
==== Front
Rheumatology (Oxford)
Rheumatology (Oxford)
brheum
Rheumatology (Oxford, England)
1462-0324
1462-0332
Oxford University Press

38552313
10.1093/rheumatology/keae190
keae190
Clinical Science
AcademicSubjects/MED00360
Systemic sclerosis-associated pulmonary arterial hypertension is characterized by a distinct peripheral T helper cell profile
https://orcid.org/0000-0003-4198-4838
Papadimitriou Theodoros Ioannis Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands
Department of Laboratory Medicine – Medical Immunology, Radboudumc, Nijmegen, The Netherlands

https://orcid.org/0000-0003-1183-5049
Lemmers Jacqueline M J Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

van Caam Arjan P M Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

Vos Jacqueline L Department of Cardiology, Radboudumc, Nijmegen, The Netherlands

Vitters Elly L Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

Stinissen Lizan Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

https://orcid.org/0000-0003-1432-069X
van Leuven Sander I Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

Koenders Marije I Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

van der Kraan P M Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

Koenen Hans J P M Department of Laboratory Medicine – Medical Immunology, Radboudumc, Nijmegen, The Netherlands

Smeets Ruben L Department of Laboratory Medicine – Medical Immunology, Radboudumc, Nijmegen, The Netherlands
Radboudumc Laboratory for Diagnostics, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, Netherlands

https://orcid.org/0000-0003-1530-6363
Nijveldt Robin Department of Cardiology, Radboudumc, Nijmegen, The Netherlands

Vonk Madelon C Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

Thurlings Rogier M Department of Rheumatology, Radboudumc, Nijmegen, The Netherlands

Correspondence to: Rogier M. Thurlings, Department of Rheumatology, Radboudumc, Geert Grooteplein Zuid 10, 6525 GA, Nijmegen, The Netherlands. E-mail: rogier.thurlings@radboudumc.nl
Theodoros Ioannis Papadimitriou and Jacqueline M.J. Lemmers contributed equally

9 2024
29 3 2024
29 3 2024
63 9 25252534
23 11 2023
17 3 2024
11 5 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

Objectives

Systemic sclerosis (SSc) is characterized by multiple clinical manifestations. Vasculopathy is a main disease hallmark and ranges in severity from an exacerbated Raynaud phenomenon to pulmonary arterial hypertension (PAH). The potential involvement of the immune system in SSc-associated vascular abnormalities is not clear. Here, we set out to study SSc-related immune parameters and determine whether and which peripheral T cell subsets associate with vascular severity in SSc patients.

Methods

Peripheral blood and clinical data were collected from 30 SSc patients, 5 patients with idiopathic PAH and 15 age and sex-matched healthy donors (HD). In this cross-sectional cohort, SSc patients with PAH (n = 15) were matched for their age, sex and medication with SSc patients with no signs of PAH (n = 15). Lymphocyte subsets were quantified by multi-colour flow cytometry.

Results

SSc patients exhibited elevated percentages of T peripheral helper cells (Tph), CD4+GZMB+ T cells and decreased levels of Th1 cells compared with HD. Increased presence of both CD4+ and CD8+ exhausted-like (CD28−) T cells, characterized by raised cytokine and cytotoxic signature, was also observed in SSc compared with HD blood. Furthermore, IL-4 expressing CD4+CD8+ T cells were significantly increased in SSc peripheral blood. Interestingly, the presence of PAH in SSc was accompanied by a distinct T helper profile, characterized by raised percentages of Th17 and Tph cells.

Conclusion

SSc patients with severe vasculopathy (presence of PAH) exhibited a distinct T cell profile, suggesting a potential role of auto-immune inflammation in SSc vascular complications.

systemic sclerosis
vasculopathy
pulmonary arterial hypertension
T lymphocytes
biomarkers
==== Body
pmcRheumatology key messages CD4+ and CD8+ exhausted-like T cells (CD28−) are elevated in SSc peripheral blood and exhibit increased cytokine and cytotoxic signature.

Presence of pulmonary arterial hypertension in SSc individuals is characterized by a distinct pro-inflammatory T helper phenotype: elevated Th17 and T peripheral helper cells.

