Student: Christien Rondaan Student number: 1870270 Supervisor: dr. Johanna Westra Location of research: University Medical Center Groningen, department of

Rheumatology and Clinical Immunology Period: September 3 2012 – February 22 2013

Humoral and cellular immunity to varicella-zoster virus

in patients with autoimmune diseases

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Dutch Summary

Achtergrond: Herpes zoster (HZ) wordt veroorzaakt door reactivatie van het varicella-zoster virus (VZV). Naast de symptomen van HZ, ontwikkelt 8-27% van de HZ-patiënten de complicatie postherpetische neuralgie (PHN). PHN veroorzaakt ernstige pijn. Met name ouderen, HIV-patiënten en patiënten die immuunsuppressieve medicatie gebruiken, hebben een verhoogd risico op HZ en PHN. Patiënten met auto-immuunziekten zoals systemische lupus erythematosus (SLE) en granulomatosis met polyangiitis (GPA) hebben een 3-20-maal verhoogd risico op HZ vergeleken met de algemene populatie. Doel: Het doel van het onderzoek was te bepalen of het verhoogde risico op HZ van patiënten met auto-immuunziekten te wijten is aan een verlaagde cellulaire en/of humorale immuunrespons tegen VZV bij deze patiënten. Methodes: Een cross-sectioneel onderzoek naar VZV-specifieke immuniteit werd uitgevoerd bij 38 patiënten met SLE, 33 patiënten met GPA en 51 gezonde controles. Bij alle deelnemers werden de antilichamen tegen VZV-glycoproteïnes (gp) gemeten in het serum met een gevalideerde gpELISA. Als controle werden antilichamen tegen difterie gemeten bij alle deelnemers. De cellulaire immuunrespons tegen VZV werd gemeten met behulp van een IFN-γ ELISpot en met CFSE-proliferatie-analyses waarbij gebruik werd gemaakt van gecryopreserveerde PBMCs en UV-geïnactiveerd VZV als antigeen. Resultaten: De hoeveelheid antilichamen tegen VZV was significant verhoogd (P=0.0004) bij SLE-patiënten (mediaan 132 IU/ml, range 21-1095) vergeleken met gezonde controles (mediaan 66 IU/ml, range 7-634). De waardes van GPA-patiënten (mediaan 75 IU/ml, range 2-1154) verschilden niet statistisch van die van gezonde controles. In tegenstelling was de hoeveelheid antilichamen tegen difterie bij zowel SLE- als GPA-patiënten significant verlaagd (P=0.012 en P=0.037) in vergelijking met gezonde controles. SLE-patiënten hadden een significant lager aantal IFN-γ spot-vormende cellen in respons op VZV (P=0.033). Bij GPA-patiënten werd geen verschil gevonden. Verder was ook de proliferatie index (PI) van CD4+ T lymfocyten significant (P=0.034) verlaagd bij SLE-patiënten in vergelijking met gezonde controles, maar dit was niet het geval bij GPA-patiënten. Conclusies: Patiënten met SLE hebben verhoogde antilichaamtiters tegen VZV vergeleken met gezonde controles. Dit kan niet verklaard worden door polyklonale hypergammaglobulinemie, aangezien de antilichaamtiter tegen difterie verlaagd was in vergelijking met gezonde controles. De cellulaire immuniteit tegen VZV was echter verlaagd bij deze patiënten. De resultaten suggereren dat de verhoogde prevalentie van HZ bij SLE-patiënten te wijten is aan een slechte cellulaire immuunrespons tegen VZV. Vaccinatiestrategieën zouden niet gebaseerd moeten worden op humorale immuniteit en zouden gericht moeten zijn op het verhogen van de cellulaire immuniteit tegen VZV.

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English Summary Background: Herpes zoster (HZ) results from reactivation of varicella-zoster virus (VZV). Besides the symptoms of HZ, 8-27% of patients with herpes zoster develop postherpetic neuralgia (PHN), leading to debilitating pains. In particular elderly, HIV-patients and patients using immunosuppressive drugs are at increased risk for developing HZ and PHN. Patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (GPA) have a 3-20-fold increased risk compared to the general population. Use of immunosuppressive drugs is associated with the development of HZ in these patients. Objectives: Aim of the study was to evaluate if the susceptibility of patients with autoimmune disease is due to decreased levels of cellular and/or humoral responses to VZV in these patients. Methods: A cross-sectional study on VZV-specific immunity was performed in 38 patients with SLE, 33 patients with GPA and 51 healthy controls (HC). In all individuals, serum antibody levels to VZV-glycoprotein (gp) was measured by a validated in-house gpELISA As control antibodies to Diphtheria were measured in all participants. Cellular responses to VZV were by IFN-γ ELIspot assay and CFSE-dye dilution proliferation assay using cryo-preserved PBMCs and UV-inactivated VZV as antigen. Results: Antibodies to VZV were significantly (P=0.0004) increased in SLE patients (median 132 IU/ml, range 21-1095) compared to HC (median 66 IU/ml, range 7-634). Values in GPA patients (median 75 IU/ml, range 2-1154) were not statistically different from values in HC. In contrast, antibodies against Diphtheria in both SLE and GPA patients were significantly (P=0.012 and P=0.037) decreased compared to HC. SLE patients had a significantly lower number of IFN-γ spot forming cells against VZV (P=0.033). In GPA patients no difference was found. Furthermore, the proliferation index (PI) of CD4+ T-lymphocytes was significantly (P=0.034) decreased in SLE patients compared to HC, but not in GPA patients. Conclusions: Patients with SLE have increased antibody levels against VZV compared to levels detected in HC, which cannot be explained by polyclonal hypergammaglobulinaemia as antibodies to Diphtheria were decreased in comparison to HC. Cellular immunity however, was decreased in these patients. The results suggest that increased prevalence of VZV in patients is due to a poor cellular response to VZV. Vaccination strategies should not be based upon humoral immunity and should aim to boost cellular immunity against VZV.

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Table of Contents Title page .................................................................................................................................... 1 Dutch summary .......................................................................................................................... 2 English summary ........................................................................................................................ 3 Table of contents ........................................................................................................................ 4 1. Introduction ............................................................................................................................ 5

Varicella-zoster virus ........................................................................................................... 5 Vaccination against VZV ...................................................................................................... 8 Autoimmune diseases relevant to this research project .................................................. 10 Problem definition and hypothesis ................................................................................... 11

2. Material and method ........................................................................................................... 12 3. Results .................................................................................................................................. 19 4. Discussion (and acknowledgements) ................................................................................... 25 5. Bibliography .......................................................................................................................... 28 Appendix................................................................................................................................... 32

Abbreviation list ................................................................................................................ 32 Tables of patient characteristics for each outcome variable ............................................ 33

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1. Introduction This research project deals with the humoral and cellular immunity against varicella-zoster virus (VZV) in patients with autoimmune diseases. In this introduction a theoretical background for this subject will be given. The introduction will start off with an elaboration on VZV and the pathogenesis of this virus. VZV virus causes two diseases: varicella and herpes zoster (HZ). More information on these diseases, in particular on HZ and it’s most common complication, postherpetic neuralgia, will be given. Thereupon, vaccination against VZV will be discussed. It can prevent both varicella and herpes zoster. There will be special attention for vaccination in patients with autoimmune diseases. The autoimmune diseases that are relevant to this research project; systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (GPA; also known as Wegener’s granulomatosis) will be shortly addressed. To conclude the introduction, the problem definition and hypothesis for this research project will be stated. An abbreviation list can be found in the appendix.

Varicella-zoster virus Varicella-zoster virus (VZV) is the neurotropic herpesvirus known for its causation of varicella and herpes zoster. Structure The varicella-zoster virion structure includes an icosadeltahedral viral nucleocapsid containing a single copy of a linear double-stranded DNA core. The virion tegument, containing viral transcriptional regulator proteins, surrounds the nucleocapsid. The envelope of the virion is composed of glycoproteins that also have important functions in pathogenesis(1,2)(Figure 1). Pathogenesis Infection with VZV can be acquired via droplets and aerosols from the nasopharynx of an infected individual. However, most transmission probably is due to contact with fluid from skin lesions in individuals infected by VZV, because of the high titres of cell-free virus they contain(1,2). VZV enters the human body via the respiratory route, amplifies in Waldeyer’s ring by infecting T cells, and is

Figure 1. Structure of varicella-zoster virus. Copied from Heininger and Seward(1).

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transported to the skin by the infected T cells. When the skin is infected, the virus probably spreads from here to the sensory nerve ganglia by retrograde axonal transport(1).

After the primary infection of VZV, the virus remains latently present for life in the cranial nerve and dorsal root sensory ganglia, causing herpes zoster when reactivating(3)(Figure 2). There are three components in the immune response to VZV infection:

1. Innate immunity. This component of the immune response is mediated by interferon-gamma (IFN-γ) and is eventually overcome by the virus.

2. Humoral immunity. During a varicella infection, VZV-specific antibodies are formed. These are important in neutralizing cell-free virus. However, children with congenital agammaglobulinaemia experience uncomplicated varicella.

3. Cellular mediated immunity (CMI). This is the most essential component of the immune response to VZV infection as VZV is a cell-associated virus(1,2). Therefore, both varicella and herpes zoster (discussed later in this introduction) are more severe in patients with defects in CMI. Herpes zoster is also more frequent in these patients(4).

The CMI mainly concerns T cells. CD4+ T cells, also known as helper T cells, recognize peptides presented by major histocompatibility class (MHC) II molecules. When VZV has infected the body, CD4+ T cells get activated and release IFN-γ. IFN-γ stimulates CD8+ T cells, also known as cytotoxic T cells, that can recognize and lyse infected cells, and up-regulates the expression of MHC II on cells that normally do not express this molecule. Because of the latter, CD4+ T cells too are able to recognize and lyse infected cells within skin lesions. However, latently infected cells do not express extra MHC II even in the presence of IFN-γ; a way in which VZV evades recognition by the immune system.

