Towards strengthening memory immunity in the ageing population
van der Heiden, Marieke
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van der Heiden, M. (2018). Towards strengthening memory immunity in the ageing population: Investigating the immunological fitness of middle-aged adults. Rijksuniversiteit Groningen.
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MEMORY IMMUNITY IN THE
AGEING POPULATION
Investigating the immunological
fitness of middle-aged adults
Printing Ridderprint BV | www.ridderprint.nl
ISBN 978-94-034-0229-1 | printed book 978-94-034-0228-4 | e-book
Printing of this thesis was kindly supported by: The Dutch National Institute of Public Health and the Environment (RIVM), The University of Groningen (RUG), and the Groningen University Institute for Drug Exploration (GUIDE).
Copyright © 2017 Marieke van der Heiden
All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission from the author, or when applicable, from the publishers of the scientific articles.
Investigating the immunological fitness of middle-aged adults
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op woensdag 10 januari 2018 om 11.00 uur
door
Copromotores
Dr. A.M. Buisman Dr. G.A.M. Berbers
Beoordelingscommissie
Prof. dr. W. van Eden Prof. dr. S.M. van Ham Prof. dr. A.L.W. Huckriede
Paranimfen
Lia de Rond
Chapter 2 Differential effects of Cytomegalovirus carriage on the immune phenotype of middle-aged males and females Sci Rep. 2016; 6: 26892
27
Chapter 3 Novel intervention in the ageing population:
a primary meningococcal vaccine inducing protective IgM responses in middle-aged adults
Front Immunol. 2017; 8: 817
57
Chapter 4 Lower antibody functionality in middle-aged adults compared to adolescents after primary meningococcal vaccination: role of IgM
Submitted
85
Chapter 5 Tetanus Toxoid carrier protein induced T-helper cell responses upon vaccination of middle-aged adults
Vaccine. 2017.08.056
103
Chapter 6 Age-dependent pre-vaccination immunity affects the immunogenicity of Varicella Zoster vaccination in middle-aged adults
Submitted
121
Chapter 7 An explorative biomarkers study for vaccine responsiveness after primary meningococcal vaccination in middle-aged adults Submitted
145
Chapter 8 General discussion 169
Appendices Nederlandse samenvatting About the author
Publication list Dankwoord 187 193 194 195
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Ageing of the world population (and other challenges)The increased life expectancy in combination with the baby boom after the Second World War leads to a rapidly ageing world population [1, 2]. The worldwide number of persons above 60 years of age is expected to be doubled in 2060, with the highest growth predicted for the oldest persons above the age of 80 [3]. This rapid ageing results in increased numbers of persons susceptible to disease and disability, causing a strong rise in health care costs [1, 2]. Of importance and in contrast with past generations, todays and future elderly are more frequently enjoying an active lifestyle, putting high demands on societal and health care facilities [4]. Furthermore, globalization and antibiotic resistance are additional challenges of the 21st century, also enhancing the infection pressure in older adults. Consequently, preventive measures, such as effective vaccination programs are of high importance to establish healthy ageing [5].
Ageing and infectious diseases
The increased susceptibility of the elderly towards infections, cancer, and autoimmune diseases, indicates that a broad range of geriatric diseases is linked to the immune system [6-10]. Importantly, the incidence of autoimmune diseases, such as rheumatoid arthritis, already strongly increases after the age of 50 [11].
Nowadays, respiratory infectious diseases cause a large disease burden in the elderly, of which influenza contributes most to morbidity and mortality [10, 12, 13]. Likewise, older adults are increasingly vulnerable to respiratory syncytial virus (RSV) [13], rhinovirus infections [10] as well as infection with Streptococcus pneumoniae. The latter pathogen is one of the major causative agents for the increasing incidence of bacterial pneumonia in the elderly [14, 15]. Along with the increased incidence of respiratory infections, the incidence of Herpes Zoster, caused by reactivation of the varicella zoster virus, is steadily increasing after the age of 50 [16]. Besides, the population herd immunity against infectious diseases in the total population may diminish as a consequence of increasing numbers of susceptible elderly [10, 17]. Consequently, protection of the elderly against infectious diseases is a prerequisite to establish healthy ageing and is therefore of high priority [18, 19]. Vaccination of the elderly
Next to the increased vulnerability of elderly persons towards infectious diseases, vaccines often induce suboptimal responses in the elderly. A vaccine routinely provided to older adults is the seasonal influenza vaccine. The effectiveness of this vaccine is strongly reduced with advancing age, resulting in an estimated efficacy of 30-50% in the elderly [23-26].
Herd immunity
Herd immunity is defined as the resistance of a population to infection by infectious pathogens [20-22]. For many pathogens, susceptible individuals are indirectly protected against infection by immunization of the surrounding individuals. The proportion of individuals that needs to be immunized to induce herd immunity differs per pathogen and depends primarily on the infectivity, spreading method and viability of the pathogen. Moreover, factors such as seasonality, demographics of the population, age of the susceptible individuals, geographical area, and social habits also determine the level of herd immunity required to prevent the spread of a particular infectious disease [20, 22]. Providing herd immunity is the primary goal of national immunization programs.
Also, despite the recommendation of pneumococcal vaccination above the age of 65 [30], pneumococcal vaccine responses drop with advancing age [30-32], leaving part of the elderly unprotected. A routine vaccination program to protect against varicella zoster is another topic of debate, due to the low effectiveness of the currently available vaccine in the elderly [33, 34]. Furthermore, serological surveillance studies reveal that increasing numbers of elderly are unprotected against tetanus and diphtheria in certain European countries. An additional booster vaccination in these unprotected elderly persons induces antibodies above the protective threshold, but unfortunately this protection is of short duration [35]. Successful tetanus and diphtheria vaccination is strongly linked to the presence of high pre-vaccination antibody levels at elderly age [35]. Finally, primary vaccine responses against hepatitis B [36, 37] and yellow fever [38] are lower in the elderly. These findings indicate general reduced vaccine responsiveness in the elderly, as a consequence of immunological ageing.
