Impact of innate lymphoid cell type 2 in chronic lymphocytic leukemia on the function of treg and CD8⁺ T cells through IL-9

Abstract

Objective

This study investigated the impact of innate lymphoid cell type 2 (ILC2s) on the function of regulatory T cells (Treg) and CD8⁺ T cells in chronic lymphocytic leukemia (CLL) through IL-9.

Methods

Peripheral blood samples were collected from CLL patients (n = 52) and healthy controls (n = 30). ILC2 proportions and IL-9 levels were assessed using flow cytometry and ELISA. Immunofluorescence staining was performed to stain GATA3, CRTH2, and IL-9 in cervical lymph nodes from CLL patients (n = 10) and control subjects with reactive lymphadenitis (n = 10). Correlation analysis between ILC2s and IL-9 was conducted using the Spearman test. ILC2s were sorted and cultured from CLL patients, followed by co-culture experiments with PBMCs of healthy controls and MEC-1 cells, with or without anti-IL-9 antibody intervention. Flow cytometry was used to measure the proportions of ILC2s, Treg cells, PD-1⁺/TIGIT⁺/CTLA-4⁺ Treg subsets, and granzyme B⁺/perforin⁺ CD8⁺ T cells, along with MEC-1 cell apoptosis.

Results

The proportions of ILC2s and Treg, along with serum IL-9 levels, were significantly elevated in CLL patients (P < 0.05). Peripheral blood ILC2s were positively correlated with IL-9 (r = 0.609, P < 0.001). The average fluorescence intensity of GATA3, CRTH2, and IL-9 in the cervical lymph nodes of CLL patients increased significantly (P < 0.001), and IL-9 showed colocalization with GATA3 and CRTH2. In vitro, IL-9 levels in the supernatant of sorted ILC2s from CLL patients increased. Treatment with anti-IL-9 antibody significantly reduced the PD-1⁺ Treg and TIGIT⁺ Treg cells while increasing granzyme B⁺ CD8⁺ T cells (P < 0.05). However, there was no significant effect on Treg, CTLA-4⁺ Treg, and perforin⁺ CD8⁺ T cells (P > 0.05). Additionally, anti-IL-9 antibody significantly increased early apoptosis (P < 0.05).

Conclusion

ILC2s affect CD8⁺ T cells and Treg cells through IL-9, weakening the anti-tumor effects of CD8⁺ T cells and enhancing the immunosuppressive effects of Treg cells, thereby contributing to CLL pathogenesis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-025-04082-4.

Introduction

Chronic lymphocytic leukemia (CLL) is a disease characterized by the clonal proliferation of CD19⁺CD5⁺ B lymphocytes [1]. In CLL, the peripheral blood, lymph nodes, and spleen are predominantly affected, accompanied by significant bone marrow hyperplasia [2]. The majority of lymphocytes are small, the number of peripheral blood lymphocytes increases significantly, and they exhibit relatively mature lymphocyte surface markers [3]. Currently, BTK inhibitors and other immunotherapies are the main treatment options for CLL [4]. However, CLL predominantly affects elderly patients, who find it challenging to tolerate the toxicity of continuous treatment [5]. Furthermore, some high-risk patients with primary resistance show poor response to current therapies [6]. Even cases initially responsive to treatment eventually develop resistance, leading to relapse [7]. Therefore, further exploration of the pathogenesis of CLL and the development of new targeted, less toxic, and resistance-independent treatment methods are pressing clinical issues.

Type 2 innate lymphoid cells (ILC2s) are non-cytotoxic ILCs characterized by high levels of transcription factor GATA binding protein 3 (GATA3). Upon stimulation by IL-25, IL-33, and thymic stromal lymphopoietin, ILC2s produce Th2-related cytokines such as IL-4, IL-5, IL-9, IL-13, and amphiregulin [8]. Therefore, they are considered an intrinsic part of Th2 cells [9]. Functionally, ILC2s can regulate inflammation and serve as a key bridge between innate and adaptive type 2 immunity [10]. Many studies [11–13] have reported that the role of ILC2s in tumors is dual-sided, exhibiting both anti-tumor effects and tumor-promoting effects. Furthermore, ILC2s can also activate regulatory T cells (Treg) by secreting amphiregulin to bind with epidermal growth factor receptors on Treg cells. This leads to the secretion of a large number of immunosuppressive factors and the expression of the lymphocyte inhibitory receptor CTLA-4, thereby promoting the formation of an immunosuppressive microenvironment [14].