Immune evasion by VZV also occurs through the down-regulation of MHC I and II expression in T cells and fibroblasts(4).

Figure 2. Establishment of VZV latency in sensory nerve ganglia. After a primary VZV infection, latent VZV infection is established in the dorsal root ganglia. Herpes zoster occurs with subsequent reactivation of the virus. (Copied from Kimberlin and Whitley(6).

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Varicella The primary infection of VZV, varicella, is also known as chickenpox and occurs mostly in children. Varicella is highly infectious and is estimated to infect 61-100% of susceptible contacts of a patient. Although the disease occurs worldwide, a higher percentage of the population in temperate countries is exposed to varicella by the age of 16 than is the case in tropical countries. In temperate countries this concerns about 90% of the population before adolescence(1,5). Approximately 99% of the population is suggested to be immune by the age of 40(5).

Symptoms of varicella are malaise, fatigue and a typical vesicular rash. Usually the primary infection of VZV is self-limiting and uncomplicated. However, it can result in severe disease, especially in certain risk groups as infants, the elderly and immunocompromised individuals(1-3). Herpes zoster Reactivation of VZV causes herpes zoster (HZ), also known as shingles. As stated before, in temperate countries 99% of the population will have experienced a VZV infection by the age of 40, so almost the whole adult population is at risk for developing HZ(5,6).

The incidence of HZ in developed countries is estimated to be 5/1000 person-years and will affect 10-30% adults in their lifetime(3,7-9). Clinically, HZ presents as an acute neurocutaneous disease characterized by severe pain and a rash in a dermatomal distribution(3,10)(Figure 3).

During reactivation of VZV, viral replication takes place in the sensory nerve ganglia, where the virus has remained latently since the primary VZV infection. After viral replication and before onset of the typical dermatomal rash, there is destruction of neurons and satellite cells. Necrosis of ganglion cells and demyelination of the corresponding axon occur(6). VZV travels along the affected sensory nerves to the skin. The histological changes in the skin lesions are similar to those of varicella. In most patients, the pain of HZ gradually resolves within 1 to 2 months(3,4,9).

The risk of HZ increases with age, beginning at about 50 years. HZ is 8 to 10 times as likely to develop in people 60 years or older than in younger people. Immunocompromised patients also are at increased risk for HZ(6,11). Postherpetic neuralgia Postherpetic neuralgia (PHN) is the most common complication of HZ. Between 8 and 27% of individuals with HZ will develop PHN(8). The risk of developing PHN when having HZ does not seem to be increased in immunocompromised patients(12). Opinions vary on the duration of pain that is required for a diagnosis of PHN but significant pain persisting more than 90 days after rash onset is the most commonly used definition(8,9,13). PHN can last for weeks, months, or even years(6). It is suggested to be caused by over-sensitive nociceptors

Figure 3. Acute Herpes Zoster. Panel A shows a cutaneous eruption in the right T7 dermatome. Panel B shows a close-up view of fresh vesicular lesions. (Copied from Kimberlin and Whitley(6)).

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resulting from peripheral nerve damage, or degradation of the nociceptors leading to central sensitization(5).

Acute pain from HZ greatly interferes with the activities of daily living. In a study by Drolet et al.(9) more than half of 215 participants suffering from HZ reported interference scores of five or greater (with a maximum of 10) in the areas of sleep (63.9%), enjoyment of life (58.2%) and general activities (52.6%). A high proportion of patients with PHN reported pain or discomfort, symptoms of anxiety or depression, and problems with mobility and self-care throughout the postherpetic period(9).

HZ and PHN also has a major impact on the productive work life of individuals. In a study by Drolet et al.(8) on productivity loss associated with HZ and PHN, 64% of the 88 employed subjects who took part in the study reported missing work and 76% reported decreased effectiveness at work due to HZ and PHN. An association between pain duration and severity and overall productivity was found(8). Other complications of herpes zoster The rate of complications other than pain in patients with HZ is higher in those who are immunocompromised. Ocular and neurological complications and skin superinfections are most common(13). Borba et al. found bacterial skin superinfections in a higher percentage, namely 10%, of SLE patients with HZ than the expected 2%. This is suggested to be due to intrinsic disease susceptibility as no clear association with drugs or disease activity was found(12).

A large part of the approximately 3% of patients with HZ that are hospitalized, consists of patients with one or more immunocompromising conditions(14). The reactivation of VZV can spread to various parts of the body, including gut, liver and other viscera. Life threatening complications as pneumonia, encephalitis, hepatitis and haemorrhagic disorders are possible in these patients. Secondary bacterial infections leading to septicaemia and meningitis also pose a serious threat(5).

Vaccination against VZV Live attenuated vaccines Vaccination can prevent varicella, the primary VZV infection. The varicella vaccine is prepared from the Oka strain of live, attenuated VZV. This strain was isolated in Japan in the early 1970s from a healthy child that had varicella. Attenuation was achieved by tissue culture passage in human embryonic lung cells, embryonic guinea pig cells, and then again in human diploid cells(2,15). In various countries immunisation programmes against VZV are already implemented. The USA were the first to do this on a large scale, in 1995. Even though vaccination is effective in reducing hospital admissions and deaths caused by varicella, the epidemiology of HZ could also be affected(1).

It is thought that vaccination programmes eventually will reduce the number of HZ cases, but will lead to a substantial increase in the interim. A 30-50% increase in the unvaccinated that will last for 30-50 years is suggested. This is because the chances of boosting immunity will reduce with the reduction of varicella outbreaks(5). In a study evaluating the incidence of HZ in 1993-2006 evidence of substantial increases in the incidence of HZ across all age groups was found. The increase in incidence of HZ however already occurred prior to the introduction of the varicella vaccination programme(16).

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Furthermore, in Canada and the United Kingdom increases in HZ incidence rates were reported that were not associated with a varicella vaccination program(17). It is not known why the incidence of HZ is rising.

In the United States of America, a vaccine to prevent HZ was licensed for use in older immunocompetent adults in 2006: Zostavax. It was proven to be safe and effective in preventing HZ and PHN in this group. Currently, Zostavax is the only approved vaccine for prevention of HZ(6,9,13,18).

Zostavax uses the same strain of virus as the childhood vaccine (Varivax) but is at least 14 times more potent(5,7). The higher potency of the adult vaccine is necessary because of the lower immune system in older people and the poor uptake. The zoster vaccine appears to reduce the risk of HZ by 51%, of PHN by 67% and to be most cost-effective in 60-69-year-olds(5). The preventive effect of the zoster vaccine is thought to be a consequence of its boosting effect on an older person’s CMI to VZV(6). Recombinant subunit vaccines An alternative to live attenuated vaccines are recombinant subunit vaccines. These are thought to be strongly immunogenic, safe in immunocompromised patients and easy to produce(19). VZV glycoprotein E (gE) is the most abundant glycoprotein in VZV virions and in VZV-infected cells and is also the principal target of VZV-specific CD4+ T cell responses. Therefore gE is a good candidate to act as the subunit antigen in an HZ vaccine(2,10,19).

In a study by Leroux-Roels et al. young and older adults were vaccinated with a gE subunit vaccine, with Zostavax or with both. Immunization with two doses of gE subunit vaccine resulted in a higher frequency of VZV-specific CD4+ T cells than following immunization with 2 doses of Zostavax in both young and older adults 12 months after the first dose of vaccine was given(19).

However, adjuvants are needed in VZV subunit vaccine formulations to enhance, direct and maintain the immune response to vaccine antigens as otherwise insufficient immune responses for protective immunity are mobilised(20). It is unclear whether adjuvants are safe to use in patients with an autoimmune disease. Vaccination in immunocompromised patients It is known that immunocompromised patients are at higher risk for developing HZ(21,22). Immunocompromisation can have various etiologies. Patients who are infected with the human immunodeficiency virus (HIV), patients with an autoimmune inflammatory rheumatic disease (AIIRD) and patients receiving immunosuppressive therapies all are exposed to an elevated HZ risk(13,23). In patients with an AIIRD the elevated HZ risk can be attributed both to the immunosuppressive effect of the underlying AIIRD and the use of immunosuppressive therapies(22).

Both systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (GPA), the diseases focused on in this project, are AIIRDs. Accordingly, SLE and GPA patients have an increased risk of HZ. As stated before, in developed countries the incidence of HZ is estimated at 3-5/1000 person-years(7). In SLE patients the risk is suggested to comprise 15-91 cases/1000 patient-years; in GPA 45 cases/1000 patient-years(5,7,12,24). So, the calculated risk of HZ in these patient groups is 3-20 times higher than in the general population. The reported prevalence of HZ varies from 13,5% to 46,6% of SLE patients in different series, with the majority of zoster reactivations occurring during mild or inactive disease(12).

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Vaccination, being the most effective management strategy for viral infections and their complications, could be of paramount importance especially in these patient groups that are at increased risk for HZ. Matter of concern however is that a live attenuated vaccine could trigger the development of HZ, however this has not been found in literature for other vaccines(12,25).

As stated before, the currently only approved vaccine to reduce the risk and severity of HZ is the live attenuated vaccine called Zostavax. It is still unclear whether this vaccine is safe to be used in autoimmune patients(12,22). The Advisory Committee on Immunization Practices (ACIP) stated in 2008 that zoster vaccine should not be administered to persons with primary or acquired immunodeficiency. However, low-dose immunomodulatory drugs are not considered severely immunosuppressive and are no contraindications for the zoster vaccine(14,15,26).