Introduction

Systemic sclerosis (SSc) is a rare auto-immune connective tissue disease and has the highest morbidity and mortality of all rheumatic diseases [1, 2]. Hallmark characteristics of this disease are auto-immune inflammation, vasculopathy and fibrosis of skin and internal organs with a variable degree of severity. The vasculopathy is marked by gradual vascular impairment, affecting both small and large blood vessels. Peripheral vascular disease is universal in individuals diagnosed with SSc and prototypically occurs as the initial symptom of the disease, manifesting as a new or exaggerated Raynaud’s phenomenon with microscopic nailfold capillary malformation. In severe cases vasoconstriction results in digital ulcers, which can also develop later in the disease course. In a subset of patients, central vasculopathy may be responsible for cardiac dysfunction and pulmonary arterial hypertension (PAH) typically later in the disease course [3].

PAH is the most severe vascular disease in SSc and a devastating and progressive complication that is among the leading causes of death [4]. To date, there is no curative treatment available. Despite progress in the treatment of PAH, the quality of life is severely reduced and survival of SSc-PAH patients remains poor [5]. Moreover, vasoactive therapy is known to be less effective in SSc-PAH compared with the idiopathic form of PAH, whereas both share clinical and haemodynamic similarities [6, 7].

Current knowledge indicates that in connective-tissue diseases such as SSc, inflammatory processes contribute to vasculopathy. Recently we and others showed that cytotoxic T cells are present in perivascular infiltrates of affected skin in SSc and cause endothelial cell damage [8–10], especially early in the disease course. Other studies indicate that T cells may also mediate progression of PAH, especially by instigating changes in the pulmonary vasculature [11, 12]. The vascular remodelling that characterizes SSc-PAH consists of intimal thickening of pulmonary arterioles and capillaries, which is caused by intimal cell proliferation and deposition of extracellular matrix. Histological analyses of pulmonary tissue from SSc-PAH patients have shown inflammatory cell infiltrates, such as of T and B lymphocytes and macrophages, in peri-vascular cell areas, and to a lesser extent in plexiform lesions [13–15]. Possibly, these immune cells contribute to the (abnormal) vascular remodelling process and dysfunction. However, to date, the potential involvement of immune cells in manifestations of severe vascular dysfunction in SSc is poorly understood.

Various studies [16–19] have reported on the aberrations of different T cell cytotoxic (CTL) and T helper (Th) subsets that are linked with inflammatory and/or fibrotic manifestations of patients with SSc. However, the SSc-associated immune cell subsets vary between disease components and between studies. Here we hypothesize that the relative contribution of distinct T cell subsets may differ according to SSc-associated vascular severity. To address that question, we performed in depth T lymphocyte immunophenotyping in a cross-sectional cohort [20] focused on the inclusion of SSc patients with or without the presence of PAH that were carefully matched for their age, sex, ethnicity and medication use.

Methods

Here, we set out to examine if and which peripheral T cell populations may associate with distinct vascular severity in patients with SSc. To answer this question, we utilized a multi-colour 17-antigen flow cytometry panel (the detected T cell populations along with the extracellular and intracellular antigens that were used for their identification are presented in Supplementary Tables S1–S3, available at Rheumatology online) to explore the relative contribution of multiple peripheral blood T cell subsets in distinct clinical manifestations of SSc. For a complete overview of the patient and healthy control characteristics and the experimental methods, see Supplementary Data S1, available at Rheumatology online. Ethics approval for the study was granted in accordance with the Dutch Code of Conduct for Health Research, the Dutch Code of Conduct for Responsible Use, the Dutch Personal Data Protection Act, and the Medical Treatment Agreement Act. The study, conducted at Radboudumc in Nijmegen, The Netherlands, was assigned File Number CMO: 2017–3979. Written informed consent was obtained from all included participants.

Results

Clinical and demographic features

Patients and healthy donors (HD) included in our study were of similar age, sex and race distribution (P-values not statistically significant). About 80% of the SSc patients and controls were female with an age range from 57 to 76 years and disease duration from a minimum of 2.4 to a maximum of 19.9 years. Patients with idiopathic pulmonary arterial hypertension (IPAH) were all Caucasian females of similar age range. A complete overview of the baseline patient characteristics including demographics, laboratory values, clinical features, autoantibody and medication status is provided in Table 1 and Supplementary Table S4, available at Rheumatology online. SSc patients were clinically classified into either limited (80%) or diffuse cutaneous (20%) SSc according to the subclassification criteria of Leroy [21]. All patients experienced symptoms of Raynaud’s phenomenon, 40% of the patients had interstitial lung disease on high resolution CT-scan despite preserved vital capacity (mean 92.5%, see Supplementary Table S4) and 50% of the patients were diagnosed with SSc-associated PAH (Supplementary Table S5, available at Rheumatology online). SSc patients without PAH enrolled in this cross-sectional cohort were matched for their age, sex, ethnic background and medication with SSc patients with PAH and HD.