IL-9 plays different roles in different tumors. In solid tumors, IL-9 mainly exerts anti-tumor effects [15, 16]. Wan et al. [15] found that IL-9 derived from ILC2s suppressed the progression of colon cancer by activating CD8⁺ T cells. However, in some solid tumors and hematological malignancies originating from T cells., IL-9 mainly promotes tumor growth [17–19]. Moreover, IL-9 acts as an activator of ILC2s, promoting tumor growth, metastasis, and angiogenesis [20].

This study is the first to explore the role of ILC2s in CLL. The effects of ILC2s through IL-9 on the function of Treg and CD8⁺ T cells in CLL were investigated. Our findings may provide new ideas for the treatment of CLL.

Materials and methods

Study participants

A total of 52 patients with newly diagnosed CLL who were hospitalized at the First Affiliated Hospital of Xinjiang Medical University from August 2023 to October 2024 were selected as the CLL group. Inclusion criteria: 1) patients were diagnosed based on the diagnostic criteria of “Diagnosis and Treatment Guidelines for Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma in China (2018 Edition)”. 2) Patients were either untreated or had not received immunotherapy within the past six months. The types of immunotherapy included BTK inhibitors (e.g., Orelabrutinib, Zanubrutinib, and Ibrutinib), PI3K inhibitors (e.g., Linperlisib and Alpelisib), BCL2 inhibitors (e.g., Venetoclax), and anti-CD20 antibodies (e.g., Rituximab, Obinutuzumab, and Ofatumumab). Exclusion criteria: patients with other immune diseases or malignant tumors. Moreover, 30 healthy individuals undergoing health examinations at the Health Management Institute of the First Affiliated Hospital of Xinjiang Medical University during the same period were selected as the healthy control group. Additionally, 10 patients who received cervical lymph node biopsy due to reactive hyperplasia of cervical lymph nodes were also included. Inclusion criteria: (1) aged 45–80 years; (2) without CLL or other immune diseases or malignant tumors. This study was performed in line with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval no. 20211015–47). All participants provided informed consent.

Sample collection

Peripheral blood samples were collected from CLL patients (n = 52) and healthy control individuals (n = 30). Serum was obtained after centrifugation of peripheral blood at 1500 r/min for 10 min. ILC2s were isolated from the peripheral blood samples of 6 CLL patients and 6 healthy control individuals, while peripheral blood mononuclear cells (PBMCs) were isolated from 6 healthy control individuals. Cervical lymph node samples were obtained from 10 CLL patients and 10 patients with reactive hyperplasia of cervical lymph nodes.

PBMC extraction

Lymphocytes were isolated from peripheral blood samples of healthy controls using lymphocyte separation fluid (#LTS1077; Tianjin Haoyang Biotechnology Co., Ltd, China). Briefly, peripheral blood was gently mixed with the separation fluid at a 1:1 ratio. Subsequent horizontal centrifugation at 350 g for 30 min was performed. The middle layer was carefully aspirated using a pipette and then re-suspended in RPMI 1640 cell culture medium (#11875101; GIBCO, Grand Land, NY, USA). After another horizontal centrifugation at 250 g for 10 min, the cells in the precipitate were collected, washed three times, and resuspended for cell counting.

Immunomagnetic bead sorting of ILC2s

The EasySep™ Human ILC2 Enrichment Kit (#17972; STEMCELL Technologies, Vancouver, Canada) was used to sort ILC2s by using a magnetic bead negative selection strategy. Briefly, peripheral blood samples of CLL patients and healthy controls were washed in a PBS solution containing 2% fetal bovine serum and then centrifuged at room temperature (15–25 °C) for 10 min at 300 g. Subsequently, red blood cells were lysed using an ammonium chloride solution, and platelets were removed through natural deceleration after centrifugation at 120 g for 10 min. The supernatant was then removed, and the cells were resuspended in PBS at a concentration of 1 × 10⁸/mL. The sample was transferred to a 14 mL round-bottom polystyrene tube, mixed with an EasySep antibody cocktail at a ratio of 100 uL/mL of cell suspension, and incubated at room temperature for 10 min. Following this, EasySep nanobeads were added at a ratio of 100 uL/mL of cell suspension, thoroughly mixed by pipetting, and incubated for 1 min at room temperature. The volume of the cell suspension was adjusted to 2.5 mL with PBS, and the cells were gently mixed by pipetting up and down 2–3 times in the tube. The tube was placed in a magnet without a cover and incubated at room temperature for 5 min. The magnet and tube were lifted together, and the supernatant was poured out by slowly inverting the magnet. After a brief pause, the tube was returned to an upright position. The tube was then removed from the magnet, 2.5 mL of PBS was added, and the cell suspension was gently mixed by pipetting 2–3 times. This process was repeated with the tube placed back in the magnet for another 5 min. Finally, the tube was removed from the magnet, and the sorted cells, with a purity over 98.01% as evaluated by flow cytometry, were used for subsequent experiments.

