Lymphocytic Prole and Ebv Serology in Sjögren’s Syndrome: Evidences for a Virally Triggered Autoimmune Epithelitis?

Sjögren's Syndrome (SjS) is characterized by lymphocytic inltration of exocrine glands, i.e. autoimmune epithelitis. Lymphocytes are central in SjS pathogenesis, with B-cell hyperactivity mediated by T-cells. B-cells are main targets of Epstein-Barr virus (EBV) infection, a frequently-suggested trigger for SjS. We aimed to evaluate how the EBV infection modulates B and T-cell subsets in SjS, including as controls Rheumatoid arthritis patients (RA) and healthy participants (HC). SjS patients presented decreased CXCR5 + T-cells, although IL21-secreting Tfh and Tfc cells were increased. Tfc were positively correlated with ESSDAI scores, suggesting their relevant role in SjS pathogenesis. As previously described, SjS patients showed expanded circulating naïve B-cell compartments. SjS patients had a higher incidence of EBV-EA-D-IgG + antibodies, characteristic of recent EBV-infection/reactivation. SjS patients with past infection or recent infection/reactivation showed increased CXCR3 + Th1 and CXCR3 + Tfh1 cells compared to those without active infection. SjS patients with a recent infection/reactivation prole presented increased transitional B-cells compared to patients with past infection and increased plasmablasts, compared to those without infection. Our results suggest EBV-infection contributes to B and T-cell differentiation towards the effector phenotypes typical of SjS. Local lymphocyte activation at ectopic germinal centres, mediated by Tfh and Tfc, can be EBV-driven, perpetuating autoimmune epithelitis which leads to gland destruction in SjS.


Introduction
Sjögrens' syndrome (SjS) is a chronic systemic autoimmune disease, with an estimated prevalence between 0,2-0,5% 1 , affecting predominantly middle-aged women. It is characterized by lymphocytic in ltration of the exocrine glands, referred to as autoimmune epithelitis 2 . Lachrymal and salivary glands (SG) are the most affected glands, originating the hallmark features of xerostomia and xerophthalmia. Extraglandular manifestations are common and can be caused by either lymphocytic in ltration of epithelial tissues, or immune complex disease 3 .
Lymphocytes are central in the pathogenesis of SjS 4 , and a lymphocyte pro le with increased naïve Bcells and decrease memory B-cells is typical 5 , re ecting the increased migratory pathway of differentiated B-cells into affected organs 6 . A deviation of B-cell differentiation towards plasma cells has also been described in SjS 7 .
In SjS, T-cells in ltrate affected organs, like the SG, and support hyperactivity of B-cells. 8 In fact, interactions between T-cells and activated B-cells occur in GC-like structures developed in target tissues, such as the SG. 9 Recently, follicular helper T-cells (Tfh) have been addressed as players in SjS pathogenesis. Tfh cells are a major source of interleukin (IL)-21, which mediates B-cell survival and promotes ectopic formation of germinal-centre(GC)-like structures 10 . SjS patients present increased circulating Tfh cells and expanded Tfh differentiation in the SG. 11,12 Tfh cells also express the chemokine receptor X5 (CXCR5), which induces their homing towards lymph nodes, particularly to B-cell sites. Since the expression of CXCR5 has been encountered in CD8 + T-cells, the existence of a follicular cytotoxic (CD8 + ) T-cell subset (Tfc) is now accepted, as well as their possible role in the regulation of GC B-cell responses and autoantibody production 13,14 .
The aetiology of SjS is still poorly understood, but the concept of an infectious trigger is widely spread.
