illustrates the changes in the absolute number of CD161⁺ Treg cells in 41 individuals with BS before and after treatment. (D) The absolute number of CD161⁺ Treg cells in 41 patients with BS after treatment was significantly lower than that before. Error bars represent the median with interquartile range. (E) The BDCAF in 41 patients with BS after treatment was significantly lower than that before treatment. Error bars represent the median with interquartile range and min/max values. (F) In the decreased CD161⁺ Treg cells group (n = 30) of 41 followed up patients, 28 patients got lower BDCAF, and in the increased CD161⁺ Treg cells group (n = 11), five patients got lower BDCAF. BDCAF, Behçet's Disease Current Activity Form; BS, Behçet syndrome. *p < 0.05; **p < 0.01.

DISCUSSION

BS is a multisystem autoimmune disease, and inadequate clinical management can result in poor prognosis. The cellular and molecular mechanisms underlying BS remain incompletely elucidated, making further exploration of its pathogenesis essential for early diagnosis and improved patient care. In this study, our results demonstrated a significant increase in CD161⁺ Treg cells in patients with BS compared to HCs. Furthermore, an elevated proportion or absolute number of CD161⁺ Treg cells in the peripheral blood was associated with neurologic involvement in BS. Notably, as disease activity decreased, the proportion and absolute number of CD161⁺ Treg cells among Treg cells also decreased after treatment.

Treg cells represent a vital suppressive subset of CD4⁺ T cells that play a critical role in maintaining immune tolerance and preventing autoimmune responses. ²⁶ CD161⁺ Treg cells are a distinct subset of Treg cells characterized by the expression of CD161, a marker associated with the modulation of proinflammatory cytokine responses. ¹⁴ , ²⁷ CD161 interacts with its ligand, lectin‐like transcript 1, which is encoded by the CLEC2D gene, to stimulate T cells to produce proinflammatory cytokines such as IL‐2, IL‐17, and IFNγ, thereby promoting inflammatory responses. ²⁸ Furthermore, the observation that patients with higher markers of inflammation (ESR and CRP) exhibited elevated peripheral blood CD161⁺ Treg cell levels in this study suggests an association between these cells and a heightened inflammatory state. Emerging evidence suggests that CD161 is involved in the development of various autoimmune diseases. Mitsialis et al conducted a single‐cell analysis of inflammatory bowel disease and demonstrated that IL17A⁺ CD161⁺ effector memory T cells were enriched in the mucosa of patients. ²⁹ Bai et al have reported that CD161 modulates human Th17 responses via acid sphingomyelinase in Crohn disease. ³⁰ While Treg cells are generally known for their suppressive roles in immune processes, CD161⁺ Treg cells have been shown to exhibit proinflammatory functions by promoting the production of inflammatory cytokines. ¹⁴ , ¹⁶ , ¹⁷ In this study, we observed a significant elevation in CD161⁺ Treg cells in patients with BS, suggesting a potential role for CD161⁺ Treg cells in the pathogenesis and inflammatory responses of BS. This finding aligns with the result of Abbasova et al, who reported increased CD161 expression on CD4⁺ T cells alongside elevated IL‐17A and IFNγ production in active BS. ³¹ Furthermore, studies on other autoimmune diseases have highlighted the relevance of CD161⁺ Treg cells. For example, Bian et al demonstrated elevated levels of CD161⁺ Treg cells in the peripheral blood of patients with IgG4‐related diseases, ¹⁹ while CD161⁺ Treg cells were enriched in the inflamed joints of patients with juvenile idiopathic arthritis ¹⁸ and psoriatic arthritis. ²⁰ Collectively, these findings underscore the potential involvement of CD161⁺ Treg cells in the inflammatory mechanisms of autoimmune diseases.

Additionally, this study revealed that patients with BS exhibit higher levels of CD161⁺ Treg cells compared to those with SLE or RA. Multiple immune cells—including Th1, Th17, neutrophil, and monocyte—are known to contribute to the pathogenesis of BS. ³ , ⁸ , ⁹ Given that CD161⁺ Treg cells can proliferate in response to inflammatory stimulation or T cell receptor activation and secrete cytokines such as IL‐17 and IFNγ, ¹⁴ , ¹⁷ they may expand in the peripheral blood of patients with BS—a systemic vasculitis—and likely contribute to the immunopathology of this disease. Although RA and SLE are also systemic autoimmune diseases, the inflammatory pathology in RA is predominantly localized to peripheral joints, ³² and its peripheral blood inflammatory immune microenvironment may differ from that of BS. SLE is primarily an autoantibody‐mediated autoimmune disease, ³³ in which CD161⁺ Treg cells may not play a dominant role. CD161⁺ Treg cells in the peripheral blood could play a more significant role in BS than in RA and SLE. Future studies should consider including other types of autoimmune vasculitis as controls for further comparison.

