Regulatory T cells: a new therapeutic link for Sjögren syndrome?

Abstract

Great advancements have been made in understanding the pathogenesis of SS, but there remain unmet needs for effective and targeted treatments. Glandular and extraglandular dysfunction in SS is associated with autoimmune lymphocytic infiltration that invades the epithelial structures of affected organs. Regulatory T (Treg) cells are a subset of CD4⁺ T lymphocytes that maintain self-tolerance during physiological conditions. Besides inhibiting excessive inflammation and autoimmune response by targeting various immune cell subsets and tissues, Treg cells have also been shown to promote tissue repair and regeneration in pathogenic milieus. The changes of quantity and function of Treg cells in various autoimmune and chronic inflammatory disorders have been reported, owing to their effects on immune regulation. Here we summarize the recent findings from murine models and clinical data about the dysfunction of Treg cells in SS pathogenesis and discuss the therapeutic strategies of direct or indirect targeting of Treg cells in SS. Understanding the current knowledge of Treg cells in the development of SS will be important to elucidate disease pathogenesis and may guide research for successful therapeutic intervention in this disease.

Introduction

SS is an autoimmune disease with exocrine gland dysfunction and at least one-third of patients experience multiorgan involvement [1, 2]. Furthermore, nearly 5% of patients may develop B lymphoma, which represents the most severe complication of the disease [3]. It includes primary SS, which occurs alone and secondary SS, which occurs following other autoimmune diseases. Understanding the mechanisms of autoimmune disease, the nature of self-tolerance and the breakdown of self-tolerance have advanced. However, effective and targeted treatments for SS are still lacking [1].

The characteristic histological feature of SS is periductal and perivascular loci of lymphocytic infiltrates in exocrine glands and other tissues, which leads to autoimmune injuries. It has been previously reported that extensive salivary gland lymphocyte infiltration is mainly represented by T cells, predominantly CD4⁺T cells, but also CD8⁺T cells [4], and that T cells predominate in mild lesions and B cells are the most represented cell subsets in the advanced lesions [5, 6]. Based on the immune function, CD4⁺T cells are sorted as regulatory T (Treg) cells and T helper (Th) cells, and the latter has been known to be distributed into Th1, Th2, Th17 and follicular helper T (Tfh) [6–8]. Th cell lineages were identified as a prime representative of the generation of proinflammatory cytokines and could also interact with B cells to produce autoantibodies upon activation [9]. Accordingly, Treg cells were confirmed as a unique population that could restrain excessive activation of effector lymphocytes [10].

Treg cells target many different immune cell subsets and tissues to suppress excessive inflammation and to support tissue repair and homeostasis. Recently, several studies about Treg cells have been carried out in experimental models and patients with SS. This review provides an overview of Treg cells in SS, which summarizes experimental and clinical data on the dysregulation of Treg cells and the current advances in the therapeutic control of Treg cells or its associated signalling to restore Treg homeostasis in SS. Articles concerning the possible role of Treg cells in SS were discussed in detail. Understanding the alterations of Treg cells and their underlying biological process in SS may help to develop successful therapeutic targets for patients.

Biology of Treg cells

Treg cells were initially identified in mice and humans according to the high surface expression of the alpha chain of IL-2 receptor (IL-2Rα, CD25) [11]. The most reliable cell-specific marker of Treg cells is forkhead box P3 (Foxp3), an important transcription factor, which is essential for the maturation and suppressive function of Treg cells [12, 13]. These cell lineages normally present in lymphoid organs. In abnormal conditions, they are recruited to the damaged sites [10, 14]. Treg cells are a suppressive T-cell subset that is required for the maintenance of self-tolerance and immune homeostasis, preventing excessive inflammation and autoimmune diseases [15]. This is best exemplified by the fact that experimental deficiency of Treg cells in mice and carrying non-functional or hypomorphic alleles of the FOXP3 gene in humans triggered severe inflammation and multi-organ autoimmune diseases [15–17]. Foxp3-deficient scurfy mice developed signs of autoimmune inflammation and eventually died at 3–4 weeks of age [18]. Risk variants in the genes encoding IL-2, the IL-2R subunits IL-2Rα or IL-2Rβ, and the downstream signalling factors STAT5A, STAT5B and PTPN2 were also associated with inflammation and autoimmunity linked to Treg cell deficiency [11, 19, 20]. In addition to being well-known suppressors of the excessive immune response, Treg cells have been recently recognized as direct and indirect regulators of tissue healing and regeneration [21, 22].

