Cytomegalovirus aggravates the autoimmune phenomenon in systemic autoimmune diseases

Accepted Manuscript Cytomegalovirus aggravates the autoimmune phenomenon in systemic autoimmune diseases Essam Mohammed Ahmed Janahi, Shukla Das, Sambit Nath Bhattacharya, Shafiul Haque, Naseem Akhter, Arshad Jawed, Mohd Wahid, Raju Kumar Mandal, Mohtashim Lohani, Mohammed Yahya Areeshi, Vishnampettai G. Ramachandran, Shaia Almalki, Sajad Ahmad Dar PII:

S0882-4010(18)30478-9

DOI:

10.1016/j.micpath.2018.04.041

Reference:

YMPAT 2920

To appear in:

Microbial Pathogenesis

Received Date: 15 March 2018 Revised Date:

17 April 2018

Accepted Date: 23 April 2018

Please cite this article as: Ahmed Janahi EM, Das S, Bhattacharya SN, Haque S, Akhter N, Jawed A, Wahid M, Mandal RK, Lohani M, Areeshi MY, Ramachandran VG, Almalki S, Ahmad Dar S, Cytomegalovirus aggravates the autoimmune phenomenon in systemic autoimmune diseases, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.04.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Cytomegalovirus aggravates the autoimmune phenomenon in systemic autoimmune diseases Authors in order of their authorship:

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Essam Mohammed Ahmed Janahia, Shukla Dasb,**, Sambit Nath Bhattacharyac, Shafiul Haqued, Naseem Akhtere, Arshad Jawedd, Mohd Wahidd, Raju Kumar Mandald, Mohtashim Lohanif, Mohammed Yahya Areeshid, Vishnampettai G. Ramachandranb,

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Shaia Almalkie, Sajad Ahmad Darb,d,* Authors Affiliations:

Department of Biology, College of Science, University of Bahrain, Sakhir, Kingdom of

Bahrain;

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a

b

Department of Microbiology, University College of Medical Sciences

(University of Delhi) & Guru Teg Bahadur Hospital, Delhi, India; cDepartment of Dermatology, University College of Medical Sciences (University of Delhi) & Guru Teg

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Bahadur Hospital, Delhi, India; dResearch and Scientific Studies Unit, College of Nursing & Allied Health Sciences, University of Jazan, Jazan, Saudi Arabia; eDepartment of Laboratory Medicine, Faculty of Applied Medical Sciences, Albaha University, Albaha,

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Saudi Arabia; fDepartment of EMS, College of Applied Medical Sciences, University of Jazan, Jazan, Saudi Arabia *

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Corresponding Author:

Sajad Ahmad Dar ([email protected]; [email protected]) Tel: +966-173174383 **

Co-corresponding Author:

Shukla Das ([email protected]) Running head: CMV aggravates systemic autoimmune diseases

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Abstract

Background: Human Cytomegalovirus (CMV), because of its ability to extensively

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manipulate host immunity during active infection, has been suggested to be involved in autoimmunity. However, its influence on T-cells and cytokines in systemic autoimmune diseases like systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) is

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indistinct.

Methods: We investigated the in-vitro response of T lymphocytes from SLE and SSc

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patients to CMV antigen. Functional activity of T lymphocytes was determined by estimating Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines. Results: We observed that CMV antigen stimulation in-vitro resulted in significant increase in CD4:CD8 T-cell ratio in peripheral blood mononuclear cells (PBMCs) from SLE and SSc patients; response dominated by CD4+ than CD8+ memory T-cells. SSc T-

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cell response was differentiated by aberrant increase in CD4+CD25+ T-cells. CMV antigen caused elevation in IL-4 and IFN-γ production in both patient PBMCs, whereas

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IL-2 was also raised in SLE PBMCs. The development of large pool of memory T-cells and overproduction of IFN-γ may result in flare-up of autoimmunity in these patients.

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Conclusion: Our study provides an insight into the immunopathological potential of CMV-reactive immune cells to develop new potential strategies for targeted therapeutic intervention.

Keywords: Cytomegalovirus; Autoimmunity; T cells; Cytokines; Systemic sclerosis; Systemic lupus erythematosus

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1. Introduction Systemic autoimmune diseases (SADs) are a heterogeneous group of immunologically mediated inflammatory disorders including multi-organ involvement. They can affect

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almost every tissue in the human body and are usually clinically serious with many complications. Two typical SADs which have been shown to share multiple genetic susceptibility loci and clinical features are systemic sclerosis (SSc) and systemic lupus

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erythematosus (SLE) [1, 2]. These two SADs share several similarities such as autoantibodies directed against nuclear antigens and overlapping clinical features.

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Autoimmune diseases are heterogeneous in nature with several intricate genetic variants and environmental triggers determining their specific manifestations [3]. Among the environmental factors, infection with human cytomegalovirus (CMV), a herpes virus, has been a matter of recent debate. In presence of encouraging genetic background, CMV

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which is normally innocuous can trigger autoimmunity. However, majority of the research in this area has been speculative.

CMV is a ubiquitous pathogen that infects 60 to 90% of the population globally. After a

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primary infection, CMV resides in latently infected monocytes or premonocytic cells, and reactivation often driven by inflammation may occur periodically [4]. The reactivation by

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an initial inflammatory insult may sustain and exacerbate inflammatory processes by the production of type I cytokines and by the specific mechanisms that induce inflammation and autoimmune reactions [5, 6]. Emerging evidence suggests that CMV proteins are common in tissues affected by autoimmunity and that both molecular mimicry and viral antigens may sustain autoimmune reactions.

