The T cell in Sjogren’s syndrome: Force majeure, not spectateur

Introduction

Sjogren’s syndrome (SS) is marked by diffuse lymphocytic infiltration of exocrine glands and other tissues. Just how these ectopic lymphocytes cause disease has been an area of active investigation, particularly over the past two decades. Unlike most other diseases of lymphocytic infiltration, the salivary glands are accessible to biopsy, thus SS serves as a model for parallel autoimmune disorders of organ infiltration. The work of Professor Youinou and others has elegantly clarified the role of the B lymphocyte in the development of ectopic lymphoid tissue, and in the characteristic B-cell hyperactivity leading to autoantibodies, hypergammaglobulinemia, and sometimes lymphoma [1]. Yet glandular tissue in SS is – in most phases of the disease – overwhelmingly infiltrated by T lymphocytes. This article will emphasize recent advances in the understanding of these cells as agents of primary SS pathogenesis.

T Cells predominate in the infiltrates

Using monoclonal antibodies with immunoperoxidase and cytofluorographic detection techniques, it was noted in 1983 that T cells constituted the majority (>75%) of lymphocytes infiltrating the salivary glands (SGL) and that CD4 T cells constituted the majority of these cells [2]. Activated T cells (expressing MHC class II and CD38) were significantly elevated in SGL when compared to the corresponding peripheral blood lymphocytes [3]. These findings have been replicated in other laboratories, although it has been emphasized that later stages of disease are often accompanied by the appearance of greater numbers of B cells in minor salivary glands [4]. It has been a challenge to deduce the function of these T cells, and to define their relationship to T cells found in the circulation of SS patients. Peripheral T cells from SS patients are generally poorly responsive to global stimulation, and efforts to define antigens that might be stimulating CD4 cells have been only partly successful (see below). What, then, is the significance of the large numbers of activated CD4 cells in lesions? Are they specific for autoantigens, or have they been recruited nonspecifically into lesions? What is their phenotype and function?

Cytokine Profiles of T cells in SS

As in other conditions, inferences have been drawn from the cytokines produced by lesional and circulating T cells, particularly as the TH1/TH2 paradigm emerged in the 1990s. The status of SS T cells may vary with disease activity and stage [5]. Many studies of intralesional T cells have found expression of predominantly TH1 cytokines [6, 7], a finding reinforced by the presence of TH1 inducible chemokines CXCL9 and CXCL10 [8, 9]. Increased levels of IL-1β, IL-6, tumor necrosis factor (TNF)-α, and IFN-γ have been reported in saliva from patients with SS in comparison with controls with histologically normal salivary glands, consistent with a role for Th1-cell-mediated pathology [10]. Yet a role for TH2-derived cytokines such as IL-10 and IL-4 is supported by studies of peripheral blood lymphocytes in SS and in some studies of intraglandular T cells and saliva [11]. It has been proposed that there is a dynamic balance between the two families of cytokines and Th1 response is favored in patients with SS and with high infiltration scores [12].

The realization that a distinct set of T helper cells characterized by secretion of IL-17 and select other cytokines plays an apparent role in multiple autoimmune and inflammatory diseases has led to a re-examination of infiltrating T cells in SS. IL-17A and IL-23 have been observed in SS, along with IL-22 [13], and IL-17 expression correlated with the severity of lesions [14]. IL-22 was derived largely from NK T cells as well as Th17 cells. IL-17 and related cytokines TGF-beta, IL-6, IL-23, and IL-12 could be detected in patient plasma, as well as in saliva [14]. Peripheral blood from SS patients has been reported to have the potential to secrete high levels of IL-7, promoting Th17 polarization, and also high levels of IL-12, which might promote Th1 cells expansion [15]. IL-10 production was found to be low in these studies. It has been suggested that IL-18 may also provide an important stimulus for IL-17 production locally in the inflamed Sjogren’s salivary gland and to contribute to high IgG1 levels [16]. The activation of TH17 cells in SS infiltrates has been hypothesized to promote the generation of germinal centers within glands and to be counter-regulated by BAFF [12]. Activated Th17 cells within tissues in patients (and perhaps elsewhere in the body) may deliver excessive T-cell help and promote B-cell activation. What triggers the intense TH17 response? Th17 polarization in humans is brought about by local exposure to TGFβ, IL-23, IL-21, and IL-6, and is mediated through the transcription factor RORγT [17]. Similar Th17 polarization is seen in inflammatory lesions of the intestinal mucosa, and in a number of autoimmune diseases [18]. Could this reflect a final common pathway for chronic inflammation of certain tissues, or does the T-cell activation come from a response to pathogens involved in multiple illnesses? For Sjogren’s syndrome, could the T-cell inflammatory process promote the activation of B cells and be responsible for the well known humoral hyperactivity and autoimmunity?