Table 1. Clinical characteristics of participants included in the cross-sectional cohort study

Baseline characteristics	SSc	IPAH	HD	P-value	
(n = 30)	(n = 5)	(n = 15)	
Age, mean (s.d.), years	67.6 (9.6)	63.0 (9.4)	63.2 (6.2)	0.46	
Female, n (%)	25 (83)	5 (100)	12 (80)	0.95	
Caucasian, n (%)	30 (100)	5 (100)	15 (100)	1.00	
SSc duration, mean (s.d.), years	10.1 (8.1)				
lcSSc, n (%)	24 (80)				
Clinical SSc features					
 PAH, n (%)	15 (50)	5 (100)			
 ILD, n (%)	12 (40)	0			
 Raynaud's phenomenon, n (%)	29 (97)	0			
 Digital ulcers, n (%)	11/29 (38)	0			
 Telangiectasias, n (%)	12/24 (50)	0			
 mRSS, mean (s.d.)	4.1 (5)				
 NYHA class, n (%)					
  I	13 (43)	0			
  II	6 (20)	5 (100)			
  III	8 (27)	0			
  IV	3 (10)	0			
Laboratory results					
 MDRD/GFR, mean (s.d.), ml/min/1.73m2	63.4 (16.2)	72.2 (14.4)			
 Creatinine, mean (s.d.), µmol/l	86.6 (23.4)	78.2 (23.5)			
 Urate, mean (s.d.), µmol/l	0.35 (0.12)	0.36 (0.13)			
 Nt-proBNP, mean (s.d.), pg/ml	794 (1253)	96.6 (42)			
 ANA positive, n (%)	26 (87)	2 (40)			
 ENA positive, n (%)	19/24 (79)	0			
Statistics were performed with ordinary one-way ANOVA with Tukey’s multiple comparisons test. ANA: Antinuclear Antibody; ENA: extractable nuclear antigen; GFR: glomerular filtration rate; HD: healthy donors; ILD: interstitial lung disease; lcSSc: limited cutaneous SSc; IPAH: idiopathic pulmonary arterial hypertension; MDRD: modification of diet in renal disease; mRSS: modified Rodnan skin score; Nt-proBNP: aminoterminal pro B-type natriuretic peptide; NYHA: New York Heart Association; PAH: pulmonary arterial hypertension.

Deep flow cytometric quantification reveals differential presence of T cell subsets in blood of SSc patients compared with healthy donors

To determine potential differences between SSc and healthy peripheral blood T cell composition, we used multi-colour flow cytometry. First, we analysed general T cell characteristics between patients and healthy donors, starting with T cell numbers (for the gating strategy see Supplementary Fig. S1, available at Rheumatology online). No differences were observed in overall and CD4+ T cell or CD8+ T cell subset percentages between SSc patients and HD. Furthermore, there was no significant effect of immunosuppressive or other medication in the percentage of these T cell populations. Of note, SSc patients exhibited an elevated expression of the CD4+CD8+ T cell population in their CD3+ compartment (3.1% SSc vs 1.3% HCs, P = 0.0021) (Fig. 1A). Next, we explored the composition of the T helper sub-groups in SSc patients vs healthy donors (Fig. 1B, Supplementary Fig. S2A–C, available at Rheumatology online). No differences were observed in the presence of Th2, Th9, Th17 and Th1–Th17 subsets in patients vs HD. However, the Th1 (CD4+CCR6−CXCR3+) compartment was significantly decreased in SSc patients compared with controls (SSc: 2.1% vs HD: 3.0% of live cells, P = 0.038). This is in line with previous reports describing a reduced ratio of Th1 vs Th2 cells [22, 23]. Hereafter we analysed the presence of a T helper subset important for B cell function, the peripheral helper cells (Tph). These cells are known for regulating B cell class switching, maturation and antibody production [24]. Tph cells, characterized as CD4+PD-1highICOShigh T cells that produce the T follicular/peripheral helper signature cytokine IL-21, were detected in both healthy and SSc blood, but the percentage of Tph in patients was twice as high as in healthy donors (SSc: 1.6% vs HD: 0.8% of live cells, P = 0.013) (Fig. 1B).