Ageing of the immune system
Immunological ageing is associated with compositional changes in immune phenotype, starting already at the level of the precursors of immune cells; the hematopoietic stem cells [39]. Most importantly, with ageing, the balance between the lymphoid and myeloid lineage shifts towards the myeloid lineage, leading to fewer precursors of the lymphoid lineage [39]. Secondly, fat deposition in the bone marrow and thymus, along with thymic shrinkage, reduces the production and development of new lymphoid cells at older age even more [19, 40-45]. Maintenance of the thymic function with age differs substantially between mice and humans, underlining the importance of human studies on immune ageing [40]. In the following paragraphs the most important changes in both the adaptive and innate human immune system are discussed, of which a schematic representation is depicted in Figure 1.
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Figure 1. The effect of ageing on the immune system.
The most profound characteristics of the ageing immune system are described for both the adaptive (blue) and innate (orange) part of the immune system. The age related changes in the primary lymphoid organs (bone marrow and thymus) are indicated in red.
Ageing of the T-cell lineage and accelerating factors
Immunological ageing primarily affects the T-cell compartment, due to shrinkage of the thymus in early adulthood. Thymic function is reduced to 10% already at the age of 50 [40, 42], resulting in diminished production of naïve T-cells in both the CD4 and CD8 T-cell lineages. As a consequence, peripheral homeostatic proliferation of already existing naïve T-cells is enhanced [40, 42, 46]. This phenomenon may be related to the observation of increased numbers of CD4+CD45RA+CD25dim cells, displaying a naïve like phenotype, in healthy elderly, possibly preserving the naïve T-cell repertoire at older age [47]. Importantly, the decreased production of naïve T-cells provokes a diminished T-cell receptor (TCR) repertoire diversity, which is likely to negatively impact responses to de novo antigens [48, 49]. As a result of the antigenic pressure throughout the life-span, naïve T-cells are (antigen specifically) stimulated and differentiated into memory cells [40, 42, 46]. Subsequently, chronic antigen stimulation, for example by latent herpes viruses such as cytomegalovirus (CMV), leads to the accumulation of late-differentiated T-cells, which are mainly found in the CD8 T-cell compartment [50-55]. These late-differentiated cells possess short telomeres and might be less able to proliferate after stimulation [42, 56]. Therefore, these cells are often referred to as exhausted or senescent cells, although the exact functionality of these
In addition, the numbers of Treg cells increase with advancing age [59, 60] and are mainly of the memory Treg phenotype [61]. These results suggest enhanced suppression of immune responses at old age, due to disturbances in the balance between Treg and effector cells in the immune system [61]. As a side note, accelerated ageing of the T-cell lineage is observed in older males, indicating large effects of sex on the immune phenotype at old age [50-52, 62].
Ageing of the B-cell lineage
Although less pronounced than shifts in the T-cell compartment, also changes are observed in the B-cell lineage with advancing age. The main changes include a reduced production of B-cell progenitors as well as an accumulation of late-differentiated CD27- memory B-cells of limited specificity [44, 63, 64]. Secondly, reduced numbers of IgM+ B-cells are found with advancing age [63, 65], whereas the numbers of long-lived plasma cells also diminish as a result of decreased bone marrow survival niches for these cells [44, 66]. Accordingly, limited B-cell receptor (BCR) diversity is noticed in older persons [67, 68]. Moreover, aged B-cells possess a reduced capacity for proliferation [44], possibly related to diminished T-cell help to the B-cells in the germinal centres, since these meeting points for cellular interaction between B- and T-cells are found to decrease with age as well [44, 69]. Likewise, a reduced capacity for class switch recombination (CSR) [65] as well as an increased production of autoreactive antibodies [44, 68, 70] are observed in old B-cells. CMV infection is found to only minimally affect B-cell numbers and frequencies [71], whereas sex hormones, mainly estrogens, are found to positively affect (auto)antibody formation and B-cell proliferation [72].
Ageing of the innate immune system
As reviewed elsewhere [73, 74], also innate immunity, the first line of defence, undergoes changes with advancing age. Most noticeable is the remodelling process of natural killer (NK) cell phenotype and function. At first, although increases in NK cell numbers are noted with advancing age, the per cell cytotoxicity is reduced. Additionally, aged NK cells are frequently of the CD56dim phenotype, in contrast to CD56bright NK cells at younger age [73, 74]. Along with the diminished NK cell functions, neutrophils, monocytes, and macrophages show reduced functional capacity, such as chemotaxis, phagocytosis and apoptosis, whereas dendritic cells show reduced TLR signalling and a reduced capacity for antigen presentation [73, 74]. More specifically, an age-dependent reduction in the expression and activation of AIM2, crucial for innate signalling against certain bacteria and dsDNA viruses, was found [75]. Nevertheless, the absolute numbers of these cells are maintained with ageing [73, 74], likely caused by the shift towards the myeloid lineage with age [39]. Thus, although the
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numbers of innate immune cells tend to increase with ageing, the functionality of thesecells is generally less effective which may underlie reduced innate sensing and responses in immunosenescence.
In addition to the altered functionality of the innate immunity, aged innate immune cells produce increased amounts of pro-inflammatory cytokines and acute phase proteins (such as C-reactive protein (CRP)), resulting in a low-grade inflammatory state [9, 76, 77]. This so called inflammageing is thought to lead to hypo responsiveness thereby rendering innate immune cells less able to clear antigens or stimulate cells of the adaptive immune system [70, 74]. Up till now, chronic infection with CMV was not found to affect the inflammatory state [78].