IL-21 has increasingly been recognized as a key mediator of help to B cells and is a potent inducer of TH17 [19]. IL-21 has been detected in the serum and within the labial salivary glands of SS patients, and levels correlated with hypergammaglobulinemia and autoantibody levels [20]. These data are of special interest with regard to the evolving understanding of IL-21 as a key mediator of T-cell help, and as a product of T follicular helper (Tfh) cells. CXCR5 and ICOS are signature Tfh surface markers. In SS lesions, CXCR5 has been observed on B cells [21], but data regarding T-cell expression are not available. It is of interest that T-cell ICOS expression is promoted when SS-derived salivary epithelial cells are cultured in vitro with autologous peripheral blood T cells, supporting the idea that Tfh cells may be of importance in SS, as they appear to be in SLE, chronic hepatitis, and other illnesses [22].

Impaired Regulatory T cells in Sjogren’s Syndrome

TH17 cells and T regulatory cells (Tregs) are linked together through a web of cytokines, notably TGFβ. Tregs are thought to exert a countervailing suppressive effect on TH17 mediated cellular immunity. Considerable evidence supports the notion that Tregs (identified through their expression of the transcription factor Foxp3) are impaired in SS patients. Christodoulou showed that in SS salivary glands, the relative numbers of Foxp3(+) cell frequency varied inversely according to disease severity, with parallel findings in the peripheral blood [23]. MSG-infiltrating Foxp3(+) cells correlated with biopsy focus score, infiltrating mononuclear cells, dendritic cells, and macrophages, and serum C4 levels, and lower Foxp3(+) cell incidence correlated with adverse predictors for lymphoma development, such as the presence of low C4 and SG enlargement [24]. These findings suggest that the Foxp3(+) T-regulatory cell frequency in the MSG lesions of SS patients correlates with the degree of inflammation and with risk factors for lymphoma development. The authors further proposed that decrease in T regulatory cells in severe lesions compared to intermediate lesions probably suggests an inability to control local immune responses, thus resulting in the ultimate loss of immune balance and systemic disease.

What triggers T-cell activation in SS tissues?

T cells in SS appear to be responding to an intense antigenic stimulus, leading to the cellular phenotypes discussed above, and probably aggravated by insufficient Treg immunoregulation. But what is the initiating insult that leads to T-cell activation? Attention has centered on the Ro and La autoantigens, known to be expressed on blebs on apoptotic cells and signature autoantigens in SS [25]. T-cell recognition of Ro antigen by blood derived T cells from patients leads to modest proliferative responses [26]. However, it is reported that the precursor frequency of T cells reactive to Ro and La is actually higher in normal controls than in SS patients [5]. In mice, immunization with certain peptides derived from the La antigen lead to a broader T-cell response and to epitope spreading with systemic autoimmunity [27, 28]. Remarkably, Scofield found that immunization with short Ro-derived peptides led to SS manifestations, including salivary gland infiltration by lymphocytes, decreased salivary flow and anti-Ro [29, 30]. Immune responses to La-derived peptides are more intense in mice with transgenic expression of human HLA-DR3/DQ2 antigens, favoring a key role for T cells in the signature autoantibodies of SS [31].

The cytoskeletal protein α-fodrin (spectrin) has been proposed as an autoantigen in SS [32]. T-cell and humoral reactivity to α-fodrin are observed in a murine post-thymectomy SS model, and have been reported in patients with SS. T-cell lines reactive to peptides derived from the antigen have been reported to induced infiltrative disease when transferred into mice. Fodrin is generated by granzyme induced proteolysis in apoptotic cells and may be made available as a consequence of cellular injury [33].