Figure 1. SSc patients are characterized by enriched presence of CD4+CD8+ T cells and Tph cells and decreased percentage of Th1 cells compared with age and sex matched HD. Percentages of circulating T cell subsets were detected with multi colour flow cytometry in the peripheral blood of SSc patients (n = 30) compared with HD (n = 15). (A) No differences in percentages of CD3+, CD4+ or CD8+ T cells between SSc and HD were observed. However an elevated percentage of CD4+CD8+ T cells was evident in SSc blood. Values are presented as percentage of total live cells. (B) Quantification of Th1, Th2, Th9, Th17 and Th1-17 cell frequencies. Percentage of Th1 cells is greatly reduced in SSc patients while percentage of Tph cells is elevated in SSc blood. Values are presented as percentage of total live cells. (C) No significant differences in percentages of naïve (CD8+CD45RA+CD27+), effector (CD8+CD45RA+CD27−) and central memory (CD8+CD45RA−CD27+) cytotoxic T cell populations were observed in SSc versus HD. Values are presented as percentage of total live CD8+ T cells. (D) Comparison of percentage of CD8+GZMB+ T cells (left) and CD4+GZMB+ T cells (right) between SSc patients and healthy donors. Values are presented as percentage of total live cells. (E) Comparison of percentages of CD4+IL-4+, CD4+IL-13+, CD8+IL-4+ and CD8+IL-13+ T cells between HD and SSc exhibits a prominent cytotoxic T cell pro-fibrotic signature in SSc blood. Values are presented as percentage of total live cells. (F) A significantly higher fraction of CD4+CD8+ T cells from SSc patients are also positive for IL-4 compared with the same cell type in HD. Statistical comparisons between groups were performed either with Student’s t-test for normally distributed data or with the Mann–Whitney U-test for non-normally distributed data. In all cases values were corrected for multiple comparisons. *P < 0.05, **P < 0.01. GZMB: granzyme B; HD: healthy donors; Tph: peripheral helper T cells

Following this, we explored CD8+ T cell function deeper by characterizing their maturation status. We observed a trend towards decreased CD8 naïve (CD8+CD45RA+CD27+) and increased effector (CD8+CD45RA+CD27−) and central memory (CD8+CD45RA−CD27+) T cells in SSc, however, none of these comparisons were statistically significant (Fig. 1C, Supplementary Fig. S3A, available at Rheumatology online).

SSc individuals exhibit an elevated pro-fibrotic and cytotoxic T cell phenotype

Since we and others previously showed a prevalent cytotoxic immune cell response in the affected skin of patients with early SSc [8–10], we next examined whether such a cytotoxic signature is also evident in patients’ blood circulation. There was no significant difference in percentage of CD8+ granzyme B (GZMB)+ T cells in circulation between SSc and HD (Fig. 2A). Considering that recent literature suggests a potential role of CD4+ cytotoxic T cells in promoting vascular dysfunction in skin of patients with early and diffuse SSc [8], we explored the presence of this subset in peripheral blood. Indeed, cytotoxic CD4+GZMB+ T cells (Supplementary Figs S3D and S4, available at Rheumatology online) could be readily detected in SSc patients, but were hardly distinguishable in HD (SSc: 1.68 vs HD: 0.54% of live cells, P = 0.05) (Fig. 2A).