Inflammageing and the senescence-associated-secretory phenotype
Inflammageing is the term used for the overall increase in (low-grade) inflammation with advancing age [9, 76, 77, 79]. This inflammageing is considered the feedback loop of life-long exposure to antigens that trigger inflammatory responses and subsequent tissue damage and production of reactive oxygen species (ROS) [9]. Alternatively, inflammageing may be the result of diminished mucosal resistance with ageing and leakage of microbial products, such as LPS, from the gut to the blood, referred to as the leaky gut syndrome [80, 81]. Also senescent cells may contribute to this low-grade inflammation, due to secretion of inflammatory mediators as a result of DNA damage, termed the senescence-associated-secretory phenotype [79].
Novel strategies for elderly vaccination
In view of the above summarized functional deterioration of the ageing immune system, referred to as immunosenescence, it is questioned at what ages specific vaccines demonstrate reduced effectivity and how the diversity of the elderly population affects vaccine responses [82]. Dedicated studies investigating the interaction between age, immune function and vaccine responses are warranted to discover alternative strategies to strengthen the memory immunity of elderly persons. Several novel approaches for elderly vaccination are proposed, such as high-dose vaccines, the use of new adjuvants, and the administration of vector based vaccines [83]. In addition, life-long vaccination schedules aiming to maintain memory immunity against infectious diseases over the entire span are a promising alternative [18, 26, 84]. We thus propose the development of life-long vaccination programs starting with childhood vaccinations, which remain extremely important to induce immunity, in combination with vaccination programs in middle-aged
and repeated vaccination might cause hypo responsiveness of the immune response [85]. Importantly, these life-long vaccination programs might strengthen the herd immunity in the total population [84]. Since the pace of immune ageing differs per individual [86], the development of chronological age based vaccination programs is challenging. Therefore, vaccination programmes should be based on biological age or even the ‘immunological age’ of individuals in so called personalised national immunization programs. Consequently, there is a large need for biomarkers that predict vaccine responses, preferably markers that can be measured earlier in life, to ensure that precautions can be taken before reaching old age [82, 87].
Immunosenescence
Immunosenescence is defined as the functional deterioration of the immune system due to ageing and or ageing mechanisms. This immunosenescence is often linked to increased susceptibility towards infectious diseases, cancer and autoimmunity [88, 89] and thus thought to contribute to increased morbidity and mortality in the elderly.
Predictive biomarkers for vaccine responsiveness
The discovery of predictive biomarkers for vaccine responsiveness is ongoing and proven to be challenging. At present, most information is obtained from influenza vaccine studies in the elderly. Several of these influenza studies, as well as a hepatitis B study, describe a positive association between the number of switched memory B-cells and vaccine responsiveness in the elderly [64, 90-92]. Moreover, high numbers of late-differentiated B-cells show a negative association with the humoral response to influenza vaccination [64], whereas also the level of activation-induced cytidine deaminase (AID) in stimulated B-cells, is found predictive for the vaccine response [93, 94]. Latent infection with CMV might negatively affect the B-cell responses towards influenza vaccination [95]. In addition, influenza vaccine responses are found to negatively associate with high numbers of late-differentiated CD4 and CD8 T-cells as well as memory Treg cells [61, 91]. Besides, associations between genetic signatures, innate immune functions, and miRNA expression levels with influenza vaccine responsiveness are observed [90, 96, 97]. In line with these influenza vaccine studies, several other vaccines were used in explorative biomarker studies. First of all, high numbers of Treg cells, as well as CMV specific late-differentiated CD4 T-cells negatively affect varicella zoster vaccine responses in the elderly [98]. In addition, the yellow fever vaccine is more successful in elderly participants possessing high numbers of recently produced naïve T-cells as well as high numbers of peripheral dendritic cells [38]. On the other hand, an explorative biomarker study using a booster vaccination against diphtheria and tetanus in the elderly, did not reveal immune markers related to the vaccine response [35]. These studies might
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indicate differences in prediction between viral and bacterial vaccines. Of importance,pre-vaccination immunity often confounds the discovery of predictive biomarkers [27, 35, 92]. Similarly, sex frequently influences vaccine responses, although the exact effects of sex at advanced age are unknown. In general, stronger humoral responses are often found in females and might be caused by stimulating effects of estrogens, contrary to suppressive effects of testosterone in males [99, 100]. Therefore, sex is an important factor that has to be taken into account in biomarkers discovery studies.
The hypothesis: vaccination of middle-aged adults
The deleterious effects of immune ageing are suggested to be more pronounced for de novo vaccine responses. Due to the early appearance of the first signs of immune ageing, it is suggested that immunizations against new antigens have to be established before the onset of immunosenescence, most probably in the 5th or 6th decade of life [17]. Consequently, we propose middle-aged adults as an interesting target group for future vaccine interventions in order to strengthen the memory immunity, both by primary and recall vaccinations, before reaching old age. Subsequently, the memory immunity against infectious diseases of these future elderly might be improved until high age (Figure 2). However, knowledge on the immunological fitness of middle-aged adults, and factors affecting their immune function, is currently limited.
Figure 2. Hypothesis of this thesis; vaccination of middle-aged adults to strengthen the memory immunity of the elderly.
1). Currently, vaccine programmes for babies and children are well accepted. 2). In view of the ageing population, prevention of the elderly against infectious diseases is a priority, but challenged by the developments of immunosenescence leading to reduced vaccine efficacy. 3). Timely vaccination of middle-aged adults might strengthen the memory immunity of the future elderly. However, knowledge on the immunological fitness of the middle-aged adults is currently limited and therefore the aim of study in this thesis.
Primary and recall vaccination
After primary vaccination, the immune system is exposed to an antigen for the first time (de novo antigen). A primary immune response thus takes time to develop and leads mainly to the production of IgM antibodies. After primary immunization, immunological memory is induced that aids a rapid and strong (IgG based) immune response after a secondary encounter with the same antigen. Active secondary immunizations by vaccination are also called recall or booster vaccinations [101].
Research aim
In this thesis, we aim to provide insight into the immunological fitness of middle-aged adults between 50 to 65 years of age. The following research questions are addressed: 1. How do sex and chronic viral infection with CMV affect the immune phenotype of
middle-aged adults?