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CMV RNA has been detected in endothelial cells in skin biopsies from patients with autoimmune sclerosis, and CMV infection has been associated with higher disease activity scores in SLE patients [4, 7, 8]. CMV may induce the vascular, fibrotic, and

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immunologic features of SSc through effects on various cell types [9, 10]. Autoantibodies specific to SSc are reactive to CMV antigen and induce apoptosis of endothelial cells [9], and activate cultured human fibroblasts [11]. Highly prevalent CMV-specific antibodies

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in SLE patients recognize nuclear structures and double-stranded DNA [12].

Infection with CMV has been implicated in both the development and progression of

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systemic autoimmunity in genetically susceptible hosts [4, 8, 12-14]. Mutations in tolllike receptor (TLR) 3 have been associated with severe CMV infection and hematopoietic autoimmune disorders among others [15]. Hyperfunctional phenotype with regard to high IFN-γ levels and low CMV-specific T cells connects to functions of TLR and, thereby,

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genetic susceptibility of CMV-induced pathologies in SLE [12]. Sustenance of CMV infection due to defective immunity in conjunction with hyperfunctional TLR phenotypes may enhance the risk of autoimmunity [12]. These findings suggest that genetic and

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immunologic defects may result in enhanced CMV replication in SLE which can trigger autoimmune phenomena. Several other studies linking CMV to SSc predisposition are of

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the opinion that specific immune reactions leads to development of the disease [16, 17]. Despite the evidence noted above, and the widely recognized clinical importance of CMV, the role of CMV in triggering, exacerbating or aggravating autoimmunity in SADs, particularly SLE and SSc, is obscure. Presence of autoantibodies and autoreactive T cells are typically associated with autoimmune diseases. Although, successful control of CMV infection in a healthy

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immune system involves anti-CMV antibodies for controlling dissemination, and interferons, natural killer cells and T cells for controlling immunity against primary and latent CMV, however, CMV still alters certain specific immune cell populations [18]. A

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hallmark of CMV infection is CD8 T cells specific for a few viral-derived epitopes [18]. Terminally differentiated effector memory cells exhibiting CD4+/CD28- phenotype, specific for CMV, are also observed in higher frequency in infected subjects [19], in

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addition to expansion of specific NK cell subset [20]. This suggests that CMV reprograms infected cells to express numerous regulator molecules for controlling

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different cellular processes including antigen presentation. However, till date the effect of CMV interference with T cell responses in systemic autoimmunity is not clear. Substantial research is required to get a better understanding of the etiopathogenic role of persistent CMV infection in triggering or aggravating these IFN-γ mediated SADs, and to

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improve the targeted therapeutics for their treatment. Keeping in view the reported similarities between SLE and SSc, a direct comparison of the T cell and cytokine profiles is warranted. We in this study investigated the in-vitro response of T lymphocytes from

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SLE and SSc patients to CMV antigen. Functional activity of T lymphocytes was also determined by estimating Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines

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released in culture supernatants.

2. Materials and Methods 2.1. Subject enrollment: Patients clinically diagnosed to have SLE (n=13) and SSc (n=20) and supported by well accepted laboratory investigations/criteria, attending the department of Dermatology at University College of Medical Sciences and Guru Teg

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Bahadur (UCMS & GTB) Hospital Delhi, were enrolled in the study. The American College of Rheumatology (ACR) revised criteria for classification of SLE [21], and the 2013 classification criteria for SSc by ACR/European league against rheumatism [22],

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were followed for diagnosis of the diseases. The protocol items specific for SLE and SSc were determined by established conventions. Clinical assessment was performed by expert dermatologists to establish a baseline index for these conditions. Seropositivity in

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each case (for SLE – positive reaction for antinuclear, anti-dsDNA, anti-smith, or antiphospholipid antibodies; for SSc – detection of anti-topoisomerase I, anti-centromere, or

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anti-RNA polymerase III antibodies) was confirmed by performing relevant assays following manufacturer’s instructions. All the cases were either newly diagnosed for the first time or were not on any steroids/immunosuppressive drugs/other therapeutic agents for at least 6 months prior to enrolment. Healthy volunteers (n=20) not known to suffer

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from any illness were also enrolled for comparisons. The study was approved by the Institutional Ethical Committee-Human Research of UCMS & GTB Hospital, Delhi and written informed consent was obtained from the subjects before enrollment in the study.

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2.2. Sample collection and processing: Peripheral venous blood (~7ml) was collected aseptically from each patient and used for isolation of serum and peripheral blood

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mononuclear cells (PBMCs). For isolation of serum, 2ml of blood was allowed to clot at room temperature for 15 to 30 minutes immediately after collection. The clot was removed by centrifugation at 3000-5000 rpm for 5 minutes in a refrigerated centrifuge. The supernatant was isolated and stored in aliquots of 300µls at -20°C until use. Isolation of PBMCs was done according to a modification of the method of Boyum, 1968 [23]. Briefly, the blood samples (5ml) were diluted 1:1 with Hank’s Balanced Salt

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Solution (HBSS) and slowly layered over equal volume of HiSep LSM (Hi-Media Laboratories Pvt. Ltd., India) at room temperature (RT) in sterile 50 ml conical centrifuge tubes. The centrifuge tube was held at 45° and sample allowed to run down on its side.

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The tubes were centrifuged at 500 x g for 30 minutes at room temperature. The cloudy layer at the interface was carefully aspirated and washed twice with HBSS and once with RPMI 1640 (Hi-Media Laboratories). During washing, centrifugation was carried out at

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250 x g for 10 minutes at RT. The viability of the cells was measured by a hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay;

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values were consistently greater than 95 percent. PBMCs obtained were re-suspended at a final concentration of 1x106 cells/ml in RPMI 1640 media supplemented with 10 percent fetal bovine serum (FBS), 100 IU penicillin/ml, 100µg streptomycin/ml, and 2mM Lglutamine (Hi-Media Laboratories).