The American College of Rheumatology (ACR) in 2012 recommended zoster vaccination in patients with rheumatoid arthritis (RA) before initiating disease-modifying antirheumatic drugs (DMARDs) and biologic agents and also during therapy with DMARDs. Zoster vaccination was not recommended during therapy with biologic agents. The recommendation of using zoster vaccine in these patients is recent as in 2008 it was not yet recommended by the ACR. However, the level of evidence for the recommendation was classified as C, the lowest level of evidence, meaning data were derived from consensus opinion of experts, case studies, or standards of care(27).

In retrospective observational cohort studies examining the incidence rate of HZ shortly after use of zoster vaccination in individuals with inflammatory and autoimmune diseases, including patients using immunosuppressive medication, the short-term risk of HZ did not appear to be increased in vaccinated patients. Among these patients were persons using biologic agents. The use of immunosuppressive therapies in the vaccinated patients was not found to be a risk factor for developing HZ shortly after vaccination. Of notice, vaccination was associated with a lower HZ incidence in these patients(23).

A prospective study of varicella vaccine in children and adolescents with SLE that were previously exposed to varicella-zoster virus resulted in a lower incidence of HZ in the vaccinated SLE patients. The frequency of flares and SLEDAI (SLE disease activity index) score were similar among vaccinated and unvaccinated patients(28).

Autoimmune diseases relevant to this research project Systemic lupus erythematosus (SLE) Systemic lupus erythematosus (SLE) is a chronic inflammatory disease that can affect the skin, joints, kidneys, lungs, nervous system, serous membranes, and/or other organs of the body(29). It is characterized by relapsing and remitting disease activity(30).

Abnormalities in immune regulation prevail in SLE. These abnormalities are thought to be secondary to a loss of self-tolerance. The mediators of SLE are autoantibodies and immune complexes they form with antigens(31). Selected antinuclear antibodies (ANAs), including anti-double-stranded deoxyribonucleic acid (dsDNA) and anti-Sm, are highly specific for the diagnosis. Antibodies directed against the U1 ribonucleoprotein (RNP) complex are markers for mixed connective tissue disease (MCTD) and may be seen in patients with SLE but also with other disorders(32).

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Phagocytosis and clearing of immune complexes, apoptotic cells and necrotic cell-derived material are defective in SLE, allowing persistence of antigen and immune complexes(31). The cause of the disease is unknown(29).

The prevalence of SLE is higher among Asians, Afro-Americans, Afro-Caribbeans, and Hispanic Americans compared with Americans of European decent in the United States, and among Asian Indians compared with Caucasians in Great Britain. In comparison, SLE occurs infrequently in Blacks in Africa(31). Especially women in their twenties and thirties are affected(29,31). Infections are one of the most common causes of morbidity and mortality in patients with SLE(11,12,30). Granulomatosis with polyangiitis (GPA) Previously granulomatosis with polyangiitis (GPA) was known as Wegener’s granulomatosis. It is an auto-immune inflammatory disease affecting small and medium-size vessels. GPA has been reported in all age groups, but is more common in older people. There is a peak age of 65-74 years(26,33,34). Both sexes are equally affected. The disease is far more common among Caucasian people(33).

Prodromal symptoms such as fever, migratory arthralgias, malaise, anorexia and weight loss may last for weeks to months without evidence of specific organ involvement. Glomerulonephritis develops in 77-85% of GPA patients. Eye, ear and nose disease manifestations are very common with an estimated frequency of 90%. Patients with GPA may present with involvement of airways or pulmonary parenchyma(33). Before immunosuppressive treatment was available, the disease had a poor prognosis with only 20% of patients surviving after 2 years after disease onset. Nowadays survival rates after 5 years are 60-80%, depending on renal function(26).

One of the hallmarks of GPA is the production of anti-neutrophil cytoplasmatic antibodies (ANCAs), that are mainly of the IgG type. Approximately 85% of GPA patients have these antibodies, which are directed against antigens present within the primary granules of neutrophils and monocytes(35).

Problem definition and hypothesis HZ is caused by reactivation of VZV after a primary infection with this virus. Patients with autoimmune inflammatory rheumatic diseases like SLE and GPA are at increased risk of HZ. This disease, and in particular it’s complication PHN, can lead to debilitating pains and loss of quality of life and productivity. Vaccination can prevent HZ, but safety and efficacy of zoster vaccine in these patients still pose questions. Before immunization of immunocompromised patients can start in order to prevent HZ in these patients, more knowledge on the baseline immune status against VZV in these patients is necessary. In the current research project, the immune status, both humoral and cellular, against VZV in SLE and GPA patients is evaluated compared to healthy controls.

It was expected to find both a lower humoral and cellular response against VZV in SLE and GPA patients compared to healthy controls, consistent with the increased risk of these patients at HZ.

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2. Material and Method Study population and study design The patient selection for this project was based on an existing data bank, series of serum samples and cryopreserved peripheral blood mononuclear cells (PBMCs) which were established during an influenza vaccination study (2005/2006) in SLE patients, GPA patients and healthy controls (HC). Both serum samples (stored at -20 °C) and PBMCs (stored in liquid nitrogen) used in this project were obtained three months after influenza vaccination. Because at this time point no reaction to influenza vaccination could be detected, both types of samples remained to be used in this project.

SLE patients eligible for the influenza study, and accordingly for this project, fulfilled at least 4 of the American College of Rheumatology (ACR) criteria for SLE(36) and had quiescent disease, defined as a SLE disease activity index (SLEDAI) of ≤5. Exclusion criteria were pregnancy, an indication for yearly influenza vaccination due to concomitant disease(37) and use of immunosuppressive drugs other than hydroxychloroquine, azathioprine, prednisone or methotrexate(30,38).

Patients with GPA eligible for the study fulfilled the criteria for GPA(39) and had quiescent disease, defined as having a Birmingham Vasculitis Activity Score (BVAS) of <2. Exclusion criteria were a BVAS of 2 or greater, indication for yearly influenza vaccination due to concomitant disease(37), the use of prednisone at a dose of more than 30 mg/day and/or cyclophosphamide at more than 100 mg/day, and pregnancy.

The healthy control group consisted of healthcare workers participating in the yearly vaccination campaign at the UMCG that were asked to participate. Pregnancy, malignancy and use of immunosuppressive drugs were exclusion criterion for participation as a control subject(34,40).

The study was approved by the institutional medical ethics committee, and informed consent was obtained from all participants. Antibody response to VZV and Diphtheria using ELISA Solid phase enzyme-linked immunosorbent assay (ELISA) is based on the sandwich principle. In this test, wells of a 96-wells plate are coated with antigen. Diluted serum of the test subject is added. After incubation and washing, specific antibodies of the sample are bound to the antigen coated well. These can be detected by a secondary enzyme conjugated antibody, specific for human IgG. After the substrate reaction, the intensity of the colour that develops is proportional to the amount of IgG-specific antibodies detected. Results of samples can be determined using an ELISA plate reader, that bases antibody quantities upon the standard curve. The standard curve is obtained by filling a number of wells of the plate with samples having a known antibody quantity. Antibody responses against VZV were measured using an in-house glycoprotein ELISA (gpELISA). This test was validated by comparing it to the Serion ELISA classic VZV IgG (produced by the Institut Virion/Serion, Würzburg, Germany). The viral antigen of this test contains VZV-specific envelope glycoproteins. The sensitivity and specificity of the Serion were already established(41).

All samples were also tested in the Vidas system, the standard diagnostic test for VZV serology in the UMCG (University Medical Center Groningen). Highly significant correlations

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were found for the in-house gpELISA compared to both Serion and Vidas. Therefore, the in-house gpELISA was considered to be usable in this project. As the student only did matching and analysis of this data and did not do these tests herself, no further information about this part of the study is given here. Antibody responses against Diphtheria were measured using two identical commercial kits (IBL International, Germany). The plates provided in the kit were already coated with

antibody. Serum of patients was prediluted (5 L of serum in 500 L of diluent buffer,

provided in kit). 100 L of the standard and diluted serum samples were pipetted into the respective wells. After the plate was covered with adhesive foil, it was incubated for 60 minutes at room temperature. After incubation, adhesive foil was removed and incubation solution discarded. The plate was washed three times with diluted wash buffer (provided in kit). Excess solution was removed by tapping the inverted plate on a paper towel. Thereupon

100 L of enzyme conjugate (provided in kit) was pipetted into each well, and the plate was covered with new adhesive foil. After an incubation period of 30 minutes at room temperature, adhesive foil was removed and the plate again was washed three times. 100

L of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (provided in kit) was subsequently added to each well and incubated for 20 minutes at room temperature in the dark. The

substrate reaction was stopped by adding 100 L of TMB stop solution (provided in kit) to each well. In this step, colour changed from blue to fellow. Optical density was measured at 450 nm shortly after this step using the VersaMax microplate reader from Molecular Devices and the according software. ELISpot ELISpot (enzyme-linked immunosorbent spot) is a modified version of the ELISA. Using ELISpot, the production of antigen-specific cytokines or other proteins by individual cells can be traced and analysed. Each spot in the test represents an activated cell. The ELISpot gives both qualitative (type of protein secreted by the cell) and quantitative (number of cells that react to the antigen) information. In the current project, the ELISpot test is used to measure the production of IFN-γ by T cells after stimulation with VZV. High-level production of IFN-γ is associated with effective host defence against intracellular pathogens like VZV. Work was executed sterile. PBMCs from patient and matched control were simultaneously processed. PBMCs were removed from the liquid nitrogen and thawed until only a small part of PBMCs was frozen and were transferred to a 15 mL tube. Further thawing was done by adding 10 mL of RPMI (Roswell Park Memorial Institute medium; Lonza, Belgium) 1640 with 0.6% gentamicin (Invitrogen/LifeTechnologies, The Netherlands) and 10% FCS (fetal calf serum; Lonza) dropwise to the PBMCs. Tubes were centrifuged for 10 minutes at 1200 rounds per minute (RPM) and brake 5. Supernatant was aspirated. Cell pellet was resuspended in 1 mL RPMI 1640 + 0.6% gentamicin + 10% FCS. Cells were counted using the Coulter Counter (Beckman Coulter) and a suspension to a maximum of 3 x 106 cells/mL was made. A trypan blue (Invitrogen) staining was done to establish the percentage of dead versus life cells. The PBMC cell suspension was allowed to rest overnight at 37 °C and 5% CO2 in polypropylene tubes (BD Falcon, BD Biosciences).