2. How do middle-aged persons respond to a primary immunization with vaccine antigens towards which no or (very) low pre-vaccination immunity exists?
3. What is the immunogenicity of the varicella zoster vaccine in middle-aged adults? 4. Can we find predictive biomarkers for vaccine responsiveness in middle-aged adults? Study outline
In order to answer these research questions, a clinical trial (study acronym: StimulAge study) with two different study arms was conducted in Dutch middle-aged adults (50-65 years of age). In total 255 middle-aged adults participated in this study. In blood samples from all participants a detailed immune phenotyping was performed, using absolute cell numbers of a comprehensive set of immune cell subsets.
Within the first study arm, 204 middle-aged adults were vaccinated with the tetravalent meningococcal vaccine conjugated to tetanus toxoid (MenACWY-TT). The meningococcus is used here as a model antigen, to initiate a primary immune response, without the interference of high pre-vaccination immunity obtained by natural contacts. These low levels of pre-vaccination immunity against the meningococcal groups was expected in the middle-aged adults since the circulation of meningococci C (MenC) is virtually absent after the mass vaccination campaign in 2002 and the historical circulation of meningococci W (MenW) and Y (MenY) has been low. This low historical circulation was confirmed by a large serological surveillance study, performed every ten years in the Netherlands, revealing low levels of meningococcal group specific IgG antibodies in Dutch adults (Figure 3) [102].
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0 0.5 1 1.5 2 2.5 3 3.5 Age at bloodsamplingMenA MenC MenW‐135 MenY
Figure 3. The age specific meningococcal IgG antibody concentrations in the Dutch population (2006-2007) after the Meningococcus C specific mass vaccination campaign [102].
The second study arm focusses on the responses towards an early varicella zoster vaccination in the middle-aged adults. The incidence of Herpes Zoster, caused by reactivation of the varicella zoster virus (VZV), strongly increases with age and is caused by a drop in VZV-specific cell mediated immunity (CMI) [16, 103, 104]. The implementation of the varicella zoster vaccination in national immunization programs is topic of fierce debate, due to low effectiveness of this vaccine in the elderly [33, 34]. Consequently, timely vaccination of middle-aged adults might be an alternative option to increase the VZV-specific CMI before reaching old age.
Thesis outline
The first research question is addressed in Chapter 2. In this chapter, effects of sex and latent infection with CMV, as well as the interaction between sex and CMV infection, on the absolute cell counts are investigated. This analysis provides a better understanding of the interaction between the immune phenotype and environmental factors such as CMV during the ageing process.
In Chapter 3, we investigate the immunogenicity of the primary meningococcal vaccination in the middle-aged adults, in order to answer the second research question. Additionally, the long-term protection of this vaccine is predicted using bi-exponential decay modelling. Subsequently, in Chapter 4, the primary vaccine responses in the middle-aged adults are
The meningococcal polysaccharides in this vaccine are conjugated to a tetanus toxoid carrier protein in order to induce T-cell help in response to the meningococcal polysaccharides that are only recognized by B-cells. The T-cell response induced by the tetanus toxoid carrier protein in the middle-aged adults is investigated in Chapter 5. Also, the relation between the humoral vaccine response and the T-cell help towards the carrier is investigated in this chapter.
The third research question is addressed in Chapter 6 and describes the immunogenicity of a varicella zoster vaccination in middle-aged adults. In addition, predictive factors for the VZV vaccine responsiveness in middle-aged adults are described in this chapter as well. In order to answer the last research question, an explorative biomarkers study is described in
Chapter 7. In this chapter, the association between the pre-vaccination immune phenotype
and vaccine responsiveness is determined using multivariate redundancy analysis (RDA). Finally, the main findings of this thesis are summarized and remaining questions and future perspectives discussed in Chapter 8.
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2
CYTOMEGALOVIRUS CARRIAGE
ON THE IMMUNE PHENOTYPE
OF MIDDLE-AGED MALES
AND FEMALES
Marieke van der Heiden1,2 Menno C. van Zelm3,4 Sophinus J.W. Bartol3 Lia G.H. de Rond1 Guy A.M. Berbers1 Annemieke M.H. Boots2 Anne-Marie Buisman1
1 Centre for Infectious Disease Control (Cib), National Institute for
Public Health and the Environment (RIVM), Bilthoven 3720 BA, The Netherlands
2 Department of Rheumatology and Clinical Immunology,
University of Groningen, University Medical Centre Groningen, Groningen 9700 RB, The Netherlands
3 Department of Immunology, Erasmus MC, Rotterdam 3000 CA,
The Netherlands
4 Department of Immunology and Pathology, Central Clinical
School, Monash University, Melbourne, Victoria 3004, Australia
Abstract
The elderly population is more susceptible to infections as a result of an altered immune response, commonly referred to as immunosenescence. Cytomegalovirus (CMV)-infection associated changes in blood lymphocytes are known to impact this process, but the interaction with gender remains unclear. Therefore, we analysed the effects and interaction of gender and CMV on the absolute numbers of a comprehensive set of naive and memory T- and B-cell subsets in people between 50 and 65 years of age.
Enumeration and characterization of lymphocyte subsets by flow cytometry was performed on fresh whole blood samples from 255 middle-aged persons. CMV-IgG serostatus was determined by ELISA.
Gender was a major factor affecting immune cell numbers. CMV infection was mainly associated with an expansion of late-differentiated T-cell subsets. CMV+ males carried lower numbers of total CD4+, CD4+ central memory (CM) and follicular helper T-cells than females and CMV- males. Moreover, CMV+ males had significantly lower numbers of regulatory T (Treg)-cells and memory B-cells than CMV+ females.
We here demonstrate an interaction between the effects of CMV infection and gender on T- and B-cells in middle-aged individuals. These differential effects on adaptive immunity between males and females may have implications for vaccination strategies at middle-age.
