Abnormalities of age-related T cell senescence in Parkinson’s disease

Abnormalities of age-related T cell senescence in Parkinson’s disease Background: A wealth of evidence implicates both central and peripheral immune changes as contributing to the pathogenesis of Parkinson’s disease (PD). It is critical to better understand this aspect of PD given that it is a tractable target for disease-modifying therapy. Age-related changes are known to occur in the immune system (immunosenescence) and might be of particular relevance in PD given that its prevalence rises with increasing age. We therefore sought to investigate this with respect to T cell replicative senescence, a key immune component of human ageing. Methods: Peripheral blood mononuclear cells were extracted from blood samples from 41 patients with mild PD (Hoehn and Yahr stages 1–2, mean (SD) disease duration 4.3 (1.2) years) and 41 age- and gender-matched controls. Immunophenotyping was performed with flow cytometry using markers of T lymphocyte activation and senescence (CD3, CD4, CD8, HLA-DR, CD38, CD28, CCR7, CD45RA, CD57, CD31). Cytomegalovirus (CMV) serology was measured given its proposed relevance in driving T cell senescence. Results: Markers of replicative senescence in the CD8+ population were strikingly reduced in PD cases versus controls (reduced CD57 expression (p = 0.005), reduced percentage of ‘late differentiated’ CD57 CD28 cells lo hi (p = 0.007) and ‘TEMRA’ cells (p = 0.042)), whilst expression of activation markers (CD28) was increased (p = 0.005). This was not driven by differences in CMV seropositivity. No significant changes were observed in the CD4 population. Conclusions: This study demonstrates for the first time that the peripheral immune profile in PD is distinctly atypical for an older population, with a lack of the CD8+ T cell replicative senescence which characterises normal ageing. This suggests that ‘abnormal’ immune ageing may contribute to the development of PD, and markers of T cell senescence warrant further investigation as potential biomarkers in this condition. Keywords: Parkinson’s disease, Immune markers, T cells, Immunosenescence Background been reported in peripheral blood including changes in There is abundant evidence implicating a role for inflam- monocyte [8]and lymphocyte subsets inPD[9, 10], which mation and immune activation in Parkinson’sdisease. maybedrivenbya specific immune response to alpha Neuroinflammatory changes including microglial activation synuclein [11]. Whilst such immune and inflammatory and increased concentrations of pro-inflammatory cyto- changes in PD might be, at least in part, secondary phe- kines are well described in PD postmortem brain [1, 2], and nomena, immune manipulation in animal models of PD microglial activation can be observed in vivo even in the has been shown to alter disease susceptibility and severity early stages of the disease using [11C]PK-11195 PET neuro- [12, 13], suggesting that immune changes may also be a imaging [3–5]. Inflammatory cytokines including IL-6 and primary contributing factor to disease development. This is IL-8 are elevated in the cerebrospinal fluid (CSF) in PD supported by the now well-established genetic association compared in controls [6, 7], and immune alterations have between the human leucocyte antigen (HLA) locus, the key genetic susceptibility factor in disorders involving patho- * Correspondence: chm27@cam.ac.uk genic adaptive immune responses, and PD risk [14, 15]. John Van Geest Centre for Brain Repair, Department of Clinical Furthermore, our own recent data has shown that a Neurosciences, University of Cambridge, Forvie Site, Cambridge CB2 0PY, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 2 of 8 pro-inflammatory cytokine profile in the serum in early PD Methods has prognostic significance, being associated with more Subjects cognitive dysfunction and faster motor progression over Patients with early-stage PD (Hoehn and Yahr stage ≤ 2, 36 months of prospective follow-up in a large incident PD no dementia) were recruited from the Parkinson’s Dis- cohort [16]. ease Research Clinic at the John Van Geest Centre for Given that PD is a condition which becomes increas- Brain Repair in Cambridge. All patients met the UKPDS ingly prevalent with age, the