Treg cells can be broadly divided into tTregs (generated in the thymus, also known as natural Treg cells) and pTregs (generated in the periphery, also known as induced Treg cells) [23]. This cellular subset exerts its suppressive function by cell-to-cell contact, as it has been reported that the in vivo imaging showed clusters of Treg cells together with activated autoreactive T cells in secondary lymphoid tissues [24, 25]. Treg cells also suppress immune responses through various mechanisms, including the secretion of inhibitory cytokines such as IL-10, transforming growth factor β (TGF-β) and IL-35, cytolysis (granzyme B/A, perforin) and modulation of the activation state and function of antigen-presenting cells via cytotoxic T lymphocyte antigen-4 and programmed cell death protein-1, as well as metabolic disruption via CD25, CD39 and CD73 expressing on Treg cells [26, 27]. Additionally, IL-2 deprivation as its binding to the IL-2 receptor on Treg cells leading to the impaired proliferation and differentiation of effector T cells is another manner to induce immune suppression. Treg cells are generated to suppress undesired and excessive immune responses, but under certain circumstances, their alterations can be also involved in the initiation and promotion of harmful immune responses, such as allergy, Mikulicz disease and SSc [27–29], as the plasticity of Treg cells depending on the different tissue microenvironments (Fig. 1).

The change of Treg cells in SS

Treg cells are key mediators of peripheral immune tolerance and are implicated in many autoimmune diseases, either because of their abnormal number or altered function. Until now, various studies have concerned the change of Treg cells in the development of SS (Table 1).

Several previous studies reported an overall reduction of circulating CD4⁺CD25^high Treg cells in pSS patients than that in healthy controls [30–33]. The accurately defined CD4⁺CD25⁺Foxp3⁺ Treg cells also showed a lower number in the peripheral blood of pSS patients, and this decreased Treg cell number was comparable between the patients with and without interstitial lung disease [34, 35]. It has also been reported that CD4⁺CD25⁺ Treg cell number and Foxp3 expression were markedly reduced in salivary gland biopsy samples of patients with SS [31], and that deficiency of follicular regulatory T (Tfr) cells, an emerging Treg cell subset, enhanced SS development [36].

Conversely, other studies showed increased CD4⁺CD25^high regulatory cells in the peripheral blood of patients with established pSS [37], and the percentages of CD25^highFoxP3⁺ Treg cells further increased in IFN-positive pSS patients [38]. The level of circulating CD4⁺Helios⁺FoxP3⁺ cells was also elevated in pSS patients compared with controls [39]. Alunno et al. reported an expansion of circulating CD4⁺CD25^lowGITR⁺ cells, a new regulatory CD4⁺ T cell subset, in pSS [40, 41]. Foxp3⁺ cells were also enriched in the lip minor salivary gland (MSG) lesions of SS patients [42, 43]. In detail, infiltrating Foxp3⁺Treg cells in MSGs predominated in intermediate lesions, and it was comparable between mild and severe lesions [4]. Another study found that CD25⁺Foxp3⁺Treg cells were increased in MSG biopsies of both pSS and secondary SS patients, and that pSS patients showed an increase of Treg cells especially at the perivascular and periductal location when compared with secondary SS [44].

In addition, similar proportion and/or absolute count of peripheral blood Treg cells were observed between pSS patients and healthy controls [43, 45–47]. We previously found that the percentage of periphery blood Treg cells (defined by CD4⁺Foxp3⁺ or CD4⁺CD25⁺CD127⁻) and the expression of FOXP3 in the MSGs of pSS patients were comparable to those in healthy subjects [48]. A recent cross-sectional study reported that the frequency of CD4⁺CD25⁺CD127⁻ Treg cells in SS was not different from healthy individuals [49].