A MultiScreen Filter Plate (Merck Millipore, USA) was filled with 50 L per well of

70% ethanol for 2 minutes. Subsequently, the plate was washed 3 times with 150 L per well

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of PBS (phosphate buffered saline; UMCG pharmacy). The plate was coated with 15 g/mL of anti-human IFN-γ (Mabtech, Sweden) and incubated overnight at 4 °C.

At day 2 the plate was washed 5 times with 150 L per well of RPMI (+0.6%

gentamicin) + 10% FCS. The plate was blocked by adding 50 L per well of RPMI (+0.6% gentamicin) + 10% FCS and incubating for a minimum of two hours at 37 °C and 5% CO2. PBMCs again were counted using the Coulter Counter and subsequently centrifuged. Supernatant was aspirated and a suspension of 2 x 106/mL was made. Blocking buffer was

discarded from the plate. In each well, 100 L cell suspension and 100 L stimulus or only medium was added. There were three conditions:

1. Negative control: 100 L of RPMI (+0.6% gentamicin) + 10% FCS was added to the cell suspension

2. Positive control: 2.5 L concanavalin A (conA) was dissolved in 12.5 mL RPMI (+0.6%

gentamicin) + 10% FCS. After vigorous mixing, 100 L of the mixture was added to the cell suspension

3. VZV stimulation: 1.5 L UV-inactivated VZV vaccine (Sanofi Pasteur, France. UV-

inactivation was done at the department of Molecular Virology) and 98.5 L RPMI (+0.6% gentamicin) + 10% FCS was added to the cell suspension

The following blueprint for the plate was used (Table 1): Table 1. Blueprint for ELISpot plate.

1 2 3 4 5 6 7 8 9 10 11 12

A Bl Bl Bl Bl Bl Bl Bl Bl Bl Bl Bl Bl

B Bl Bl Bl Bl Bl Bl Bl Bl Bl Bl Bl Bl

C ConA ConA ConA ConA ConA ConA ConA ConA ConA ConA ConA ConA

D VZV VZV VZV VZV VZV VZV VZV VZV VZV VZV VZV VZV

E VZV VZV VZV VZV VZV VZV VZV VZV VZV VZV VZV VZV

F

G

H Bl = blank = negative control, ConA = stimulation with concanavalin A = positive control, VZV = stimulation with UV-inactivated varicella-zoster virus vaccine

So in each ELISpot plate, samples of 12 subjects could be processed.

After adding of medium and stimuli to the cell suspension in each well, the plate was wrapped in tinfoil and placed on the shaker for a few minutes. Then the plate was placed in the incubator at 37 °C and 5% CO2 for 48 hours.

From day 4 it was no longer necessary to work sterile. The plate was washed 6 times

with 150 L per well of PBS. After tapping off excess PBS, 50 L/well 1:1000 biotinylated mouse monoclonal antibody to human IFN-γ (Mabtech) in PBS was added. The plate was placed on the shaker at room temperature for 3 hours. After this period, again the plate was

washed 6 times with PBS and 50 L 1:1000 streptavidin-ALP (alkaline phosphatase; Mabtech) was added to each well. After 1.5 hours on the shaker at room temperature, again

the plate was washed 6 times with PBS. Now 100 L BCIP/NBT (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt/nitro-blue tetrazolium chloride; Mabtech) was added to the wells. After 15 minutes reaction was stopped by washing 3 times with tap water.

15

The plate was left to dry at room temperature in the dark. Subsequently it was scanned and number of IFN-γ spot forming cells determined using the A.EL.VIS ELISPOT Reader (A.EL.VIS, Germany). The mean of the number of spot-forming cells of the negative control was subtracted from the mean of the number of spot-forming cells in the VZV-stimulated wells. In figure 4 an example is given of ELISpot results for one subject. The well with the positive control is very dark, meaning there were a lot of IFN-γ spot-forming cells in this well and so the positive control in fact is positive. The mean of number of IFN-γ spot-forming cells in the wells in which only medium was added to the wells (negative control) is 16 and the mean of the VZV stimulated wells is 40.5. When the mean of the negative control is subtracted from the mean of the VZV stimulated wells, a number of 24.5 IFN-γ spot-forming cells remain that have evolved in response to stimulation with UV-inactivated VZV vaccine.

Proliferation of T cells With a proliferation assay the proliferating capacity of T cells in response to a stimulus can be determined. This is done by staining PBMCs with carboxyfluorescein succinimidyl ester (CFSE), which is a fluorescent dye. CFSE is taken up by the cells. When the cell divides, the amount of CFSE intensity halves (Figure 5). By means of CFSE fluorescence intensity it can be determined how many times a cell has divided. All the work was executed sterile. PBMCs from patient and matched control were simultaneously processed. PBMCs were removed from the liquid nitrogen and thawed until only a small part of PBMCs was still frozen and were transferred to a 15 mL tube. Further thawing was done by adding 10 mL of RPMI 1640 with 0.6% gentamicin and 10% FCS dropwise to the PBMCs. Tubes were centrifuged for 10 minutes at 1200 RPM. Supernatant was aspirated. Cell pellet was resuspended in 1 mL PBS. A trypan blue staining was done to establish the percentage of dead versus life cells. CFSE (Molecular Probes/LifeTechnologies, The Netherlands) was diluted in 1:4000 in PBS (2

L in 4 mL) and mixed vigorously. PBMCs were counted using the Coulter Counter (Beckman Coulter). A suspension of 10 x 106 cells/mL in diluted CFSE was made. Tin foil was wrapped around the tubes to protect them from light. The mixture of PBMCs and CFSE was vigorously mixed and incubated in a bath of water of 37 °C. Every two minutes the mixture was mixed.

Negative control Positive control VZV stimulated

Figure 4. Example of ELISpot results of one subject. Please see text for explanation.

Figure 5. CFSE intensity decreased with subsequent cell divisions.

16

Then 10 mL RPMI 1640 + 0.6% gentamicin + 10% FCS was added to stop the reaction. PBMCs were washed 2 times with RPMI 1640 + 0.6% gentamicin + 10% FCS. Again PBMCs were counted using the Coulter Counter. A suspension of 1 x 106 cells/mL in RPMI 1640 + 0.6% gentamicin + 10% HPS (human pool serum; produced from pool serum of 12 blood donors, Sanquin) was made. A sterile U-bottom 96 wells plate (Cellstar/Greiner bio-one, Germany)

was filled with 100 L cell suspension and stimulus/medium. There were three conditions:

1. Negative control: 100 L of RPMI (+0.6% gentamicin) + 10% FCS was added to the cell suspension

2. Positive control: 20 L anti-CD3, 20 L anti-CD28 (obtained from hybridomas in the

laboratory) and 60 L RPMI 1640 + 0.6% gentamicin + 10% HPS was added to the cell suspension

3. VZV stimulation: 1.5 L UV-inactivated VZV vaccine and 98.5 L RPMI (+0.6% gentamicin) + 10% HPS was added to the cell suspension

The following blueprint for the plate was used (Table2): Table 2. Blueprint for T cell proliferation plate

1 2 3 4 5 6 7 8 9 10 11 12

A Bl Bl CD3 VZV VZV Bl Bl CD3 VZV VZV

B Bl Bl CD3 VZV VZV Bl Bl CD3 VZV VZV

C Bl Bl CD3 VZV Bl Bl CD3 VZV

D Bl Bl CD3 VZV Bl Bl CD3 VZV

E Bl Bl CD3 VZV Bl Bl CD3 VZV

F Bl Bl CD3 VZV Bl Bl CD3 VZV

G Bl Bl CD3 VZV Bl Bl CD3 VZV

H Bl Bl CD3 VZV Bl Bl CD3 VZV Bl = blank = negative control, CD3 = stimulation with antibodies to CD3 and CD28 = positive control, VZV = stimulation with UV-inactivated varicella-zoster virus vaccine

So in each T cell proliferation plate, samples from 2 subjects could be processed. After filling of the plate, it was placed on the shaker for a few minutes and subsequently put in the incubator at 37 °C and 5% CO2 for 7 days.

After 7 days of proliferation 10 L 40 mM EDTA (ethylenediaminetetraacetic acid; Merck, Germany) was added to each well and PBMCs were harvested from the plate. It was no longer necessary to work sterile at this point. All of the PBMCs from one subject and condition were harvested in one tube. The wells were washed with PBS. Tubes were centrifuged for 4 minutes at 1500 rounds per minute (RPM) and brake 5. Supernatant was aspirated and cell pellet was resuspended in 2 mL FLS (fluorescence-activated cell sorting lysing solution; BD Biosciences USA) diluted 1:10 in water (Versylene/Fresenius, France). The mixture was vigorously mixed before incubating 10 minutes in the dark. Subsequently, tubes were placed in the centrifuge for 4 minutes at 1500 RPM and brake 5. Supernatant was aspirated. Cells were washed with PBS + 0.3% heparin (UMCG pharmacy) and centrifuged (1500 RPM, brake 5, 4 minutes). After aspiration of supernatant, the pellet was resuspended in 1 mL PBS +0.3% heparin and put in the freezer (-20 C°) until cells could be measured using flow cytometry.