relevance of age-related Brain Bank criteria for the diagnosis of PD [29] and were immune dysregulation (known as immunosenescence) aged between 55 and 80. Controls without neurological in this disorder warrants further investigation. One of disease were recruited from the NIHR Cambridge BioR- the hallmarks of immunosenescence is the loss of esource. MAPT (microtubule-associated protein tau) naïve T cells, especially CD8+ cells, and the reciprocal H1/H2 genotypes were available for all subjects. Con- accumulation of memory cells, particularly terminally trols were age-, sex- and MAPT genotype-matched to differentiated effector CD8+ cells (TEMRAs), as a patients. Exclusion criteria included current infections, result of age-related thymic involution and repeated chronic inflammatory or autoimmune disorders, vaccin- exposure to pathogens [17–19]. In vitro, progression ation within the past 3 weeks, surgery within the past of CD8 T cells to a senescent state is characterised by month, and recent use of immunosuppressive therapy the loss of surface expression of the co-stimulatory (< 1 year), oral or intravenous steroids (< 3 months), molecule CD28 and expression of CD57 [17, 20]. In non-steroidal anti-inflammatories (< 2 weeks) or as- vivo, both TEMRA and CD28 CD57 CD8+ cells are pirin > 75 mg (< 2 weeks). Control subjects had no lo hi increased in frequency in patients with chronic infec- history of neurological disease, no self-reported memory tions, e.g. HIV, as well as in older age [21–23]. Indeed, problems and no depression. PD cases underwent full clin- accumulation of late differentiated CD8 cells has been ical assessment including medical history and comorbidities, proposed to be a major characteristic of an ‘immune the Movement Disorder Society-Unified Parkinson’sDisease risk profile’ in patents > 85 years which is associated Rating Scale (MDS-UPDRS), Hoehn and Yahr Stage, with higher mortality [18, 24]. Infection with cyto- Addenbrooke’s Cognitive-Examination-Revised (ACE-R), megalovirus (CMV) has been proposed as the key assessment of semantic fluency (animal naming in 90 s) and pathogenic driver of this senescent CD8 subset [18], pentagon copying and the Beck Depression Inventory. with T cell receptor (TCR) restricted, CMV-specific T Levodopa equivalent daily doses were calculated [30]. Cases cells comprising a large proportion of the CD8 T cells were stratified in terms of prognosis using factors in the elderly [25, 26]. Within the CD4 population, identified in our previous CamPaIGN study: they were similar phenotypic changes occur with repeated anti- classified as high dementia risk if they carried the genic stimulation, but late differentiated CD4 cells MAPT (microtubule-associated protein tau) H1/H1 accumulate at a much lower frequency [27]. Loss of genotype and performed poorly on tests of semantic thymic T cell production can also be measured in the fluency (< 20 animals in 90 s) and/or pentagon copy- CD4 pool as a loss of CD4+ recent thymic emigrants ing (< 2/2), intermediate risk if they carried one of (RTEs), CD4+ naïve cells that co-express CD31 [19]. these factors and low risk if they carried no risk factors [31]. The functional impact of CD8+ T cell replicative senescence is an overall impairment of the adaptive Sample processing immune response, with a consequent reduction in the Up to 50 ml of venous blood was collected from each ability to fight novel infections and impairment in re- participant in a combination of lithium heparin tubes for sponses to vaccinations in the elderly [17]. The impact peripheral blood mononuclear cell (PBMC) isolation, of CD8+ cell senescence on the development and plain tubes for serum separation and ethylenediamine- progression of PD is unknown. Such changes may tetraacetic acid (EDTA) tubes for measurement of full reduce responses to disease-related antigens such as blood count and differential count. Samples for misfolded alpha synuclein, thus limiting clearance of patient-control pairs were collected on the same day (9– toxic proteins and potentially contributing to PD. 11 am) and processed in parallel to minimise inter-assay Alternatively, they may be protective through reducing variability. Plain tubes were left to clot for 15 min before the capacity of the immune system to become