ICOS⁺ T helper 2 cells promote the pulmonary fibrosis in systemic sclerosis

Abstract

Background

Interstitial lung disease (ILD) is the most frequent complication of systemic sclerosis (SSc), associated with a poor prognosis and limited treatments. T helper 2 (Th2) cells play a key role in SSc pathogenesis, with T cell inducible co-stimulator (ICOS) involved in Th2 differentiation and IL-4 expression. However, the specific role of ICOS⁺ Th2 cells in SSc-ILD remains unclear.

Methods

Peripheral blood mononuclear cells (PBMCs) from SSc patients and healthy controls were analyzed via mass cytometry (CyTOF). The CD4⁺CXCR5⁻ICOS⁺IL-4⁺ T cell subset was further characterized using flow cytometry. In vitro, human lung fibroblasts were co-cultured with differentiated CD4⁺ICOS⁺IL-4⁺ cells, and fibrotic gene expression was measured. An in vivo SSc-ILD model was induced via bleomycin instillation, and the frequency of CD4⁺CXCR5⁻ICOS⁺IL-4⁺ cells was assessed in the lung. Functional contributions were evaluated by transferring CD4⁺ICOS⁺IL-4⁺ cells into Rag2^−/− mice, followed by histological analysis of lung inflammation and fibrosis.

Results

CyTOF identified a CD4⁺CCR4⁺ICOS⁺ T cell subset significantly increased in SSc-ILD patients, defined as ICOS⁺ Th2 cells by the absence of CXCR5, CXCR3, and CCR6 and the presence of CCR4. Elevated CD4⁺CXCR5⁻ICOS⁺IL-4⁺ T cells were confirmed in SSc-ILD patient PBMCs and in the lungs of bleomycin-treated mice. Functionally, ICOS⁺ Th2 cells induced high expression of Vimentin, Fibronectin, and α-SMA in fibroblasts when co-cultured with TGF-β. Adoptive transfer of ICOS⁺ Th2 cells into Rag2^−/− mice caused lung inflammation and upregulated fibrosis-related genes, including α-SMA, Col1a1, Col1a2, and Col3a1.

Conclusion

This study identifies ICOS⁺ Th2 cells as a key subset contributing to SSc-ILD inflammation and fibrosis, highlighting their potential as therapeutic targets.

Supplementary Information

The online version contains supplementary material available at 10.1186/s10020-026-01455-y.

Introduction

Systemic sclerosis-associated interstitial lung disease is the most frequent and clinically significant visceral complication, representing the primary driver of mortality in the SSc patient population, accounting for over 35% of SSc-related mortality (Tyndall et al. 2010). More than 50% of SSc patients develop ILD within the first 5 years of diagnosis, with disease progression marked by irreversible lung fibrosis and functional decline (Hoffmann-Vold et al. 2021). Despite advancements in immunosuppressive therapies such as mycophenolate mofetil (MMF) and cyclophosphamide (CYC), treatment responses remain variable, and no disease-modifying agents are currently approved for SSc-ILD(Cottin and Brown 2019). This therapeutic impasse underscores the urgent need to dissect the molecular drivers of fibrosis and identify actionable biomarkers for patient stratification.

A critical component in the pathogenesis of SSc-ILD is the dysregulated immune response, characterized by the overactivation of T cells, which plays a significant role in the initiation and progression of pulmonary fibrosis. Patients demonstrate a pronounced Th1/Th2 immune imbalance, with elevated Th2 levels being associated with interstitial pulmonary fibrosis (Meloni et al. 2009; Jin et al. 2022). This condition is further evidenced by increased levels of Th2 cell-associated cytokines, which correlate with reduced lung function and heightened disease severity (Pellicano et al. 2023), indicating that Th2 polarization may exacerbate lung tissue damage and fibrotic remodeling through the promotion of inflammatory and pro-fibrotic factors, such as interleukin-4 (IL-4) and interleukin-13 (IL-13) (Desai et al. 2018; Mattoo et al. 2023). Additionally, Th2 cells may modulate the direction and intensity of the immune response through interactions with other immune cells. For instance, Th2 cells promote the fibrotic process by interacting with macrophages to induce M2 polarization. These M2 macrophages then facilitate the transformation from fibroblasts to myofibroblasts by releasing key growth factors, including transforming growth factor-beta (TGF-β), which ultimately advances fibrosis (Gordon and Martinez 2010; Xu et al. 2024). Recent studies have shown that Th2 cells are not a single population, but are composed of multiple subpopulations with different functions and phenotypes (Nakayama et al. 2017). For example, in allergic diseases, specific Th2 cell subpopulations are considered key factors in disease onset. These subpopulations not only differ functionally from traditional Th2 cells but also display unique phenotypic characteristics(Wambre, et al. 2017; Irvin et al. 2014). Studies have found that certain memory Th2 cell subpopulations can produce large amounts of interferon-gamma (IFN-γ), which contradicts the typical characteristics of Th2 cells. This phenomenon may be related to the phosphorylation state of Gata3 (Hosokawa et al. 2016). Nevertheless, the diversity of Th2 subsets and their specific roles in SSc-ILD remain inadequately elucidated.