Virally-triggered autoimmunity in SjS possibly results from an antigen-driven CD4 + T-cell activation. Combined with a genetic predisposition to loss of tolerance, this activation process elicits a migration of both CD4 + T-cells and B-cells towards exocrine glands, where the expansion and formation of plasma cells occurs. 8 Lymphotrophic viruses, namely Cytomegalovirus (CMV) and Epstein-Barr virus (EBV), are strong candidates for triggering the disease. 15 EBV primary infection occurs in B lymphocytes of the oropharyngeal mucosa, where lytic and latent phases of the viral cycle take place. In the active lytic phase, EBV replicates and propagates, while in the latent phase it remains inactive in B-cells. 16 Viral agents also interfere with T-cell mediated responses. 17 In chronic viral infections, helper T-cells sustain cytotoxic T-cell responses as long as viral antigens persist. 18 Moreover, the proin ammatory Th1 pro le, usually present in acute viral infections, is somehow replaced by Tfh in response to viral persistence and prolonged T-cell receptor stimulation. 19 The implication of EBV in SjS is widely accepted. Mechanisms such as molecular mimicry and genetic susceptibility to EBV infection can overlap with T-cell costimulatory overactivity, impaired EBV-speci c Tcell response, cross-reactivity of anti-EBV antibodies or inhibition of B-cell apoptosis, often associated with stimulation-driven polyclonal and monoclonal lymphoproliferation (recently reviewed by Máslínska 20 ). Nonetheless, the impact of viral infection on the typical B-cell pro le and hypergammaglobulinemia observed in SjS patients needs further clari cation. Thus, we aimed to evaluate circulating B and T-cell subsets of SjS patients and to assess their relation to the EBV background of patients.

Population
Fifty-seven SjS patients were recruited along with 20 RA patients and 24 HC. From our cohort, we assessed EBV serology in 34 SjS patients, 20 RA patients, and 20 HC. Participants' characteristics are presented in Table 1 and Supplementary Table 1.

T-cell subsets
SjS patients presented lower CD4 + T-cell percentages and absolute counts than HC (p=0.002 and p<0.0001, respectively). Accordingly, the absolute counts of CXCR5 + Tfh cells were also lower in the SjS group compared to both HC (p<0.0001) and RA patients (p=0.038). However, the percentages of IL21secreting CD4 + T-cells were increased in SjS patients when compared to both RA patients and HC (p<0.0001). Nonetheless, a positive correlation between the percentages of IL21 + CD4 + T-cells and the percentages of CXCR5 + Tfh cells (r=0.281, p=0.034) was observed.
CD8 + T-cells percentages were higher in SjS patients compared to HC (p=0.001), but not absolute counts.

B-cell subsets
Considering the IgD/CD27 classi cation, the percentages of IgD + CD27 -B-cells (naïve) were higher in SjS patients when compared to HC (p=0.028) and RA patients (p=0.043), and SjS patients also presented higher absolute counts of this subset compared to RA (p=0.015). Total memory B-cells (CD27 + IgD +/-) and unswitched memory B-cells (CD27 + IgD + ) were lower in SjS patients compared to HC (p=0.001). However, only absolute counts of switched memory B-cells (CD27 + IgD -) were lower in SjS compared to HC (p<0.001), and no differences were observed towards RA patients.
Using the Bm1-5 classi cation, B-cells were classi ed as Bm1, Bm2, Bm2', Bm3+4, eBm5, and Bm5 subsets 21 . The percentages of Bm1 cells were signi cantly lower in SjS compared to RA (p=0.005) and HC (p=0.008), and the absolute counts were also signi cantly lower in SjS (p=0.002) compared to HC. Bm2 (naïve) and Bm2' (transitional) percentages were signi cantly higher in SjS compared to RA patients (p=0.015 for Bm2 and p=0.041 for Bm2'), and Bm2' absolute counts followed the same trend (SjS vs RA; p=0.003). Lower percentages (p=0.037) and absolute values (p<0.001) of eBm5 cells were found in SjS when compared to HC. As for Bm5 cells, SjS patients presented lower percentages than RA (p=0.011), and lower absolute counts than HC (p=0.001). The results are summarized in Table 2 and Supplementary  Table 2. EBV serological markers All patients and controls were negative for anti-VCA IgM and anti-EA IgA, except for 1 HC that presented borderline levels for anti-EA IgA. All samples were positive for anti-VCA IgG, except for 2 SjS patients, who 75.0% of RA and 80.0% of HC), without signi cant differences between groups. Interestingly, for anti-EA IgG, signi cant differences were observed between SjS patients and HC (32.4% in SjS; 20.0% in RA; 5.0% in HC). The results are presented in Table 3.