Culture of ILC2s

The sorted ILC2s from CLL patients and healthy controls were resuspended in the RPMI-1640 culture medium, respectively. The recombinant human IL-2 (500 ng/mL) (#JN0335; BioLab, Beijing, China) was added and incubated in a CO2-free culture chamber for rotation culture at 37 °C. Cell density was observed daily and maintained at 3–4 × 10⁵ cells/mL. After 48 h of culture, the supernatant was collected for IL-9 level measurement.

Co-culture of ILC2s with PBMCs and MEC-1 cells

ILC2s (1 × 10⁵/mL) sorted from CLL patients and healthy control-derived PBMCs (5 × 10⁵/mL) were co-cultured in two Transwell wells of the upper chambers, with 1 mL in each well and 3 replicates per group. MEC-1 cells (5 × 10⁵/mL; #CBP60514; Cobioer, Nanjing, China) that were pre-cultured for 24 h were seeded into the two Transwell wells of the lower chambers, with 1 mL in each well. After co-culturing for 24 h, one well was treated with 1 μL of anti-IL-9 antibody (30 ng/mL; #AB-209-NA; R&D Systems, Minneapolis, MN, USA) while the other well was not treated. The cells were then co-cultured for an additional 72 h. The cells in the upper chambers were collected for flow cytometry analysis, the supernatant was used for ELISA experiments, and the MEC-1 cells in the lower chambers were subjected to apoptosis assays. The experiment was independently repeated three times.

Flow cytometry analysis

For the analysis of ILC2, Treg, PD-1⁺ Treg, TIGIT⁺ Treg, and CTLA-4⁺ Treg, the 100 μL of anticoagulant whole blood or resuspended cultured cells (1 × 10⁷ cells/mL) were incubated with corresponding antibodies: 1) Lin (CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, FcεΡΙα)-FITC-A, CD45-APC, CD127-PE, CRTH2-PE-CF595, and CD117-PE-Cy^TM5 for ILC2s; 2) CD4-PE-Cy^TM7, CD25-BB515, and CD127-PE for Treg; 3) CD4-PE-Cy^TM7, CD25-BB515, CD127-PE, and PD-1-PC5.5-A for PD-1 on Treg; 4) CD4-PE-Cy^TM7, CD25-BB515, CD127-PE, and TIGIT-PC7-A for TIGIT on Treg; and, 5) CD4-PE-Cy^TM7, CD25-BB515, CD127-PE, and CD152-ECD-A for CTLA-4 on Treg. The antibody incubation was performed in the dark at room temperature for 15–30 min. The supplier details for these antibodies are presented in Supplementary Table S1. Negative controls were set up. Following this, the BD FACS™ Lysing Solution 10X Concentrate (#555899; BD, San Jose, CA, USA) was diluted with dH2O to obtain a 1 X solution, and 2 mL of this solution was added to every 100 μL of whole blood. After incubating at room temperature for 8–12 min, the samples were centrifuged at 500 g for 5 min. The cells in the precipitate were re-suspended in 500 μL of PBS and then analyzed using the DXflex flow cytometer (Beckman Coulter, Brea, CA, USA). The data were processed with Kaluza software (Beckman Coulter, USA). CD45⁺Lin⁻CD127⁺CRTH2⁺CD117⁺ cells were specified as ILC2s, while CD4⁺CD25⁺CD127^low was defined as Treg. The PD-1⁺ Treg was defined as CD4⁺CD25⁺CD127^lowPD-1⁺, the TIGIT⁺ Treg was identified as CD4⁺CD25⁺CD127^lowTIGIT⁺, and, CTLA-4⁺ Treg was determined as CD4⁺CD25⁺CD127^lowCD152⁺.

For the analysis of granzyme B⁺ CD8⁺ T cells and perforin⁺ CD8⁺ T cells, the cultured cells (100 μL; 1 × 10⁷ cells/mL) were resuspended and incubated with CD8-FITC-A antibody at room temperature in the dark for 20 min. Subsequently, the cells were washed with PBS twice and then incubated with 500 μL of BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution (#554722; BD, USA) at room temperature in the dark for 30 min. After another round of washing with PBS twice, perforin-APC-A antibody (5 μL) and granzyme B-ECD-A antibody (5 μL) was added, and the cells were incubated at room temperature in the dark for 30 min. The supplier details for these antibodies are provided in Supplementary Table S1. Negative controls were set up. Finally, after a final wash with PBS twice, the cells were suspended in 500 μL of PBS and analyzed on the DXflex flow cytometer (Beckman Coulter, USA). The data obtained were analyzed with Kaluza software (Beckman Coulter, USA).