If T-cell recognition of self antigens is crucial to the cascade of events leading to SS immunopathology, what environmental agent might be implicated as the initiating factor? Viruses have been considered as possible causes, without definitive proof [34]. The tendency of certain viruses, especially those of the herpes group, to localize to salivary glands is well known and fits well with their possible role in pathogenesis. Recent data concerning gut commensal bacteria in the generation of Th17 responses [35] may be relevant to SS, as the oral flora could also contain filamentous bacteria that could result in chronic Th17 predominant responses.

Other environmental effects may trigger T-cell autoreactivity leading to SS. Niederkorn et. al. have demonstrated that desiccating stress (DS) elicits T cell-mediated inflammation of the cornea, conjunctiva, and lacrimal glands, but not other organs in mice [36]. The lacrimal keratoconjunctivitis (LKC) was mediated by CD4(+) T cells, which, when adoptively transferred to T cell-deficient nude mice, produced inflammation in the LG, cornea, and conjunctiva, but not in any other organ. Adoptively transferred CD4(+) T cells produced LKC even though recipients were not exposed to DS. LKC was exacerbated in euthymic mice depleted of Foxp3+ CD4 regulatory T cells. The local production of interferon γ was shown to be of key importance in activating the extrinsic apoptotic pathway [37]. The results suggest that DS exposes shared epitopes in the cornea, conjunctiva, and LG that induce pathogenic CD4(+) T cells that produce LKC, which under normal circumstances is restrained by Foxp3+ regulatory T cells. Surprisingly, in a follow up study, TGFβ was shown to have a role in ocular injury, as TGFβ deficient mice were protected from ocular injury [38]. These studies imply that any stimulus leading to drying of ocular tissue (or other mucosa, in principle) might progress to an autoimmune response with epitope spreading and possibly immune mediated glandular pathology. As desiccation or other stress might lead to apoptosis with expression of autoantigens on cell surfaces, Sjogren’s related antigens might come to the attention of the immune system.

Irrespective of the triggering cause, elegant work has implicated salivary glandular epithelial cells as active participants in T-cell mediated immune responses and in the perpetuation of exocrine gland inflammation. Glandular epithelial cells inappropriately express class II MHC molecules HLA-DR antigens [39, 40]. Salivary epithelial cells upon activation by IFNγ are fully capable of expressing high levels of co-stimulatory molecules B7.1 and B7.2 [41]. By virtue of their capacity to express B7 co-stimulatory molecules along with MHC proteins after activation, activated SG epithelia in SS lesions appear to be suitably equipped for the presentation of antigens to CD28- expressing T lymphocytes. The paucity of macrophages in the infiltrate also supports role of epithelial cells acting as antigen presenting cells [39] for the T cells.

T cells in SS lesions seem oligoclonal

As in other infiltrative lesions, analysis of the clonality of lymphocytes is a potential clue to the etiology. Assessment of the T cell receptor (TCR) repertoire of infiltrating T cells in the salivary glands of SS patients has been done using antibodies directed against the TCR β chain variable region families (TCR Vβ) and molecular genetic techniques and results have been interpreted to indicate restricted clonality consistent with an antigen driven response [42]. Analysis of clonality is inherently limited when there are small numbers of infiltrating T cells, but these studies do favor clonal expansion of T cells within the affected glands.

Homing of T cells to Salivary Glands

T cells may proliferate locally in the salivary glands, but very likely much of the infiltration is due to the migration to glands of T cells from the circulation. This migration is T-cell infiltration of chemokine-mediated. Whether their action is primarily on antigen-specific T cells or on nonspecific bystander cells is difficult to discern. Ogawa et al [43] in 2002 showed that two chemokines, IFN-γ inducible 10 kd protein (IP-10, also called CXCL10) and a monokine induced by IFN γ (Mig, also called CXCL9) are important in attracting T-cells to the salivary glands and also showed that most of the CD3+ infiltrating lymphocytes in the dense foci expressed CXCR3, the receptor for IP-10 and Mig. Hasegawa [44] constructed an IP-10 antagonist by deleting 5 NH2 terminal amino acid residues and adding methionine at the NH2 terminal. This molecule decreased the infiltration of CXCR3+ T cells, predominantly Th1 cells into the salivary glands and eventual amelioration of sialadenitis in MRL/lpr mice.

