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Basic and translational research
Extended report
IL-17-producing CD4−CD8− T cells are expanded in the peripheral blood, infiltrate salivary glands and are resistant to corticosteroids in patients with primary Sjögren's syndrome
  1. Alessia Alunno1,
  2. Onelia Bistoni1,
  3. Elena Bartoloni1,
  4. Sara Caterbi1,
  5. Barbara Bigerna2,
  6. Alessia Tabarrini2,
  7. Roberta Mannucci3,
  8. Brunangelo Falini2,
  9. Roberto Gerli1
  1. 1Rheumatology Unit, Department of Clinical & Experimental Medicine, University of Perugia, Perugia, Italy
  2. 2Department of Clinical & Experimental Medicine, Institute of Hematology, University of Perugia, Perugia, Italy
  3. 3Laboratory of Confocal Microscopy and Image Analysis, University of Perugia, Perugia, Italy
  1. Correspondence to Professor Roberto Gerli, Rheumatology Unit, Department of Clinical & Experimental Medicine, University of Perugia, Via Enrico Dal Pozzo, Perugia I-06122, Italy; gerlir{at}unipg.it

Abstract

Objectives It has been recently observed that a T-cell subset, lacking of both CD4 and CD8 molecules and defined as double negative (DN), is expanded in the blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of primary Sjögren's syndrome (SS) patients, we investigated whether DN T cells may be involved in the pathogenesis of salivary gland damage.

Methods Phenotypic characterisation of peripheral blood mononuclear cells from SS patients and controls was performed by flow cytometry in freshly isolated and anti-CD3-stimulated cells. SS minor salivary glands were processed for immunofluorescence staining.

Results CD3+CD4−CD8− DN T cells were major producers of IL-17 in SS and expressed ROR-γt. They were expanded in the peripheral blood, spontaneously produced IL-17 and infiltrated salivary glands. In addition, the expansion of αβ-TCR+ DN T cells was associated with disease activity. Notably, IL-17-producing DN T cells from SS patients, but not from healthy controls, were strongly resistant to the in vitro effect of dexamethasone.

Conclusions These findings appear to be of great interest since the identification of a peculiar T-cell subset with pro-inflammatory activity, but resistant to corticosteroids, in an autoimmune disorder such as SS may help to design new specific treatments for the disease.

  • Sjögren's Syndrome
  • T Cells
  • Corticosteroids

https://doi.org/10.1136/annrheumdis-2012-201511

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    Introduction

    Primary Sjögren's syndrome (SS) is a systemic autoimmune disease primarily characterised by chronic inflammation of exocrine glands that leads to compromised secretory function and tissue damage.1 Surprisingly, despite the recognised autoimmune nature of the disease, there is no evidence for the efficacy of corticosteroids (CS) or other traditional immunosuppressive drugs in affecting the natural course of chronic inflammatory infiltration and its destructive potential of exocrine glands.2

    It is thought that T helper 1 (Th1) cells are major players in the induction of glandular damage. However, the discovery of Th17 cells challenged the long-standing Th1–Th2 paradigm and prompted a re-evaluation of glandular infiltrate in SS. Th17 cells, indeed, represent a main pathogenic effector subset involved in inflammation and autoimmunity3–5 and are actively involved in glandular tissue damage of SS.6–9