acti- centrifugation at 2000 rpm and removal of serum, which vated, hence limiting an important driver of central was stored at − 80 °C prior to subsequent analysis for neuroinflammation [28]. Here, we sought to answer CMV IgG using a LIAISON® CMV IgG II chemilumines- this question by investigating whether there was an as- cent immunoassay (DiaSorin, Italy). PBMCs were sepa- sociation between an immunosenescent T cell pheno- rated by centrifugation over a Ficoll gradient and type and PD and to determine the relevance of CMV washed and divided into aliquots for immunophenotyp- infection to this. ing and cell separation into T cell subsets. Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 3 of 8 PBMCs for phenotyping were blocked with 2% Table 1 Demographic and clinical characteristics of subjects mouse serum prior to labelling with two panels of Variable PD (n = 41) Controls p (n = 41) fluorochrome-conjugated monoclonal antibodies to T Age (years) 68.4 (6.3) 68.1 (5.6) 0.85 cell surface markers of interest [panel 1: CD3(FITC), CD4(PECy5), CD8(APCCy7), HLA-DR(V500), CD38(PE); Gender (% male) 68.3 68.3 1.0 panel 2: CD3(APC), CD4(V500), CD8(APCCy7), Disease duration (years) 4.3 (1.2) CD28(PECy7), CCR7(FITC), CD45RA(PECy5), CD57(PE), MDS-UPDRS motor score 34.9 (12.4) CD31(V450), BD Biosciences). Cells were fixed with 2% ACE-R 93.0 (8.3) paraformaldehyde. Flow cytometry was performed on a Levodopa equivalent daily dose (mg) 591.5 (292.9) BD Biosciences FACS CantoII with BD FACS Diva soft- CMV IgG (% positive) 46.3 61.0 0.18 ware. Isotype-matched mouse monoclonal antibodies were used where appropriate to exclude non-specific anti- Values shown are mean (SD) unless otherwise stated. Continuous variables compared using Student’s t tests and categorical variables compared using body binding. Unstained lymphocytes were also used as chi-square tests or Fisher’s exact test as appropriate negative controls. MDS-UPDRS Movement Disorder Society Unified Parkinson’s Disease Rating Scale, ACE-R Addenbrooke’s Cognitive Examination-Revised Data analysis Immunophenotyping data was analysed using Flowjo. Lym- PD and 20 controls revealed lower total lymphocyte phocytes were gated using forward scatter and side scatter, counts in PD cases than controls (1.4 (0.4) versus 1.9 and single cells were identified using forward-scatter area (0.6) × 10 /L; p = 0.01) with no significant differences in versus width. CD4 and CD8 subsets were identified by gat- monocyte or neutrophil numbers. Flow cytometric data ing on CD3-high CD4-high and CD3-high CD8-high cells from the full cohort indicated no difference in the respectively. Naïve, central memory, effector memory CD4:CD8 ratio between PD cases and controls [3.8 (2.3) and terminally differentiated memory CD45RA+ cells versus 3.3 (3.0); p > 0.05]. (TEMRA) were identified based on CCR7 and CD45RA However, there was a reduction in the number and expression [19]. Senescent ‘late differentiated’ cells were proportion of CD28 CD57 CD8+ T cells in individuals lo hi defined as CD28-low CD57-high cells within the CD8 with PD compared to controls, along with a marginally population. RTEs were identified as CD31+ cells within significant reduction in CD8+ TEMRA cells and accom- the CD4 naïve (CCR7+CD45RA+) population. Expres- panying small increase in CD8+ central memory cells sion of markers of activation (CD28, CD38, HLA-DR) (Table 2 and Fig. 1a, b). Expression of the activation and senescence (CD57) were quantified within CD4+ markers CD38 and HLA-DR on CD8+ T cells was not and CD8+ populations as the ratio of median fluores- different between patients and controls, but expression cence intensity in labelled versus unlabelled lympho- of CD57 was reduced and expression of CD28 was in- cytes (MFI ratio), to allow for variations in voltage creased in PD patients (Table 3; Fig. 1c), in keeping with between samples. the CD8+ subset data (Table 3; Fig. 1c). No differences The primary statistical analyses involved comparison of T Table 2 T lymphocyte subsets cell subsets (as a percentage of lymphocytes) and MFI ratios for relevant markers in PD cases versus matched Subset (% of lymphocytes) PD Control p controls using paired t tests. CMV positivity in patients CD8 Naïve 3.4 (2.3) 3.1 (2.4) 0.511 versus controls was compared