Foxp3⁺ Treg cells are widely acknowledged for their critical role in immune regulation and suppression. In this study, we observed that an increase in CD161⁺ Treg cell levels was accompanied by elevated Teff cells and a reduction in total Foxp3⁺ Treg cells. This pattern suggests that a high proportion of CD161⁺ Treg cells may reflect a proinflammatory state. However, the relatively small correlation coefficient suggests that this association should be interpreted with caution. Given previous findings that CD161⁺ Treg cells can secrete inflammatory cytokines such as IL‐2, IL‐17, and IFNγ, we propose that these cells may contribute to immune dysregulation by promoting Teff cell responses and impairing Foxp3⁺ Treg cell differentiation, thereby shifting the immune balance toward inflammation. Future in vitro studies are warranted to clarify the interactions between CD161⁺ Treg cells and other immune cells and to further investigate their role in autoimmune disease mechanisms.

Neurologic BS (neuro‐BS) is a severe and complex clinical subtype of BS, characterized by parenchymal nervous system involvement and nonparenchymal nervous system involvement. ¹ , ² Our study revealed that patients with BS with neurologic involvement exhibited a higher proportion within both total Treg cells and higher absolute number of CD161⁺ Treg cells and elevated proportion of CD161⁺ Treg cells among Treg cells may serve as a risk factor for neurologic involvement in BS. These findings highlight the potential role of CD161⁺ Treg cells in the pathogenesis of neurologic lesions in BS. Our results showed that both CNS parenchymal lesions and IVST were associated with CD161⁺ Treg cells. The mechanisms underlying nervous system injury in BS are believed to involve the activation of peripheral blood immune cells, which release various cytokines that disrupt the blood‐brain barrier (BBB). This disruption facilitates the influx of immunopathogenic factors into the brain parenchyma, leading to neuronal demyelination or degeneration. ³⁴ , ³⁵ Elevated levels of IL‐17 and increased retinoic acid–related orphan receptor γt/Foxp3 ratios have been observed in the cerebrospinal fluid (CSF) of patients with neuro‐BS. ³⁴ , ³⁶ CD161⁺ Treg cells may contribute to this process by disrupting the balance between Teff cells and Foxp3⁺ Treg cells in peripheral blood. Besides, the concentration of IL‐10 is significantly increased in the CSF of patients with neuro‐BS, ³⁷ and CD161⁺ Treg cells are known to promote IL‐10 production. ³⁸ We hypothesize that the increase in CD161⁺ Treg cells in peripheral blood may drive the production of inflammatory cytokines, which could cross the BBB and contribute to parenchymal nervous system injury. Furthermore, vascular endothelial damage induced by chronic inflammation may underlie nonparenchymal nervous system involvement. In addition, we also found that CD161⁺ Treg cells were associated with peripheral neuropathy of BS, but the sample size of such patients is too small. Future samples need to be expanded to verify the relationship between peripheral neuropathy and immune cell subsets in BS. Except for neurologic involvement, this study did not find a correlation between elevated CD161⁺ Treg cell levels and other systemic manifestations of BS. When patients were divided into two groups according to the Krause score based on disease severity, no significant difference in CD161⁺ Treg cell levels was observed. We, therefore, propose that the increase in CD161⁺ Treg cells is associated specifically with neurologic involvement rather than overall disease severity.

However, the roles of CD161⁺ Treg cells are complex and multifaceted. In addition to their proinflammatory functions, CD161⁺ Treg cells have been shown to exhibit suppressive effects on conventional T cells and play a role in promoting bowel wound healing. ³⁸ Expression of PD‐1, BTLA, and ICOS‐1, which possess immunomodulatory and immunosuppressive capabilities, is up‐regulated in CD161⁺ Treg cells. ³⁹ , ⁴⁰ , ⁴¹ , ⁴² Therefore, although CD161⁺ Treg cells are associated with proinflammatory effects, one possibility is that their elevation in autoimmune diseases, such as BS, may represent a compensatory response to the impairment of the inflammatory process. The exact role of CD161⁺ Treg cells in the pathogenesis of BS requires further validation through in vitro studies.