    Th17 lymphocytes are developmentally and functionally divergent from classic Th1 and Th2 cells.10–12 They can derive from CD4 T cells activated by interleukin (IL)-6 and transforming growth factor (TGF)-β stimulation.10–12 However, CD4 expression by Th17 cells is not mandatory, since a small CD3 cell population, lacking both CD4 and CD8, can produce IL-17 following T cell receptor (TCR) stimulation.13 These cells, known as CD4−CD8− double negative (DN), represent less than 5% of the total peripheral blood (PB) T-cell pool and express either αβ- or γδ-TCR. The conflicting data that have been published about DN T-cell functional role could be explained by their identification by exclusion and, therefore, may include T cells with different functions, including suppressive activity.14–16 Several lines of evidence, however, appear to suggest that DN T cells play a major role in the pathogenesis of autoimmune disorders. DN T-cell accumulation is associated with the development and severity of autoimmune disease in animal models.17 In addition, deficiency of Fas or its ligand causes dramatic accumulation of DN T cells in autoimmune lymphoproliferative syndrome.18 Increased DN T-cell proportions, associated with anti-DNA production and kidney involvement, have been described in systemic lupus erythematosus (SLE).19 ,20 In these patients, DN T cells can promote immunoglobulin B cell production.21 ,22 More importantly, DN T lymphocytes represent a large component of kidney-infiltrating T cells during lupus nephritis and also represent a major source of IL-17 in SLE.23

    The evidence of a pro-inflammatory role of DN T cells in autoimmune disorders and the recognition of a potential pathogenic involvement of IL-17 in SS inflammatory glandular damage prompted us to investigate a possible involvement of DN T lymphocytes in SS pathogenesis and to verify whether these T cells may contribute to increased production of IL-17 in this disorder.24

    Methods

    Patients

    Thirty consecutive patients with SS, classified according to the Euro-American criteria,25 were enrolled. In all, 16 sex- and age-matched healthy donors (HD) and 10 subjects with sicca syndrome, but without evidence of SS (sicca-non-SS), acted as controls. All patients were female subjects (age: 54.2±13 years, disease duration: 7.9±5 years). Disease activity was measured using the EULAR Sjögren's syndrome disease activity index (ESSDAI).26 Fifteen patients were taking hydroxycloroquine 200 mg/day and the others were using only topic medications for sicca symptoms. None of the patients was taking immunosuppressive drugs or CS. The study was approved by the local Ethics Committee (CEAS) and written informed consent was obtained in accordance with the declaration of Helsinki.

    Cell isolation and flow cytometry

    Peripheral blood mononuclear cells (PBMCs) were isolated by gradient separation. For surface staining Phycoerythrin (PE), Fluorescein isothiocyanate (FITC), Alexa Fluor 647, Allophycocyanin (APC) or Pe-Cy5 labelled antihuman CD3, CD4, CD8, αβ-TCR, γδ-TCR, CD56, CD69, CD25, Human leukocyte antigen DR (HLA-DR) and respective isotypes were used (BD, San Jose, CA, USA; eBioscience San Diego, CA, USA; BioLegend, San Diego, CA, USA). When required, cells were permeabilised with 0.1% saponin blocking buffer after 4% paraformaldehyde fixation or with fixation/permeabilisation concentrate (eBioscience). Six-hour in vitro stimulation with 25 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 µg/ml ionomicyn and 0.1 mg/ml brefeldin in complete medium was performed prior to performing intracellular staining. For intracellular staining, APC labelled antihuman IL-17, ROR-γt and their isotypes were used (BD and R&D). Debris was excluded by back-gating to CD3 T cells in forward scatter/side scatter (FSC/SSC) plots. Samples were analysed using FACScalibur flow cytometer (BD) and CellQuestPro software (BD).

    Cell cultures

    Untouched PBMCs from five SS patients and five HD were cultured for 5 days with 20 U recombinant human IL-2 on cross-linked anti-CD3 coated plates in the presence or absence of dexamethasone phosphate (Dex) (Soldesam).27–29 As negative control, cells were cultured with complete medium alone. Cell proliferation was assessed by 3H thymidine assay. Cell viability was determined by Trypan Blue staining. A cell aliquot was collected at appropriate time-points that allowed consecutive 6 h stimulation for IL-17 expression assessment among CD4 and DN T cells, as described above.