The inducible T-cell costimulator (ICOS), a costimulatory molecule, has been extensively investigated in the context of T follicular helper (Tfh) cells, where it is integral to the facilitation of germinal center reactions and antibody class switching. Furthermore, ICOS has been implicated in Th2 polarization and the selective production of IL-4 in models of allergic responses and parasitic infections (Wikenheiser and Stumhofer 2016). ICOS signaling enhances T cell receptor (TCR)-driven activation, sustains Th2 survival, and amplifies IL-4 transcription through pathways involving STAT6 (Harada et al. 2003; Watanabe et al. 2005). However, despite its established role in Th2 biology, the identity and functional relevance of ICOS-expressing Th2 cells in human autoimmune fibrosis, particularly SSc-ILD, remain unexplored.

To address this research question, the present study employs a comprehensive approach by integrating high-dimensional immune profiling, functional assays, and preclinical models to examine the role of a novel ICOS⁺ Th2 subset in SSc-ILD. The identification of this subset not only enhances the immunological classification of SSc but also reveals novel therapeutic targets, thereby facilitating the development of personalized immunomodulatory strategies for the management of SSc-ILD.

Method

Patients enrollment and sample collection

A cohort comprising 12 SSc-ILD, 11 SSc without interstitial lung disease (SSc-nILD) and 15 healthy volunteers participated in this study. The classification of patients referred to a previous report (Raghu et al. 2024). Patients were diagnosed with SSc by applying the 2013 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria (Hoogen et al. 2013). The exclusion criteria include (1) pregnancy, breastfeeding, or planning to become pregnant within half a year; (2) serious heart-related diseases, like arrhythmia, Class III-IV congestive heart failure (NYHA), unstable angina, uncontrolled high blood pressure, or a pericardial effusion causing symptoms; (3) concurrent bacterial or viral infections; (4)coexistence of other autoimmune diseases; (5) current tumors or other cancerous lesions. All patients signed the informed consent form with full notification prior to sample collection. The study was approved by the Ethics Committee of Nanjing Drum Tower Hospital (Approval No. SC-2022–008) and the Institutional Review Board (IRB). Following collection, the isolated PBMCs from patient peripheral blood were resuspended in PBS containing 1% fetal bovine serum (FBS, Gibco) for subsequent experiments.

Mouse models

Female C57BL/6 J and Rag2^−/− mice (aged 7–8 weeks, weighing 20–25 g) were sourced from GemPharmatech (Nanjing, China). All animals were subsequently housed in an SPF facility. The C57BL/6 J mice were randomized into either the BLM group or the PBS control group. The BLM group received an intratracheal infusion of bleomycin (2.5 U/kg) on day 0, while the control group was given the same volume of PBS. 1 × 10⁶ ICOS⁺ Th2 cells was administered to Rag2^−/− mice via the tail vein. 21 days or four weeks later, the mice were sacrificed and samples were collected for subsequent experiments.

PBMC preparation for CyTOF

PBMCs were isolated from patient blood samples, which had been collected in EDTA-anticoagulated tubes, by means of Ficoll density gradient centrifugation. Following isolation, the cell concentration was normalized to 1 × 10⁷ cells/mL for subsequent experiments. A volume of 1 × 10⁶ PBMCs per sample was aliquoted, and a pre-configured antibody cocktail containing metal isotope-tagged antibodies (42 markers; panel details provided in Table 1) was added. The metal-tag conjugation protocol was performed as recommended by the manufacturer’s manual. For staining, 100 μL of cell suspension was mixed with an antibody cocktail (pre-optimized for concentration) and incubated at room temperature in the dark for 30 min. Unbound antibodies were removed by two washes with pre-chilled PBS (HyClone). Subsequently, 1 mL of fixation solution (Fixation/Permeabilization Kit, eBioscience) was added to the cell pellet, followed by incubation at 4 °C in the dark for 45 min. Residual fixative was eliminated by two washes with 1 × Permeabilization Buffer (eBioscience). For intracellular protein staining (e.g., Foxp3, IFN-γ, TNF-α), cells were incubated with metal-conjugated antibodies (Huaying Biology) at 4 °C in the dark for 30 min, followed by additional washes. Finally, stained cells were resuspended in PBS containing 0.1% BSA. Samples were analyzed using the Helios CyTOF2 system (Fluidigm) with a detection rate set to 500 cells per second. Approximately 300,000 cells per sample were acquired over a 10-min acquisition period. Daily instrument calibration was performed using EQ Four Element Calibration Beads (Fluidigm).