SjS patients with recent infection/reactivation markers (G2) had earlier disease manifestations and shorter disease duration.
Half of the patients from group G1 and 2/3 of those in group G2 had active disease at the time of recruitment, with G1 patients showing higher ESSDAI scores than G2 patients. The lowest ESSDAI scores were observed in G3 patients, though none of the abovementioned differences reached statistical signi cance.
These data are presented in Table 4 and Supplementary Table 3.

Discussion
Our study aimed to explore the relation between the EBV serological pro le of SjS patients and the distribution of circulating B and T-lymphocyte subsets. First, we report interesting differences in follicular T-cell subsets between SjS patients and both HC and RA patients. Despite circulating CXCR5 + T cell subsets were decreased in SjS patients, functionally IL21-secreting Tfh and Tfc cells seem to be more pronounced in these patients. Tfc were even positively correlated with ESSDAI scores, suggesting their relevant role in SjS pathogenesis. Moreover, we con rmed the enriched circulating naïve B-cell compartment of SjS patients (compared to both control groups, healthy and autoimmune), previously reported in the literature 5 .
The major observation of our study, however, comes from the EBV pro le, with SjS patients presenting a greater incidence of EBV-EA-D-IgG positivity, a pro le characteristic of recent infection/reactivation of EBV infection. Furthermore, SjS patients with either serological evidence of past EBV infection or recent infection/reactivation presented higher values of CXCR3 + Th1 cells and CXCR3 + Tfh1 compared to those without serological evidence of active infection. Also, the B-cell compartment was distinctive in SjS patients with signs of recent EBV infection/reactivation: showing higher levels of transitional Bm2' cells compared to patients with past infection and increased plasmablasts, compared to patients without serological evidence of infection.
The factors underlying the onset and development of SjS are still uncertain. Nevertheless, typical immune pro les have been characterized in these patients, which can be relevant to unveil important links to other triggering players in this autoimmune disease. Despite B-cells are the main target for EBV latent infection, T-cells have also a role in this play, and have been studied in autoimmune diseases for which EBV is considered a potential trigger. For instance, EBV-speci c CD8 + T-cells are increased during B-cell transformation and in the productive viral replication phases of EBV in infected RA 22 and SLE patients 23 .
Additionally, the EBV-speci c CD8 + T-cell pool is reduced by immunosuppressive therapy 24 .
Our study supports the presence of a promoted follicular T-cell environment in SjS patients, traduced by the increased secretion of IL21 by both CD4 + and CD8 + T-cells. We found no differences in the percentages of circulating CXCR5 + follicular T-cells between groups, and absolute counts for this subset were even decreased in SjS patients, possibly due to the decreased absolute counts of CD4 + T-cells observed in these patients. Similar data had been described in the work of Brokstad 25 , which reported no differences for total CXCR5 + CD4 T-cell percentages, but only changes in particular subsets of these cells in SjS patients, such as the increase in Tfh-like ICOS + PD-1 + cells. Interestingly, in our study, another Tfhlike subset was increased in SjS patients, the IL21 + Tfh cells. Indeed, both CD4 + and CD8 + T-cells were more prone to produce IL21, the Tfh modulating cytokine. If the lower absolute numbers may indicate retention of CXCR5 + follicular T-cells at the exocrine glands, as supported by previous studies showing a T-cell predominance in lymphocytic in ltrates of these organs 26 , SjS patients seem to be predisposed to promote Tfh differentiation. Also, when naïve T-cells and salivary gland epithelial cells are co-cultured, Tfh differentiation is observed, i.e. T-cells acquire a classical Tfh phenotype and are able to secrete IL21 27 . Thus, this systemic follicular function may be overexpressed in SjS patients, also as an effect of the local altered interplay. We have previously reported that the ESSDAI score, which is a measure of disease activity in SjS, seemed to be correlated with Tfc cells levels 28 . In fact, patients with more active disease present increased circulating Tfc cells, though the causative link between these observations is still to be clari ed (i.e. whether higher levels of Tfc cells lead to increased disease severity or, on the contrary, happen in response to disease aggravation).