    Salivary gland specimens

    Subjects with sicca syndrome symptoms underwent labial minor salivary gland (MSG) biopsy and 5/6 lobules were analysed. The presence of at least one inflammatory focus within 4 mm2 allowed the diagnosis of SS.30 ,31 Serial sections of five SS-MSG and five sicca-non-SS-MSG were cut at 3 µm thickness, deparaffinised and rehydrated. Envision flex target retrieval solution high ph (DAKO) was used for antigen retrieval and unspecific binding was avoided by 30 min incubation with 10% FCS. For double immunofluorescence staining, anti-CD8 (DAKO), anti-CD3 (NeoMarkers) and anti-CD4 (BD) were used diluted in appropriate buffer. Alexa Fluor 488 antirabbit and Alexa Fluor 568 antimouse were used as secondary antibodies. Nuclear counterstaining was performed with 4',6-diamidino-2-phenylindole (DAPI). Slides were mounted with Mowiol 4–88 (Sigma Aldrich, St Louis, MO, USA) and analysed with Olympus AX70 microscope (Olympus America, Center Valley, PA, USA).

    Statistical analysis

    SPSS V.13.0 package was used and either Mann–Whitney U test or Spearman correlation coefficient was calculated. All values are indicated as mean±SD. p Values ≤0.05 were considered significant.

    Results

    Circulating DN T cells are expanded in SS patients

    We initially sought to investigate whether DN T cells were expanded in PB of SS patients. As shown in figure 1A, the percentage of total circulating DN T cells was increased in SS patients with respect to HD. Notably, DN T cells did not include CD56 cells and circulating DN T cell number in sicca-non-SS controls were comparable with those of HD (see online supplementary text 1). This result was subsequently analysed by the evaluation of DN T-cell subset according to the coexpression of the surface CD3 molecule with either αβ-TCR or γδ-TCR. The results demonstrated that the high number of DN T cells in SS was attributable to an expansion of both αβ-TCR+ and γδ-TCR+ cells. Representative flow cytometry dot plots are shown in figure 1B–E.

    Figure 1
    Figure 1

    (A) Percentage of total and both αβ-TCR+ and γδ-TCR+ DN T cells in the peripheral blood of 30 Sjögren’s syndrome patients and 16 healthy donors. Freshly isolated peripheral blood mononuclear cells were stained for CD3, CD4 and CD8 and respective isotypes. Graphs indicate mean±SD. **p<0.01 and ***p<0.001. (B–E) Representative flow cytometry dot plots of freshly isolated T cells: (B) isotype; (C) total DN T cells; (D) αβ-TCR+ DN T cells; (E) γδ-TCR+ DN T cells.

    Freshly isolated DN T cells produce IL-17 and express ROR-γt

    Next, we moved to the assessment of IL-17 expression among total CD3 T cells and both CD4 and DN T-cell subpopulations (figure 2). The fraction of freshly isolated IL-17-producing CD3 T lymphocytes was slightly, but not significantly, increased in SS (figure 2A). Within the CD3+CD4+ cell population, the proportion of IL-17+ cells represented a very small subset in both patient and HD (about 5%). However, a consistent fraction (>60%) of freshly isolated DN T cells from either HD or SS displays intracellular IL-17. In brief, our data demonstrate that DN T cells represent a small cell subpopulation able to spontaneously produce IL-17. In addition, since CD4 T cells represented up to 55%, whereas DN T cells were about 5%, of total freshly isolated PBMC, the present results also suggest that the absolute number of circulating DN and CD4 T cells producing IL-17 was similar. We subsequently investigated whether DN T cells resemble a Th17 cell phenotype by the expression of the orphan nuclear receptor ROR-γt, the peculiar transcription factor displayed by this cell subpopulation. Interestingly, almost all DN T cells (percentages ranging from 98% to 100%) show consistent amounts of intracellular ROR-γt both in SS and in HD (figure 2B), hence confirming overlapping phenotype between Th17 and DN T cells.