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Introduction
Evidence is accumulating that the increased morbidity, risk for infections, and reduced vaccination responses in elderly are associated with changes in immune function [1-4]. Several heritable and non-heritable factors, such as chronological age, cytomegalovirus (CMV) infection, and gender have been documented to affect this process [5], which is termed immunosenescence [1-4].
Chronological age is primarily associated with alterations in the adaptive part of the immune system, especially the T-cell compartment. With age, thymic output of naive T-cells decreases to less than 10% of the original function by the age of 50 years [6,7]. This leads to increased peripheral replication of T-cells [7, 8], a reduction in naive T-cell numbers, and an expansion of memory T cells [9-13]. Combined, these changes result in a diminished diversity of the T-cell receptor (TCR) repertoire, which may negatively impact on the recognition of novel antigens with age [14]. In addition, the numbers of several other lymphocytes are affected by age. Multiple studies have shown higher numbers of regulatory T-(Treg) cells [15-17] and CD4+CD45RA+CD25dim naive T-cells [8, 18] in elderly than in young adults. Moreover, an inverted CD4/CD8 T-cell ratio is observed with age, and has been proposed to be an immune risk indicator [19, 20]. Finally, multiple studies showed an age-associated decline in the numbers of B-cells, both of the naive and the memory subsets [2, 3, 21, 22].
Multiple intrinsic and extrinsic factors may affect the immune status and infection with cytomegalovirus (CMV) has been associated with enhanced immunosenescence [23-25]. This herpes virus remains persistent upon primary infection and is actively suppressed by the immune system [23]. CMV infection primarily results in accumulation of late-differentiated memory T-cells, both in the CD4 and CD8 T-cell lineages [24-26]. These effects are already apparent in CMV-infected children [27]. CMV has limited effects on B-cell numbers, but might affect B-cell function as it is associated with high mutation frequencies in IgM and IgG transcripts [28].
Gender is a major intrinsic factor that affects circulating immune cell numbers and immune function [17, 19, 29, 30]. These effects can be mediated by hormone levels [30-33], as well as by genes on sex chromosomes [33]. However, the impact of gender on naive and memory T- and B-cell numbers remains incompletely understood [29]. Recent studies suggest that T-cell senescence might be more pronounced in elderly men than in women [17, 29].Furthermore, the impact of persistent viruses, including CMV, might differ between males and females. For a better understanding of immunosenescence, it is necessary to dissect the individual and combined effects of age, CMV infection and gender on numbers of circulating T- and B-cell subsets. Insights into these effects can be directly translated into early markers for
In an effort to understand the effects and interaction of gender and CMV on the immune phenotype in a Dutch middle-aged population (defined as 50-65 years of age), we have enumerated a comprehensive set of T- and B-cell subsets including Treg cells, follicular helper T- (TFH) cells, and the ageing-associated CD4+CD45RA+CD25dim naive T-cells. The characterisation of these ‘immune markers’ may help in the identification of persons being at risk of impaired immune function and thereby higher susceptibility to disease. Our data reveal that CMV infection differentially affects the immune phenotype in middle-aged males and females.
Methods
Study subjects and blood sampling
Peripheral blood was collected after written informed consent was obtained from 255 healthy middle-aged persons, aged 50-65 years of age, who were equally distributed over three 5-year intervals: 50-54, 55-59, and 60-65 years. The study was approved by the Medical Ethical Committee: Verenigde Commissie Mensgeboden Onderzoek (VCMO) in Nieuwegein, the Netherlands and registered at the Dutch trial register (NTR4636). All procedures were in accordance with the Declaration of Helsinki.
Subjects were excluded if they had fever or used antibiotics within the last 14 days, had a serious immune related disease such as cancer, received immunosuppressive treatment within the last 3 months (e.g. steroids), had a known or suspected immune deficiency, a coagulation disorder or a neurologic disorder, used hormone treatment, or were administered blood products within the last 6 months. All participants completed a short questionnaire concerning health status, medication use, smoking, and physical activity. Weight and height obtained from the short health questionnaire were used to calculate the Body Mass Index (BMI) according to the formula BMI= Weight (kg) / Height (m)2. Blood samples were collected during evening hours in tubes containing lithium heparin (BD Biosciences, Franklin Lakes, New Jersey) for detailed cellular immune phenotyping within 24 hours after collection. Additionally, serum was collected using serum clotting tubes (BD Biosciences) and processed within 6 hours for storage at -20°C.
CMV and EBV serology
Serum CMV IgG was determined by an enzyme-linked immunoassay (ETI-CYTOK-G Plus, P002033, Diasorin, Salugga, Italy) according to the manufacturer’s indications. The threshold for CMV seropositivity was 0.4 IU/ml. Serum Epstein Barr Virus (EBV)-capsid antigen IgG was determined by an enzyme-linked immunoassay (EUROIMMUN, Lubeck, Germany) according to the manufacturer’s indications. The threshold for EBV seropositivity was 20 relative units (RU)/ml.
2
Biochemical parameter measurementsSerum levels of C-reactive protein (CRP), Rheumatoid Factor (RF), and Reactive Oxygen Metabolites (ROM) were measured with a clinical auto-analyser (Dx5, Beckman-Coulter). The kits used were from Beckman-Coulter, Fullerton, CA (CRP), Roche Diagnostics, Almere, The Netherlands (RF), and Diacron, Grosseto, Italy (ROM). Dehydroepiandrosterone Sulphate (DHEAs), a precursor for most major sex hormones [36], was measured using the kit and immuno-analyser Access-2 from Beckman Coulter.
Flow cytometric immune phenotyping
The absolute numbers of lymphocytes, CD3+ T-cells, B-cell subsets, NK-cells, monocytes, and granulocytes were determined with a lyse-no-wash protocol using TruCOUNT tubes (BD Biosciences, San Jose, CA, USA). The following fluorochrome-conjugated antibodies were used: CD3(UCHT1)-BV711, CD16(B73.1)-PE, and CD38(HB7)-APC-H7 (all from BD Biosciences), CD45(GA90)-OC515 and CD56(C5.9)-PE (both from Cytognos, Salamanca, Spain), CD27(M-T271)-BV421 and IgD(IA6-2)-FITC (both from Biolegend, San Diego, CA), and CD19(J3-119)-PE-Cy7 (Beckman Coulter, Fullerton, CA).