Table 1.

Markers to phenotype total PBMCs by CyTOF

Antibodies	Clone	Identifier
Anti-CD45 conjugated to 89Yb	HI30	Biolegend (Cat#304,002)
Anti-CD3 conjugated to 115In	UCHT1	Bioxcell (Cat#BE0231)
Anti-CD68 conjugated to 139La	Y1/82A	Biolegend (Cat#333,802)
Anti-CD56 conjugated to 141Pr	NCAM16.2	BD (Cat#559,043)
Anti-CD19 conjugated to 142Nd	HIB19	Biolegend (Cat#302,268)
Anti-CD196 conjugated to 143Nd	G034E3	Biolegend (Cat#353,402)
Anti-CD38 conjugated to 144Nd	HIT2	Biolegend (Cat#303,502)
Anti-CD195 conjugated to 145Nd	J418F1	Biolegend (Cat#359,102)
Anti-TNF-α conjugated to 146Nd	Mab11	Biolegend (Cat#502,902)
Anti-CD15 conjugated to 147Sm	W6D3	Biolegend (Cat#323,002)
Anti-CD69 conjugated to 148Nd	FN50	Biolegend (Cat#310,902)
Anti-CD25 conjugated to 149Sm	24,212	R&D (Cat#MAB1020)
Anti-CD73 conjugated to 150Nd	AD2	Biolegend (Cat#344,002)
Anti-CD278 conjugated to 151Eu	C398.4A	Biolegend (Cat#313,502)
Anti-CD34 conjugated to 152Sm	581	Biolegend (Cat#343,502)
Anti-CD194 conjugated to 153Eu	L291H4	Biolegend (Cat#359,402)
Anti-CD152 conjugated to 154Sm	14D3	eBioscience (Cat#14–1529-82)
Anti-CD45RA conjugated to 155Gd	HI100	Biolegend (Cat#304,102)
Anti-CD204 conjugated to 156Gd	351,615	R&D (Cat#MAB2708)
Anti-CD39 conjugated to 157Gd	A1	Biolegend (Cat#328,202)
Anti-CD197 conjugated to 158Gd	G043H7	Biolegend (Cat#353,256)
Anti-CD33 conjugated to 159 Tb	WM53	Biolegend (Cat#303,419)
Anti-CD28 conjugated to 160Gd	CD28.2	Biolegend (Cat#302,934)
Anti-CD1c conjugated to 161Dy	L161	Biolegend (Cat#331,502)
Anti-VEGF conjugated to 162Dy	G153-694	BD(Cat#555,036)
Anti-CD24 conjugated to 163Dy	ML5	Biolegend (Cat#311,102)
Anti-CD185 conjugated to 164Dy	RF8B2	BD (Cat#552,032)
Anti-IFN-γ conjugated to 165Ho	B27	Biolegend (Cat#506,502)
Anti-CD183 conjugated to 166Er	G025H7	Biolegend (Cat#353,750)
Anti-CD326 conjugated to 167Er	9C4	Biolegend (Cat#324,202)
Anti-CD31 conjugated to 168Er	WM59	Biolegend (Cat#303,144)
Anti-CD133 conjugated to 169Tm	170,411	R&D (Cat#MAB11331)
Anti-CD366 conjugated to 170Er	F38-2E2	Biolegend (Cat#345,010)
Anti-CD14 conjugated to 171Yb	M5E2	Biolegend (Cat#301,862)
Anti-FOXP3 conjugated to 172Yb	PCH101	eBioscience (Cat#14–4776-82)
Anti-CD86 conjugated to 173Yb	FUN-1	BD (Cat#555,655)
Anti-CD279 conjugated to 174Yb	EH12.2H7	Biolegend (Cat#329,926)
Anti-CD16 conjugated to 175Lu	3G8	Biolegend (Cat#302,057)
Anti-HLA-DR conjugated to 176Yb	L243	Biolegend (Cat#307,648)
Anti-CD4 conjugated to 197Au	RPA-T4	Biolegend (Cat#300,570)
Anti-CD8a conjugated to 198pt	RPA-T8	Biolegend (Cat#301,074)
Anti-CD11b conjugated to 209Bi	M1/70	Biolegend (Cat#101,202)