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2.3. In-vitro stimulation of PBMCs: PBMCs (1x106 cells/ml) in RPMI 1640 were seeded into wells of a 12-well cell culture plate (Nalgene Nunc, Rochester, NY), precoated with 1ml of anti-human CD3/CD28 antibody (BD Biosciences, Gurgaon, India) at

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a concentration of 1µg/ml in phosphate buffered saline (PBS) for 18 hours at 37ºC. The cells were treated with CMV antigen (Microbix Biosystems, Inc., Canada) at 1/100

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dilution of 3.4mg/ml antigen, which was found to be the optimal concentration for generating best possible T cell responses on the basis of preliminary experiments. Antigen was first added into the wells followed by purified PBMCs at 1x106 cells/ml in RPMI 1640. All the antigen treatments were done in triplicate and cells were incubated at 37°C in humidified air containing 5 percent CO2 for 72 hrs. Subsequent to incubation, the PBMCs were harvested by centrifugation at 500xg for 5 minutes and the cell-free culture

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supernatants were removed and frozen at -80°C for cytokine estimation through ELISA. Centrifuged PBMCs were washed and used for staining. 2.4. PBMC staining and Fluorescence-activated cell sorting (FACS): Harvested

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PBMCs were washed three times with wash buffer (0.5% BSA + 0.1 percent NaN3 in 1X PBS [pH7.4]) and stained with conjugated monoclonal antibodies (mAbs; BD Biosciences, Gurgaon, India). Each mAb was used at appropriate concentration, worked

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out previously through titration, in different combinations - (a) anti-human CD3 peridinchlorophyll (PerCP) + CD4 fluorescein isothiocyanate (FITC) + CD8 phycoerythrin (PE)

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+ CD45RA allophycocyanin (APC); (b) anti-human CD3 PerCP + CD4 FITC + CD8 PE + CD45RO APC; and (c) anti-human CD4 FITC + CD25 APC. Staining was done on ice for 30 -60 minutes in the dark after which cells were washed thrice with wash buffer, fixed with 300µl ice-cold 2 percent paraformaldehyde in 1X PBS and stored at 4ºC till

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analysis. Flow cytometry was performed within 18hrs on a BD FACSCaliburTM system (BD Biosciences, Gurgaon, India). A minimum of 20,000-30,000 events was collected per condition. All data were analyzed using WinMDI 2.9 software. Each experiment

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included isotype-matched control antibodies to establish the specificity of antibody binding on an identically gated set of cells.

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2.5. ELISA for cytokine estimation: Enzyme-linked immunosorbent assays (ELISA) kits for estimating IL-2, IL-4, IL-10, and IFN-γ cytokines were obtained from BD Biosciences, Gurgaon, India. Levels of all these cytokines were estimated in cell-free culture supernatants following manufacturer’s instructions. Briefly, microtitre plate wells were coated overnight at 4°C with capture antibody (100µl per well) for IL-2, IL-4, IL10, and IFN-γ. After washing, the wells were blocked with assay diluent (≥200µl/well)

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for 1 hour at room temperature. The plates were washed and various sample and standard dilutions (100µl each) were added to different wells and plates incubated at room temperature for 2 hours. After aspiration and washing, 100µl of biotinylated anti-human

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IL-2, IL-4, IL-10, and IFN-γ mAb plus streptavidin-horseradish peroxidase conjugate was added to each well for 1 hour, followed by 100µl of substrate solution (tetramethylbenzidine and hydrogen peroxide) for 30 minutes. The reaction was stopped

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by adding 50µl of 2N H2SO4 to each well and absorbance was read at 450nm within 30 minutes. Concentrations of IL-2, IL-4, IL-10 and IFN-γ in samples were determined by

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plotting the standard curves with concentration versus absorbance. For diluted samples, concentration was multiplied by the dilution factor. The lower level of detection of all the kits was ≥ 4 pg/ml.

2.6. Post-therapy enrollment of patients: Immunosuppressive therapy was initiated for

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each patient after inclusion into the study, by concerned specialists, as per standard protocol/accepted regimen suitable for his or her clinical condition. Both SLE and SSc patients

mainly

received

immunosuppressive

therapy

with

corticosteroids.

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Mycophenolate mofetil, cyclophosphamide, methotrexate, azathioprine, and cyclosporine were alternative add on steroid sparing disease modifying agents used. The therapeutic

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regimen was documented as also the clinical outcomes at follow-up. Six months after induction of clinical remission/disease inactivity, as determined by clinical as well as laboratory assessment, repeat blood samples were collected from each patient for isolation of serum and PBMCs. All the assays done at the time of initial enrollment (pretherapy) were repeated to measure any differences in T cell responses to CMV antigen stimulation. One of the SLE and 3 of the SSc patients were lost to follow-up.

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2.7. Data analysis: The two-way analysis of variance (ANOVA) was used, followed by Tukey’s test, to analyze the variations between percentages of different T cell subpopulations and cytokines between patients and healthy controls. The level of

IL, USA) was used to perform all the statistical analyses.

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3. Results

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significance was maintained as p<0.05. The SPSS 16.0 for windows (SPSS Inc., Chicago,

3.1. Patient profile: All the SLE (n = 13) and SSc (n = 20) patients enrolled in the study

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fulfilled the American College of Rheumatology classification criteria for these diseases. Most of the SLE patients (65-70%) had mucocutaneous lesions and non-erosive polyarthritis. Twenty five percent of the SLE patients were nephropathic, 15% had neuropsychiatric disorder, and 10.5% presented with anti-phospholipid syndrome.