using chi-square tests, and Central memory 1.5 (1.2) 1.1 (0.9) 0.015 analyses of variance (ANOVA), including age as a covariate, Effector memory 6.6 (3.6) 7.2 (5.0) 0.516 were used for patient-control comparisons of T cell markers TEMRA 6.2 (6.3) 9.3 (7.9) 0.042 in CMV-positive and CMV-negative subgroups. Relation- CD28 57 5.3 (4.5) 9.0 (8.0) 0.007* lo hi ships between relevant markers and clinical measures of motor and cognitive functions were explored using Pear- CD4 Naïve 17.5 (9.0) 16.0 (8.2) 0.406 son’s correlations. Statistical analysis was performed using Central memory 18.0 (6.7) 15.8 (6.7) 0.052 GraphPad Prism version 6.0 and SPSS version 25 (IBM). Effector memory 14.2 (6.5) 12.8 (6.4) 0.245 Results TEMRA 2.1 (2.4) 2.0 (2.3) 0.747 Forty-one patients with PD and 41 age/gender-matched RTE 2.2 (3.3) 2.1 (2.2) 0.827 controls were recruited. Demographic and clinical char- T lymphocyte subsets are expressed as a percentage of the acteristics of the subjects and CMV status are shown in lymphocyte population. TEMRA terminally differentiated effector memory CD45RA+ve cells, RTE recent Table 1. Nine PD cases were designated high dementia thymic emigrants risk, 18 were low risk and 14 were intermediate risk. *p value (from paired t test) which remains < 0.05 following Bonferroni Analysis of full blood and differential counts in n =20 correction for multiple testing Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 4 of 8 Fig. 1 CD8 immunophenotyping in PD cases (n = 41) versus age-matched controls (n = 41). a Following gating of the lymphocyte and singlet populations, CD8 cells were gated on the basis of high CD3 and CD8 staining, and the CD28 CD57 subset was identified as a discrete lo hi population of CD8+ cells using 5% contour plots. Quadrant gates were used to divide the CD8 population into naïve, central memory, effector memory and TEMRA (terminally differentiated effector memory CD45RA+ve cells) subsets on pseudocolour dot plots. b Individual-level data for CD8 cell subsets (shown as percentage of lymphocytes). c Individual-level data for surface marker expression in CD8 T cells (median fluorescence intensity (MFI) ratio versus unstained lymphocytes). Horizontal lines denote means; *p < 0.05, **p < 0.01 in paired t tests were identified in the CD4+ T cell pool between patients and controls. For cell subsets/markers reaching significance (p < 0.05), ANOVA were performed to assess the effect of dementia risk group on the observed case-control differences (with Table 3 T cell surface marker expression ‘case-control status’ and ‘risk subgroup’ included as fixed Marker PD Control p factors and age and gender as covariates). Main effects of CD8 CD28 188.6 (102.5) 131.5 (88.3) 0.005* ‘case-control status’ were confirmed for the markers previ- CD38 1.9 (1.2) 2.3 (1.6) 0.117 ously identified, but there was no interaction with risk subgroup. Amongst the PD cases, no significant correla- HLA-DR 1.8 (0.4) 2.0 (0.6) 0.055 tions were found between T cell subset percentages, or CD57 38.2 (69.9) 161.0 (261.7) 0.005* surface markers of activation and senescence, and either clinical measures of motor and cognitive function or CD4 CD28 334.6 (97.3) 299.8 (89.1) 0.067 equivalent daily levodopa dose. CD38 6.1 (3.0) 7.1 (4.0) 0.057 CMV IgG seropositivity was not significantly different HLA-DR 1.1 (0.2) 1.2 (0.2) 0.413 between PD cases (19/41) and controls (25/41) (p =0.18). CD57 1.8 (1.5) 2.0 (1.3) 0.493 Nonetheless, given the previously described association Values shown are mean (SD) ratio of median fluorescent intensity versus between CMV exposure and CD8 immunosenescence, unstained lymphocytes (MFI ratio) we explored this relationship further. As anticipated, CD8+ *p value which remains < 0.05 following Bonferroni correction for multiple testing senescence markers were elevated in CMV-positive versus Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 5 of 8 CMV-negative subjects overall, including CD57 expression [14, 34], as well as more widespread genomic associations (ANOVA with age as covariate, F = 4.66, p = 0.03), in loci affecting T cell regulation and function [35, 36]. CD28 CD57 cells (% of lymphocytes, F = 18.75, p < 0.001) Furthermore, specific T cell responses to epitopes of alpha lo hi and TEMRA cells (% of lymphocytes, F =12.71, p = 0.001). synuclein have recently been described at a higher fre- However, this effect was more apparent for controls than quency in PD patients than controls by Sulzer and col- for PD patients, with significantly higher CD57 ex- leagues [11] and were closely associated with possession pression (p = 0.017) and CD28 CD57 cells (% of of known PD risk alleles at the HLA locus. In addition, lo hi lymphocytes, p = 0.028) in controls versus PD cases in alpha synuclein peptides have been shown to bind to these the CMV-positive group (Fig. 2). HLA ‘risk variant’ molecules in vitro, thus revealing a potential functional mechanism through which the Discussion well-described HLA association may contribute to the This study demonstrates for the first time that the per- development of PD. Whilst the authors of this work ipheral immune profile in PD is distinctly atypical for a highlighted the importance of the Th2-type CD4+ more elderly population, with a lack of the CD8+ T cell cell-mediated response observed in their experiments, replicative senescence which characterises normal age- they also found evidence of a significant CD8+-mediated ing. Although other authors have reported phenotypic response when stimulating cells with shorter alpha synu- alterations in the T lymphocyte population in PD, these clein peptides, and in fact, the strongest association be- alterations have mainly been restricted to CD4+ cell sub- tween alpha synuclein-specific T cell responses and HLA sets with conflicting findings [9, 32, 33] and we found was seen for the MHC class I allele HLA A*11.02, thus no changes in the CD4+ T cell pool in this study. No underlining the relevance of the CD8 (MHC class 1 re- previous studies have specifically explored markers of T stricted) response [11]. The latter would be in keeping cell replicative senescence in PD. CMV infection has with our observations that PD-associated immunophe- been strongly implicated in CD8+ replicative senescence notypic changes in our cohort were within the CD8+ [18], but this does not appear to be confounding our ob- population. Other authors have similarly shown an servations, with similar levels of CMV IgG positivity in elevated CD8+ T cell response to alpha synuclein both patient and control groups. Instead, the senescent peptides and herpes simplex virus peptides in PD shift typically induced by chronic latent CMV infection versus controls [37]. is not observed in this PD cohort. This is an intriguing The mechanism by which alterations in peripheral finding, raising the possibility that there are inherent CD8+ T cell function and CNS pathology interact in PD differences in the adaptive immune response to chronic is still not fully elucidated. However, it is now clear that viral infections in PD. We hypothesise that these differ- there is an abundance of T lymphocytes within the men- ences may contribute to a more active T cell-mediated inges which keep the brain under immune surveillance immune response to PD-relevant antigens such as alpha and which drain, together with CNS antigens, via the synuclein, leading in turn to an exaggerated neuroin- CNS lymphatic system to the local lymph nodes [38]. flammatory response which may contribute to the These meningeal T cells may be activated by ‘alarm neuropathology of PD. signals’ released into the CSF by degenerating CNS neu- In keeping with this hypothesis, there is well-established rones [39] and in turn produce inflammatory cytokines evidence from genetic studies of an immune-mediated which can diffuse into the brain parenchyma and lead to susceptibility to PD, with association between the HLA further activation of already ‘primed’ microglia [40]and/ locus, which is critical in antigen recognition, and PD risk or impact directly on neuronal function [41]. Fig. 2 CD8+ senescence markers in CMV-positive versus CMV-negative subjects. The figure shows CD57 expression on CD8+ lymphocytes (median fluorescence intensity (MFI) ratio versus unstained lymphocytes), CD8+ CD28 CD57 cells (percentage of lymphocytes) and TEMRA cells lo hi (percentage of lymphocytes) in PD versus age-matched controls stratified by CMV seropositivity. Horizontal lines denote means; *p < 0.05 and ***p ≤ 0.001 Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 6 of 8 Furthermore, the