Detailed immune phenotyping of T-cell subsets was performed separately in fresh whole blood samples using additional antibodies: CD4(RPA-T4)-BV510, CD45RA(HI100)-BV605 and CD28(CD28.2)-PerCP- Cy5.5 (all from Biolegend), CCR7(150503)-PE-CF594, CD8(SK1)-APC-H7, CD25(2A3)-FITC, and TCRgd(11F2)-PE-Cy7 (all from BD Biosciences), and CXCR5(51505)-APC (R&D systems, Minneapolis, MN). Absolute numbers of T-cell subsets were calculated using the CD3+ T-cell numbers from the TruCOUNT analysis. Gating strategies for T-cells [37], Treg cells [38], and B-cells [39] were applied as described previously, and shown in
Figure 1a and c, 4a and S. Figure 2a, respectively. In short, CCR7+ T cells were separated
into CD45RA+ naive and CD45RA- central memory (CM) subsets as described by Sallusto et al. [40]. Furthermore, CCR7- effector memory T cells (Tem) were separated into CD45RA- TemRO and CD45RA+ TemRA cells. Within TemRO and TemRA, early, intermediate and late subsets were defined on the basis of differential expression of CD27 and CD28, as described by Appay et al. [41]. In previous studies, CD45RA- T cells were confirmed to be CD45RO+ [27]. Flow cytometric analyses were performed on a 4-laser LSRFortessa (BD Biosciences) using standardized measurement settings as described by Kalina T et al. [42], and data analysis using FacsDiva V8 (BD Biosciences) and FlowJo V10 (FlowJo company, Ashland, OR). Statistics
The Mann Whitney U test was used to compare the CMV+ and CMV- groups, and males versus females on single immune cell subsets. To determine significant differences of one cell subset between two groups, p-values < 0.05 were considered significant. CMV+ males, CMV- males, CMV+ females, and CMV- females were compared for every immune cell subset using the Kruskal-Wallis test adjusted for multiple comparisons with the Bonferroni correction. The following comparisons were made: CMV+ males vs CMV+ females, CMV+ males vs CMV- males, CMV+ females vs CMV- females, CMV- females vs CMV- males. These statistical tests were performed in GraphPad Prism v6.05 for Windows (GraphPad Software Inc., La Jolla, CA). To conclude whether gender, CMV or the interaction between gender and CMV had an effect on the immune phenotype, a multiple comparison correction was included, since 36 immune cell subsets were tested. Only p-values < 0.0014 (p= 0.05/36) were considered significant. The statistical tests were supplemented with an Enter linear regression method in SPSS V22.0 to determine the individual effects of CMV status, gender, age, and the interaction between CMV status and gender on the immune cell subsets. Non-normally distributed data were log-transformed. To confirm the differential effects of CMV in males and females, an interaction term, gender*CMV, was included in the analysis. In this model, the p-value indicates whether a variable was significantly associated with the absolute number of the respective immune cells. The β coefficient indicates the strength of the association; the higher the value of β, the larger the deviation between groups that were compared. A negative value indicates a lower number of cells within males, CMV+ individuals, or CMV+ males, whereas a positive value indicates a higher absolute number of cells in these groups. The R2 of the model explains the strength of the model in predicting the absolute number of the respective immune cells; the closer the R2 is to 1, the stronger the predictive value of the model.
Results
Characteristics of study participants
A total of 255 persons participated in the study with mean age: 57.7 (50-65) years, and of which 140 were male (54.9%). About half of the participants were seropositive for CMV. Baseline characteristics are shown in Table 1. Five participants were excluded from the analysis due to CMV status ambiguity. Mean age and BMI were similar between males and females, as well as between CMV+ and CMV- individuals.
Effects of CMV on the absolute numbers of circulating leukocyte subsets CMV infection significantly influenced 8 out of the 28 (28.5%) leukocyte subsets depicted in Table 1. A significantly lower number of NK-cells (p= 0.046) was observed in CMV+ persons (Table 1).