Isolation and differentiation of ICOS⁺ Th2 cells

The isolated PBMCs from patient peripheral blood were resuspended in PBS containing 1% FBS, then incubated with anti-human CD4 MicroBeads (Miltenyi Biotec, Cat#130–045–101, 20 μL per 1 × 10⁷ cells) at 4 °C for 15 min, followed by positive selection using LS columns (Miltenyi Biotec, Cat#130–042–401) on a MACS separator (Miltenyi Biotec, Cat#130–042–303). For mouse spleen samples, minced spleen tissue was digested with collagenase IV (1 mg/mL, Gibco) for 30 min at 37 °C, filtered to obtain single-cell suspensions, and subjected to positive selection with anti-mouse CD4 MicroBeads (Miltenyi Biotec, Cat#130–117-043). Isolated CD4⁺ T cells (1 × 10⁶ cells/mL) were seeded in 24-well plates pre-coated with anti-human/mouse-CD3 (1 μg/mL, BioLegend) and anti-human/mouse-CD28 (1 μg/mL, BioLegend) antibodies, cultured in RPMI 1640 (Gibco) containing 10% FBS, 100 U/mL penicillin–streptomycin (Gibco), 2 mM L-glutamine (Gibco), 10 μg/mL anti-IFN-γ antibody (BioLegend), and 10 ng/mL recombinant IL-4 (PeproTech) at 37 °C with 5% CO₂ for 4 days. Activated cells were washed with PBS, labeled with biotinylated anti-human/mouse ICOS antibody (BioLegend, Cat#313,504, 1 μg per 1 × 10⁶ cells) at 4 °C for 15 min, then incubated with anti-biotin MicroBeads (Miltenyi Biotec, Cat#130–090–485, 20 μL per 1 × 10⁷ cells) for 10 min, and ICOS⁺ Th2 cells were isolated using MS columns (Miltenyi Biotec, Cat#130–042–201).

Flow cytometry

For surface marker detection, 1 × 10⁶cells were washed in PBS with 0.5% BSA and stained with the following fluorochrome-conjugated antibodies for 30 min at 4 ℃ in the dark. Human samples used: anti-human CD3-BV421 (BioLegend, Cat#300,434, clone UCHT1), anti-human CD4-FITC (BioLegend, Cat#317,408, clone OKT4), anti-human CXCR5-PE (BioLegend, Cat#356,903, clone J252D4), anti-human ICOS-APC (eBioscience, Cat#17–9948-42, clone ISA-3), and anti-human CCR4-PE-Cy7 (BD Biosciences, Cat#561,034, clone 1G1). Mouse samples used: anti-mouse CD3-FITC (BioLegend, Cat#100,306, clone 145-2C11), CD4-APC (BioLegend, Cat#100,412, clone GK1.5), CXCR5-PE (BD Biosciences, Cat#561,988, clone 2G8), ICOS-BV605 (BD Biosciences, Cat#745,254, clone 7E.17G9).

For intracellular cytokine staining, cells were stimulated with PMA (50 ng/mL)/ionomycin (1 μg/mL, Sigma-Aldrich) and brefeldin A (10 μg/mL, BioLegend) for 4 h, fixed with 4% paraformaldehyde (Thermo Fisher), permeabilized using 0.1% saponin buffer (BD Biosciences), and stained with cytokine-specific antibodies. Human samples used: anti-human IL-4-PerCP-Cy5.5 (BioLegend, Cat#500,822, clone MP4-25D2), anti-human IL-21-Alexa Fluor 647 (BD Biosciences, Cat#562,043, clone 3A3-N2.1), anti-human IFN-γ-BV605 (BD Biosciences, Cat#562,974, clone B27), and anti-human IL-17A-PE/Dazzle 594 (BioLegend, Cat#512,335, clone BL168). Mouse samples used: anti-mouse IL-4-PE (BD Biosciences, Cat#554,435, clone 11B11), IL-21-AF647 (Sony Biotechnology, Cat#3,184,015, clone BL25168), IFN-γ-FITC (BioLegend, Cat#505,806, clone XMG1.2), and IL-17A-PE-Cy7 (BioLegend, Cat#506,922, clone TC11-18H10.1). All samples were analyzed on a BD LSRFortessa flow cytometer, and data were processed using FlowJo v10.8. Positive populations for intracellular cytokines were determined based on Fluorescence Minus One (FMO) controls.

Statistical analysis

The R package cytofWorkflow (version 1.14.0) was employed for processing all CyTOF data. Statistical analyses were performed using GraphPad Prism V8.0.2 (Graph Software, San Diego, CA, USA), with data shown as mean ± SD. The correlation between the percentage of ICOS⁺ Th2 cells and modified Rodnan skin score (mRSS) was analyzed using Spearman’s rank correlation test. P values were derived from one-way ANOVA or unpaired t-tests where applicable, and significance was established at a threshold of P < 0.05.