17

When it was possible to measure, cells were thawed and centrifuged at 1500 RPM and brake

5 for 4 minutes. Supernatant was aspirated and pellet loosened. 5 L APC (allophycocyanin)

mouse anti-human CD3 (BD Pharmingen/BD Biosciences, USA) and 5 L PerCP (peridinin chlorophyll protein) mouse anti-human CD8 (BD Pharmingen/BD Biosciences). Tubes were vigorously mixed and incubated for 45 minutes at room temperature in the dark. After incubation, 2 mL PBS + 0.3% heparin was added and the tubes were centrifuged (1500 RPM,

brake 5, 4 minutes). Supernatant was aspirated and pellet resuspended in 300 L PBS + 0.3% heparin.

Samples were measured using BD FACS (fluorescence-activated cell sorting) Calibur (BD Biosciences, USA) and CellQuest Pro Software (BD Biosciences, USA). Analysis was done using ModFit (Verity Software House, USA). CD4+ T cells were gated being CD3+/CD8-. CD8+ T cells were gated being CD3+/CD8+.

ModFit calculates proliferation indices (PIs). This is the sum of the cells in all generations divided by the calculated number of original parent cells. In figure 6 examples of ModFit results are given. When a PI of <1.2 was found for the positive control, the proliferation assay was repeated for the relevant subject.

A B C

Statistical analysis Data were analysed using IBM SPSS Statistics version 20 (IBM, The Netherlands) and graphs were made using GraphPad Prism 5 (GraphPad Software, USA).

For testing of normality in distribution of the various variables, a Q-Q plot was made. The variables tested were age, VZV titre, number of IFN-γ spot forming cells (ELISpot data), and the proliferation indices for both CD4+ and CD8+ T-lymphocyte proliferation data. Age was normally distributed, therefore differences in age between groups were tested using a Student’s t-test. Differences in sex (a nominal variable) between groups were tested using Fisher’s exact test. For other, not normally distributed variables, differences between groups were tested using the Mann-Whitney U test. For correlations, Spearman’s ρ was used.

CFSE intensity

Figure 6. Examples of ModFit results for different conditions. (A) Example of a negative control of CD4+ T cell proliferation. A single peak of CFSE intensity is seen (PI = 1.00). (B) Example of a positive control of CD4+ T cell proliferation. CFSE intensity has shifted compared to the negative control (PI = 10.53). (C) Example of VZV stimulated CD4+ T cell proliferation (PI = 1.74).

18

Because of the different patient groups and number of subjects for each test (humoral data, ELISpot data, CD4+ T-lymphocyte data and CD8+ T-lymphocyte data, for each category a separate table with patient characteristics was made and differences between patient groups and HC tested. The table made for the CD4+ T cell proliferation data is shown in the results section as this table contains the largest number of subjects of the tables made for each test. In the appendix, all tables with patient characteristics are shown.

Because of the small number of SLE patients not using immunosuppressive drugs (n=3/4 for the different outcome variables), it was not possible to test if there was a difference in the different outcome variables between SLE patients using immunosuppressives and those not using these drugs. Data on the antibody titres against VZV and PI (proliferation index) of the positive controls of the T cell proliferation data were logarithmically transformed, and ELISpot data (number of IFN-γ spot-forming cells in response to VZV) square root transformed, to be able to use linear regression to test the influence of different immunosuppressive drugs and the SLEDAI score on the height of the antibody titre against VZV in SLE patients. The predictors used in the model were prednisone, azathioprine, hydroxychloroquine, other immunosuppressive drugs and SLEDAI score. The conditions of using linear regression; normally distributed residues and homogeneous spread of residues, were checked using a P-P plot and a scatterplot. First each predictor was univariately tested and then multivariate analysis was done.

For the VZV stimulated CD4+ T-lymphocyte and CD8+ T-lymphocyte data it was not possible to make a linear regression model as even with logarithmic or square root transformation, residues were not normally distributed. As in GPA patients there was both a substantial group of patients using immunosuppressives and a group of patients not using these drugs (see appendix for numbers for each test) the Mann-Whitney U test was used to test whether a difference in the height of antibody titres against VZV, the number of IFN-γ spot-forming cells and PIs existed between these patients. Using linear regression for this purpose was not possible in GPA patients as the conditions of using it were not met. Two-sides P-values <0,05 were considered statistically significant. Power In the application form for this scientific internship, a power of 90% was intended. To reach this, 38 patients in each group and 38 controls had to be included. Unfortunately, it was not possible to reach this number in every test. First it was intended only to use patients and matched controls in tests. But by using data of more controls, while differences in age and sex between patient and control groups were not present, power of the study is maintained as high as possible. Using G*Power 3.1.4 (free statistical analysis program) and basing on the ELISpot tests, that are considered a more sensitive tests than the proliferation assays, the power of the study at this point is 60%. This is lower than intended. However, more ELISpot data are in progress, so the ultimate power of the study will be larger.

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3. Results

Characteristics of patients and controls Humoral responses against VZV were evaluated in 33 SLE patients and 33 age and sex matched healthy controls (HC) and 31 GPA patients and 31 matched age and sex matched HCs. Cellular immunity against VZV was measured using both ELISpot and T cell proliferation tests. Using ELISpot, data on cellular immunity against VZV was obtained in 45 HC, 34 SLE patients and 28 GPA patients. Data on cellular immunity as tested with proliferation assays in CD4+ T cells is available for 51 HC, 38 SLE patients and 33 GPA patients. For CD8+ T cell proliferation tests, data are available for 35 HC, 23 SLE patients and 25 controls.

There were no statistically significant differences found between age and sex between patient and control groups for all different tests. Use of immunosuppressives is shown in the tables as well as disease activity scores (SLEDAI = systemic lupus erythematosus disease activity index for SLE patients and BVAS = Birmingham vasculitis activity score for GPA patients. All patients were included having quiescent disease, however, one GPA patient developed a BVAS of 5 three months after influenza vaccination, from which time point samples were used in this study. In table 3 characteristics of patients are shown. This table is made for patients and subjects used in the CD4+ T cell proliferation data. However, tables with characteristics of patients and controls made for the other outcome variables show no striking differences (see Appendix tables A1-A5). Table 3. Characteristics of patients and healthy controls (participating in the CD4+ T-lymphocyte proliferation

data)

HC, n=51 SLE, n=38 GPA, n=33

Sex, males, n (%) 16 (31) 9 (24), p=0.481* 12 (36), p=0.644*

Age, mean SD, in years 45.1(10.4) 43.3 (10.3), p=0.428* 48.0 (8.9), p=0.188*

Patients not using immunosuppressives, n (%)

NA 4 (10.5) 18 (54.5)

Prednisone, n (%) In users, median (range) mg/day

NA 13 (34.2) 5,0 (2,5-10)

11 (33.3) 5 (2,5-10)

Azathioprine, n (%) In users, median (range) mg/day

NA 11 (28.9) 125 (50-200)

14 (42.4) 87.5 (14.3-150)

Hydroxychloroquine, n (%) In users, median (range) mg/day

NA 18 (47.4) 400 (200-800)

NA

Other immunosuppressive drugs, n (%) NA 4 (10.5)† 3 (9.1)‡

Use of immunosuppressives unknown, n (%)

NA 7 (18.4) 0 (0)

SLEDAI/BVAS, median (range) NA 2 (0-7) 0 (0-6)**

*Given p-values are for the differences between HC&SLE or HC&GPA. Differences between the two patients groups were not tested. † three patients used methotrexate 15 mg/week and one patient used methotrexate 7,5 mg/week. ‡ one patient used mycophenolate mofetil 2 g/day, one patient used ciclosporin 150 mg/day, and one patient used prednisolone eye drops and eye ointment **Only one patient had a BVAS>0. This patient had a score of 6. NA=not applicable; HC=healthy controls; SLE=systemic lupus erythematosus; GPA=granulomatosis with polyangiitis; SLEDAI=systemic lupus erythematosus disease activity index; BVAS=Birmingham vasculitis activity score.

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Humoral data The VZV in-house gpELISA, Serion and Vidas of were not executed by the student herself. As stated in the materials and methods section, the in-house gpELISA was validated by another student. However, the student did the matching of patients and controls, followed by the analysis of data in the matched groups. Therefore, only the data of matched patients and controls are shown in this report. The student, however, executed the Diphtheria IgG ELISAs herself. SLE patients had significantly higher antibody titres against VZV than their matched HC as tested with all three tests (gpELISA, Serion and Vidas). The median antibody titre as measured with the in-house gpELISA in SLE patients was 132 IU/ml (range 21-1095), in GPA patients 75 IU/ml (range 2-1154) and in HC 66 (range 7-634). The gpELISA gave a p-value of 0.0004. Both Serion and Vidas resulted in a highly significant p-value too. In figure 7A the results of the in-house gpELISA in SLE patients and their age and sex-matched HCs are shown. Both other tests (Serion and Vidas) resulted in very similar graphs, and as the student did not execute these tests herself, the graphs are not shown.

Between the antibody titres against VZV of GPA patients and their age and sex-matched HCs in all three tests no differences were found. The results of the gpELISA in GPA patients are shown in figure 7B.

A B

Figure 7. Antibody titres against VZV measured using an in-house gpELISA. (A)Antibody titres against VZV in patients with SLE and their matched HCs. (B) Antibody titres against VZV in GPA patients and their matched HCs. Bars show the median and interquartile range.

The linear regression model made to test for influence of immunosuppressive drugs and SLEDAI scores on the height of IgG antibody titres against VZV in SLE patients in response to VZV stimulation showed no statistically significant results. The p-value for the model as a whole was 0.463.

When the height of IgG antibody titres against VZV in GPA patients without immunosuppressive drugs (n=16) were compared with the number in GPA patients using any immunosuppressive drug (n=15), a trend towards a higher antibody titre was found in GPA patients using immunosuppressives (p=0.086). However, there was no statistically significant difference present. Influence of BVAS on the titre could not be assessed, as only one patient had a score >0.