In our study, we observed a decrease in the proportion and absolute number of CD161⁺ Treg cells in patients with BS, which corresponded to a reduction in BDCAF score after treatment. Bian et al also reported a reduction in the number of CD161⁺ Treg cells following treatment in patients with IgG4‐related diseases. ¹⁹ These findings suggest that the elevated levels of CD161⁺ Treg cells in patients can be reversed and that in a low‐inflammatory state, the immune system may reduce the differentiation of CD4⁺ T cells into CD161⁺ Treg cells. The upstream regulatory pathways governing CD161⁺ Treg cells require further investigation to better understand their role. Across the treatment groups analyzed, mycophenolate mofetil was associated with a significant reduction in CD161⁺ Treg cells. Given the relatively small sample size of this study, further validation in larger cohorts is warranted.

The limitations of this study include the following: First, the limited number of follow‐up cases reduced the robustness of our conclusions, emphasizing the need for larger longitudinal studies. Second, the lack of in vitro cellular or animal experimental validation highlights the necessity for further research to confirm our findings.

In conclusion, patients with BS exhibit an increased CD161⁺ Treg cells in peripheral blood. Patients with BS with neurologic involvement had higher CD161⁺ Treg cells and a high proportion of CD161⁺ Treg cells may be a risk factor for neurologic involvement in BS. Following treatment, the level of CD161⁺ Treg cells in peripheral blood decreased, along with a reduction in BDCAF scores, suggesting its potential as a biomarker for disease activity in BS.

AUTHORS CONTRIBUTIONS

All authors contributed to at least one of the following manuscript preparation roles: conceptualization AND/OR methodology, software, investigation, formal analysis, data curation, visualization, and validation AND drafting or reviewing/editing the final draft. As corresponding authors, Drs Z. Li and Liu confirm that all authors have provided the final approval of the version to be published and takes responsibility for the affirmations regarding article submission (eg, not under consideration by another journal), the integrity of the data presented, and the statements regarding compliance with institutional review board/Declaration of Helsinki requirements.

Supporting information

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Figure S1: Gating strategy for the identification of CD161‐expressing regulatory T (Treg) cells.

Table S1: Characteristics of patients with BS

Figure S2. Comparison of CD161 ⁺ Tregs between BS patients with different treatment. (A) There was no significant difference in the proportion of CD161⁺ Tregs among Tregs between treatment naïve group and treated with glucocorticoids alone group, treated with immunosuppressants/immunomodulators group, treated with glucocorticoids and immunosuppressants/immunomodulators group and treated with biologic agents group. (B) There was no significant difference in the absolute number of CD161⁺ Tregs between treatment naïve group and other groups. GC, glucocorticoids. IS, immunosuppressants. IM, immunomodulators. Error bars represent the median with interquartile range and min/max values.

Table S2: Association of different CD161⁺Treg and different clinical manifestation of patients with BS

Figure S3. Proportion of CD161 ⁺ Treg among total Tregs in BS patients with varying system involvement and severity. The level of CD161⁺ Treg/Treg in BS patients with articular (A), gastrointestinal (B), ocular (C), or cardiovascular (D) involvement showed no significant difference compared to BS patients without involvement of these systems. (E) The level of CD161⁺ Treg/Treg in BS patients with neurological involvement was significantly higher than that in patients without neurological involvement. (F) There was no significant difference in the CD161⁺ Treg/Treg ratio between patients with a Krause's score of ≤5 and those with a Krause's score of ≥6.

Figure S4. Absolute number of CD161 ⁺ Treg in BS patients with varying system involvement and severity. The absolute number of CD161⁺ Treg in BS patients with articular (A), gastrointestinal (B), ocular (C), or cardiovascular (D) involvement showed no significant difference compared to BS patients without involvement of these systems. (E) The absolute number of CD161⁺ Treg in BS patients with neurological involvement was significantly higher than that in patients without neurological involvement. (F) There was no significant difference in the absolute number of CD161⁺ Treg between patients with a Krause's score of ≤5 and those with a Krause's score of ≥6.

Table S3: Logistic regression of neurological involvement in patients with BS

Table S4: Information of followed up BS patients

Table S5: The association between different treatments and the decline of CD161⁺ Treg cells