Results

A new ICOS⁺ Th2 cell subpopulation was identified

To comprehensively profile the immune cells in SSc, we analyzed the peripheral blood of SSc-ILD and SSc-nILD patients using CyTOF. We first isolated the T cell populations for a separate cluster analysis. By applying the t-random neighbor embedding (t-SNE) dimensionality reduction method, we successfully delineated 33 T cell subpopulations (Figs. 1A, 1B). Among the 33 subsets, clusters 1 to 17 are all CD4⁺ T helper cells. Among CD4⁺ T helper cells, cluster 9 was defined as Th2 cells, as they highly expressed CCR4 but did not express biomarkers of Tfh cells (CXCR5, PD-1), Th1 cells (CXCR3), Th17 cells (CCR6), and Treg cells (CD25, Foxp3) (Fig. 1C). The frequency of cluster 9 significantly increased in SSc-ILD patients compared with HC (Fig. 1D). We generated a heatmap to display the unique molecular expression patterns for each identified subgroup. Among these, cluster 9 stood out due to its particularly strong expression of ICOS (Figs. 1E, 1 F). Therefore, we defined this group of CD4⁺CCR4⁺ICOS⁺ T cells as ICOS⁺ Th2 cells, a new subgroup of Th2 cells in SSc. Additionally, beyond its defining high ICOS expression, this new subpopulation exhibited distinct cytologic phenotypes among SSc-ILD, SSc-nILD patients and HCs, characterized by altered mean expression levels of other functional markers (Supplementary Fig. 1).

Fig. 1.

T cell type transition based on the CyTOF analysis (A) TSNE projection of T cells derived from CyTOF data. B The TSNE maps of the SSc-ILD group, SSc-nILD group and HC group. C The TSNE maps of antibodies for cell typing. D The percentages of cluster 9 in T cells among three groups. E Heatmap of antibodies expressed on 33 T cell subpopulations in SSc-ILD group. F The TSNE plot of ICOS. SSc1: SSc-ILD, SSc2: SSc-nILD, HC: healthy control. * p < 0.05

ICOS⁺ Th2 cells were related with fibrotic characteristics in SSc

Next, we labeled cluster 9 subpopulation by using anti-CXCR5, anti-CCR4, anti-ICOS, anti-IL-4, and anti-IL-21 antibodies. As ICOS and IL-21 were also biomarkers of Tfh cells and IL-4 was preferentially secreted by Th2 cells, we tried to detect CD4⁺CXCR5⁻ICOS⁺IL-4⁺IL-21⁻ cells in peripheral blood of SSc patients by flow cytometry, and found that CD4⁺CXCR5⁻ICOS⁺CCR4⁺ cells secreted more IL-4 than IL-21, and nearly 90% of CD4⁺CXCR5⁻ICOS⁺IL-4⁺IL-21⁻ expressed CCR4. So we tried to define this new ICOS⁺ Th2 subset as CD4⁺CXCR5⁻ICOS⁺IL-4⁺ cells (Fig. 2A). The population of ICOS⁺ Th2 cells (IL-4⁺IL-21⁻) was notably expanded in SSc patients. Interestingly, we also observed a concomitant increase in IL-21-expressing cells within the CD4⁺CXCR5⁻ICOS⁺ Th2 population in SSc samples compared to HC (Fig. 2B), although our primary focus in this study remains on the IL-4 predominant subset. Consequently, the percentage of the defined ICOS⁺ Th2 cells was significantly increased in patients with SSc as compared with HC (Figs. 2B, 2 C). We then analyzed the proportions of ICOS⁺ Th2 cells according to SSc characteristics and subtypes and observed that this subset was significantly expanded in the diffuse cutaneous systemic sclerosis (dcSSc) as compared with limited cutaneous systemic sclerosis (lcSSc) (Fig. 2C). ICOS⁺ Th2 cells frequency was also increased in patients with ILD (Fig. 2C). The frequency of ICOS⁺ Th2 cells was positively correlated with mRSS (r = 0.8962, p < 0.0001, Fig. 2D). These results indicated that ICOS⁺ Th2 cells were involved in the pathogenesis of fibrosis in SSc patients.

Fig. 2.

ICOS⁺ Th2 expansion and clinical correlation in SSc (A) Flow cytometry gating plot of CD4⁺CXCR5⁻ICOS⁺IL-4⁺IL-21⁻ cells. B The percentages of ICOS⁺ Th2 cells between HC and SSc patients. C The percentages of ICOS⁺ Th2 cells were compared between groups. D Spearman correlation analysis of the percentage of ICOS⁺. Th2 cells and mRSS score. mRSS: modified Rodnan skin score, r = 0.8962, p < 0.0001. All data were expressed as means ± SD. P value was calculated using unpaired t test. * p < 0.05, ** p < 0.01