2
Table 1. Individual effects of CMV and gender on the absolute numbers of different leukocyte subsets
CMV- CMV+ Male Female
Demographic factors
No. (%) 127 (50.8) 123 (49.2) 140 (54.9) 115 (45.1) Mean age in years
(range) 57.6 (50- 65) 57.8 (50- 65) 57.8 (50- 65) 57.5 (50- 65) Mean BMI (range) 25.7 (19- 39) 26.0 (18- 36) 25.8 (19- 35) 25.9 (18- 39)
Leukocyte subsets (cells/µl blood)
Granulocytes 2275 [2075- 2495] 2198 [2018- 2394] 2249 [2060- 2457] 2240 [2041- 2459] Monocytes 198.5 [182.6- 215.8] 182.5 [168.8- 197.3] 192.8 [178.0- 208.8] 188.5 [173.0- 205.5] NK cells 143.5 [126.0- 163.3] 118.2 [103.0- 135.6]* 131.6 [115.7- 149.8] 131.3 [114.3- 150.9] Lymphocytes 2179 [2039- 2329] 2174 [2030- 2328] 1937 [1799- 2087] 2367 [2204- 2541]**** T-cells 1548 [1428- 1678] 1428 [1293- 1577] 1317 [1201- 1445] 1732 [1599- 1875]**** γδ T-cells 18.7 [16.01- 21.85] 17.4 [14.95- 20.17] 16.2 [14.0- 18.8] 20.6 [17.6- 24.1]* CD4/CD8 ratio 3.3 [3.0- 3.6] 2.5 [2.3- 2.7]**** 2.7 [2.5- 2.9] 3.1 [2.8- 3.3] CD8 T-cells 331.7 [299.2- 367.8] 368.0 [328.4- 412.3]* 317.8 [285.0- 354.3] 393.8 [354.9- 437.0]* CD8 naive 74.0 [63.3- 86.4] 64.7 [53.4- 78.4] 51.3 [43.2- 61.0] 100.9 [87.2- 116.8]**** CD8 CM 13.8 [11.7- 16.2] 10.0 [8.5- 11.7]* 11.1 [9.3- 13.1] 13.0 [11.2- 15.1] CD8 TemRO 142.4 [126.8- 160.0] 151.3 [134.7- 169.9] 144.6 [128.7- 162.5] 151.9 [135.5- 170.2] CD8 TemRA 63.2 [54.9- 72.9] 102.3 [87.8- 119.2]**** 72.3 [62.5- 83.7] 88.9 [75.9- 104.0] CD4 T-cells 1084 [994.5- 1181] 911.3 [817.0- 1016] 852.0 [769.4- 943.3] 1205 [1109- 1309]**** CD4 naive 414.0 [363.6- 471.4] 310.3 [265.5- 362.7]* 290.6 [251.1- 336.3] 460.2 [405.3- 522.5]**** CD4 CM 262.1 [234.8- 292.6] 206.7 [182.9- 233.5]* 213.8 [188.4- 242.7] 260.5 [235.3- 288.4]* CD4 TemRO 202.2 [184.0- 222.2] 206.3 [182.7- 232.8] 181.2 [162.9- 201.5] 239.5 [216.4- 265.1]*** CD4 TemRA 38.1 [32.8- 44.3] 44.5 [37.8- 52.3] 32.2 [28.0- 37.0] 53.9 [45.9- 63.3]**** TFH cells 214.3 [189.4- 242.5] 205.3 [178.9- 235.6] 186.4 [162.7- 213.5] 241.7 [215.9- 270.6]* Treg cells 107.3 [97.5- 118.1] 90.7 [80.4- 102.4] 89.0 [79.7- 99.4] 113.6 [102.4- 126.0]*** naive Treg 12.6 [10.7- 14.7] 11.08 [9.5- 13.0] 9.9 [8.4- 11.5] 14.7 [12.7- 17.0]*** memory Treg 4.7 [4.0- 5.6] 3.2 [2.7- 3.8]** 3.9 [3.2- 4.7] 4.0 [3.4- 4.7] CD4+CD45RA +CD25dim 27.0 [23.1- 31.5] 21.7 [18.3- 25.6] 20.3 [17.3- 23.9] 29.7 [25.5- 34.4]*** B-cells 286.7 [257.8- 318.9] 265.8 [241.0- 293.0] 259.2 [234.4- 286.5] 301.8 [271.8- 335.0]* Transitional 9.5 [8.1- 11.2] 7.7 [6.5- 9.1] 7.9 [6.8- 9.1] 9.7 [8.1- 11.5] naive mature 175.3 [154.2- 199.2] 162.2 [142.1- 185.1] 160.9 [144.2- 179.4] 183.2 [161.6- 207.7] natural effector 22.1 [18.4- 26.6] 26.6 [22.3- 31.8] 21.7 [18.7- 25.2] 23.8 [20.0- 28.5] CD27- memory 15.5 [13.1- 18.3] 15.3 [13.4- 17.6] 14.8 [12.8- 17.0] 19.2 [16.8- 22.0]** CD27+ memory 32.8 [28.1- 38.3] 36.6 [32.1- 41.8] 29.0 [25.5- 33.0] 36.7 [32.0- 42.1]**
Geometric mean of cells/μl blood [95% CI]. CMV- and CMV+ persons were compared, as well as males and females. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Bold numbers indicate significant differences within one subset. After a multiple testing correction only values with p< 0.0014 are considered significant. Significant differences after correction for multiple testing are underlined.
T-cells (p=0.036) in CMV+ individuals. The numbers of total CD4 T-cells were not different, because higher numbers of late-differentiated memory cells were accompanied by significantly lower numbers of naive (p= 0.023) and central memory (p=0.030) CD4 T-cells (Table 1 and Figure 1d). No differences were observed for Tfhand Treg cell numbers. Finally,
as a consequence of the above-mentioned changes, the CD4/CD8 ratio was significantly lower in CMV+ individuals (p<0.0001, Table 1).
Figure 1. Effect of CMV on the CD8 and CD4 T-cell lineages
a) Gating strategies for the CD8 T-cell subsets. A representative example is shown. b) A cumulative schematic overview of the geometric mean values of absolute numbers of CD8 naive, CM, TemRO, and TemRA cells in CMV- and CMV+ participants. TemRO and TemRA cells are split into early, intermediate, and late differentiation subsets. c) Gating strategies for the CD4 T-cell subsets. A representative example is shown. d) A cumulative schematic overview of the geometric mean values of absolute numbers of CD4 naive, CM, TemRO, and TemRA cells in CMV- and CMV+ participants. TemRO and TemRA cells are were split into early, intermediate, and late differentiated subsets. The Mann Whitney U test was used for statistical analysis. * p<0.05, ** p<0.01, **** p<0.0001 (n=250).
2
Effects of gender on the absolute numbers of circulating leukocyte subsetsGender was associated with significant differences in 17 out of 28 (60.7%) leukocyte subsets (Table 1). First, males showed significantly (p<0.0001) lower numbers of total lymphocytes, resulting from low numbers in multiple subsets, especially the T-cells (p<0.0001). As both numbers of CD4 T-cells (p<0.0001) and CD8 T-cells (p= 0.006) were affected, no difference was observed in the CD4/CD8 ratios between males and females. In addition, males showed lower numbers of naive cells within both these lineages (both p<0.0001). Except for the memory Treg cells, gender was associated with significant changes in the absolute numbers of all measured CD4 T-cell subsets, including the CD45RA+CD25dim cells (p= 0.0008). Finally, males carried lower numbers of B-cells (p= 0.019) than females, which was mostly due to lower numbers of memory B-cells (CD27-, p=0.007; CD27+, p= 0.004).