In addition, the increase in IL21-expressing T-cells resembles the pro le of a chronic active viral infection, as proposed by Fahey and collaborators, who showed that viral persistence redirects T-cell differentiation towards the Tfh pro le in animal models 19 . Moreover, patients with infectious mononucleosis show an increase in a particular subset of Tfh cells in peripheral blood 29 , which supports our hypothesis that viral triggers may take part in the modulation of the immune responses also in SjS patients 29 .
Interestingly, Fahey and colleagues 19 proposed that viral-induced Tfh cells deviate from an original Th1 pro le. In line with this, we observed that SjS patients with serological evidence for recent infection/reactivation presented increased Th1 and Tfh1 subsets. Thus, the autoimmune background of SjS patients could provide T-cells with alternate activation signals leading them to assume both Th1 and Tfh1 pro les under viral persistence, since it is accepted that the pathogenesis of SjS is mediated by Th1derived responses 27 .
Strikingly, the implication of Tfc cells in SjS pathogenesis is supported by their increase in patients with higher disease activity. In line with our results, serum levels of IL21 had already been associated with systemic disease activity in SjS 30 , but our results seem to highlight a role for CD8 T-cells in this scenario.
Initially, CXCR5 + CD8 T-cells were described as early effector memory CD8 T-cells present in B-cell follicles of human tonsils 13 . Recently these cells were implicated in the control of chronic viral infections 29,31,32 .
Also, associations between humoral responses and CXCR5 + CD8 T-cells 32,33 were identi ed, as these cells express co-stimulatory molecules. In fact, increased immunoglobulin production by B-cells occurs when they are co-cultured with CXCR5 + CD8 T-cells, suggesting these cells have other immune functions besides cytotoxic activities 33 . Considering that dysregulated humoral responses are present in SjS, Tfc cells, along with Tfh cells, may induce the atypical antibody production of SjS patients. Nevertheless, the major function of the follicular CD8 + T-cells may still be limiting the replication of viral agents in B-cell follicles, as these cells show increased cytotoxic capacities 34 .
As for B-cells, it is accepted they are EBV's main target 35 . Several studies tried to relate SjS pathogenesis with a speci c clonality of B-cells. One of the hallmarks of Sjögren's syndrome is, in fact, the formation of ectopic lymphoid structures (ELS) in the SG. ELS are composed of B-cell/T-cell follicles, supported by networks of stromal follicular dendritic cells, which support ectopic GC reactions 36 . Active EBV infection has been associated with ELS in the SG of SjS patients and appears to contribute to local growth and differentiation of disease-speci c autoreactive B-cells 37 . Despite the possibility of an EBV-triggered B-cell proliferation in SjS, EBV-infected memory B-cells were found to express lower levels of self-and polyreactive antibodies than their uninfected counterparts 38 .
As corroborated by our data, SjS patients present a typical circulating B-cell compartment, enriched in transitional/naïve subsets, in opposition to memory subsets. If we consider the observations from Coleman and colleagues on the effect of EBV in B-cells 39 , we may also suggest a possible role for EBV in the alterations observed in the B-cell compartment of SjS patients. In fact, these authors have recognized that murine transitional B-cells from the spleen can be reservoirs for gammaherpesvirus like EBV, which can remain latent in these cells, prolonging their life span inde nitely. Our results are in line with this hypothesis, as SjS patients with serological evidence of recent infection/reinfection presented higher percentages of transitional Bm2'. Whether this is an effect of EBV or other concomitant viral infection, remains to be elucidated. We acknowledge that the assessment of the viral genome in different B-cell subsets could clarify this idea.
Regarding the serological EBV markers, we found an increased prevalence of Anti-EBV EA-D IgG in SjS patients, compared to both RA and HC, as described in previous works 37,40 . The anti-EBV EA-D IgG prevalence in our SjS patients (about 33% vs 5% in HC) was very close to the one reported by Pasoto and colleagues 41 (36% vs 4.5% in HC), which strengths our data, and led us to further assess the immune compartments according to the EBV serological pro le of SjS patients. Interestingly, patients with evidence of EBV infection, and particularly those with recent infection/reactivation (EA-D IgG positive) had earlier disease manifestations, but also a distinct immune pro le, with a shift towards proin ammatory Th1/Tfh1 subsets in the T-cell compartment. Furthermore, SjS patients with evidence of recent infection/reactivation exhibited higher levels of transitional B-cells and plasmablasts, which may traduce the importance of EBV in the modulation of the immune responses in SjS patients, with possible clinical impact, as sustained by precocious clinical manifestations.