Figure 2. SSc patients exhibit a prevalent pro-fibrotic and cytotoxic T cell phenotype. Percentages of circulating T cell subsets were detected with multi colour flow cytometry in the peripheral blood of SSc patients (n = 30) compared with HD (n = 15). Expression of GZMB, IL-4 and IL-13 was measured with intracellular staining after cells were stimulated with PMA, ionomycin and BFA for 4 hours at 37°C. (A) Comparison of percentage of CD8+GZMB+ T cells (left) and CD4+GZMB+ T cells (right) between SSc patients and healthy donors. Values are presented as percentage of total live cells. (B, C) Comparison of percentages of CD4+IL-4+, CD4+IL-13+, CD8+IL-4+ and CD8+IL-13+ T cells between HD and SSc exhibits a prominent cytotoxic T cell pro-fibrotic signature in SSc blood. Values are presented as percentage of total live cells. (D) A significantly higher fraction of CD4+CD8+ T cells from SSc patients are also positive for IL-4 compared with the same cell type in HD. (E) Classification of SSc and HD peripheral blood CD3+ T cells based on hierarchical clustering of the protein markers CD3, CD4, CD8, CCR4, CD28, CCR6, CXCR3, CD45RA, CD27, PD-1, ICOS, IL-4, IL-13, IL-21 and GZMB was used to detect cell populations of comparable marker expression and their abundance utilizing the CITRUS tool provided by Cytobank. Five thousand events were sampled per individual donor. The size of each node depicts the cell frequency. Prediction analysis for microarrays (PAM) clustering was performed to detect cell clusters contributing to SSc patients (red clusters) compared with HD (blue clusters). Citrus hierarchical ‘clustering trees’ exhibiting the relationships of the detected nodes are also shown for selected markers and the colour scales illustrates intensity of expression of each depicted marker per cluster and the size of each node shows the event frequency. Statistical comparisons between groups were performed either with Student’s t-test for normally distributed data or with the Mann–Whitney U-test for non-normally distributed data. In all cases values were corrected for multiple comparisons. *P < 0.05, **P < 0.01. BFA: Brefeldin A; GZMB: granzyme B; HD: healthy donors; PMA: phorbol 12-myristate 13-acetate

Furthermore, accumulating evidence supports the pro-fibrotic role of the cytokines IL-4 and IL-13 in connective tissue diseases [25]. As expected, expression of both IL-4 and IL-13 was significantly up-regulated in the CD4+ compartment of patients with SSc, revealing a prominent pro-fibrotic response (SSc: 6.63% CD4+IL-4+, 1.50% CD4+IL-13+vs HD: 1.94% CD4+IL-4+, 0.85% CD4+IL-13+ of live cells, P = 0.002 and 0.007, respectively) (Fig. 2B, Supplementary Fig. S5, available at Rheumatology online). Interestingly, CD8+ T cells from SSc patients also exhibited an elevated expression of pro-fibrotic cytokines such as IL-4 and IL-13, suggesting their potential involvement in disease-related manifestations such as fibrosis (Fig. 2C, Supplementary Fig. S5). In addition, we previously observed an increased presence of CD4+CD8+ T cells in SSc blood. The functional and phenotypical properties of these double positive cells are still poorly understood. Of note and in accordance with earlier studies in SSc skin [26], this double positive population expressed significantly higher levels of the pro-fibrotic cytokine IL-4 in SSc compared with HD (18.7% SSc vs 12.9% HCs, P = 0.033) (Fig. 2D).

To further investigate phenotypic features of the different T cell populations, we utilized the CITRUS tool on the Cytobank platform [27], employing unsupervised clustering based on similar descriptive features such as marker expression, correlating with assigned sample types (SSc vs HD and IPAH). The top four clusters with the highest stratification for predicting SSc disease (Fig. 2E, Supplementary Fig. S6, available at Rheumatology online) were characterized by elevated expression of CD8, PD-1, IL-4, IL-21 and GZMB (Fig. 2E, Supplementary Fig. S7, available at Rheumatology online). Collectively and in line with our supervised analysis, these results illustrate that SSc circulating T cells exhibit an increased pro-fibrotic (IL-4), cytotoxic (GZMB) and peripheral helper (IL-21) signature.

SSc blood is characterized by an increased exhausted-like T cell signature that is accompanied by an elevated cytokine and cytotoxic phenotype