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Among the SSc patients, 95% showed Raynaud phenomenon, 90% presented with skin sclerosis and pigmentation. Sixty five percent of the SSc patients had finger contracture, 60% had digital ulceration, 55% dyspnea, 50% mouth opening restriction, 40% joint

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complaint, and 30% dysphagia. The mean Rodnan skin score of SSc patients was 27.4±4.31 with a range of 9 to 51. Four of the SSc patients were CRP positive (>6

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mg/dl), 4 were ASO positive (>200 IU/ml), and 10 were RF positive (>20 IU/ml). All the patients were receiving treatment at the University College of Medical Sciences & GTB Hospital, Delhi, India. Healthy volunteers (n = 20) had no signs and symptoms of any autoimmune disease. The profile and characteristics of patients and healthy volunteers is given in Table 1.

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3.2. Changes in T cell subpopulations on CMV antigen stimulation in-vitro: Stimulation of PBMCs with CMV antigen showed a significant increase in CD4+:CD8+ ratio in both SLE and SSc patients when compared with healthy controls (Figure 1A). We

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found that higher CD4+ T cell response to CMV antigen stimulation was dominated by memory CD4+ T cells (CD4+CD45RO+) in both groups of patients (Figure 1B). However, a decreased CD8+ T cell response in patient PBMCs as compared to control PBMCs was

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conspicuous. Subgroup analysis of CD8+ T cells for naïve (CD8+CD45RA+) and memory (CD8+CD45RO+) cells revealed a significantly decreased naïve but increased memory

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CD8+ T cell response in SSc (Figure 1C). The increase in memory component of CD8+ T cells was lesser as compared to increase in memory CD4+ T cells in SLE. In case of healthy controls, a significant number of CD4+ T cells showed a revertant phenotype with high-level expression of CD45RA (data not shown).

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We found a significant decrease in expression of CD25 on CD4+ T cells in SLE patients, however, an unusual increase in this T cell subpopulation was observed in SSc patients, both in unstimulated and CMV stimulated conditions, compared to healthy controls.

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Healthy control PBMCs depicted a significant increase in CD4+CD25+ T cells in response to CMV antigen stimulation (Figure 2A).

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Post-therapy response of T cells to in-vitro CMV antigen stimulation, after six months of immunosuppressive therapy given to these patients, was relatively the same as pretherapy except an increase in SLE, and a decrease in SSc, of CD4+CD25+ T cell subpopulation (Figure 2B). 3.3. Changes in expression of IL-2, IL-4, IL-10, and IFN-γ on in-vitro stimulation with CMV antigen: Unstimulated PBMCs from SLE patients produced significantly

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lower levels of IL-2 and IFN-γ than cells from healthy controls. However, IL-4 level was similar to controls but IL-10 was found to be higher (Table 2). SSc patient PBMCs on the other hand secreted higher levels of IL-4 and IL-10 but lower levels of IFN-γ when

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unstimulated, with no significant differences in IL-2 secretion (Table 3). A comparison between the SLE and SSc patients demonstrates that both the groups produce higher IL10 but lower IFN-γ compared to healthy controls. Also, IL-2 was reduced in SLE and IL-

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4 was elevated in SSc (Figure 3).

In-vitro CMV antigen stimulation resulted in an enormous increase in IL-2 and IFN-γ

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expression in SLE (Table 2), but only IFN-γ was elevated in SSc patients (Table 3), relative to baseline level among unstimulated counterpart cells. The increase in IFN-γ secretion in SLE PBMCs with was the highest. Health control PBMCs showed either no significant change or a decrease in production of these cytokines upon CMV stimulation

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(Table 2 & 3).

Both IL-4 and IL-10 cytokines showed high expression in SLE PBMCs induced by CMV antigen. Increase in IL-4 secretion was higher but IL-10 was lower comparative to

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healthy controls (Table 2). In contrast SSc patient PBMCs showed elevation only in IL-4 expression, more significantly than control PBMCs (Table 3).

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Post-therapy, unstimulated SLE patient PBMCs depicted a decline in secretion of IL-2, IL-10 and IFN-γ. A further decrease in production of IFN-γ was observed, with no significant change in other cytokines, upon CMV antigen stimulation in these patients (Table 2). Unstimulated SSc patient PBMCs also showed reduction in expression of IL-2 and IL-10 post-therapy, but an elevation in IFN-γ levels. Similar to SLE PBMCs, IFN-γ

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expression was significant decreased in SSc PBMCs when stimulated with CMV antigen post-therapy (Table 3).

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4. Discussion

Association of CMV infection with autoimmune pathologies has been debated for long but absence of a clear link has remained conspicuous. Most of the studies done in

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European population report a link between CMV seroprevalence and autoimmune diseases [7, 8, 24-27], however, such association in other studied and other populations

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has remained ambiguous [28-31]. Furthermore, systemic CMV infection could lead to the activation of CD4+ T cells [32, 33] which can cross-react via molecular mimicry and lead to autoimmunity [34]. We in this study present a direct comparison of the possible role played by CMV in dysregulating T cell response and cytokine secretion in patients of

similarities [35-38].