presence of CD8+ cells within the age-related neurodegenerative disorders such as PD, brain parenchyma in postmortem tissue from PD pa- through limiting immune activation to aberrant peptides tients raises the possibility of direct interaction with associated with these diseases. On a practical level, this neurones presenting relevant antigen in the context of work suggests that the CD8+ CD28 CD57 immunophe- lo hi MHC class 1, with consequent cytotoxic effects [12]. notype warrants further evaluation as a biomarker in PD, In this study, we adopted a hypothesis-driven ap- which could be relevant for patient stratification for clin- proach and so restricted our T cell phenotyping to ical trials of immunomodulating therapies. Furthermore, specifically explore cell subsets and markers relevant to whilst immunomodulatory strategies targeting the CD4 activation and replicative senescence. A consequent limi- population are being developed [43], the CD8 population tation of the study is that we were not able to fully char- may also warrant consideration as a target for immune acterise the T cell population in our cohort, nor assess therapies in PD. changes in T regulatory cells, which have been reported Abbreviations to be reduced in both number, and functional ability to ACE-R: Addenbrooke’s Cognitive Examination-Revised; ANOVA: Analysis of suppress effector T cells in PD patients [9]. Another po- variance; CMV: Cytomegalovirus; CSF: Cerebrospinal fluid; EDTA: Ethylenediaminetetraacetic acid; HLA: Human leucocyte antigen; tential limitation of our study is that the majority of our MAPT: Microtubule-associated protein tau; MDS-UPDRS: Movement patients were taking dopaminergic medication, which Disorder Society Unified Parkinson’s Disease Rating Scale; MFI: Median has been implicated as a potential confounding factor in fluorescence intensity; PBMC: Peripheral blood mononuclear cell; PD: Parkinson’s disease; RTE: Recent thymic emigrant; SD: Standard deviation; TEMRA: Terminally alterations in lymphocyte populations in PD cohorts differentiated effector memory CD45RA+ cells [33]. In particular, in vitro data indicates that dopamine is an activator of effector T cells and an inhibitor of Acknowledgements We gratefully acknowledge the participation of all our patient volunteers and regulatory T cells [42]. However, studies specifically NIHR Cambridge BioResource volunteers. We thank the Cambridge BioResource comparing medicated and unmedicated PD patients have staff for their help with volunteer recruitment. found no relationship between levodopa treatment and We thank members of the Cambridge BioResource SAB and Management Committee for their support of our study. We also acknowledge the support changes in T cell subsets [32]. Similarly, we found no as- of the Cambridge NIHR BRC Cell Phenotyping Hub. sociations between T cell phenotypes or surface marker expression and equivalent levodopa dose in our patient Funding Grant funding from the Academy of Medical Sciences, UK, the Rosetrees cohort suggesting that medication was not a significant Trust, the Stevenage Biosciences Catalyst and Addenbrooke’s Charitable Trust factor driving the observed reduction in markers of CD8 contributed to this work. The research was also supported by the NIHR replicative senescence. Cambridge Biomedical Research Centre (Cambridge University Hospitals NHS Trust/University of Cambridge). C. H. Williams-Gray is supported by a fellowship Although we found clear differences in T cell senes- from the Medical Research Council. R. S. Wijeyekoon was supported by a cence markers between patient and control groups, we fellowship from Addenbrooke’sCharitableTrust.K.M. Scott andJ.L. were not able to demonstrate a relationship between Jones are supported by fellowships from the Wellcome Trust. R. A. Barker is supported by the Wellcome-MRC Cambridge Stem Cell Institute. these markers and dementia risk status or clinical mea- The funding bodies had no role in the design of the study; the collection, sures of disease severity within the PD group. This may analysis and interpretation of data; or the writing of the manuscript. be because the observed immunophenotypic differences Availability of data and materials are most relevant to the earliest stages of the disease, Datasets