Often, in patients with cancer and chronic inflammation, the early elevated cytotoxic T cell responses are attributed to exposure to certain (auto)-antigens and the inflammatory microenvironment. Antigen-specific T cell activation is a critical regulator of their effector functions. A key determinant of T cell activity is exhaustion [28]. Long-term exposure to persistent antigen stimulation is usually associated with T cell anergy/exhaustion. T cell exhaustion is observed in many rheumatic diseases including rheumatoid arthritis and is often correlated with disease manifestations [29]. Therefore, we next looked at T cell exhaustion in our dataset. Exhausted/anergic T cells are characterized by loss of the co-stimulatory molecule CD28. In our cohort, the percentage of CD8+CD28− T cells (Fig. 3A, Supplementary Fig. S3B) was expanded in SSc patients compared with HD (7.92% in SSc vs 4.88% in HD, percentage of total live cells, P = 0.06). It has been shown that in rheumatoid arthritis, T cell exhaustion leads to atypical cytotoxic properties [30]. Indeed, in SSc blood, CD8+CD28− T cells showed on average a 6-fold higher GZMB expression compared with their CD28+ counterpart (Fig. 3B). In line with this observation, our unsupervised analysis after dimensionality reduction and cell clustering with the CITRUS algorithm, we found that among the distinct CD8+ T cell clusters there was a clear distinction between clusters that were either GZMB+CD28− or GZMB−CD28+ (Supplementary Fig. S8, available at Rheumatology online). In addition, in SSc CD8+CD28− T cells, an elevated percentage of GZMB, IL-4 and IL-21 positive cells was detected compared with HD (Fig. 3C). This observation points to a potential role of exhausted T cells in facilitating SSc disease processes related to cytotoxicity, fibrosis and auto-antibody production.

Figure 3. Increased presence of exhausted T cells in early SSc may facilitate key disease manifestations, such as cytotoxicity mediated tissue destruction, fibrosis and auto-antibody production. (A) CD8+CD28− T cells are expanded in SSc patients compared with HD. Values are presented as percentage of total live PBMCs. Representative flow cytometry plots of one HD and one SSc patient to identify CD8+CD28− T cells are illustrated. (B) Exhausted-like CD8+CD28− T cells show increased GZMB positivity in comparison to their effector CD8+CD28+ counterpart. Values are expressed as percentage of cells that are GZMB+ within CD8+CD28− and CD8+CD28+ T cell populations. (C) CD8+CD28− T cells from SSc patients exhibit significantly more GZMB+, IL-21+ and IL-4+ cells compared with the same cell type in HD. Values are presented as percentage of GZMB+/IL-21+/IL-4+ cells among the total CD8+CD28− T cells. (D) SSc patients are characterized by an elevated percentage of CD4+CD28− T cells compared with HD. Values are presented as percentage of total live PBMCs. Right: representative flow cytometry plots of one HD and one SSc patient to identify CD4+CD28− T cells are also depicted. (E) Exhausted-like CD4+CD28− T cells show increased GZMB positivity in comparison to their effector CD4+CD28+ counterpart. Values are expressed as percentage of cells that are GZMB+ within CD4+CD28− and CD4+CD28+ T cell populations. (F) CD4+CD28− T cells from SSc patients exhibit significantly more GZMB+, IL-21+ and IL-4+ cells compared with the same cell type in HD. Values are presented as percentage of GZMB+/IL-21+/IL-4+ cells among the total CD4+CD28− T cells. Statistical comparisons between groups were performed either with Student’s t-test for normally distributed data or with Mann–Whitney U-test for non-normally distributed data. In all cases values were corrected for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. GZMB: granzyme B; HD: healthy donors; PBMCs: peripheral blood mononuclear cells

Furthermore, in patients with SSc increased percentage of exhausted-like CD4+ T cells (CD4+CD28−) compared with HD was also observed (6.03% in SSc vs 4.38% in HD, percentage of total live cells, P = 0.032) (Fig. 3D). Similarly to the anergic cytotoxic T cells, in CD4+CD28− T cells an elevated GZMB expression was observed in comparison to CD4+CD28+ T cells (Fig. 3E). In addition, more CD4+CD28− T cells of SSc patients were positive for GZMB, IL-21 and IL-4 than those of healthy donors (Fig. 3F). In conclusion, both CD4+ and CD8+ T cells were expanded in SSc blood and exhibited an increased cytotoxic and cytokine expressing profile compared with HD.

Presence of PAH in SSc individuals is associated with a distinct T helper profile: increased frequency of Th17 and Tph cells