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SLE and SSc, the two important systemic autoimmune diseases with many possible

We observed a high proliferation of CD4+CD45RO+ among CD4+ and CD8+CD45RO+

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among CD8+ T cells, with a higher CD4:CD8 ratio, upon in-vitro CMV antigen stimulation in both SLE and SSc patient PBMCs. The CD45RO isoform is a marker of

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antigen experience [39, 40]. It is possible that the development of such large memory T cell expansions in patient PBMCs may be associated with previous CMV exposure causing an impairment of the immune response, by weakening the number of naıve T cells, particularly within the CD4+ T cell repertoire. The onset of autoimmune disorders has been linked with the time course of active CMV infection in previously healthy individuals. Notably, the presence of CMV replication has been associated with the

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development of autoimmune vasculitis, SLE and SSc or Scleroderma, implicating the virus as a trigger or exacerbator of autoimmunity [41-44]. Lot of inconsistency is existent in studies reporting frequency of CD4+CD25+ regulatory

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T cells (Tregs) in autoimmune diseases like SLE, SSc and RA as reviewed by Michelsvan Amelsfort, et al. in 2011 [45]. Functionally suppressive Tregs have been found to exist in SLE patients but persistent inflammatory cytokine environment has been

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associated with the failure of these cells to control disease progression [46, 47]. SSc patients instead have mostly been reported to have increased amounts of CD4+CD25+ T

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cells which are not effective and are lacking in immunosuppressive activity [48]. In the present study, we observed an unusual increase in CD4+CD25+ T cells of SSc patients, but a decrease in SLE patients, both in unstimulated and CMV stimulated conditions. This is in agreement with the previous findings reporting a decreased percentage of

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CD4+CD25+ T cells in SLE [49, 50] and an increased percentage in SSc [48]. Stimulation with CMV did not alter significantly the CD4+CD25+ T cells in SLE but increased their frequency nearly two-fold in SSc. This indicates that CMV may be a culprit for causing

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substantial alterations in Tregs in these systemic autoimmune diseases. High production of IFN-γ by CMV antigen activated PBMCs of both SLE and SSc

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patients were conspicuous in our study. SLE and SSc have been reported to belong to the same spectrum of IFN-γ mediated diseases [38]. The overproduction of IFN-γ coincides with the disease flare in SLE patients [51], but has a protective effect. This may lead to the release of IL-2 by autoaggressive T cells, thereby resulting in autoimmunity [52]. SSc patient PBMCs activated by CMV antigen also showed a high secretion of IL-4 in comparison to control PBMCs. There is abundant evidence which suggests a critical role

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for activated T cells in the pathogenesis of SSc. In fact, several cytokines secreted by activated T cells may contribute to modulate fibrosis and promote vascular damage [5355].

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Elevated level of IL-10 has been associated with various autoimmune diseases and this cytokine plays an important role in autoimmune pathogenesis [56-59]. Both SLE and SSc patient PBMCs produced higher IL-10 in unstimulated state, in comparison to healthy

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controls, but its secretion decreased when PBMCs were stimulated with CMV antigen. Because of immune dysregulation in autoimmune patients, CMV appears to keep IL-10

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production in check for its own advantage [60], which may be aided by suppression of NK cell function [61]. Raised production of IL-10 may play a protective role in autoimmunity [57, 62]. CMV driven cascade of immunological events (typical alterations in T cells and cytokines) could thus be a triggering or exacerbating factor of

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autoimmunity in genetically predisposed and infected patients.

Poor immune control of CMV infection may lead to sustained periods of infectivity, thereby enhancing the risk of autoimmunity in genetically susceptible persons, perhaps

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especially in patients who develop CMV-specific antibodies that recognize nuclear structures and double-stranded DNA, which are highly prevalent in these patients [12].

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CMV is known to incorporate host cell RNA in its virion [63]. RNA is not normally free in circulation but its access to immune system can only occur after cell lysis. As latent CMV is not lytic, reactivation of the virus may cause the virus bound host cell RNA, and RNA-protein complex to function as better antigens. These events may be responsible for dysregulation of T cell response and cytokine production, thereby, playing a role in development of autoimmunity in SLE and SSc. However, the remarkable complexity of

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pathogenesis of autoimmune disorders suggests that no single trigger or genetic factor by itself is likely to be responsible for their development. Microbial products like CMV antigens could synergize with other environmental and genetic factors, predisposing to

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autoimmunity development.

Post six months of immunosuppressive therapy we observed a decreased CD4:CD8 T cell ratio in unstimulated SLE and SSc patient PBMCs. Depletion of CD4:CD8 T cell ratio in

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patient PBMCs, in conjunction with augmentation of CD4+CD45RO+ T cells, post immunosuppressive therapy may indicate diminished autoimmune response but activated

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CMV-specific response in these patients. Insignificant alterations observed in response of different T cell subpopulations and cytokines to CMV antigen stimulation post-therapy indicates that steroid delivered global suppression of active immune cells cannot be a permanent solution. Patients treated with only immunosuppressive/steroid therapy are at

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high risk for CMV reactivation and thereby triggering disease exacerbations or flares. However, post-therapeutic increase in CD4+CD25+ T cell subpopulation in SLE, and a decrease in these cells in SSc, may be attributed to corticosteroid(s) treatment given to

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these patients [64]. Although, further investigations are required to elucidate the corticosteroid effect on the Treg cell suppressor activity. Despite all the discrepancies

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observed in frequencies of Tregs in systemic autoimmune patients, these cells hold a promising potential for development of future effective therapy [64]. In summary, the rarity of systemic autoimmune diseases and the heterogeneity of their clinical presentation have undermined the power of previous interventional studies to reach conclusive evidence regarding treatment efficacy [65]. Hence, selective immunological tuning targeting infectious agents like CMV and discreet containment of

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activity of overzealous cells is required to contain these systemic autoimmune disorders. Our study provides a clue towards this direction. Further studies are warranted to validate these observations.