To our knowledge, our study is the rst to report an association between EBV serological patterns and the immune pro le of SjS patients, despite EBV EA (early antigen) had already been correlated with autoantibodies production 40 . Although we observed no further differences in the clinical manifestations of SjS patients according to their EBV serology, as reported by Pasoto and colleagues, who reported more frequent joint involvement in anti-EBV EA IgGs positive patients 41 , we were able to identify distinct immune pro les according to the EBV serological pattern. Still, we must recognize the low number of patients considered and the absence of other con rmatory methodologies for the effective viral infection. Nonetheless, our data support the idea that different EBV serological pro les affect circulating B and Tcells in SjS patients. In fact, other authors have supported the hypothesis that reactivation in the lytic phase of EBV infection promotes immunological dysfunction in SjS 37 . Considering our results, we also believe special attention should be given to the group of SjS patients with serological evidence for recent infection/reactivation, which present a pro-in ammatory pro le, with increased Th1/Tfh1 ratio cells along with elevated transitional B-cells and increased plasmablast differentiation.
We acknowledge limitations in our study, such as the absence of standard molecular biology assays to con rm EBV infection. In future studies, it would be relevant to assess not only serology, but also EBV viral load, and eventually other viruses with potential impact in SjS development, such as CMV. Also, our study was performed exclusively in peripheral blood, and we realize it may not properly re ect the numbers and interactions of immune cells at exocrine glands. For instance, SG biopsies would not only clarify the hypotheses on cell tra c between affected organs and the circulating lymphocyte pool but would also allow us to prove the presence of EBV in such organs.
Nevertheless, from our results, it is possible to suggest that EBV plays a role in inducing B and T-cells towards an effector phenotype. EBV enters the replicating phase in the exocrine glands, where this facilitated interaction between EBV antigens and effector T-cells might lead to a breakdown of tolerance. The ensuing autoimmune response mediated by effector B and T-cells might lead to a localized lymphocyte activation with the formation of ectopic GC or GC-like structures. This process, mediated by Tfh and Tfc, can thus perpetuate the autoimmune epithelitis and result in gland destruction.
Our work provides a new perspective on how EBV might be involved in lymphocytic alterations known to be a feature in SjS. Clarifying the role of follicular CD4 and CD8 T-cells in the context of viral infection can be of great value in con rming a viral-triggered autoimmune response in SjS, but a speci c strategy for the characterization of these cells in peripheral blood and target organs is still needed.
Our study can also constitute a starting point for approaching the role of CXCR5 + and IL21 + CD8 T-cells in the context of autoimmunity. The association between CXCR5 + CD8 T-cells and disease activity in SjS observed in our study may be an indicator of their involvement in the pathophysiology of autoimmune epithelitis. In the current scenario where Tfc cells involvement in autoimmune pathologies is yet to be elucidated, our study pioneers the association of Tfc cells with human autoimmunity and paves the way for further studies regarding Tfc cells in autoimmune diseases.  44 . Clinically active disease was de ned as activity in any ESSDAI domain, except the hematologic and biologic.
The healthy control group (HC) consisted of women without symptoms or signs of xerostomia or xerophthalmia, or any history of autoimmune rheumatic diseases, selected from the Ophthalmology outpatient clinic of Hospital CUF Descobertas.
Informed consent was obtained from all participants. The study was approved by the Ethics committees of both recruiting institutions, and NOVA Medical School Ethics Committee (no.17/2016/CEFCM).