ELISA

The IL-9 level in the serum samples and cell culture supernatants was detected using an IL-9 ELISA kit (#EK109; MULTISCIENCES(LIANKE) BIOTECH, CO., LTD, Hangzhou, China). The sensitivity of the IL-9 ELISA kit was 0.06 pg/ml, with a detection range between 7.81 pg/mL and 500 pg/mL. Following the instructions strictly, standard wells, blank wells, and sample wells were set up. Double-wavelength detection was performed using a microplate reader (Multiskan™ FC Microplate Photometer; Thermo Fisher Scientific; San Jose, CA, USA) to determine the optical density values at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm.

Apoptosis detection

MEC-1 cells were resuspended in PBS at a density of 1 × 10⁶ cells/mL. Subsequently, 100 μL of the cell suspension was stained with 5 μL of Annexin V-FITC from the Annexin V-FITC Apoptosis Detection Kit (#556547; BD, USA) in the dark at room temperature for 15 min, followed by the incubation with 5 μL of PI staining solution at room temperature in the dark for 5 min. Finally, the samples were analyzed using flow cytometry.

Immunofluorescence staining

GATA3, CRTH2, and IL-9 expressions in lymph nodes were detected through immunofluorescence staining. In detail, the samples were sequentially placed in xylene I for 15 min, xylene II for 15 min, ethanol I for 5 min, ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then rinsed with distilled water. The samples were then subjected to antigen retrieval in a microwave with citrate antigen retrieval buffer (pH 6.0; #ZLI-9064; ZSGB-BIO, Beijing, China) or EDTA antigen retrieval buffer (pH 9.0; #ZLI-9069; ZSGB-BIO, China). After serum blocking, rabbit anti-human CRTH2 polyclonal antibody (#bs-13537R; Bioss, Beijing, China), anti-human IL-9 monoclonal antibody (#66144–1-1 g; Proteintech Group Inc., Wuhan, China), and mouse anti-human GATA3 monoclonal antibody (#66400–1-1 g; Proteintech Group Inc., China) were added and incubated at 4 °C overnight. Following washing, the samples were incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (#4412; Cell Signaling Technology, Inc., Danvers, MA, USA), Alexa Fluor 555-labeled goat anti-mouse IgG (#4409; Cell Signaling Technology, USA), and Alexa Fluor 594-labeled goat anti-mouse IgG secondary antibodies (#bs-0296G-AF594; Bioss, China) at room temperature for 50 min. DAPI (#C02-04002; Bioss, China) was added for nuclear staining. The images were observed under a microscope (OLYMPUS DP26; Tokyo, Japan) and analyzed using Image J software.

Statistical analysis

Data analysis was performed using SPSS 27.0. The normality of data was analyzed using the Shapiro–Wilk test, while the homogeneity of variance was tested using the Levine test. Measurement data of normal distribution and homogeneous variance are expressed as mean ± standard deviation and analyzed using a t-test. Non-normally distributed data were analyzed using non-parametric tests. Categorical data were analyzed using the chi-square test. Spearman correlation analysis was used for correlation analysis of non-normally distributed data. Graphs were generated using GraphPad Prism 9.5 software. A significance level of P < 0.05 was considered statistically significant.

Results

Clinical data of participants

The baseline clinical data of participants are presented in Table 1. In 52 cases of CLL, 14 were classified as stage A, 18 as stage B, and 20 as stage C, according to the Binet staging system. Compared to the control group, the white blood cell count, lymphocyte count, and lymphocyte percentage were significantly higher in CLL patients (P < 0.001), while hemoglobin and platelet levels were significantly lower (P < 0.001). Among the 10 CLL cases subjected to lymph node biopsy, 2 were at stage A, 4 at stage B, and 4 at stage C. The white blood cell count, lymphocyte count, and lymphocyte percentage in these cases showed significant elevation compared to the control group (P < 0.001), while hemoglobin and platelet levels were substantially lower (P < 0.001). There was no statistically significant difference in age between the CLL group and the control group (P > 0.05).

Table 1.