Taken together, these data suggest that the change of Treg cells and its association with SS are often controversial. Such discrepancies may be attributed to the different disease duration employed to assess Treg cells, as different disease stages show various ESSDAI disease activity and heterogeneity of the clinical feature with/without multiorgan involvement and lymphoma. Subsequently, the definition of Treg cells changes over time with inconsistent markers. Finally, the in vitro functional assays about the suppressive activity of Treg cells may not ascertain the consistent function in vivo, as the plasticity of Treg cells is usually affected by the local inflammatory microenvironment. Thus, further studies with conclusive data are needed to reflect the dynamic change of Treg cells in SS.

Treg cells in the pathogenesis of SS

Quantitative and functional defects of Treg cells have been documented in various rheumatic diseases, including RA, SLE, SSc and others [11, 16, 19, 50, 51]. Recently, several studies have explored the potential role of Treg cells in SS pathogenesis.

The association of Treg cells with SS development has been investigated in various studies. In detail, Alunno et al. found that CD4⁺CD25^lowGITR⁺ regulatory T-cell subset expansion was more relevant in SS patients with inactive rather than active disease [40, 41]. Liu et al. reported inverse correlations between the levels of CD4⁺CD25^bright T cells and the clinical parameters of pSS including erythrocyte sedimentation rate, C-reactive protein, IgG and rheumatoid factors [32]. Liu et al. found that anti-SSB negative patients presented with a higher level of Helios⁺Foxp3⁺ cells than anti-SSB positive patients and that elevated circulating CD4⁺Helios⁺Foxp3⁺ cell percentage was inversely correlated with ESR, IgG, IgM and ESSDAI in pSS patients [39]. These results suggest an anti-inflammatory and immunosuppressive role of Treg cells in the pathogenesis of SS.

Interestingly, it has also been reported that functional CD4⁺CD25^high regulatory cells are present and increased in the peripheral blood of patients with established pSS, despite ongoing autoimmunity [37]. Another study found that CD4⁺CD25⁺ Treg cell percentage showed a negative correlation with sialometry values in patients, and this Treg cell exhibited lower suppression activity in vitro functional assay [33]. In the salivary glands of pSS patients, the increased Foxp3⁺Treg cell number was positively correlated with the gland histopathologic Chisholm score [43]. A recent study showed a weakened immunosuppressive function of Treg cells with decreased STAT5 activation in pSS patients, especially in non-severe SS with a normal/high salivary flow [49]. Changes in the investigated subsets of Treg cells in pSS suggest a contribution of them to the development and progression of the disease. Moreover, Li et al. reported that the inhibitory function of CD4⁺CD25⁺ T cells in pSS was unchanged and that peripheral CD4⁺CD25^high T-cell numbers in pSS were not correlated with Schirmer's test, salivary flow rate, anti-SSA/SSB antibodies or immunoglobulin level [31]. Our previous work found that the upregulation of Th17 generation in pSS did not depend on the low dose IL-2-associated Treg function [48].

An imbalance between Th17 and Treg cells has been postulated as a significant contributor to the immunopathological lesion in SS, as Iizuka et al. reported that both RORγt-overexpressed CD4⁺T cells and reduced Treg cells contributed to the development of SS-like sialadenitis [52, 53]. Generally, IL-2 promotes Treg cell survival and function but inhibits Th17 cell differentiation via binding to IL-2R and subsequent phosphorylation and activation of STAT5, while IL-6 favours Th17 cell differentiation but suppresses Treg cell generation via phosphorylation and activation of STAT3 [49, 54]. Our previous work found that the IL-2 level was reduced in the plasma of pSS patients accompanied by reduced phosphorylated-STAT5 (p-STAT5) and enhanced p-STAT3 level in the MSGs and peripheral CD4⁺ T cells, and that in vitro IL-2 treatment-induced STAT5 competed with STAT3 binding in human Il17a locus, leading to decreased Th17 differentiation, while the FOXP3 expression was unchanged after IL-2 stimulation [48], as it has been reported in a murine model that IL-2-induced STAT5 activation limited Th17 cell differentiation and related autoimmunity in a Foxp3-independent fashion [55]. Other studies conducted in pSS patients found that low-dose recombinant IL-2 administration promoted an amplification of Treg compartment [34, 56]. These findings may suggest that low-dose IL-2 functions via direct suppression of Th17 cell differentiation and/or through Treg cells expansion to restore immune tolerance, which needs further confirmation.