Flow Cytometry Procedures
For the immunophenotyping protocols, peripheral blood samples collected in EDTA-coated tubes were processed and analyzed within 24h of collection. A pre-validated panel of monoclonal antibodies (mAbs) was used for the characterization of T and B-cell subsets, including CD3, CD4, CD8, CD19, CD24, CD27, CD38, CCR6, CCR7, CXCR3, CXCR5, Anti-IgD, and Anti-IgM. A lyse-wash protocol was performed for both T and B-cell characterization. A lyse-no wash single platform strategy with BD Trucount tubes (BD Biosciences, San Diego CA, USA) was used to obtain absolute counts of all cell subsets. All samples were acquired in a 4-color BD FACS-Calibur cytometer (BD Biosciences).
CellQuest Pro™ (BD Biosciences) software was used for acquisition and analysis purposes, and In nicyt™ 2.0 (Cytognos S.L., Salamanca, Spain) software was also used for more differentiated subset analysis.
Whenever appropriate, uorescence-minus-one control tubes were prepared to assess the positivity of dimer expressions. The subsets analyzed, and the respective gating strategies, are displayed in gure 1.
Within T-cells, we characterized CD4 + and CD8 + (CD4-) subsets, including CXCR5 + Tfh and Tfc cells, and the Tfh1 and Tfh17 pro les, according to the expression of CXCR3 and CCR6, respectively. B-cells' subsets were addressed according to the classical IgD/CD27 classi cation, and the Bm1-5 classi cation, often used in autoimmunity settings 21 .
Functional assays for the evaluation of IL21 production by T-cells Heparinized peripheral blood samples were used to assess IL21 and IL-17 production by CD4 + and CD8 + T-cells.
In brief, cells were stimulated with PMA and ionomycin, for 5h at 37ºC in a 5% CO 2 atmosphere in the presence of brefeldin-A. After stimulation, cells were lysed, washed and incubated with anti-CD3 and anti-CD8 mAbs for surface staining. For intracellular stain, cells were treated according to the protocol de ned by the manufacturer for the BD Fixation/Permeabilization Solution Kit with BD GolgiPlug™ (BD Biosciences) and then marked with anti-IL21 and anti-IL-17 mAbs, after cell xation and permeabilization. For each patient, stimulated and unstimulated tubes were run in parallel to assure proper stimulation and staining controls ( gure 2).

EBV serological markers
Enzyme-linked immunosorbent assays (ELISA) were used for the assessment of IgG, IgA and IgM antibodies (Abs) against EBV antigens (Ags). All ELISA kits were obtained from Euroimmun (Euroimmun, Luebeck, Germany) and used according to the manufacturers' instructions. The following Abs for EBV Ags were determined: IgG for diffuse early Ag (EA-D), IgG for viral capsid Ag (VCA), IgG for nuclear Ag-1 (EBNA1), IgA for EA-D, IgA for VCA and IgM for VCA. All tests for IgG Abs were quantitative, while IgA and IgM were semiquantitative. In quantitative assays, sample concentration was determined using 3-point calibration curves constructed with ELISA-Logit software, available at https://ednieuw.home.xs4all.nl/Calibration/Logit/Logit.htm (V24May2017). The cut-off level for all IgG antibodies assayed was 20 RU/ml. For semiquantitative assessments, a single calibrator was determined in triplicate per assay. The ratio sample/calibrator was used to assess positivity levels (Negative: ratio<0.8; Borderline: ratio≥0.8 to <1.1; Positive: ratio≥1.1). Patients were randomly assigned to undergo EBV serology evaluation.

Statistics
Graph Pad Prism™ 6.0 (Graph Pad Software, San Diego, CA, USA) was used for statistical analysis. The normality of data sets was assessed using D'Agostino & Pearson omnibus and Shapiro-Wilk normality tests. ANOVA and Kruskal-Wallis tests were made for multiple analyses among groups, followed by Dunn's multiple comparisons test. When a signi cant difference was found, comparisons were done using Unpaired Student's t-test with Welch's correction or Mann-Whitney test, for every two groups. For categorical variables, Fischer's or Chi-square tests were applied to assess differences between groups. Statistical signi cance was considered for p-values <0.05. All of the authors contributed to data analysis and interpretation. FB and RM drafted the manuscript, and all of the authors revised it and contributed to it intellectually. All of the authors have approved the nal version of the manuscript.

Competing Interests
The authors declare no competing nancial and non-nancial interests.