Since several T cell subsets were found to be expanded in SSc blood, we further combined these data with clinical features of the included patients to understand how such immunological differences may be related to distinct SSc clinical patterns (such as PAH) and disease severity. First, subgroup analysis showed that SSc individuals with active disease (patients that exhibited elevated modified Rodnan skin score or developed a new organ complication that was attributed to SSc over a 6-month period before inclusion in our study) exhibited elevated numbers of CD4+ T cells (Fig. 4A), suggesting that T helper responses may be associated with disease severity. Furthermore, in our patient cohort half of the SSc patients were diagnosed with PAH (SSc-PAH), while the rest were not (SSc-noPAH). These two patients groups were carefully matched for their sex, age and underlying medication and further compared with age and sex matched healthy controls (n = 15) and also with patients with IPAH (n = 5). We aimed to detect T cell subsets that were specifically related to patients with or without PAH, which could serve as candidate biomarkers. Strikingly, percentages of Th17 (CD4+CCR6+CXCR3−CCR4+) and Tph (PD-1+ICOS+IL-21+) cells were specifically up-regulated in SSc-PAH patients (Fig. 4B). This reveals a distinct pro-inflammatory/peripheral T helper phenotype associated with the presence of PAH in SSc patients. Of note, no differences in CD8+ T cell subsets were found. Patients with IPAH, did not show elevated percentages of Th17 nor Tph cells. On the other hand, Th2 cells characterized as CD4+CCR6−CXCR3−CCR4+ were significantly expanded in patients with IPAH, while the rest comparing groups shared similar percentages (Supplementary Fig. S9, available at Rheumatology online). In conclusion, CD4 T cell inflammatory responses (Th17, Tph) are evident in SSc patients with prevalent vascular disease (PAH), suggesting a potential role of autoimmune inflammation in SSc-associated vascular dysfunction.

Figure 4. Presence of PAH in SSc is accompanied by a distinct T helper profile. (A) Comparison of percentages (%) of CD4+ T cells in patients with active versus not active disease shows elevated percentages of CD4+ T helper cells in patients with progressive disease. Patients with active disease exhibited elevated modified Rodnan skin score or developed a new organ complication that was attributed to SSc over a 6-month period before inclusion in our study. (B) Comparison of percentages of T cell subsets in SSc patients with and without the presence of PAH shows elevated percentages of Th17 and Tph cells in patients with SSc-associated PAH. Statistical comparisons between groups were performed with two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. HD: healthy donors; IPAH: idiopathic pulmonary arterial hypertension; PAH: pulmonary arterial hypertension; Tph: peripheral helper T cells

Discussion

Immune dysregulation seems to play an important role in SSc pathogenesis. Recent observations suggest a prevalent T cell cytotoxic signature in the affected skin of patients with early diffuse SSc [8, 10]. However, the potential involvement of T cell abnormalities in relation to prolonged SSc disease duration, severity and vascular complications such as PAH remains elusive. In the present study, we performed an in-depth flow cytometric analysis in SSc peripheral blood to explore the potential involvement of specific T cell subpopulations in SSc pathology and its distinct clinical manifestations such as PAH.

In accordance with previous studies [31, 32], we confirmed expansion of IL-4/IL-13 expressing CD4+ and CD8+ T cell populations in SSc blood circulation. For the first time, in SSc peripheral blood, we detected a CD4 and CD8 double positive population that may contribute to SSc pathology by producing excessive amounts of pro-fibrotic cytokines such as IL-4. Such a population has been previously described as exhibiting a very high IL-4 production in SSc lesional skin [26]. Strikingly, a CD4+GZMB+ cytotoxic T cell population was almost exclusively present in SSc individuals but was hardly detectable in healthy blood.

T cell exhaustion was prevalent in SSc blood for both T helper and cytotoxic cells. T cell dysfunction is increased with ageing, cancer and chronic viral infections, and the function of these cells can range from effector to immunosuppressive [33]. In immune mediated inflammatory diseases such as systemic erythematosus lupus, type I diabetes mellitus and ANCA-associated vasculitis, their presence is often associated with improved disease prognosis [34–36]. Our analysis showed that exhausted T cells in SSc blood had elevated cytotoxic and cytokine producing phenotype compared with those present in healthy blood. This suggests that SSc blood contains ‘dysfunctional’ T cells that are functionally active probably due to chronic auto-antigen presentation. Such a phenotype has been described before for the exhausted CD4+ T cell compartment in SSc blood [37]. Of note, in our study we also observed elevated percentages of cytotoxic (CD8+) exhausted-like T cells in patients with SSc. Our data suggest that exhausted CD8+ T cells are functionally adapted and may potentially exacerbate tissue destruction rather than being anergic. However, a closer examination of their functional role and their potential association with better or worse disease progression in further longitudinal analyses is needed.