T cells mediate direct glandular injury in SS

T cells may mediate pathology by their bulk and disruption of architecture, and by their potential to activate B cells. T-cells may also mediate glandular destruction by inducing apoptosis via FasL, by direct cytotoxic action via perforin and via cytokine secretion [45]. Fas-mediated apoptosis may also be important in the salivary glands of patients with SS. Fas, Fas ligand (FasL), and bcl-2 in salivary gland biopsy material were demonstrated in situ by immunohistochemical staining and reverse transcriptase-polymerase chain reaction [46]. DNA fragmentation in apoptotic cells was assessed by the enzymatic incorporation of labeled nucleotides (digoxigenin-dUTP). Their results indicate that the infiltrating lymphocytes in the focal lesions of the salivary glands of patients with SS are blocked in their ability to commit to apoptosis, even though they may express Fas and this in turn might be due to the presence of bcl-2 in these cells. The acinar epithelial cells, in contrast, may undergo Fas-mediated apoptosis.

The Fas/FasL pathway is not the only functional apoptotic pathway in the MSGs of patients with SS. Substances released by cytotoxic lymphocytes such as perforin and granzymes constitute a second extrinsic pathway of apoptosis. Cytotoxic T lymphocytes (CTL), either CD4 or CD8, which are scattered in the infiltrates are activated, release perforin and Granzyme B, and may play a significant role in the induction of epithelial cell apoptosis [47]. The increased expression of pro-apoptotic signals (Fas and Bax) and decrease in anti-apoptotic signals (Bcl-2) in the epithelial cells of SS patients may tip the balance towards apoptosis. TUNEL assay shows a significant increase in the percentage of apoptotic ductal and acinar epithelial cells in MSG biopsies of patients with SS compared with control individuals.

Lessons from SS Animal models

T-cell mediation of disease through elaboration of various cytokines is important in different animal experiments. In the NOD (non-obese diabetic) model of SS, IL-4 seems to be important in mediating disease. IL-4 knockout NOD mice have focal salivary gland infiltrates similar to conventional NOD mice, yet do not develop salivary secretory insufficiency [48]. IL-4 deficient NOD mice also fail to produce anti-muscarinic acetylcholine type-3 receptor (M3R) autoantibodies. Transfer of wild type T cells into young NOD IL-4 knockoutmice reconstituted the production of anti-M3R autoantibodies and subsequent decline in salivary secretory activity. In the related, NOD-derived Aec1/Aec2 model, glandular disease is preceded by the appearance in spleens and lymph nodes of activated CD4+ T cells bearing markers of effector-memory cells [49]. These cells are present before the appearance of activated B cells and autoantibodies and before salivary flow is reduced. In the salivary glands, most CD4 bearing cells also bear the CD44 marker (see Figure 1), indicative of a function as effector-memory cells.

Figure 1.

CD4+CD44+ memory cells in a murine model of SS. Submandibular gland of a 12 week old Aec1/Aec2 mouse (expressing NOD SS susceptibility intervals) shows CD4+ cells (green), most of which also express CD44. These dual staining cells have the phenotype of effector-memory cells. Unpublished data of P. Cohen and Y. Li.

In support of studies of human SS, Nguyen at al [50] have shown a role for IL-17 in Sjogren’s syndrome in mice Adenovirus serotype 5 (Ad5) vectors expressing IL-17A were infused into the salivary glands of C57BL/6J mice of different ages and the mice were characterized for SS phenotypes. SS phenotype was reproduced in mice receiving Ad5-IL17A vector, with lymphocytic infiltration, decrease in salivary flow, increased cytokine levels as well as a change in antinuclear antibody profiles.

Conclusions and Perspectives

Research over the past three decades has gone beyond phenomenology to establish an important role for the T cell in the etiology and pathogenesis of Sjogren’s syndrome. It seems likely that T cells undergo activation in response to environmental triggers: viral infection, or even physical desiccation – and that their further activation is facilitated by the immunological capabilities of the resident glandular epithelial cells. Activated T cells with TH1 and TH2 phenotypes arise, but Th17 and Th21 helpers predominate and likely provide a stimulus for B lymphocytes. T cells also are directly involved in the destruction of the gland through cytotoxicity.