ICOS⁺ Th2 cells could promote fibrosis in vitro

Given that myofibroblasts are typically absent in healthy lung tissue and emerge primarily during injury and repair processes, we sought to assess the direct functional effects of the aforementioned ICOS⁺ Th2 cells on pulmonary fibrosis. To directly evaluate the translational relevance of these findings to human pathology, we performed in vitro assays using human pulmonary fibroblasts (IMR-90). To this end, IMR-90 cells were co-cultured with ICOS⁺ Th2 cells that had been differentiated in vitro (Fig. 3A). Under polarizing conditions and magnetic cell sorting, human naive CD4⁺ T cells up-regulated a substantial subset coexpressing IL-4 and ICOS (Figs. 3B, 3 C). These cells were co-cultured with IMR-90 at a 1:4 ratio in the presence or absence of suboptimal TGF-β and soluble anti-CD3/anti-CD28 antibodies. After 3 to 4 days in co-culture, ICOS⁺ Th2 cells upregulated the mRNA expression of Vimentin and Fibronectin in IMR-90 cells. At the same time, in the presence of both TGF-β and ICOS⁺ Th2 cells, the expression of α-SMA was also significantly increased in IMR-90 cells (Fig. 3D). Similarly, at the protein level, the combined action of TGF-β and Th2 cells can significantly increase the levels of Vimentin, Fibronectin and a-SMA (Fig. 3E). ICOS⁺ Th2 cells initiate moderate myofibroblast activation, but classical full differentiation and immense matrix deposition strictly require synergistic interaction with TGF-β.

Fig. 3.

ICOS⁺ Th2-driven fibrosis in SSc-ILD co-culture model (A) Flow chart of IMR-90 co-cultured with ICOS⁺ Th2 cells of SSc-ILD patient. B The expression of IL-4, IL17A and IFN-γ by naive CD4⁺ T cells before and after polarization. C Sorting efficiency of human ICOS⁺. Th2 cell. D The mRNA expression of Vimentin, Fibronectin and α-SMA in IMR-90 cells. (E) The protein level of Vimentin, Fibronectin and α-SMA in IMR-90 cells. All data were expressed as means ± SD. P value was calculated using one way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

ICOS⁺ Th2 cells increased in bleomycin-induced pulmonary fibrosis

To investigate the ICOS⁺ Th2 cells in the lung, we established SSc-ILD mouse models by tracheal instillation of BLM as previously reported (Schniering et al. 2019). With disease progression, the number of inflammatory infiltrates and fibrotic severity increased in the lungs of BLM-treated mice (Fig. 4A), accompanied by enhanced collagen deposition (Fig. 4C). Furthermore, CD4⁺ T cell infiltration (Fig. 4A) and overall IL-13 levels (Fig. 4D) were also elevated in the lungs. Meanwhile, we analyzed the mRNA expression of fibrotic gene in lung sections of BLM-treated mice. Both of them were markedly elevated in model mice compared with the control group (Fig. 4B). Then we further investigated the cytokine expression of ICOS⁺ Th2 cells in lung tissues of SSc-ILD model and found that in comparison with the PBS group, IL-4 expression was significantly increased in CD4⁺CXCR5⁻ICOS⁺ T cells. However, there were no significant differences in IL-21, IL-17A and IFN-γ expression between PBS group and BLM group (Figs. 4E, F). These results indicated that the ICOS⁺ Th2 cells might play a role in SSc-ILD by producing IL-4.

Fig. 4.

BLM-induced lung fibrosis and ICOS⁺ Th2 inflammation (A) H&E, Masson staining and CD4 IHC in the lungs of mice treated by PBS and BLM. B The mRNA expression of α-SMA, Col1α1, Col1α2, Col3α1 in lung tissus between PBS and BLM groups. C The hydroxyproline in lung tissue between PBS and BLM groups. D The level of IL-13 in lung tissue between PBS and BLM groups detected by ELISA. E Flow cytometry representative plots and (F) proportions of inflammatory cytokines IL-4, IFN-γ, IL-17A, and IL-21 expressed on ICOS⁺. Th2 cells in lung tissues of PBS and BLM groups. All data were expressed as means ± SD. P value was calculated using one way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

ICOS⁺ Th2 cells facilitate fibrosis in vivo

Next, in vitro differentiated mouse ICOS⁺ Th2 cells were injected into Rag2^−/− mice to investigate their profibrotic role in vivo without the compounding interference of endogenous adaptive immunity(Fig. 5A). Under polarizing conditions and magnetic cell sorting, mice naive CD4⁺ T cells up-regulated a substantial subset coexpressing IL-4 and ICOS (Figs. 5B,C). We found extensive interstitial collagen deposition in the lung interstitium with accumulated inflammatory cells on day 28 after cellular injection, while not detected on day 14 (Fig. 5D). We additionally analyzed the mRNA expression of and fibrotic mediators in lungs. Both of them were markedly elevated on day 28 after cellular injection (Fig. 5E). These data indicated that adoptive transfer of ICOS⁺ Th2 cells could induce inflammatory responses and fibrosis progression in the lung of Rag2^−/− mice, and its effect was closely related to the expansion of IL-4/ICOS co-expressing T cell subsets.