    Figure 2
    Figure 2

    IL-17 production in freshly isolated CD3, CD4 and DN T cells from Sjögren's syndrome (SS) and healthy donors (A); representative histogram of ROR-γt expression among freshly isolated DN T cells from an SS patient (B); IL-17 production in resting or anti-CD3-triggered CD3, CD4 and DN T cells after 5 day-cultures (C). Bars indicate mean±SD. *p<0.05 and **p<0.01. Representative flow cytometry dot plot of T cells following stimulation (D).

    Activation effect on IL-17 production of DN T cells

    Unlike data observed in freshly isolated T cells, the percentage of CD3+IL-17+ cells after 5 day culture in medium alone was higher in SS (17.4±4.3%) than in HD (10.4±0.7%; p≤0.05). This finding was essentially due to a further increase in the percentage of IL-17+ DN T cells in SS (64±16% vs 82.6±3%; p<0.05), since the proportion of SS CD4+IL-17+ cells after 5 day culture without activatory stimuli remains similar to that observed in freshly isolated cells (5.9±3.7 vs 9.2±3.%; NS). Interestingly, anti-CD3-triggering led to a strong increase of IL-17-producing cells within both CD3 and CD4 cells, but not among DN T cells which were characterised by an already maximal production of IL-17 (figure 2C). A representative dot plot following T-cell activation is shown in figure 2D. Taken together, these findings appear to support the idea that DN T cells represent a fraction of already in vivo activated T cells able to spontaneously produce IL-17, whereas CD4 T cells include a number of potential IL-17-producing cells that can exert their pro-inflammatory action after stimulation. This hypothesis was supported by the phenotypic analysis of activation markers (see online supplementary text 2 and online supplementary figure S1).

    αβ-TCR+, but not γδ-TCR+, DN T-cell expansion in SS is dependent on disease activity

    In order to evaluate a possible association between disease activity and DN T-cell PB expansion, we subdivided SS patients according to ESSDAI values in two groups: subjects with active (ESSDAI≥2; n° 15) and inactive (ESSDAI<2; n° 15) disease. Patients with either active or inactive disease had percentages of total circulating DN T cells higher than HD (figure 3). The γδ-TCR+ DN T-cell population was expanded in SS patients apart from disease activity. Noteworthy, however, when αβ-TCR+ DN T-cell subset was analysed, only patients with active disease displayed an expansion of this cell population. No significant differences in the proportion of DN T cells were found according to age, disease duration, focus score, parotid gland swelling occurrence, extra-glandular involvements or treatment (data not shown).

    Figure 3
    Figure 3

    Percentage of total and both αβ-TCR+ and γδ-TCR+ DN T cells in the peripheral blood of Sjögren's syndrome (SS) patients according to EULAR SS disease activity index (ESSDAI) score. Patients with an ESSDAI score <2 (n. 15) were classified as inactive, whereas a score≥2 identified patients with active disease (n. 15). Bars indicate mean±SD. *p<0.05 and **p<0.01. HD, healthy donors.

    DN T cells are present in SS salivary gland infiltrate

    We then verified the presence of DN T cells within MC infiltrate of SS sialoadenitis. CD3 cells lacking both CD4 and CD8 molecule on their surface were present in SS salivary glands together with other CD3 T cells (figure 4). They were interspersed within both periductal infiltrate and ductal epithelium (intraepithelial lymphocytes). On the opposite, no DN T cells were observed within the mild infiltrate of sicca-non-SS controls (see online supplementary text 3 and online supplementary figure S2).

    Figure 4
    Figure 4

    DN T cells are present in the salivary gland infiltrate of patients with Sjögren's syndrome. (A) H&E staining was performed to identify mononuclear cell infiltrate (20× magnification). (B and C) A consecutive minor salivary glands section from the same specimen was stained with anti-CD3 (green) and anti-CD4/CD8 (red). Nuclear counterstaining was performed with DAPI. The area in the square shown at 20× magnification in panel B is displayed at 40× magnification in panel C. DN T cells are identified as CD3+CD4−/CD8− (white arrows in panel C).