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5. Conclusions

Autoimmune diseases like SLE and SSc are characterized by the development of large pool of memory T cells and the overproduction of IFN-γ in response to repeated CMV

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antigen exposure. This may lead to the release of IL-2 by auto-aggressive T cells, thereby, impairing immune response to heterologous agents by abating the naıve T cell

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pool, particularly within the CD4+ T cell repertoire, and resulting in triggering or flare up of autoimmunity. Furthermore, overall suppression of active immune cells in such diseases is not a permanent solution which also needs targeted therapy against infectious agents like CMV. Our study provides an insight into the immunopathological potential of

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CMV-reactive immune cells to develop new potential strategies for targeted therapeutic

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intervention in these systemic autoimmune diseases.

Acknowledgements: The department of Biology, College of Science, University of

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Bahrain, Sakhir, Kingdom of Bahrain is acknowledged for providing software related support in the data analysis. Funding: This work was supported by the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India, New Delhi (grant no.: F.No. SR/SO/SH-82/2005). Competing interests: The authors declare that they have no competing interests.

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Authors' contributions: EMAJ, SH, NA, AJ, MW, RKM, and SAD performed the experiments, collected and analyzed the data; ML, MYA, SA, SD and SAD interpreted the data and were the major contributors in writing the manuscript; SD, SNB, VGR and

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SAD designed and supervised the study. All authors read and approved the final

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manuscript for publication.

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6. References

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[18] Sylwester AW, Mitchell BL, Edgar JB, Taormina C, Pelte C, Ruchti F, et al. Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J Exp Med. 2005;202:673-85. [19] van Leeuwen EM, Remmerswaal EB, Vossen MT, Rowshani AT, Wertheim-van Dillen PM, van Lier RA, et al. Emergence of a CD4+CD28- granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection. J Immunol. 2004;173:1834-41. [20] Guma M, Budt M, Saez A, Brckalo T, Hengel H, Angulo A, et al. Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts. Blood. 2006;107:3624-31. [21] Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40:1725. [22] van den Hoogen F, Khanna D, Fransen J, Johnson SR, Baron M, Tyndall A, et al. 2013 classification criteria for systemic sclerosis: an American college of rheumatology/European league against rheumatism collaborative initiative. Ann Rheum Dis. 2013;72:1747-55. [23] Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77-89. [24] Rider JR, Ollier WE, Lock RJ, Brookes ST, Pamphilon DH. Human cytomegalovirus infection and systemic lupus erythematosus. Clin Exp Rheumatol. 1997;15:405-9. [25] Neidhart M, Kuchen S, Distler O, Bruhlmann P, Michel BA, Gay RE, et al. Increased serum levels of antibodies against human cytomegalovirus and prevalence of autoantibodies in systemic sclerosis. Arthritis Rheum. 1999;42:389-92. [26] Stratta P, Canavese C, Ciccone G, Santi S, Quaglia M, Ghisetti V, et al. Correlation between cytomegalovirus infection and Raynaud's phenomenon in lupus nephritis. Nephron. 1999;82:145-54. [27] Palafox Sanchez CA, Satoh M, Chan EK, Carcamo WC, Munoz Valle JF, Orozco Barocio G, et al. Reduced IgG anti-small nuclear ribonucleoprotein autoantibody production in systemic lupus erythematosus patients with positive IgM anti-cytomegalovirus antibodies. Arthritis Res Ther. 2009;11:R27. [28] Vaughan JH, Shaw PX, Nguyen MD, Medsger TA, Jr., Wright TM, Metcalf JS, et al. Evidence of activation of 2 herpesviruses, Epstein-Barr virus and cytomegalovirus, in systemic sclerosis and normal skins. J Rheumatol. 2000;27:821-3. [29] Pandey JP. Immunoglobulin GM genes and IgG antibodies to cytomegalovirus in patients with systemic sclerosis. Clin Exp Rheumatol. 2004;22:S35-7. [30] Arnson Y, Amital H, Guiducci S, Matucci-Cerinic M, Valentini G, Barzilai O, et al. The role of infections in the immunopathogensis of systemic sclerosis--evidence from serological studies. Ann N Y Acad Sci. 2009;1173:627-32. [31] Esen BA, Yilmaz G, Uzun S, Ozdamar M, Aksozek A, Kamali S, et al. Serologic response to Epstein-Barr virus antigens in patients with systemic lupus erythematosus: a controlled study. Rheumatol Int. 2012;32:79-83. [32] Oldstone MB. Molecular mimicry and immune-mediated diseases. FASEB J. 1998;12:125565. [33] Ufret-Vincenty RL, Quigley L, Tresser N, Pak SH, Gado A, Hausmann S, et al. In vivo survival of viral antigen-specific T cells that induce experimental autoimmune encephalomyelitis. J Exp Med. 1998;188:1725-38. [34] Hiemstra HS, Schloot NC, van Veelen PA, Willemen SJ, Franken KL, van Rood JJ, et al. Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase. Proc Natl Acad Sci U S A. 2001;98:3988-91.