Besides IL-2, various factors could affect Treg cells in pathogenic conditions. Gut microbiota of pSS patients, presented with less beneficial bacteria and a higher proportion of opportunistic pathogens, was associated with the inflammatory processes of pSS by increasing proinflammatory cytokine production and decreasing anti-inflammatory cytokine IL-10 release and peripheral FOXP3 mRNA expression [57]. Interleukin-35 promoted the proliferation and activation of Treg cells and suppressed the function of Th17 cells and other inflammatory cells during the pathogenesis of connective tissue diseases, including pSS [58]. Furthermore, indoleamine 2,3-dioxygenase (IDO), a master regulator of self-tolerance, is essential for Treg generation [27]. Blockade of IDO, in the presence of IL-6 induced conversion of Treg into Th17-like cells in rodent tumor-draining lymph node of mice [59]. It has been shown that increased IDO activity in conjunction with enhanced percentages of CD25^highFoxp3⁺ Treg cells was observed in IFN-positive pSS patients, and these parameters were significantly correlated with each another [38]. In addition, IL-33 receptor ST2 is preferentially expressed on colonic Treg cells, IL-33 signalling could enhance TGF-β1-mediated differentiation of Treg cells and promote Treg cell accumulation and maintenance in inflamed tissues to control intestinal chronic inflammation responses in mice [60]. The same could be said for mucosal immunity-associated tissues, the association of IL-33 signalling with Treg cells in the salivary glands of SS needs to be investigated.

Treg cells are well known to suppress undesired and excessive immune responses to facilitate inflammation resolution, as Treg depletion obviously correlates with the disease severity [61]. Based on new technology, a new understanding of the heterogeneity of Treg cell populations in the lymphoid and non-lymphoid tissue has evolved in the past decades. It has been confirmed that Treg subsets gained effector functions to enhance autoimmunity and inflammatory processes upon loss of Foxp3 expression in different settings, such as in EAE, SSc, infection and others [27, 29, 62–64].

Under certain circumstances, Treg cells have been shown to participate in the repair and regeneration of various organ systems, such as skeletal and heart muscle, skin, lung, bone marrow, and the central nervous system [21]. For instance, skin-resident Treg cells directly facilitated hair follicle stem cell (HFSC) activation and HF regeneration without affecting immune homeostasis, as glucocorticoid receptor and Foxp3 in Treg cells cooperated to induce TGF-β3-mediated Smad2/3 activation and subsequent proliferation in HFSCs [65]. CCN3 from Treg enhanced oligodendrocyte differentiation in mixed glial cultures and depletion of Treg cells impeded myelin regeneration in vivo [66]. In the gland microenvironment of SS, the role of Treg cells, which may present with heterogeneity during disease progress, still needs further investigation.

Potential implications of Treg cells in other rheumatic diseases

Restoration of the immune balance by manipulating Treg cells represents the basis of therapeutic perspectives to alleviate autoimmune responses. Considerable evidence has indicated that increasing CD4⁺CD25⁺Foxp3⁺ Treg cell number and/or function thus holds promise for developing immunotherapy to improve autoimmune and inflammatory diseases [67]. The associated therapeutic strategies include Treg-based cellular therapies or therapies which aim to restore the balance of Treg and effector Th cells such as low-dose IL-2 and other indirect therapeutic approaches.

The concept of using Treg cells as a cell-based therapeutic approach was first demonstrated in murine models. IL-2 knockout, IL-2Rα knockout and scurfy mice lacking Treg cells could develop severe inflammation and immune response in multiple organs [61]. Another two mouse models for Treg depletion, in which Treg was depleted by purified rat anti-mouse CD25 antibody in C57 mice or by diphtheria toxin (DT) in inserted diphtheria toxin receptor (DTR) Foxp3^DTR mice, were also usually described [68, 69]. Depletion of Treg cells in mice is considered a representative animal model of autoimmune disease, while transfer of Treg cells could ameliorate the disease [70]. Due to the low number of Treg cells during Treg cell transfer, manufacturing protocols were developed recently to expand Treg cells in vitro or ex vivo to produce a large number of polyclonal Treg cells. Some clinical trials have been designed to test the efficacy of ex vivo expanded polyclonal Treg cells on treating type 1 diabetes and active cutaneous lupus [71–73]. Furthermore, several studies using ex vivo or in vivo expanded antigen-specific Treg cells have shown improved potency and lower risk of pan-immunosuppression [74, 75]. Expansion of autologous or heterologous glucocorticoid-induced TNF receptor-related protein⁺ (GITR⁺) Treg cells in vitro by GITR-triggering drugs was recognized as a potential new approach for treating autoimmune diseases, as GITR is superior to other cell surface markers to differentiate Treg cells from other CD4⁺T cells [76]. Additionally, chimeric antigen receptor (CAR) Treg cells technology has been applied to induce immune suppression against soluble antigens, such as citrullinated vimentin and insulin, avoiding the cytolytic activity of CAR Treg cells by direct targeting the antigen-expressing cells, and membrane antigens, such as CD19 [77–79].