Fig. 5.

ICOS⁺ Th2-driven fibrosis in Rag2^−/− mice over time (A) Schematic diagram of ICOS⁺ Th2 cells inducing fibrosis in Rag2^−/− mice. B The expression of IL-4, IL17A and IFN-γ by naive CD4⁺ T cells before and after polarization. C Sorting efficiency of mouse ICOS⁺ Th2 cell. D H&E and Masson staining in the lungs of mice treated by ICOS⁺ Th2 cells at day 14 and 28. E The mRNA expression of α-SMA, Col1α1, Col1α2, Col3α1 in lung tissus of mice treated by ICOS⁺. Th2 cells at day 14 and 28. All data were expressed as means ± SD. P value was calculated using one way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Discussion

Our study established ICOS⁺ Th2 cells (CD4⁺CXCR5⁻CXCR3⁻CCR6⁻ICOS⁺IL-4⁺) as pivotal drivers of fibrogenesis in SSc-ILD. High-dimensional immune profiling via CyTOF revealed a marked expansion of CD4⁺CCR4⁺ICOS⁺ T cells in SSc-ILD patients compared to healthy controls. This population’s pathological relevance was further corroborated in a bleomycin-induced murine SSc-ILD model, where analogous ICOS⁺ Th2 cell expansions in the lungs mirrored human disease progression. In vitro and in vivo experiments have further demonstrated that ICOS⁺ Th2 cells directly promote pulmonary fibrosis through an IL-4-dependent mechanism. These findings not only reveal the importance of Th2 cell heterogeneity in the pathogenesis of SSc, but also provide a theoretical basis for precise therapy targeting the ICOS/IL-4 axis.

While the pathogenic involvement of type 2 immune responses in fibrosis is well-established (Gieseck et al. 2018; Fattahi, et al. 2024; Sorkhdini, et al. 2024; Kokubo et al. 2022), our study refines this paradigm by delineating a distinct ICOS⁺ Th2 subpopulation. Historically, high expression of ICOS has been primarily regarded as a hallmark of follicular helper T (Tfh) cells, which localize to germinal centers to aid B cells (Marafioti et al. 2010; Crotty 2019). However, the population we identified lacks CXCR5, clearly distinguishing it from classical Tfh cells. This phenotype resonates with the recently characterized "peripheral helper T" (Tph) cells observed in autoimmune tissues (Rao et al. 2017); yet, unlike the broad spectrum of Tph cells, our subset is uniquely defined by a potent Th2-skewed cytokine profile (IL-4^hi). Altogether, these phenotypic features outline a novel, highly specialized effector subpopulation uniquely equipped to promote autoimmune-driven tissue fibrosis.

Specifically, the role of ICOS in lung pathology likely depends on the specific disease stage and the outcomes evaluated. A previous study by Hrusch et al. demonstrated that ICOS exerted a protective effect against mortality in a BLM-induced lung injury model by promoting IL-5 production, which helped preserve tissue integrity (Hrusch et al. 2018). This seemingly contrasts with our current findings, which suggest a profibrotic role of ICOS in SSc-ILD(Taylor, et al. 2018). This discrepancy is likely due to the different phases of disease progression being studied. Hrusch et al. focused on the acute lung injury phase, where ICOS-induced IL-5 is necessary to protect tissue and prevent acute death. In contrast, our study focuses on the later fibrotic remodeling phase. During this chronic stage, persistent ICOS activation drives profibrotic responses and exacerbates fibrosis. Therefore, ICOS may be protective against acute mortality early on, but during long-term tissue remodeling, the expanded ICOS⁺ Th2 cells fuel a chronic, excessive IL-4 production, thereby shifting the balance from tissue repair to progressive fibrosis.

T cell imbalance is a key feature of SSc-ILD (Fuschiotti 2016; Yu et al. 2025). Although Th2 cells are known to drive fibrosis (Barron and Wynn 2011), our study highlights the specific clinical importance of the ICOS⁺ Th2 subset. We found that the frequency of these cells is significantly expanded in SSc patients compared to healthy controls. More importantly, this expansion was more pronounced in patients with dcSSc than in those with lcSSc, and was also significantly elevated in patients with ILD. Furthermore, we observed a direct positive correlation between ICOS⁺ Th2 cell levels and mRSS scores. These results indicate that the abundance of ICOS⁺ Th2 cells closely tracks with fibrotic severity. Thus, this specific subset could serve as a useful biomarker for monitoring disease progression (Khanna et al. 2020; Qin, et al. 2023). These clinical associations led us to investigate how these cells drive fibrosis.