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[35] Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, et al. Interferoninducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A. 2003;100:2610-5. [36] Tan FK, Zhou X, Mayes MD, Gourh P, Guo X, Marcum C, et al. Signatures of differentially regulated interferon gene expression and vasculotrophism in the peripheral blood cells of systemic sclerosis patients. Rheumatology (Oxford). 2006;45:694-702. [37] Duan H, Fleming J, Pritchard DK, Amon LM, Xue J, Arnett HA, et al. Combined analysis of monocyte and lymphocyte messenger RNA expression with serum protein profiles in patients with scleroderma. Arthritis Rheum. 2008;58:1465-74. [38] Assassi S, Mayes MD, Arnett FC, Gourh P, Agarwal SK, McNearney TA, et al. Systemic sclerosis and lupus: points in an interferon-mediated continuum. Arthritis Rheum. 2010;62:58998. [39] Zola H, Flego L, Macardle PJ, Donohoe PJ, Ranford J, Roberton D. The CD45RO (p180, UCHL1) marker: complexity of expression in peripheral blood. Cell Immunol. 1992;145:175-86. [40] Merkenschlager M, Terry L, Edwards R, Beverley PC. Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHL1: implications for differential CD45 expression in T cell memory formation. Eur J Immunol. 1988;18:1653-61. [41] Meyer MF, Hellmich B, Kotterba S, Schatz H. Cytomegalovirus infection in systemic necrotizing vasculitis: causative agent or opportunistic infection? Rheumatol Int. 2000;20:35-8. [42] Sekigawa I, Nawata M, Seta N, Yamada M, Iida N, Hashimoto H. Cytomegalovirus infection in patients with systemic lupus erythematosus. Clin Exp Rheumatol. 2002;20:559-64. [43] Zandman-Goddard G, Shoenfeld Y. Infections and SLE. Autoimmunity. 2005;38:473-85. [44] Magro CM, Crowson AN, Ferri C. Cytomegalovirus-associated cutaneous vasculopathy and scleroderma sans inclusion body change. Hum Pathol. 2007;38:42-9. [45] Michels-van Amelsfort JM, Walter GJ, Taams LS. CD4+CD25+ regulatory T cells in systemic sclerosis and other rheumatic diseases. Expert Rev Clin Immunol. 2011;7:499-514. [46] Golding A, Hasni S, Illei G, Shevach EM. The percentage of FoxP3+Helios+ Treg cells correlates positively with disease activity in systemic lupus erythematosus. Arthritis Rheum. 2013;65:2898-906. [47] Ohl K, Tenbrock K. Regulatory T cells in systemic lupus erythematosus. Eur J Immunol. 2015;45:344-55. [48] Liu X, Gao N, Li M, Xu D, Hou Y, Wang Q, et al. Elevated levels of CD4(+)CD25(+)FoxP3(+) T cells in systemic sclerosis patients contribute to the secretion of IL-17 and immunosuppression dysfunction. PLoS One. 2013;8:e64531. [49] Liu MF, Wang CR, Fung LL, Wu CR. Decreased CD4+CD25+ T cells in peripheral blood of patients with systemic lupus erythematosus. Scand J Immunol. 2004;59:198-202. [50] Crispin JC, Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun. 2003;21:273-6. [51] Barcellini W, Rizzardi GP, Borghi MO, Nicoletti F, Fain C, Del Papa N, et al. In vitro type-1 and type-2 cytokine production in systemic lupus erythematosus: lack of relationship with clinical disease activity. Lupus. 1996;5:139-45. [52] Miller A, Lider O, Weiner HL. Antigen-driven bystander suppression after oral administration of antigens. J Exp Med. 1991;174:791-8. [53] Famularo G, Procopio A, Giacomelli R, Danese C, Sacchetti S, Perego MA, et al. Soluble interleukin-2 receptor, interleukin-2 and interleukin-4 in sera and supernatants from patients with progressive systemic sclerosis. Clin Exp Immunol. 1990;81:368-72.

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[54] Kahaleh MB. Raynaud's phenomenon and the vascular disease in scleroderma. Curr Opin Rheumatol. 1995;7:529-34. [55] Giacomelli R, Cipriani P, Danese C, Pizzuto F, Lattanzio R, Parzanese I, et al. Peripheral blood mononuclear cells of patients with systemic sclerosis produce increased amounts of interleukin 6, but not transforming growth factor beta 1. J Rheumatol. 1996;23:291-6. [56] Lalani I, Bhol K, Ahmed AR. Interleukin-10: biology, role in inflammation and autoimmunity. Ann Allergy Asthma Immunol. 1997;79:469-83. [57] Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin10 receptor. Annu Rev Immunol. 2001;19:683-765. [58] Hudson LL, Rocca KM, Kuwana M, Pandey JP. Interleukin-10 genotypes are associated with systemic sclerosis and influence disease-associated autoimmune responses. Genes Immun. 2005;6:274-8. [59] Godsell J, Rudloff I, Kandane-Rathnayake R, Hoi A, Nold MF, Morand EF, et al. Clinical associations of IL-10 and IL-37 in systemic lupus erythematosus. Sci Rep. 2016;6:34604. [60] Kotenko SV, Saccani S, Izotova LS, Mirochnitchenko OV, Pestka S. Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10). Proc Natl Acad Sci U S A. 2000;97:1695-700. [61] Liu Y, Mu R, Gao YP, Dong J, Zhu L, Ma Y, et al. A Cytomegalovirus Peptide-Specific Antibody Alters Natural Killer Cell Homeostasis and Is Shared in Several Autoimmune Diseases. Cell Host Microbe. 2016;19:400-8. [62] Soderberg-Naucler C. CMV and NK Cells: An Unhealthy Tryst? Cell Host Microbe. 2016;19:277-9. [63] Greijer AE, Dekkers CA, Middeldorp JM. Human cytomegalovirus virions differentially incorporate viral and host cell RNA during the assembly process. J Virol. 2000;74:9078-82. [64] Azab NA, Bassyouni IH, Emad Y, Abd El-Wahab GA, Hamdy G, Mashahit MA. CD4+CD25+ regulatory T cells (TREG) in systemic lupus erythematosus (SLE) patients: the possible influence of treatment with corticosteroids. Clin Immunol. 2008;127:151-7. [65] Manno R, Boin F. Immunotherapy of systemic sclerosis. Immunotherapy. 2010;2:863-78.