used during the current study are available from the corresponding impacting on susceptibility but not on the rate of pro- author on reasonable request. Access to Cambridge BioResource volunteers gression. However, the power of our study to detect and their data and samples is governed by the Cambridge BioResource SAB. Documents describing access arrangements and contact details are available at differences between dementia risk subgroups was likely http://www.cambridgebioresource.org.uk/. suboptimal given the size of our cohort. In terms of measures of disease severity, the spread across the group Authors’ contributions CHW-G contributed to the study design, acquisition of data, analysis and was relatively minimal (Table 1) which consequently lim- interpretation of data and drafting of the manuscript. RW contributed to the ited our ability to explore correlations with disease stage. acquisition of data and critical revision of the manuscript. KMS contributed Further studies exploring these markers both across a to the acquisition of data and critical revision of the manuscript. SH contributed to the acquisition of data and critical revision of the manuscript. range of disease stages, and longitudinally in individual RAB contributed to the study design and supervision and critical revision of patients, would be of interest. the manuscript. JLJ contributed to the supervision of study, study design, interpretation of data and critical revision of the manuscript. All authors read and approved the final manuscript. Conclusions Our data reveal that there is a reduction in age-associated Ethics approval and consent to participate replicative senescence of CD8+ T cells in PD patients This study was approved by the East of England—Cambridge Central compared with strictly age-matched controls. Hence, Research Ethics Committee (ref 03/303). All participants provided written informed consent. whilst immunosenescence may be detrimental in terms of increased susceptibility to infections and cancers with in- Competing interests creasing age, it may provide a degree of protection against The authors declare that they have no competing interests. Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 7 of 8 Publisher’sNote 17. Chou JP, Effros RB. T cell replicative senescence in human aging. 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Abnormalities of age-related T cell senescence in Parkinson’s disease

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Biomedicine; Neurosciences; Neurology; Neurobiology; Immunology
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Abstract

Background: A wealth of evidence implicates both central and peripheral immune changes as contributing to the pathogenesis of Parkinson’s disease (PD). It is critical to better understand this aspect of PD given that it is a tractable target for disease-modifying therapy. Age-related changes are known to occur in the immune system (immunosenescence) and might be of particular relevance in PD given that its prevalence rises with increasing age. We therefore sought to investigate this with respect to T cell replicative senescence, a key immune component of human ageing. Methods: Peripheral blood mononuclear cells were extracted from blood samples from 41 patients with mild PD (Hoehn and Yahr stages 1–2, mean (SD) disease duration 4.3 (1.2) years) and 41 age- and gender-matched controls. Immunophenotyping was performed with flow cytometry using markers of T lymphocyte activation and senescence (CD3, CD4, CD8, HLA-DR, CD38, CD28, CCR7, CD45RA, CD57, CD31). Cytomegalovirus (CMV) serology was measured given its proposed relevance in driving T cell senescence. Results: Markers of replicative senescence in the CD8+ population were strikingly reduced in PD cases versus controls (reduced CD57 expression (p = 0.005), reduced percentage of ‘late differentiated’ CD57 CD28 cells lo hi (p = 0.007) and ‘TEMRA’ cells (p = 0.042)), whilst expression of activation markers (CD28) was increased (p = 0.005). This was not driven by differences in CMV seropositivity. No significant changes were observed in the CD4 population. Conclusions: This study demonstrates for the first time that the peripheral immune profile in PD is distinctly atypical for an older population, with a lack of the