Besides the development of Treg cell-based therapies, various existing therapies have been described to affect Treg cell numbers and function, such as anti-CD3, CTLA-4Ig, anti-CD25 monoclonal antibody (basiliximab), rapamycin and tumor necrosis factor receptor 2 blockers [64, 80–83]. It has also been reported that HDAC inhibitors increased CTLA-4 expression and the suppressive function of Treg cells in RA [84]. Moreover, extracellular IL-33 exerted an important role in recruitment and function of Treg cells in different settings [60, 85].

Owing to the constitutive expression of high-affinity trimeric variants of IL-2R on Treg cells, low-dose IL-2 allowing a preferential activation and expansion of Treg cells was used as a treatment strategy for autoimmune diseases [11, 86]. Promising results have been observed in patients with type I diabetes and SLE [87, 88]. A study with 61 SLE patients demonstrated that complete remission could be achieved in 50% of patients with lupus nephritis in the IL-2 treated group [89]. Recently, Zhao et al. showed that low-dose IL-2 together with rapamycin restored the balance of Th17/Treg cells in SLE patients along with a reduction of disease activity [90]. The potential use of low-dose IL-2 was also investigated in 11 different autoimmune diseases, including RA, ankylosis spondylitis, SLE, psoriasis, Behçet's disease, granulomatosis with polyangiitis, Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis and sclerosing cholangitis. In general, low-dose IL-2 therapy was well tolerated and led to an expansion and activation of Treg cells [91].

Potential implications of Treg cells in SS

Until now, evidence has shown a potential of Treg-associated immunoregulatory therapy in SS (Table 2). Several animal models for SS have been used to confirm the therapeutic role of IL-2 signalling and Treg cells [61]. It has been reported that IL-2 knockout and IL-2Rα knockout mice showed severe lymphocyte infiltration in the salivary and lacrimal glands and a decrease in salivary secretory function, and that transfer of lymph node cells from scurfy mice, which have dormant salivary gland-specific autoreactive lymphocytes, to RAG-1 knockout recipients rapidly and effectively induced inflammation and dysfunction of salivary glands [61]. Furthermore, daily LPS feeding in scurfy mice also induced inflammation in the salivary glands [61]. On the contrary, low dose of IL-2 treatment in female NOD/ShiLtJ mice induced a higher proportion of CD4⁺CD25⁺Foxp3⁺Treg cells in both spleen and cervical-lymph-node and salivary gland tissues and reduced effector Th and Tfh cells, resulting in decreased gland inflammation scores [92].

In clinical trials, the efficacy of low-dose recombinant IL-2 treatment was also evaluated in pSS patients. Miao et al. reported that short-term and low-dose IL-2 therapy did not change the disease activity of pSS; however, this IL-2 treatment restored CD4⁺Treg cells and the ratio of Th17/Treg to control disease progress accompanied by reduced glucocorticoid and hydroxychloroquine use [34, 52]. Results from a randomized and placebo-controlled clinical trial of subcutaneous administration of low-dose IL-2 in 30 pSS patients showed an expansion of Treg cells and greater resolutions of dryness, pain and fatigue and with no severe adverse events [56].