Our study strongly implies that the profibrotic function of ICOS + Th2 cells is closely linked to their potent IL-4 production. We found that ICOS⁺ Th2 cells produce much higher levels of IL-4 compared to other cytokines. IL-4 is a critical signal that activates fibroblasts directly (Arndt, et al. 2023; Jakubzick et al. 2003) and a known inducer of M2 macrophage polarization, which in turn promotes the release of profibrotic mediators such as TGF-β1 and platelet derived growth factor (PDGF) (Rao et al. 2021; Pechkovsky et al. 2010). Consistent with this, our in vitro co-culture assays demonstrated that the presence of ICOS⁺ Th2 cells induced early fibrotic markers, such as Vimentin, in human lung fibroblasts. Crucially, we found that these cells act synergistically with TGF-β, a master regulator of fibrosis (Trinh-Minh, et al. 2024; Massagué and Sheppard 2023). While TGF-β alone initiates fibrotic signaling, the addition of ICOS⁺ Th2 cells significantly amplified α-SMA expression, indicating a robust promotion of myofibroblast differentiation. This synergistic interplay suggests that ICOS⁺ Th2 cells do not merely initiate fibrosis but rather potentiate the responsiveness of fibroblasts to TGF-β, thereby accelerating the transition from tissue repair to progressive, irreversible scarring.

Several limitations should be noted. Our clinical samples were derived from SSc patients, and additional comparisons with non-SSc fibrotic controls or longitudinal sampling would further define whether this subset is specific to SSc-ILD or reflects a shared fibrotic program. In addition, while IL-4 emerges as a dominant cytokine in this subset, we also observed increased IL-21 within the CXCR5⁻ICOS⁺ compartment, which may contribute to disease through effects on B-cell responses and fibrosis; defining the contributions of IL-4⁺IL-21⁺ and IL-21⁺ subsets will require further work. Despite these limitations, our data collectively support ICOS⁺ Th2 cells as a clinically relevant, profibrotic immune population and highlight the ICOS/IL-4 axis as a potential target for precision immunomodulation in SSc-ILD.

Conclusions

We identified a novel profibrotic ICOS⁺ Th2 subset (CD4⁺CXCR5⁻ICOS⁺CCR4⁺) expanded in SSc, particularly in SSc-ILD and diffuse cutaneous disease. In vitro, ICOS⁺ Th2 cells synergize with TGF-β to amplify α-SMA and fibrotic gene expression in IMR-90 cells. In vivo, these cells are enriched in bleomycin-induced pulmonary fibrosis models, and their adoptive transfer into Rag2^−/− mice triggers robust lung inflammation and fibrogenesis.

Abbreviations

SSc

Systemic sclerosis

ILD

Interstitial lung disease

SSc-ILD

Systemic sclerosis associated Interstitial lung disease

SSc-nILD

SSc without interstitial lung disease

Th2

T helper 2

ICOS

Inducible co-stimulator

PBMC

Peripheral blood mononuclear cell

CyTOF

Mass cytometry

H&E

Hematoxylin and eosin

TSNE

T-random neighbor embedding

MMF

Mycophenolate mofetil

CYC

Cyclophosphamide

IL-4

Interleukin-4

IL-6

Interleukin-6

IL-13

Interleukin-13

TGF-β

Transforming growth factor-beta

IFN-γ

Interferon-gamma

Tfh

T follicular helper

TCR

T cell receptor

lcSSc

Limited cutaneous systemic sclerosis

dcSSc

Diffuse cutaneous systemic sclerosis

mRSS

Modified Rodnan skin score

PDGF

Platelet derived growth factor

Authors’ contributions

HM Z and YX C were responsible for experimental visualization, data validation, methodology design, data organization and the writing of the first draft. YY W, Y Z, SS L, N Y, M L and ZK W participated in data collection, experimental operation and methodological implementation; DD W led data analysis, method optimization, fund acquisition, experimental supervision and paper revision; LY S proposed the research concept, coordinated project resources, coordinated financial support, supervised the research progress throughout the process, and was responsible for the review and finalization of the final manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82572069), Natural Science Foundation of Jiangsu Province (BK20231119), the Medical Research Program of Jiangsu Provincial Health and Wellness Commission (H2023087), Clinical Trials from Nanjing Drum Tower Hospital (2022-YXZX-MY-03) and Bethune Charitable Foundation (BCF-QYWL-FS-2024–3).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All investigations received approval from the Ethics Committee of the Nanjing Drum Tower Hospital (Approval No. SC-2022–006). All patients provided informed consent prior to being enrolled.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.