21

As a control for the IgG antibody titres against VZV, a Diphtheria IgG ELISA was executed. In both patient groups a lower IgG antibody against Diphtheria was found (p=0.012 for SLE patients versus HC; p=0.037 for GPA patients versus HC) (Figure 8).

A B

Figure 8. IgG antibody titres against Diphtheria measured using a commercial kit ELISA. (A)Antibody titres against Diphtheria in patients with SLE and their matched HCs. (B) Antibody titres against Diphtheria in GPA patients and their matched HCs. Bars show the median and interquartile range.

ELISpot data The median number of IFN-γ spot forming cells per 2 x 105 cells in response to VZV stimulation was 19.5 in HC (range 0-74); 8.5 in SLE patients (range 0-61) and 12.8 in GPA patients (range 0-66.8).

The number of IFN-γ spot forming cells in response to VZV was significantly lower in SLE patients than in HC (p=0.033). For GPA patients there was no significant difference with HC (p=0.152) (Figure 9).

Figure 9. Enzyme-linked immunospot assay of interferon-γ (IFN-γ) spot-forming cells per 2 x 10

5 peripheral

blood mononuclear cells in patients with systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (GPA) and healthy controls (HC) in response to VZV stimulation. Bars show the median and interquartile range.

The linear regression model made to test for influence of immunosuppressive drugs and SLEDAI scores on the number of IFN-γ spot forming cells per 2 x 105 cells in SLE patients in

22

response to VZV stimulation showed no statistically significant results. The p-value for the model as a whole was 1.00.

When the number of IFN-γ spot forming cells per 2 x 105 cells in GPA patients without immunosuppressive drugs (n=15) were compared with the number in GPA patients using any immunosuppressive drug (n=13), no differences were found (p=0.821). Influence of BVAS on the number of IFN-γ spot forming cells could not be assessed, as only one patient had a score >0. No correlation between the number of IFN-γ spot forming cells and antibody titre against VZV was found when tested in all subject groups (Spearman’s ρ=-0.37, p=0.704). When correlation was tested only in SLE patients (to test if there is an association between high antibody titres and a low cellular immune response against VZV), again no correlation was found. A weak correlation between the number of IFN-γ spot forming cells and age of subjects was found (Spearman’s ρ=0.201, p=0.037). CD4+ T cell proliferation data In both SLE and GPA patients and in HC, the median of PIs (proliferation indices) of the negative controls of the CD4+ T cell proliferation was 1.01. Ranges were also very similar. For HC the range was 1.00-1.11. For both patient groups this was 1.00-1.11. Therefore, no figure is shown.

The PIs of the positive controls in SLE patients (3.175; range 1.22-10.84) and in GPA patients (3.4; range 1.35-12), were significantly lower than in HC (4.19; range 1.32-9.65). For both the differences between SLE patients and HC, and GPA patients and HC there was a p-value of 0.023 (Figure 10A).

The median PI of the VZV stimulated CD4+ T-lymphocytes in SLE patients was 1.065 (range 1.00-2.86), in GPA patients 1.08 (range 1.00-3.84) and in HC 1.17 (range 1.00-4.36). The PIs of the VZV stimulated CD4+ T-lymphocytes were significantly lower than that in HC (p=0.034) (Figure 10B). A B

Figure 10. (A) Proliferation indices (PIs) for CD4+ T cell proliferation in response to CD3 and CD28 stimulation (positive control) in patients with SLE and GPA and in control subjects. (B) PIs for CD4+ T cell proliferation in response to VZV stimulation in patients with SLE and GPA and in control subjects. Bars show the median and interquartile range.

23

The linear regression model made to test for influence of immunosuppressive drugs and SLEDAI scores on PIs of the positive control and VZV stimulation of the CD4+ T-lymphocyte data in SLE patients cells in showed no statistically significant results. The p-value for the model as a whole was 0.097 for the PIs of the positive controls. When the PIs of the positive controls in GPA patients without immunosuppressive drugs (n=18) were compared with the PIs in GPA patients using any immunosuppressive drug (n=15), no differences were found (p=0.382). For the PIs of the VZV stimulated cells also no difference was found (p=0.709). Influence of BVAS on PI could not be assessed, as only one patient had a score >0. No correlation between the PIs of the VZV stimulated CD4+ T cells and the antibody titre against VZV was found when tested in all subject groups (Spearman’s ρ=0.021, p=0.820). No correlation between the PIs of the VZV stimulated CD4+ T cells and age of the subjects was found (Spearman’s ρ=0.112, p=0.220). The same held true for the PIs of the positive controls and age of the subjects (Spearman’s ρ=-0.003, p=0.974). CD8+ T cell proliferation data In SLE patients the median of PIs of the negative controls of the CD8+ T cell proliferation data was 1.02 (range 1.00-1.13). This number was 1.01 in GPA patients (range 1.00-1.06) and in HC the median was 1.01 (range 1.00-1.11). Because the results for all groups were very similar, no figure is shown.

The PIs of the positive controls of CD8+ T lymphocyte data in SLE patients (median 2.64; range 1.27-5.54) were significantly lower than in HC (median 3.97; range 1.4-7.78) (p= 0.002). In GPA patients (median 2.79; range 1.53-11.18) a trend towards a lower PI was found in comparison with HC (p=0.063) (Figure 11A).

Between the PIs of the VZV stimulated CD8+ T lymphocytes in both patients groups and HC no statistically significant difference was found. However a trend towards a lower PI was observed in SLE patients versus HC (p=0.071). The median PI in SLE patients was 1.05 (range 1.00-2.02) and the median PI of the HC 1.13 (range 1.00-3.15). Between the PIs of GPA patients (median 1.11; range 1.00-2.79) and HC no difference was found (p=0.793) (Figure 11B).

A B

Figure 11. (A) Proliferation indices (PIs) for CD8+ T cell proliferation in response to CD3 and CD28 stimulation (positive control) in patients with SLE and GPA and in control subjects. (B) PIs for CD8+ T cell proliferation in response to VZV stimulation in patients with SLE and GPA and in control subjects. Bars show the median and interquartile range.

24

The linear regression model made to test for influence of immunosuppressive drugs and SLEDAI scores on PIs of the positive controls and VZV stimulated cells of the CD8+ T-lymphocyte data in SLE patients cells in showed no statistically significant results. The p-value for the model as a whole was 0.624 for the PIs of the positive controls.

When the PIs of the positive controls in GPA patients without immunosuppressive drugs (n=11) were compared with the PIs in GPA patients using any immunosuppressive drug (n=14), no differences were found (p=0.434). For the PIs of the VZV stimulated cells also no difference was found (p=0.647). Influence of BVAS on PI could not be assessed, as all GPA patients had a score of 0.

No correlation between the PIs of the VZV stimulated CD8+ T cells and the antibody titre against VZV was found when tested in all subject groups (Spearman’s ρ=-0.053, p=0.634). No correlation between the PIs of the VZV stimulated CD8+ T cells and age of the subjects was found (Spearman’s ρ=0.111, p=0.319). The same held true for the PIs of the positive controls and age of the subjects (Spearman’s ρ=0.147, p=0.186).

25

4. Discussion In this study, humoral and cellular immunity in both SLE and GPA patients was evaluated. Humoral responses were measured using ELISA and cellular immunity using ELISpot assays and flow cytometry. ELISpot is considered as the most sensitive method of the latter two(38). It was found that SLE patients have higher VZV antibody titres while having decreased cellular immune responses against VZV compared to healthy controls. In GPA patients no differences in the immune response against VZV compared to healthy controls were found. No influence of immunosuppressives could be determined in both patient groups. Both SLE and GPA patients showed lower T cell proliferating capacity in response to CD3 and CD28 antibodies, the positive control, compared to healthy controls.

SLE patients were found to have a higher antibody IgG titres against VZV compared to age and sex-matched healthy controls. It is known that many of the patients with SLE, especially with active disease, have increased numbers of antibody-forming cells of the IgG class(46). The high VZV antibody level in SLE patients in this study cannot be explained by polyclonal hypergammaglobulinaemia, as SLE patients were found to have a lower Diphtheria IgG antibody titre. That SLE patients have a higher humoral immune response against VZV has been reported previously(42). Also for other viruses a higher antibody titre was found in SLE patients(43,44). In the influenza vaccination study of Holvast et al.(30,45) of which samples were used in the present study, an increased VZV antibody titre to various influenza strains was found in SLE patients, but this was then explained by the higher rate of previous influenza vaccination in SLE patients compared to controls.

It is now hypothesized that the increased humoral response against VZV in SLE patients is caused by the higher incidence of HZ periods in these patients, leading to a higher antibody titre. It was not possible to test this hypothesis in this study as data on HZ periods in patients and controls participating are lacking. Nagasawa et al. reported that SLE patients without a history of HZ had higher antibody titres as well(42). However, determining the occurrence of HZ periods retrospectively has been found problematic(13,47). Subclinical periods of HZ might also be causative. The existence of subclinical HZ periods is controversial(48-51). More research into the cause of higher antibody titres against VZV and other viruses is needed. For this research project there are plans to determine the total IgG of patients and controls to check if this too is increased in SLE patients. Furthermore, it may be clarifying to determine the antibody titre against a viral agent that is not endemic in The Netherlands in SLE patients and healthy controls as an extra control. Patients with SLE were shown to have fewer IFN-γ spot-forming cells and decreased T cell proliferation in response to stimulation with VZV. This is in accordance with the influenza vaccination study in which SLE patients were shown to have considerably fewer IFN-γ spot-forming cells against the different influenza strains(30,38).