Inducing autoantigen-specific Treg cells is a valuable attempt to inhibit gland injury in SS. Obviously, RNA-binding nuclear antigens, such as Ro/SSA and La/SSB, are a major class of self-antigen to which immune tolerance is lost in rheumatic diseases, especially in SS. Xu et al. established an immunotherapy for SS-like NOD/Ltj mice by combining a transient depletion of CD4+T cells by anti-CD4 mAb together with the administration of autoantigen-specific peptide Ro480 (belongs to Ro/SSA), which generated Ro480 antigen-specific Treg cells in these mice, leading to reduced IFN-γ production from CD4+T cells and decreased inflammation infiltration in glands [93]. Mechanistically, another study found that expansion of Foxp3+Treg cells specific for La/SSB was mediated by antigen-presenting plasmacytoid dendritic cells in a type 1 IFN-dependent manner to induce peripheral immune tolerance [75].

Several other strategies have been attempted to improve SS which focused on Treg cells. A previous study found that mesenchymal stem cells (MSCs) treatment directed T cells towards Treg and Th2, while suppressing Th17 and Tfh responses, and alleviated disease symptoms in both SS-like NOD/Ltj mice and SS patients [94]. Subsequently, various studies reported that MSCs Extract (MSCsE) secreted factors from cultured dental pulp stem cells, and labial gland-derived MSCs and their exosomes were able to improve SS syndrome which was linked to the Treg-associated peripheral tolerance [95, 96]. Lactococcus lactis expressing enterotoxigenic E. coli colonization factor antigen 1 treatment also maintained salivary flow in a SS model which was associated with an increase of Foxp3⁺ and IL-10- and TGF-β-producing Treg cells [97]. Inhibition of B7-H4 with anti-B7-H4 mAb exacerbated SS in NOD/Ltj mice presented with increased lymphocyte infiltration in salivary glands and inflammatory cytokines production and lower level of CD4⁺Foxp3⁺/CD4⁺ T cells in the spleen, while B7-H4Ig treatment obviously alleviated SS-associated symptoms [98]. Clinical data reported that sirolimus therapy restored the PD-1⁺ICOS⁺Tfh cells/CD45RA^-Foxp3^high activated Tfr ratio (Tfr cell subsets may resemble Treg cell lineages) and controlled disease activity in pSS [99]. In addition, traditional Chinese medicine was reported to be a useful and promising alternative strategy for the treatment of SS, which was partially associated with the balance of Th17/Treg cells [100].

Conclusions and future perspectives

Treg cells play an important role in preventing autoimmune disease and maintaining immune homeostasis. This is best exemplified by the fact that complete loss of Foxp3 protein and a subsequent lack of Treg cells have fatal effects in animals and that inactivating mutations in the FOXP3 gene in humans are associated with severe autoimmunity and associated syndrome. Reports on the number and function of Treg cells in SS are contradictory and the definitive role of Treg cells in SS remains unclear. Here we provide an overview of current knowledge on Treg cells in SS pathogenesis and current therapies to reconstitute the balance between Treg cells and effective T cells in SS. We believe that these insights into SS pathogenesis may provide the basis for successful therapeutic intervention in this disease.

However, several future challenges exist concerning the role of Treg cells, especially in salivary gland infiltrates and tissue damage, in SS pathogenesis. Current data about Treg cells in SS are still insufficient and could not obtain a consistent conclusion. It will be important to determine the physiological relevance and the mechanism that drives heterogeneity, plasticity and stability of Treg cells in autoimmune response to disease pathology, as findings in peripheral blood do not completely predict the phenotype and function of tissue Treg cells, and findings from in vitro suppression assays may not reflect the in vivo function. This is ultimately necessary for the new and exciting treatment approaches that target Treg cells as a direct Treg-based therapy or indirect strategies. A more detailed understanding of the exact role of Treg cells in pathogenic microenvironments during SS development will help to refine a personalized treatment approach.

Data availability

Data are available upon reasonable request by any qualified researchers who engage in rigorous, independent scientific research, and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). All data relevant to the study are included in the article.

Contribution statement

B.M. and Y.Z. contributed equally to search data for the article, write the manuscript and draw the figures. B.M., Y.Z., J.Z. and L.D. organized and revised the paper and substantially discussed of the content. All authors reviewed and approved the submitted version.

Funding

This work was supported by grants from the National Natural Scientific Foundation of China (No. 81901586 and No. 82271847) and Tongji Hospital Clinical Research Flagship Program (No. 2019CR206).

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

References

Author notes