    DN, but not CD4, IL-17-producing T cells are in vitro resistant to Dex

    Initially, in order to optimise culture conditions, Dex concentration was titrated from 10−5 to 10−7 M and cell viability and proliferation were assessed at different time-points from 48 h to 15 days. According to experiment results, Dex concentrations of 10−6 M and 5×10−6 M and culture duration of 5 days were identified as optimal in vitro conditions to evaluate Dex effects (data not shown). Although ROR-γt expression was not affected by Dex addition in any cell population studied (data not shown), figure 5 shows that Dex was able to strongly reduce IL-17 production among anti-CD3-stimulated CD4 cells in both HD and SS in a dose- and time-dependent manner (see also online supplementary text 4). However, the most relevant finding was that Dex was able to almost completely abolish the IL-17 production from anti-CD3-triggered DN T cells in HD, whereas it was absolutely ineffective on IL-17 production from activated DN T cells in SS.

    Figure 5
    Figure 5

    Effects of two different concentrations of Dex on CD3-triggered CD4 and DN T-cell subsets from Sjögren's syndrome patients and healthy donors. Bars indicate mean±SD. Results were generated in separate experiments from those shown in figure 2. *p<0.05 and **p<0.01.

    Discussion

    It is now evident that IL-17 represents a dominant cytokine involved in the immunopathogenesis of SS.1 ,5 ,6 Indeed, elevated PB levels and pronounced expression of this cytokine at local tissue level, correlated with focus score, have been shown in SS.7 Moreover, IL-17 pathogenic effect in inducing exocrine gland damage has been also confirmed in experimental models of the disease.8 Although CD4 T cells, which have been shown to be major producers of IL-17 in SS, should be sensitive to CS and immunosuppressive drug effects, both IL-17 levels and SS clinical manifestations do not appear to respond to commonly employed treatments, including anti-TNF agents.2 ,24

    The present study demonstrated that DN T cells are expanded in the PB and infiltrate the salivary glands of SS patients. Although our data did not allow to verify the functional activity of DN T cells infiltrating salivary glands, the demonstration that circulating DN T cells produce IL-17 supports the idea that this T-cell subset may be actively involved in the immunopathogenesis of SS. In this setting, our results are in line with data from a number of studies suggesting that DN T cells are key players in autoimmune and inflammatory disorders in humans.32

    Along with conventional αβ-TCR+, the DN T-cell population also includes cells bearing the γδ-TCR.33 According to our data, both these two DN T-cell subsets are expanded in SS, although possible differences in their origin and functional role remain, at least in part, uncertain. It has been proposed that circulating DN T cells originate from the thymus where the expression of γδ- or αβ-TCR is fundamental in regulating positive and negative selection of DN T cells.34 Spontaneous development of IL-17-producing γδ-TCR+ T cells arise in the thymus via a TGF-β-dependent mechanism in naive animals.35 Several lines of evidence, however, suggest that DN T cells could be also directly generated in the periphery rather than in the thymus during autoimmune and inflammatory disorders.36–39 Interestingly, the αβ+ DN T cells, apparently derived from activated CD8 T cells,13 seem to be major players in autoimmune disorders, where they are expanded in the PB and accumulated in inflamed tissues.17 ,20 ,23 In this context, our finding showing an association between disease activity and number of circulating αβ+, but not γδ+, DN T cells is particularly intriguing and needs further investigations.