General clinical data of patients with CLL and healthy controls

Clinical variables	CLL for peripheral blood collection	Control for peripheral blood collection	P value	CLL subjected to lymph node biopsy	Control subjected to lymph node biopsy	P value
Number	52	30		10	10
Age (years)	63.65 ± 10.95	64.77 ± 6.91	0.617	66.00 ± 10.53	59.90 ± 7.55	0.156
Gender (male/female)	29/23	17/13		6/4	7/3
White blood cell (× 109/L)	69.4 ± 89.59	6.23 ± 1.20	< 0.001	113.54 ± 102.56	6.58 ± 1.78	< 0.001
Lymphocyte (× 109/L)	58.86 ± 78.13	2.13 ± 0.52	< 0.001	103.58 ± 98.04	2.48 ± 0.65	< 0.001
Lymphocyte percentage (%)	74.01 ± 19.87	33.55 ± 5.57	< 0.001	87.70 ± 8.60	37.06 ± 4.99	< 0.001
Hemoglobin (g/L)	111.6 ± 29.83	152.50 ± 9.73	< 0.001	106.90.08 ± 35.70	157.00 ± 11.02	< 0.001
Platelet (× 109/L)	141.04 ± 61.92	289.10 ± 45.15	< 0.001	121.50 ± 49.14	258.20 ± 62.87	< 0.001

Note: CLL, chronic lymphocytic leukemia

ILC2s percentage and IL-9 level in peripheral blood and lymph nodes as well as the correlation between ILC2s and IL-9

To determine the proportion of ILC2s in peripheral blood, flow cytometry was performed. The gating strategy for ILC2s is presented in Fig. 1A. Statistically, there was a markedly higher percentage of ILC2s in the CLL group (6.23% ± 6.60%) compared to the healthy control group (2.78% ± 2.22%) (P < 0.01) (Fig. 1B). ELISA results showed a significantly elevated concentration of IL-9 in the CLL group (13.68 ± 2.75 pg/ml) relative to the healthy controls (8.67 ± 1.59 pg/ml) (P < 0.001) (Fig. 1C). Moreover, a positive correlation was observed between the levels of ILC2s in peripheral blood and IL-9 in serum (r = 0.609, P < 0.001) (Fig. 1D). Additionally, immunofluorescence staining of lymph nodes indicated a substantial increase in the average fluorescence intensity of GATA3, CRTH2, and IL-9 in CLL patients (P < 0.001), along with evidence of co-localization (Fig. 2).

Fig. 1.

Analysis of ILC2s percentage, IL-9 level, and their correlation. A Representative flow cytometry images illustrating the gating strategy for ILC2s. ILC2 was defined as CD45⁺Lin⁻CD127⁺CRTH2⁺CD117⁺. B The percentage of ILC2s in peripheral blood. C ELISA analysis of IL-9 levels in serum. D Correlation between IL-9 in serum and ILC2s in peripheral blood. ^**P < 0.01, ^***P < 0.001

Fig. 2.

Immunofluorescence staining of CRTH2, GATA3, and IL-9 in cervical lymph node. A Representative immunofluorescence staining results. Scale bar: 25 μm. B The mean fluorescence intensities of CRTH2, GATA3, and IL-9. ^***P < 0.001

Differences in peripheral blood Treg proportions between CLL patients and the control group

Flow cytometry results showed that the percentage of Treg in the peripheral blood of the CLL group (8.88% ± 5.51%) was significantly higher than that of the healthy control group (5.81% ± 2.73%) (P < 0.01, Fig. 3).

Fig. 3.

Flow cytometry analysis of Treg percentage. The gating strategy of Treg, which was defined as CD4⁺CD25⁺CD127^low, is present on the left panel. The percentage of Treg in the peripheral blood is shown on the right panel.^**P < 0.01

Analysis of IL-9 level in the culture supernatant of sorted ILC2s and the co-culture of ILC2s and PBMCs

The ILC2s were sorted from the peripheral blood of CLL patients and healthy controls using immunomagnetic beads. The purity was identified to be over 98.01% (Fig. 4A). After culture for 48 h, the levels of IL-9 in the culture supernatant were measured with ELISA. The results revealed that the level of IL-9 in the supernatant of ILC2s from the CLL patients was significantly higher than that from the healthy control group (P < 0.001) (Fig. 4B). Subsequently, ILC2s from the CLL patients were co-cultured with PBMCs of the healthy control group and MEC-1 cell line for 72 h. The anti-IL-9 antibody was added for intervention. As shown in Fig. 4C, the level of IL-9 in the supernatant decreased significantly when compared between the group stimulated with anti-IL-9 antibody and the group without anti-IL-9 antibody stimulation (P < 0.05).

Fig. 4.