Correlation between the number of IFN-γ spot forming cells and VZV antibody titre was poor. This finding fits with the findings of Vermeulen et al(52) who did a vaccination

study against HZ in healthy adults 60 years of age and with the findings of Holvast et al.(38) for influenza in SLE and GPA patients. Holvast et al. furthermore did not find an influence of immunosuppressive drugs on cellular immunity against influenza at baseline.

Both CD4+ and CD8+ T cell responses to CD3 and CD28 antibodies (positive control) were decreased in patients compared to controls. This finding seems to indicate there is a lower proliferating capacity of T cells in these patient groups in general. As no influence of

26

immunosuppressive drugs was found, this seems to be intrinsic to disease. The finding of a lower T cell proliferating capacity in SLE and GPA patients seems to be in line with the higher prevalence of infections in patients with systemic autoimmune diseases. Infections are a common cause of morbidity and mortality in these patients. The reason for that has not been fully elucidated yet, but the characteristic immune dysregulation of such diseases is thought to account at least in part (53).

A general lower T cell proliferating capacity could mean the lower response to VZV in SLE patients is not VZV-specific, but a reflection of the lower proliferating capacity of T cells in SLE patients in general. However, this does not change the assumption that the increased frequency of HZ in these patients is caused by a decreased cellular immune response. In the influenza vaccination study of Holvast et al(34,38), a normal cytokine response to Staphylococcal enterotoxin B (SEB; their positive control) was found in both patient groups. The different findings may be caused by the distinct chemicals that were used. SEB probably elicits a stronger reaction than CD3 and CD28 antibodies do.

No differences in both cell mediated and humoral immune responses to VZV were found between patients with GPA and healthy controls. This is in accordance with the influenza vaccination study of Holvast et al(34,40), in which also no difference in both types of immunity were found between GPA patients and healthy controls.

This study has some limitations. First, the standard range of the commercial kits used to determine the Diphtheria IgG antibody titre was limited to 1 IU/mL. Above this level, the

antibody level of a subject cannot be certified and was noted as 1. There are plans to buy

another kit and retest the samples that gave a result 1, in a diluted form to be able to obtain an exact antibody level for these samples.

Second, as stated in the Material and Method section, for the VZV stimulated T-lymphocyte proliferation data it was not possible to make a linear regression model to test for influence of immunosuppressives and SLEDAI score on the results. However, as for both ELISpot data and the results of the positive control of CD4+ T-lymphocyte data there was no such influence found, it is not likely that this is the case for the VZV stimulated T-lymphocyte data.

Third, the exclusion criteria of the influenza vaccination study could have had impact on the conclusions that can be drawn from this study. The medication use in both patient groups was restricted to a small number of agents and limited dose. The inclusion of merely patients with quiescent disease could also have had an influence on the medication use. In this study no influence of immunosuppressive drugs was found on the immune response to VZV. In a retrospective study by Sayeeda et al.(54) it was found that SLE patients with HZ were more likely to use cyclophosphamide, intravenous methylprednisolone pulse therapy or mycophenolate mofetil than were control SLE patients. These types of immunosuppressives were not used by the patients participating in this study. The inclusion of only patients with quiescent disease furthermore hampered the ability to assess the influence of disease activity on humoral and cellular immunity against VZV in both patient groups.

Fourth, the group of GPA patients studied in this study may give a distorted image of the immune status against VZV in these patients. The GPA patient group is relatively young and consists for a larger part of women than is typical for GPA patients. This imbalance probably is caused by the wish to match patients and controls in age and sex. More female and relatively young healthy controls were available to be included in this study.

27

Fifth, only a limited amount of data on CD8+ T cell proliferation was obtained. This is probably caused by the lack of use of EDTA in the first part of tests that were done. After starting to use EDTA more cells, including CD8+ T cells, could be measured using flow cytometry. Especially small numbers of CD8+ T cell proliferation data were obtained in SLE patients. This could be caused by the disease(55). The small number of data on CD8+ T cell proliferation could have led to the absence of a statistically significant difference in the VZV stimulated T cells between SLE patients and healthy controls. The p-value found was 0.071; with a higher number of SLE patients and healthy controls this trend towards a lower PI possibly would be statistically significant. The same holds true for the trend towards a lower PI in response to CD3 and CD28 antibodies (positive control) in GPA patients compared to controls (p = 0.063). To obtain this, more T cell proliferation test need to be done.

Despite these limitations, this study has contributed to the knowledge about the humoral and cellular immune responses to VZV in patients with autoimmune diseases. A higher humoral immune response against VZV was found in SLE patients, while cellular immunity (as determined with T cell proliferation tests and ELISpot tests) against VZV was found to be decreased in these patients compared to healthy controls. As SLE patients are known to have an increased risk of HZ(5,7,12,24) it can be concluded vaccination strategies in these patients should not be based upon humoral immune status and should aim to boost cellular immunity.

As in SLE patients a decreased cellular immune response was found and vaccination against HZ was shown to increase cellular immunity(38), vaccination against HZ in these patients could be of use. The live attenuated vaccine Zostavax probably is the appropriate vaccine to be used in autoimmune patients instead of a VZV subunit vaccine. More experience has been gained with the live attenuated vaccine than with the subunit vaccine(6,18,52,56-60). The adjuvant needed in VZV subunit vaccines has not been proven to be safe in patients with autoimmune diseases and might cause increased disease activity in these patients considering the nature of an adjuvant. The live attenuated zoster vaccine on the other hand has retrospectively been shown to be safe in patients with an autoimmune disease(23,61) and the American College against Rheumatism (ACR) is already positive about HZ vaccination in patients with rheumatoid arthritis, an autoimmune disease(27). A prospective study of varicella vaccine in children and adolescents with SLE that were previously exposed to varicella-zoster virus resulted in a lower incidence of HZ in the vaccinated SLE group. The frequency of flares and SLEDAI score were similar among vaccinated and unvaccinated patients(28). However, before recommendations can be made for the use of zoster vaccine in specific autoimmune patient groups, prospective randomised trials are necessary. Acknowledgements I would like to thank the members of the vaccination group for the very useful weekly discussion meetings; Hannie Westra (my supervisor), Aalzen de Haan, Nico Bos, Sander van Assen and Marc Bijl. Special thanks to Gerda Horst for teaching me all the laboratory skills and for her untiring optimism.

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350. (47) Klompas M, Kulldorff M, Vilk Y, Bialek SR, Harpaz R. Herpes zoster and postherpetic neuralgia surveillance using structured electronic data. Mayo Clin Proc 2011 Dec;86(12):1146-1153. (48) Malavige GN, Rohanachandra LT, Jones L, Crack L, Perera M, Fernando N, et al. IE63-specific T-cell responses associate with control of subclinical varicella zoster virus reactivation in individuals with malignancies. Br J Cancer 2010 Feb 16;102(4):727-730. (49) Kronenberg A, Bossart W, Wuthrich RP, Cao C, Lautenschlager S, Wiegand ND, et al. Retrospective analysis of varicella zoster virus (VZV) copy DNA numbers in plasma of immunocompetent patients with herpes zoster, of immunocompromised patients with disseminated VZV disease, and of asymptomatic solid organ transplant recipients. Transpl Infect Dis 2005 Sep-Dec;7(3-4):116-121. (50) Mehta SK, Cohrs RJ, Forghani B, Zerbe G, Gilden DH, Pierson DL. Stress-induced subclinical reactivation of varicella zoster virus in astronauts. J Med Virol 2004 Jan;72(1):174-179. (51) Birlea M, Arendt G, Orhan E, Schmid DS, Bellini WJ, Schmidt C, et al. Subclinical reactivation of varicella zoster virus in all stages of HIV infection. J Neurol Sci 2011 May 15;304(1-2):22-24. (52) Vermeulen JN, Lange JM, Tyring SK, Peters PH, Nunez M, Poland G, et al. Safety, tolerability, and immunogenicity after 1 and 2 doses of zoster vaccine in healthy adults >/=60 years of age. Vaccine 2012 Jan 20;30(5):904-910. (53) Doria A, Zampieri S, Sarzi-Puttini P. Exploring the complex relationships between infections and autoimmunity. Autoimmun Rev 2008 Dec;8(2):89-91. (54) Sayeeda A, Al Arfaj H, Khalil N, Al Arfaj AS. Herpes Zoster Infections in SLE in a University Hospital in Saudi Arabia: Risk Factors and Outcomes. Autoimmune Dis 2010 Sep 13;2011:174891. (55) Iliopoulos AG, Tsokos GC. Immunopathogenesis and spectrum of infections in systemic lupus erythematosus. Semin Arthritis Rheum 1996 Apr;25(5):318-336. (56) Oxman MN. Zoster vaccine: current status and future prospects. Clin Infect Dis 2010 Jul 15;51(2):197-213. (57) Oxman MN. Herpes zoster pathogenesis and cell-mediated immunity and immunosenescence. J Am Osteopath Assoc 2009 Jun;109(6 Suppl 2):S13-7. (58) Oxman MN. Vaccination to prevent herpes zoster and postherpetic neuralgia. Hum Vaccin 2007 Mar-Apr;3(2):64-68. (59) Oxman MN, Levin MJ, Johnson GR, Schmader KE, Straus SE, Gelb LD, et al. A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med 2005 Jun 2;352(22):2271-2284. (60) Weinberg A, Zhang JH, Oxman MN, Johnson GR, Hayward AR, Caulfield MJ, et al. Varicella-zoster virus-specific immune responses to herpes zoster in elderly participants in a trial of a clinically effective zoster vaccine. J Infect Dis 2009 Oct 1;200(7):1068-1077. (61) Zhang J, Xie F, Delzell E, Chen L, Winthrop KL, Lewis JD, et al. Association between vaccination for herpes zoster and risk of herpes zoster infection among older patients with selected immune-mediated diseases. JAMA 2012 Jul 4;308(1):43-49.