In many respects, the role of T cells in SS remains cryptic, despite recent knowledge. Are most of the cells in the infiltrate specific, or are many of them bystanders recruited from the periphery? Do T cells undergo expansion within the gland, or does this occur elsewhere with subsequent migration? Is there traffic in and out of the gland, or do T cells remain in the infiltrates once they arrive? What is the principal antigen or antigen driving T-cell expansion? And to what extent are T cells involved in glandular destruction versus their role as provocateurs of B-cell activation and differentiation? Answers to these questions seem within reach using model systems and through study of human tissue.

Table.

The Role of T cells In Sjogren's syndrome

Principal Findings	Reference(s)
T cells, mostly CD4 T cells, predominate in the salivary gland lesions	2
The activated T cells are elevated in the SGL compared to peripheral blood Lymphocytes	3
In later stages of disease, B cells are present in greater numbers in the minor salivary glands	4
Status of SS T cells may vary with disease activity and stage	5
Salivary gland pathology is mainly Th1-mediated especially with high infiltration Scores	6–10, 12
Th2-derived cytokines are important in SS	11
Activation of Th17 cells in SS infiltrates promote generation of germinal centers within glands	12
IL-17 family of cytokines is present in SGL as well as patient plasma and saliva	13, 14
IL-18 may stimulate IL-17 production locally and provoke B-cell IgG1 production	16
IL-21 mediates B-cell help and stimulates Th17 cells	19
IL-21 is present in SG and levels correlate with hypergammaglobulinemia and autoantibody levels	20
T follicular helper cells are important in SS	22
T regulatory cell frequency varies inversely with disease severity, parallel findings in peripheral blood	23
Lower Foxp3 cell incidence correlates with adverse predictors for lymphoma development, such as low C4 and SG enlargement	24
T cell recognition of Ro antigen leads to modest proliferative responses	26
In mice, immunization with peptides derived from La antigen leads to broad T cell response and epitope spreading with systemic autoimmunity	27, 28
Immunization with short Ro-derived peptides leads to SS manifestations	29, 30
Immune responses to La-derived peptides are more intense in mice with transgenic expression of human HLA-DR3/DQ2 antigens	31
Alpha fodrin proposed as an autoantigen in SS	32
Viruses have been considered as possible environmental trigger of T cell- autoimmunity without definite proof	34
Gut commensal bacteria lead to generation of Th17 responses	35
Desiccating stress (DS) elicits T cell-mediated lacrimal keratoconjunctivitis (LKC). Adoptively transferred CD4(+) T cells produce LKC in recipients not exposed to DS	36
Salivary gland epithelial cells are active participants in T cell mediated responses- express class II MHC	39, 40
After activation by IFN-G, salivary epithelial cells express co-stimulatory molecules B7.1 and B7.2	41
Paucity of macrophages in the SGL supports role of epithelial cells as APC for the T cells	39
In SGL, T cell receptor expression is restricted, indicating an antigen driven response	42
IP-10 and CXCL10 and Mig (CXCL9) are important in homing of T cells to the salivary glands	43
In mice, sialadenitis can be ameliorated by decreasing influx of T cells to SGL by IP-10 antagonist	44
T cells mediate glandular destruction by inducing apoptosis via FasL	45
Infiltrating lymphocytes in SGL are blocked in their ability to undergo apoptosis, might be due to presence of bcl-2	46
Cytotoxic lymphocytes also mediate injury by releasing perforin and granzymes	47
Role of Th2 cytokine, IL4, in SS through generation of IL-4 KO mice	48,49
Role of IL-17 in SS in mice	50

Abbreviations: SS- Sjogren’s Syndrome; SGL- Salivary gland

• T cells are the majority of lymphocytes in Sjogren’s infiltrates

• T cells secrete Th1 and Th2 cytokines

• Th17 T cells actively contribute to Sjogren pathology

• Defective T regulatory cells play a role in disease

• T cells can directly injure glands through cytotoxicity