Currently, PAH is one of the most severe complications of SSc that usually develops in later disease stages, and although treatment options have proven to improve quality of life and survival, it remains one of the main causes of death in SSc. The pathogenesis of SSc-PAH is still poorly understood and the intricate pathological characteristics of SSc make it challenging to address the role of the immune and vascular systems in the development of SSc-PAH. Here, we identified a prominent T helper phenotype in SSc PAH patients that is addressed by elevated numbers of circulating Th17 and Tph cells and thus suggests a potential role of T helper autoimmune inflammation in SSc vascular complications. Expansion of Th17 cells in SSc blood has been previously described [22, 38, 39]; however, we are the first to report increased presence of these cells in SSc-PAH. Apart from their central role in SSc-related fibrosis [40], IL-17 producing cells were shown to be involved in SSc vasculopathy through the recruitment of inflammatory cells to vascular endothelial cells leading to small vessel vasculopathy and fibrosis due to a faulty repair response. Possibly a similar mechanism occurs in the pulmonary arteries leading to its thickening and decreased lumen [18, 41].

We also observed increased T peripheral helper cells in SSc-PAH. This T cell subtype lacks CXCR5 expression but has a transcriptionally similar phenotype with T follicular helper cells that produce IL-21 and play an essential role in stimulating B cell differentiation to immunoglobulin secreting plasma cells. Interestingly, we recently identified IL-21 as one of the key cytokines defining SSc subtypes in a cohort of 346 patients [42]. Importantly, in previous studies, (auto)- antibodies have been linked to risk of the development of PAH and this could be partially explained by the observed elevated Tph numbers in these patients [43–45]. Increased numbers of circulating T follicular helper cells that promote plasmablast differentiation in an IL-21-dependent manner have also been reported in SSc-PAH patients [46]. Taken together, these findings support a role of the immune system in SSc-PAH development. This is in line with current views on PAH pathophysiology [47].

This study has several strengths and limitations. First, most of the included patients received medication, including immunomodulatory and PAH modifying drugs. Such medication may affect T cell populations and skew the data. To address this point, we compared SSc patients with or without the presence of PAH that were matched for their medication use. Second, immune ageing and sex ratio is often an important confounding factor in immunological studies. To account for this, we took special care to age and sex match the healthy volunteers included in our study with the enrolled SSc patients. Importantly, in this study we used cryopreserved peripheral blood mononuclear cells of the enrolled participants, and that enabled us to avoid potential batch effects since all samples were processed, stained and acquired for flow cytometry on the same day. Finally, the lack of in vitro functional assays due to limited amount of cells from each donor is hampering a more in-depth investigation of the mechanisms behind the pathogenic role of Th17 and Tph expansion in SSc-PAH. In this regard, examining the presence and function of these cells in the affected tissue would be essential in determining their potential pathogenicity.

To conclude, our results shed light on SSc immune pathogenesis by determining several T cell aberrations in the peripheral blood of SSc patients compared with carefully age and sex matched healthy individuals. Of note, patients with PAH exhibited a distinct expansion of the pro-inflammatory Th17- and auto-antibody-related Tph subsets, highlighting a potential role of auto-immune inflammation in SSc vascular complications. Our findings suggest the potential use of therapeutic approaches that target inflammation in the treatment course of PAH in SSc.

Supplementary Material

keae190_Supplementary_Data

Acknowledgements

We want to thank Bram van Cranenbroek and the Radboud Technology Centre Flow Cytometry facility for their technical support in flow cytometry experiments.

Supplementary material

Supplementary material is available at Rheumatology online.

Data availability

Data are available upon request.

Author contributions

T. I. Papadimitriou (Conceptualization, Formal Analysis, Investigation, Writing-Original Draft, Writing—Review & Editing, Visualization), J. Lemmers (Conceptualization, Patient Inclusion and clinical data collection, Writing—Review), A. van Caam (Conceptualization, Formal Analysis, Investigation, Writing—Review & Editing, Supervision), J. Vos (Patient Inclusion, Writing—Review), E. Vitters (Technical support), L. Stinissen (Technical support), M. Koenders (Writing—Review, Supervision), P. van der Kraan (Writing—Review, Project administration), H. Koenen (Supervision), R. Smeets (Supervision), R. Nijveldt (Supervision, Writing—Review), M. C. Vonk (Conceptualization, Supervision, Writing—Review, Project Administration) and R. Thurlings (Conceptualization, Supervision, Writing—Review, Project Administration).

Funding

No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.

Disclosure statement: The authors have declared no conflicts of interest.
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