Analysis of IL-9 level in the culture supernatant. A Identification of the sorted ILC2s by flow cytometry. B IL-9 level in the culture supernatant of ILC2s from CLL patients and healthy controls. C IL-9 level in the culture supernatant of the co-culture system of ILC2s from CLL patients, PBMCs of the healthy control group, and MEC-1 cell line, in the presence or absence of anti-IL-9 antibody. ^*P < 0.05, ^***P < 0.001

The proportions of Treg, PD-1⁺ Treg, TIGIT⁺ Treg, CTLA-4⁺ Treg, granzyme B⁺ CD8⁺, and perforin⁺ CD8⁺ T cells after co-culture of ILC2s, PBMCs, and MEC-1

To further demonstrate the effects of ILC2s on Treg cells, CD8⁺ T cells, and MEC-1 cells, ILC2s were sorted from the peripheral blood of CLL patients and co-cultured with PBMC from healthy controls and MEC-1 cells. After co-culture for 72 h, the cells in the upper chamber were collected and subjected to flow cytometry analysis. The results indicated that the proportion of Tregs decreased in the group treated with anti-IL-9 antibody compared to the group without anti-IL-9 antibody stimulation, although without statistical significance (P > 0.05) (Fig. 5A, G). Furthermore, the percentages of PD-1⁺ Treg (Fig. 5B, G) and TIGIT⁺ Treg cells (Fig. 5C, G) exhibited a significant decrease (both P < 0.05), whereas the proportion of CTLA-4⁺ Treg cells (Fig. 5D, G) decreased without statistical significance (P > 0.05). Additionally, there was a significant increase in the proportion of granzyme B⁺ CD8⁺ T cells (Fig. 5E, G) (P < 0.05), while the proportion of perforin⁺ CD8⁺ T cells (Fig. 5F, G) increased insignificantly (P > 0.05).

Fig. 5.

Analysis of Treg, PD-1⁺ Treg, TIGIT⁺ Treg, CTLA-4⁺ Treg, granzyme B⁺ CD8⁺, and perforin⁺ CD8⁺ T cells after co-culture. After co-culture of ILC2s of CLL patients, PBMCs of the healthy control group, and MEC-1 cell line for 72 h, the cells in the upper chamber were collected and subjected to flow cytometry analysis. Representative flow cytometry images illustrating the gating strategy for Treg (A), PD-1⁺ Treg (B), TIGIT⁺ Treg (C), CTLA-4⁺ Treg (D), granzyme B⁺ CD8⁺ (E), and perforin⁺ CD8⁺ T cells (F). CD4⁺CD25⁺CD127^low was defined as Treg. The PD-1⁺ Treg was defined as CD4⁺CD25⁺CD127^lowPD-1⁺, the TIGIT⁺ Treg was identified as CD4⁺CD25⁺CD127^lowTIGIT⁺, and, CTLA-4⁺ Treg was determined as CD4⁺CD25⁺CD127^lowCD152⁺. G The relative percentages of Treg, PD-1⁺ Treg, TIGIT⁺ Treg, CTLA-4⁺ Treg, granzyme B⁺ CD8⁺, and perforin⁺ CD8⁺ T cells are shown. ns, not significant; ^*P < 0.05

Analysis of MEC-1 cell apoptosis after co-culture

To analyze cell apoptosis of MEC-1 cells after co-culture, we stained cells with Annexin V/PI and then conducted flow cytometry analysis. The rates of early apoptosis (Fig. 6A, B) and total apoptosis (Fig. 6A, C) increased significantly in the group stimulated with anti-IL-9 antibody compared to the group without anti-IL-9 antibody stimulation. Although the proportion of late apoptotic cells also increased, there was no statistical difference (P > 0.05).

Fig. 6.

MEC-1 cell apoptosis rate. Flow cytometry detected cell apoptosis. A Representative flow cytometry results. B Comparison of the early and late apoptosis rates. C Comparison of the total apoptosis rate. ^*P < 0.05

Discussion

In the innate immune response, ILCs play crucial roles in infection resistance, anti-tumor responses, and other functions [21, 22]. ILC2s exert either promoting or inhibitory effects on different tumors. For instance, in colorectal and pancreatic cancers, ILC2s exhibit anti-tumor activities [11, 12], while in breast and lung cancers, they tend to promote tumor growth, possibly due to their involvement in the formation of an immunosuppressive tumor microenvironment [13, 23, 24]. Trabanelli et al. [25] observed elevated levels of ILC2s in the peripheral blood of acute promyelocytic leukemia patients, with the function of recruiting more MDSCs and enhancing tumor cell proliferation. However, a more in-depth investigation is warranted to elucidate the involvement of ILC2s in CLL. Here, this study demonstrated higher expression levels of ILC2s in the peripheral blood and cervical lymph nodes of newly diagnosed CLL patients compared to controls, implying a potential promoting role of ILC2s in the occurrence and progression of CLL.