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Figure Legends Figure 1: Response to CMV antigen was CD4+ T cell dominated. (A) CD4+:CD8+ ratio in SLE and SSc patient PBMCs in comparison to healthy controls (HC) [*p<0.05 in

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comparison to unstimulated HC cells; #p<0.05 in comparison to CMV stimulated HC cells]. (B) Representative FACS plots showing elevated CD4+ T cell response to CMV antigen stimulation was dominated by memory CD4+ T cells (CD4+CD45RO+) in both

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SLE and SSc patients. (C) Representative FACS plots showing reduced CD8+ T cell response to CMV antigen stimulation was also dominated by memory CD8+ T cells

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(CD8+CD45RO+) in both SLE and SSc patients.

Figure 2: Percentage of CD4+CD25+ T cells was decreased in SLE and increased in SSc patients. (A) CD4+CD25+ T cell percentages in unstimulated and CMV stimulated

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PBMCs from SLE and SSc patients pre-therapy in comparison to healthy controls (HC) [*p<0.05 in comparison to unstimulated HC cells; #p<0.05 in comparison to CMV stimulated HC cells; ◊p<0.05 in comparison to unstimulated HC cells]. (B) CD4+CD25+ T

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cell percentages in unstimulated and CMV stimulated PBMCs from SLE and SSc patients post-therapy [&p<0.05 in comparison to unstimulated SLE cells; ©p<0.05 in comparison

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to CMV stimulated SLE cells].

Figure 3: Higher IL-10 and lower IFN-γ production in unstimulated PBMCs of SLE and SSc patients. A comparison of IL-2, IL-4, IL-10 and IFN-γ secretion by PBMCs of SLE and SSc patients with healthy controls (HC) is shown [*p<0.05 in comparison to unstimulated HC cells].

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Table 1: Profile and characteristics of SSc and SLE patients, and healthy controls in the study.

SLE patients 13

controls 20

24.16 [±2.89]

34.6 [±3.86]

25.85 [±0.66]

Sex (M/F )

03/10

04/16

0/20

Mean duration of Disease (months [mean ±SE])

6.50 [±1.31]

39 [±18]

0

18

NDe

0

ND

Age (years, mean [±SE ]) b

c

ANA positive

13

Anti-dsDNAd positive

09

standard error;

b

male ⁄female;

anti-nuclear antibody;

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antibody; e not detected

c

d

anti-double stranded DNA

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Healthy

20

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Number (n)

SSc patients

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Table 2: Levels of cytokines (pg/ml) in SLE patients pre-therapy (SLE(BT)), SLE patients post-therapy (SLE(PT)) and healthy controls (HC) after stimulation with CMV antigen.

IL-4

IL-10

IFN-γ

p-value

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SLE(BT)a

SLE(PT)a

stimulant

(n=20)

(n=13)

(n=12)

[HC vs

SLE(BT)] *

[SLE(BT) vs

SLE(PT)]

None

340.28±26.17

53.44±21.08

38.54±14.15

<0.001

0.051

CMV

314.32±33.61

327.41±41.81

321.27±15.37

0.145

0.342

None

5.57±3.73

4.75±1.14

4.58±1.17

0.369

0.717

CMV

7.43±1.62

9.54±1.51

10.36±2.58

0.064

None

105.41±16.08

CMV

326.17±27.38

None

828.39±197.20

CMV

653.43±181.43

0.424 *

264.35±68.78

165.84±27.06

<0.001

<0.001*

305.44±52.21

288.33±11.30

0.068

0.486 *

68.15±14.96

41.35±11.05

<0.001

<0.001*

622.13±142.23

511.32±106.40

0.057

<0.001*

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value in terms of mean (±SD); * mean difference is significant at the indicated p-value

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a

HCa

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IL-2

Antigen/

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Cytokines

p-value

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Table 3: Levels of cytokines (pg/ml) in SSc patients pre-therapy (SSc(BT)), SSc patients post-therapy (SSc(PT)) and healthy controls (HC) after stimulation with CMV antigen.

IL-4

IL-10

IFN-γ

(n=17)

SSc(BT) (n=20)

SSc(PT)

p-value

(n=17)

[HC vs

SSc(BT)]

None

340.67±27.59

319.20±64.45

288.66±65.46

0.188

CMV

303.27±45.71

293.43±41.22

261.54±13.72

0.088

None

5.58±3.75

CMV

13.5±1.67

7.61±1.34

17.15±6.1

None

105.25±17.31

287.58±81.66

CMV

322.32±32.43

283.12±32.10

p-value [SSc(BT)

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stimulant

13.01±1.97

16.25±3.28

vs

SSc(PT)] 0.163 0.108

*

0.410

*

0.132

*

<0.001

<0.001

235.88±35.37

<0.001

0.016*

300.62±18.54

0.074

0.324

None

848.64±194.58

248.10±37.66

274.75±30.91

<0.001

0.026*

CMV

645.23±182.43

542.42±33.81

467.86±48.54

<0.001*

0.025*

*

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value in terms of mean (±SD); * mean difference is significant at the indicated p-value

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a

HC

a

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IL-2

Antigen/

a

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Cytokines

a

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Highlights: •

Elevation in CD4:CD8 T cell ratio in SLE and SSc patient PBMC on CMV stimulation. Aberrant increase in percentages of CD4+CD25+ T cells differentiates SSc from

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•

SLE.

IFN-γ overproduction and large memory T cell pool may lead to IL-2 release.

•

Abating naıve T cell pool, particularly of CD4 repertoire, flares up autoimmunity.

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•