Combined effects of CMV and gender on the absolute numbers of major lymphocytes subsets
To determine whether CMV infection was associated with differential effects on the immune phenotype between males and females, the participants were divided into four different groups: CMV- males, CMV+ males, CMV- females, and CMV+ females (Table 2). Mean age (57.7 years) and BMI (mean 25.9 range 18-39) were similar between the four study groups. Data on general health, lifestyle and biochemical parameters are reported in S. Table 1. Furthermore, CMV infected males and females showed equal levels of CMV-specific IgG (S.
Figure 1).
Although CMV- males and females had similar total lymphocyte numbers in blood, these were significantly lower in CMV+ males than in CMV+ females (p< 0.0001; Table 2). This difference was mostly due to significantly lower numbers of CD4 T-cells in CMV+ males than in CMV+ females (p < 0.0001) and in CMV- males (p= 0.037) and to a lesser extent due to lower B-cell numbers compared to CMV+ females (p = 0.029). As a consequence, CMV+ males showed a significantly lower CD4/CD8 ratio than CMV- males (p<0.0001) and CMV+ females (p= 0.011). Moreover, only within the CMV+ males, 5/61 participants had a CD4/ CD8 ratio below 1, which has been referred to as an immune risk profile [19, 20]. The lower numbers of B-cells in CMV+ males were mainly due to lower numbers of CD27- (p= 0.028) and CD27+ (p= 0.0066) memory B-cells (Table 2 and S. Figure 2).
Table 2. Combined effects of CMV and gender on the absolute numbers of different leukocyte subsets
Male CMV- Male CMV+ Female CMV- Female CMV+
Demographic factors
No. (%) 76 (30) 61 (24) 51 (20) 62 (25) Mean age (range) 57.9 (50- 65) 57.9 (50- 65) 57.2 (50- 65) 57.8 (50- 65) Mean BMI (range) 25.8 (19- 39) 26.1 (18- 36) 25.7 (19- 35) 25.9 (19- 35)
Leukocyte subsets (cells/µl blood)
Lymphocytes 2043 [1854- 2251] 1820 [1624- 2041] ****a 2367 [2078- 2696] 2405 [2223- 2602] NK cells 145.9 [122.8- 173.5] 110.1 [90.7- 133.6] 139.8 [114.2- 171.2] 126.7 [103.9- 154.5] Monocytes 202.1 [182.0- 224.4] 177.8 [158.6- 199.3] 193.0 [167.5- 222.5] 187.2 [167.9- 208.8] Granulocytes 2392 [2140- 2675] 2059 [1810- 2342] 2105 [1792- 2473] 2344 [2092- 2625] T-cells 1422 [1292- 1567]*b 1164 [991.8- 1367]**** a 1756 [1532- 2012] 1746 [1583- 1927] CD4 T-cells 989.4 [891.0- 1099]*b 701.1 [589.4- 833.8]****a/*c 1242 [1076- 1432] 1180 [1065- 1306] CD8 T-cells 304.7 [267.3- 347.3] 329.0 [274.8- 394.0] 376.5 [318.8- 444.7] 410.9 [357.4- 472.3] CD4/CD8 ratio 3.2 [2.9- 3.6] 2.1 [1.9- 2.4]*a/ ****c 3.3 [2.9- 3.8] 2.9 [2.5- 3.2] Treg cells 100.8 [89.5- 113.6] 74.7 [62.0- 90.0]**a 117.8 [100.2- 138.5] 109.8 [95.2- 126.8] γδ T-cells 16.3 [13.4- 19.8] 16.3 [13.0- 20.4] 22.9 [17.8- 29.5] 18.5 [15.1- 22.7] B-cells 273.9 [237.3- 316.1] 235.9 [207.2- 268.6]*a 306.9 [261.2- 360.6] 298.8 [259.0- 344.7] transitional 8.2 [6.6- 10.1] 7.1 [5.7- 8.8] 12.0 [9.3- 15.3] 8.4 [6.5- 10.7] naive mature 167.8 [143.4- 196.4] 149.1 [129.1- 172.3] 194.3 [162.5- 232.3] 174.0 [145.4- 208.1] natural effector 21.6 [18.0- 26.0] 20.0 [15.6- 25.8] 20.9 [15.7- 27.9] 27.3 [21.9- 34.1] CD27- memory 15.7 [12.6- 19.5] 13.3 [11.3- 15.6]*a 19.7 [15.8- 24.5] 19.2 [16.2- 22.7] CD27+ memory 29.7 [24.8- 35.5] 27.4 [22.9- 32.9]**a 33.3 [26.2- 42.2] 41.5 [35.6- 48.3]
Geometic mean of cells/μl blood [95% CI]. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a. Male CMV+ versus Female CMV+; b. Male CMV- versus Female CMV-; c. Male CMV+ versus Male CMV-; Bold numbers indicate significant differences within one subset. After a multiple testing correction only values with p< 0.0014 are considered significant. Significant differences after correction for multiple testing are underlined.
Combined effects of CMV and gender on the composition of the CD8 T-cell lineage
Besides relatively stable absolute numbers of total CD8 T-cells, the composition of the CD8 lineage was affected by the combination of CMV and gender (Figure 2a).
Naive CD8 T-cell numbers were lower in males than in females, both in CMV+ (p=0.0008) and CMV- (p= 0.0001) individuals (Figure 2b). Moreover, the numbers of CD8 CM T-cells were significantly lower in CMV+ males than in CMV- males (p= 0.002) and in CMV+ females (p= 0.010) (Figure 2c). Slightly different conclusions might be drawn when reporting on proportional data (S. Figure 3b). The higher numbers of late-differentiated CD8 TemRA cells in CMV infected individuals was equal in males and females (Figure 2e). Furthermore, the distributions of CD8 TemRA early, intermediate, and late cells were similar between CMV-infected males and females (Figure 2d).