IL-9 is a pleiotropic cytokine that exerts dual regulatory functions in the immune responses of various cell types [26]. Its role in modulating the immune response in tumor progression has also been reported [27, 28]; nevertheless, its effects on tumorigenesis vary depending on the tumor type. Particularly in solid tumors like melanoma, lung cancer, colorectal cancer, and breast cancer, IL-9 demonstrates potent anti-tumor capabilities [29–31]. In contrast, IL-9 can promote the development of hematologic malignancies derived from T-cells, likely attributed to its role as a growth factor for T cells [19]. Wan et al. [15] reported that in colorectal cancer tissues, the proportion of ILC2s was higher than in the adjacent tissues, and IL-9 in colorectal cancer tissues mainly originated from ILC2s. Our study found that in newly diagnosed CLL patients, the proportions of ILC2s and IL-9 in peripheral blood and lymph nodes were higher than those in the control group, and the levels of IL-9 in peripheral blood were positively correlated with ILC2s. Furthermore, ILC2s and IL-9 co-localized in lymph nodes. Additionally, the IL-9 levels in the culture supernatant of ILC2s sorted from CLL patients were higher than those from the control individuals. These results suggest that ILC2s in CLL patients may have a higher capacity to secrete IL-9 than in healthy individuals. Moreover, ILC2s may promote the progression of CLL by secreting IL-9.

Studies have shown that ILC2 and IL-9 have an impact on Treg and CD8⁺ T cell function in different tumors [14, 15, 32, 33]. In this study, we also identified Treg overexpression in the peripheral blood of newly diagnosed CLL patients, which is consistent with the results of Pang et al. [34]. To further verify our hypothesis that the ILC2s may regulate Treg and CD8⁺ T cells through IL-9, ILC2s sorted from CLL patients were co-cultured with PBMCs and MEC-1 cells, followed by the stimulation with anti-IL-9 antibody to block the effects of ILC2s mediated by IL-9. The results indicated that the proportions of PD-1⁺ Treg and TIGIT⁺ Treg cells after anti-IL-9 antibody stimulation decreased significantly, while the proportions of CTLA-4⁺ Treg decreased without statistical significance. Additionally, MEC-1 cell apoptosis increased significantly after treatment with anti-IL-9 antibody. In tumor immunity, Treg cells participate in tumor occurrence and development by inhibiting anti-tumor immunity [35]. PD-1 is instrumental in inhibiting immune responses and promoting self-immune tolerance by regulating T cell activity, facilitating antigen-specific T cell apoptosis while inhibiting Treg cell apoptosis [36, 37]. In vitro experiments with Treg cell suppression have shown that Treg cells lacking PD-1 had reduced inhibitory effects on CD8⁺ T cell proliferation and cytokine production [38]. Several in vivo experiments have demonstrated that the conversion of CD4⁺ T cells to pTreg in peripheral tissues is highly dependent on PD-1 [39, 40]. These studies suggest that PD-1 not only negatively regulates the function of conventional T cells but also promotes the formation of pTreg to induce immune tolerance. TIGIT is an inhibitory receptor expressed on resting Treg cells, T cells, natural killer cells, natural killer T cells, and memory T cells [41], showing structural and mechanistic similarities with PD-1 and CTLA-4. TIGIT is reported as a marker of CD8⁺ T cell exhaustion and a characteristic feature of Treg cells in the tumor microenvironment [42, 43]. Kurtulus et al. demonstrated that in melanoma, TIGIT on tumor-infiltrating lymphocytes was predominantly expressed on FOXP3⁺ Tregs. TIGIT⁺ Tregs from tumor-infiltrating lymphocytes were the main producers of IL-10 and exhibited more immunosuppressive properties compared to TIGIT⁻ Tregs. Chen et al. [44] found that IL-9 reduced MEC-1 apoptosis. Based on our results and previous findings, we hypothesize that ILC2s may play a promoting role in the occurrence and development of CLL by IL-9.

Moreover, we observed a significant increase in the proportion of granzyme B⁺ CD8⁺ T cells when treated with anti-IL-9 antibodies. Although the levels of perforin also elevated, the difference was not statistically significant. Cytotoxic T lymphocytes and natural killer cells play pivotal roles in immune defenses against tumor cells and virally-infected cells. They can release cytoplasmic granules containing cytolytic proteins like perforin and granzyme B, which induce target cell death by disrupting the plasma membrane and/or triggering apoptosis. Granzyme B-deficient mice exhibit reduced efficacy in eliminating allogeneic transplanted tumors [45]. Additionally, in a study on non-small cell lung cancer conducted by Chung et al. [46], patients with a programmed death-ligand 1 tumor proportion score > 50% and low granzyme B levels did not show clinical benefits from immune checkpoint inhibitor therapy. Our study indicated that blocking ILC2s via anti-IL-9 antibodies resulted in significantly increased granzyme B⁺ CD8⁺ and non-significantly increased perforin⁺ CD8⁺ T cells, suggesting augmented anti-tumor capabilities in the absence of IL-9 secretion by ILC2s. Moreover, elevated IL-9 secretion levels in ILC2s of CLL patients compared to healthy controls further demonstrate the negative regulatory role of ILC2s and IL-9 in CLL.