    Noteworthy, DN T cells appear to be functionally heterogeneous with a dual identity. Indeed, a regulatory function of DN T cells has been described in mice and humans.14 ,15 ,40 On the other hand, there is also evidence that these cells can display inflammatory capacity mediated by production of pro-inflammatory mediators, including IL-17.13 In this context, our data demonstrated that, unlike circulating CD4 T cells, a high number of freshly isolated DN T cells from HD and SS patients spontaneously produced IL-17 and the number of IL-17+ DN T cells was further increased after 5 days of culture in the absence of activation stimuli. Interestingly, the IL-17 production of resting DN T cells was comparable with that of CD4 T cells generated following in vitro activation. This supports the claim that DN T cells circulating in controls and SS represent the consequence of in vivo activation and/or decreased removal of activated DN T cells. In addition, they display a pro-inflammatory Th17 phenotype, as supported by ROR-γt expression.

    These observations agree with recent data postulating a direct link between DN T cells and development of autoimmune diseases. Increased number of circulating DN T cells, indeed, have been shown in patients with myasthenia gravis and lymphofollicular hyperplasia, where they appear to play a key role in immunoregulation and antibody production.41 In addition, an emerging role for DN T cells as main IL-17-producing effector cells in SLE has been shown.42 Indeed, increased frequency of IL-17-producing DN T cells in PB and kidney of SLE subjects has been described.23 ,43 Consistent with human studies, increased expression of IL-17 has been found in DN T cells from lupus-prone mice.44 Furthermore, in line with their ability to produce high amounts of IL-17, DN, rather than CD4, T cells have been reported to play a major role also in different infectious disorders.45–47

    Conventional therapeutic approaches for SS are mainly aimed to improve glandular function. Among commonly employed compounds, CS might represent a potential tool to affect also histological abnormalities of inflamed glands. However, apart from a case report describing infiltrate reduction in an SS subject taking CS high doses, there is no evidence of CS efficacy in reducing gland inflammation.2 ,48 ,49 In brief, it is widely accepted that CS represent a symptomatic approach in SS not able to affect the natural history of chronic sialoadenitis. Therefore, on the basis of the present findings showing a strong involvement of DN T cells in the production of IL-17 as well as their presence among glandular cell infiltrate in SS, we verified the effects of CS on IL-17-producing T cells in SS. We showed Dex capability to dramatically reduce IL-17 production of activated CD4 T cells from both healthy and SS subjects. Similar results were observed in DN T cells from controls. Noteworthy, however, Dex did not influence IL-17 production by DN T cells from SS.

    These observations appear to be particularly intriguing in the setting of the well-described CS resistance in several common inflammatory disorders.50 Poor clinical response to CS is also commonly observed in some patients with autoimmune/inflammatory rheumatic disorders, such as rheumatoid arthritis and SLE.51 ,52 Interestingly, in vitro unresponsiveness of Th17-cytokine production to Dex has been reported in the context of antigen-induced airway inflammation.53 In addition, recent findings showed that Dex is able to decrease IL-17 expression in bronchial epithelial cells from normal, but not asthmatic, subjects.54

    In conclusion, our findings support the concept that IL-17-producing DN T cells are actively involved in the pathogenesis of SS and are resistant to CS action. It is now evident that an imbalance between regulatory T (Treg) cells and Th17 cells may be of great pathogenic relevance in systemic autoimmune diseases, including SS.5 In this setting, a great deal of attention has been recently given to the concept of T-cell plasticity. According to this theory, T-cell subsets can switch from one phenotype to another. Thus, Th17 cells can convert into Th1 cells and Treg cells can become Th17. Whether pro-inflammatory DN T cells could acquire a regulatory phenotype and vice versa is still a matter of debate. We believe that the identification of in vivo activated pathogenic DN T cells, not sensitive to CS, in a systemic autoimmune disease such as SS may represent a finding of great interest. Indeed, further insight into the mechanisms of DN T-cell activation could be used to investigate what makes these cells so hyper-reactive and pathogenic at the biochemical level. This information could be used for designing specific drugs to limit pro-inflammatory DN T-cell activation or, alternatively, favour DN Treg-cell development and/or expansion.

    Acknowledgments

    SC was supported by a postdoctoral research fellowship from Regione Umbria.

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