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Appendix

Belonging to the scientific internship report of Christien Rondaan:

“Humoral and cellular immunity against varicella-zoster virus in patients with autoimmune diseases”.

Abbreviation list ACIP: Advisory Committee on Immunization Practices ACR: American College of Rheumatology AIIRD: Autoimmune Inflammatory Rheumatic Disease ALP: Alkaline Phosphatase ANA: Antinuclear Antibody ANCA: Anti-Neutrophil Cytoplasmatic Antibody APC: Allophycocyanin BCIP/NBT: 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt/nitro-blue tetrazolium

chloride Bl: Blank BVAS: Birmingham Vasculitis Activity Score CD: Cluster of Differentiation CFSE: Carboxyfluorescein Succinimidyl Ester CMI: Cellular Mediated Immunity CO2: Carbon dioxide ConA: Concanavalin A DMARD: Disease Modifying Anti-Rheumatic Drug DNA: Deoxyribonucleic Acid dsDNA: double-stranded Deoxyribonucleic Acid EDTA: Ethylenediaminetetraacetic Acid ELISA: Enzyme-linked Immunosorbent Assay ELISpot: Enzyme-linked Immunosorbent Spot FACS: Fluorescence-Activated Cell Sorting FCS: Fetal Calf Serum FLS: FACS lysing solution gE: glycoprotein E gp: glycoprotein GPA: Granulomatosis with Polyangiitis HC: Healthy control HIV: Human Immunodeficiency Virus HPS: Human Pool Serum HZ: Herpes Zoster IFN-γ: Interferon-gamma IgG: Immunoglobulin G IU: International Units MCTD: Mixed Connective Tissue Disease MHC: Major Histocompatibility Class PBMC: Peripheral Blood Mononuclear Cell PBS: Phosphate Buffered Saline

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PerCP: Peridinin Chlorophyll Protein PHN: Postherpetic Neuralgia PI: Proliferation Index RNP: Ribonucleoprotein RPM: Rounds Per Minute RPMI: Roswell Park Memorial Institute SEB: Staphylococcal Enterotoxin B SLE: Systemic Lupus Erythematosus SLEDAI: SLE Disease Activity Score TMB: 3,3’,5,5’-tetramethylbenzidine UMCG: University Medical Center Groningen UV: Ultraviolet VZV: Varicella-Zoster Virus Tables with patient characteristics for each outcome variable

Table A1. Characteristics of SLE patients and matched healthy controls participating in the humoral data tests

HC, n=33 SLE, n=33

Sex, males, n (%) 7 (21.2) 7 (21.2)

Age, mean SD, in years 43.8 (10.4) 43.1 (10.0)

Patients not using immunosuppressives, n (%)

NA 3 (9.1)

Prednisone, n (%) In users, median (range) mg/day

NA 12 (36.4) 5,0 (2,5-10)

Azathioprine, n (%) In users, median (range) mg/day

NA 10 (30.3) 112.5 (50-175)

Hydroxychloroquine, n (%) In users, median (range) mg/day

NA 14 (42.4) 400 (200-800)

Other immunosuppressive drugs, n (%) NA 4 (12.1)* Use of immunosuppressives unknown, n (%)

NA 7 (21.2)

SLEDAI, median (range) NA 2 (0-7)

* three patients used methotrexate 15 mg/week and one patient used methotrexate 7,5 mg/week.

TableA 2. Characteristics of GPA patients and matched healthy controls participating in the humoral data tests

HC, n=31 GPA, n=31

Sex, males, n (%) 11 (35) 11 (35)

Age, mean SD, in years 47.6 (8.3) 47.5 (8.8)

Patients not using immunosuppressives, n (%)

NA 16 (51.6)

Prednisone, n (%) In users, median (range) mg/day

NA 9 (29.0) 7.5 (5-15)

Azathioprine, n (%) In users, median (range) mg/day

NA 11 (35.5) 75 (14,3-150)

Other immunosuppressive drugs, n (%) NA 3 (9.7)* BVAS, median (range) NA 0 (0-6)**

* One patient used mycophenolate mofetil 2 g/day, one patient used ciclosporin 150 mg/day, and one patient used prednisolone eye drops and eye ointment ** Only one patient had a BVAS>0. This patient had a score of 6. NA=not applicable; HC=healthy controls; GPA=granulomatosis with polyangiitis; BVAS=Birmingham vasculitis activity score.

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Table A3. Characteristics of patients and controls participating in the ELISpot data

HC, n=45 SLE, n=34 GPA, n=28

Sex, males, n (%) 15 (33) 8 (24), p=0.45* 11 (39), p=0.624*

Age, mean SD, in years 45.4 (11.0) 43.2 (10.7), p=0.39* 48.5 (8.8), p=0.20 *

Patients not using immunosuppressives, n (%) NA 3 (8,8) 15 (53,6) Prednisone, n (%) In users, median (range) mg/day

NA 14 (41,2) 5,0 (2,5-7,5)

10 (35,7) 5,625 (2,5-7,5)

Azathioprine, n (%) In users, median (range) mg/day

NA 12 (35,3) 112,5 (50-200)

11 (39,3) 100 (14,3-150)

Hydroxychloroquine, n (%) In users, median (range) mg/day

NA 18 (52,9) 400 (200-800)

NA

Other immunosuppressive drugs, n (%) NA 4 (11,7)† 3 (10,7)‡

Use of immunosuppressives unknown, n (%) NA 5 (14,7) 0 (0) SLEDAI/BVAS, median (range) NA 2 (0-7) 0 (0-6)**

*Given p-values are for the differences between HC&SLE or HC&GPA. Differences between the two patients groups were not tested. † three patients used methotrexate 15 mg/week and one patient used methotrexate 7,5 mg/week. ‡ one patient used mycophenolate mofetil 2 g/day, one patient used ciclosporin 150 mg/day, and one patient used prednisolone eye drops and eye ointment **Only one patient had a BVAS>0. This patient had a score of 6. NA=not applicable; HC=healthy controls; SLE=systemic lupus erythematosus; GPA=granulomatosis with polyangiitis; SLEDAI=systemic lupus erythematosus disease activity index; BVAS=Birmingham vasculitis activity score. Table A4. Characteristics of patients and healthy controls participating in the CD4+ T-lymphocyte proliferation data

HC, n=51 SLE, n=38 GPA, n=33

Sex, males, n (%) 16 (31) 9 (24), p=0.481* 12 (36), p=0.644*

Age, mean SD, in years 45.1(10.4) 43.3 (10.3), p=0.428* 48.0 (8.9), p=0.188*

Patients not using immunosuppressives, n (%)

NA 4 (10.5) 18 (54.5)

Prednisone, n (%) In users, median (range) mg/day

NA 13 (34.2) 5,0 (2,5-10)

11 (33.3) 5 (2,5-10)

Azathioprine, n (%) In users, median (range) mg/day

NA 11 (28.9) 125 (50-200)

14 (42.4) 87.5 (14.3-150)

Hydroxychloroquine, n (%) In users, median (range) mg/day

NA 18 (47.4) 400 (200-800)

NA

Other immunosuppressive drugs, n (%) NA 4 (10.5)† 3 (9.1)‡

Use of immunosuppressives unknown, n (%)

NA 7 (18.4) 0 (0)

SLEDAI/BVAS, median (range) NA 2 (0-7) 0 (0-6)**

*Given p-values are for the differences between HC&SLE or HC&GPA. Differences between the two patients groups were not tested. † three patients used methotrexate 15 mg/week and one patient used methotrexate 7,5 mg/week. ‡ one patient used mycophenolate mofetil 2 g/day, one patient used ciclosporin 150 mg/day, and one patient used prednisolone eye drops and eye ointment **Only one patient had a BVAS>0. This patient had a score of 6. NA=not applicable; HC=healthy controls; SLE=systemic lupus erythematosus; GPA=granulomatosis with polyangiitis; SLEDAI=systemic lupus erythematosus disease activity index; BVAS=Birmingham vasculitis activity score.

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Table A5. Characteristics of patients and healthy controls participating in the CD8+ T-lymphocyte proliferation data

HC, n=35 SLE, n=23 GPA, n=25

Sex, males, n (%) 12 (34) 6 (26), p=0.573* 9 (36), p=0.492*

Age, mean SD, in years 48.0 (10.5) 43.0 (10.4), p=0.161* 48.0 (9.4), p=1.00 *

Patients not using immunosuppressives, n (%)

NA 3 (13.0) 11 (44.0)

Prednisone, n (%) In users, median (range) mg/day

NA 6 (26.1) 5,0 (2,5-10)

9 (36.0) 5.0 (2.5-10)

Azathioprine, n (%) In users, median (range) mg/day

NA 7 (30.4) 125 (75-175)

12 (48.0) 75 (14,3-150)

Hydroxychloroquine, n (%) In users, median (range) mg/day

NA 12 (52.2) 400 (200-800)

NA

Other immunosuppressive drugs, n (%) NA 1 (4.3)† 3 (12.0)‡

Use of immunosuppressives unknown, n (%)

NA 4 (17.4) 0 (0)

SLEDAI/BVAS, median (range) NA 2 (0-7) 0 (NA)**

*Given p-values are for the differences between HC&SLE or HC&GPA. Differences between the two patients groups were not tested. † the patients used methotrexate 15 mg/week. ‡ one patient used mycophenolate mofetil 2 g/day, one patient used ciclosporin 150 mg/day, and one patient used prednisolone eye drops and eye ointment **All GPA patients had a BVAS of 0. NA=not applicable; HC=healthy controls; SLE=systemic lupus erythematosus; GPA=granulomatosis with polyangiitis; SLEDAI=systemic lupus erythematosus disease activity index; BVAS=Birmingham vasculitis activity score.