Additionally, the present study also observed a positive correlation between ILC2s and IL-9 levels. It has been shown that IL-9 signals through JAK/STAT pathways, and plays a critical role in the survival, development, and maintenance of ILC2s [47]. IL-9 is an autocrine factor that promotes the survival of ILC2s by inducing the expression of the anti-apoptotic protein BCL3, thereby maintaining their functional activity in vivo [33]. Elevated levels of ILC2s and IL-9 have been associated with immune evasion in several cancers, including CLL. This suggests that targeting the ILC2s-IL-9 axis could be a promising therapeutic strategy. In our future research, we plan to conduct more in-depth studies, potentially using in vivo models or patient samples, to better understand the molecular mechanisms and clinical implications of the ILC2s-IL-9 axis in CLL progression.

In this study, we focused on IL-9 due to its emerging role in the regulation of immune responses in CLL. However, several other cytokines, such as IL-2, IL-10, and TGF-β, also play significant roles in modulating Treg and CD8⁺ T cell function. For instance, IL-2 is crucial for the maintenance and proliferation of Tregs [48], while IL-10 and TGF-β are key immunosuppressive cytokines that can enhance Treg function [49, 50] and inhibit CD8⁺ T cell activity [51, 52]. Since ILC2s do not secrete the aforementioned cytokines, they were not included in our experiments. In future studies, we will further investigate the roles of these cytokines to better understand the immune regulation mechanism of CLL. Moreover, we obtained some negative results in this study. This may be caused by various factors, including sample size, ethnic differences, patient heterogeneity, and other underlying clinical conditions. These factors resulted in tumor microenvironment differences and affected the immune responses observed in our experiments, therefore affecting the results. In future research, additional variables that may contribute to the observed results should be investigated.

This study is limited by its relatively small sample size, which may restrict the generalizability of the findings. Moreover, the reliance on in vitro models may not fully replicate the complex in vivo immune microenvironment, potentially limiting the clinical relevance of the results. To address this limitation, we plan to establish CLL models in mice to further explore the clinical implications of our in vitro observations. Additionally, while IL-9 was the primary focus, the roles of other relevant cytokines were not thoroughly investigated. The mechanisms underlying the observed correlations between ILC2s and IL-9 require further exploration. Overall, these limitations suggest the need for further research to validate and expand upon our findings.

Conclusion

Our findings reveal a significant increase in ILC2s and IL-9 levels in CLL patients compared to healthy controls. The positive correlation between ILC2s and IL-9 further emphasizes their potential involvement in CLL pathogenesis. Moreover, the co-culture experiments demonstrated that blocking IL-9 by anti-IL-9 antibody leads to alterations in Treg cell subsets, indicating a potential regulatory role of IL-9 and ILC2s in the tumor microenvironment. Additionally, the upregulation of granzyme B⁺ CD8⁺ T cells upon IL-9 blocking suggests potentially enhanced anti-tumor immunity. Overall, this study provides novel insights into the complex interplay between ILC2s, IL-9, Treg cells, and CD8⁺ T cells in CLL. By unraveling the mechanisms through which ILC2s modulate immune responses in CLL, our findings pave the way for potential targeted therapeutic approaches that disrupt this pathway to enhance anti-tumor immune responses. Further research in this area is warranted to fully elucidate the therapeutic potential of targeting ILC2s and IL-9 in CLL management.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Ruixue Yang and Xuejiao Zeng contributed to the study conception and design. Ruixue Yang, Xuejiao Zeng, and Xierenguli Alimu collected the data. Ruixue Yang analyzed the data. Ruixue Yang and Jianhua Qu interpreted the data. Ruixue Yang wrote the manuscript. Xuejiao Zeng, Xierenguli Alimu, and Jianhua Qu revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82160034), the Science and Technology Program of Xinjiang Uygur Autonomous Region (2022D14008), and the Tianshan Medical and Health Talents Training Program of Xinjiang Uygur Autonomous Region (TSYC202301A006).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval no. 20211015–47).

Consent to participate

All participants provided informed consent.

Consent to publish

Not applicable.