CD8+ T cell replicative senescence which characterises normal ageing. This suggests that ‘abnormal’ immune ageing may contribute to the development of PD, and markers of T cell senescence warrant further investigation as potential biomarkers in this condition. Keywords: Parkinson’s disease, Immune markers, T cells, Immunosenescence Background been reported in peripheral blood including changes in There is abundant evidence implicating a role for inflam- monocyte [8]and lymphocyte subsets inPD[9, 10], which mation and immune activation in Parkinson’sdisease. maybedrivenbya specific immune response to alpha Neuroinflammatory changes including microglial activation synuclein [11]. Whilst such immune and inflammatory and increased concentrations of pro-inflammatory cyto- changes in PD might be, at least in part, secondary phe- kines are well described in PD postmortem brain [1, 2], and nomena, immune manipulation in animal models of PD microglial activation can be observed in vivo even in the has been shown to alter disease susceptibility and severity early stages of the disease using [11C]PK-11195 PET neuro- [12, 13], suggesting that immune changes may also be a imaging [3–5]. Inflammatory cytokines including IL-6 and primary contributing factor to disease development. This is IL-8 are elevated in the cerebrospinal fluid (CSF) in PD supported by the now well-established genetic association compared in controls [6, 7], and immune alterations have between the human leucocyte antigen (HLA) locus, the key genetic susceptibility factor in disorders involving patho- * Correspondence: chm27@cam.ac.uk genic adaptive immune responses, and PD risk [14, 15]. John Van Geest Centre for Brain Repair, Department of Clinical Furthermore, our own recent data has shown that a Neurosciences, University of Cambridge, Forvie Site, Cambridge CB2 0PY, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 2 of 8 pro-inflammatory cytokine profile in the serum in early PD Methods has prognostic significance, being associated with more Subjects cognitive dysfunction and faster motor progression over Patients with early-stage PD (Hoehn and Yahr stage ≤ 2, 36 months of prospective follow-up in a large incident PD no dementia) were recruited from the Parkinson’s Dis- cohort [16]. ease Research Clinic at the John Van Geest Centre for Given that PD is a condition which becomes increas- Brain Repair in Cambridge. All patients met the UKPDS ingly prevalent with age, the relevance of age-related Brain Bank criteria for the diagnosis of PD [29] and were immune dysregulation (known as immunosenescence) aged between 55 and 80. Controls without neurological in this disorder warrants further investigation. One of disease were recruited from the NIHR Cambridge BioR- the hallmarks of immunosenescence is the loss of esource. MAPT (microtubule-associated protein tau) naïve T cells, especially CD8+ cells, and the reciprocal H1/H2 genotypes were available for all subjects. Con- accumulation of memory cells, particularly terminally trols were age-, sex- and MAPT genotype-matched to differentiated effector CD8+ cells (TEMRAs), as a patients. Exclusion criteria included current infections, result of age-related thymic involution and repeated chronic inflammatory or autoimmune disorders, vaccin- exposure to pathogens [17–19]. In vitro, progression ation within the past 3 weeks, surgery within the past of CD8 T cells to a senescent state is characterised by month, and recent use of immunosuppressive therapy the loss of surface expression of the co-stimulatory (< 1 year), oral or intravenous steroids (< 3 months), molecule CD28 and expression of CD57 [17, 20]. In non-steroidal anti-inflammatories (< 2 weeks) or as- vivo, both TEMRA and CD28 CD57 CD8+ cells are pirin > 75 mg (< 2 weeks). Control subjects had no lo hi increased in frequency in patients with chronic infec- history of neurological disease, no self-reported memory tions, e.g. HIV, as well as in older age [21–23]. Indeed, problems and no depression. PD cases underwent full clin- accumulation of late differentiated CD8 cells has been ical assessment including medical history and comorbidities, proposed to be a major characteristic of an ‘immune the Movement Disorder Society-Unified Parkinson’sDisease risk profile’ in patents > 85 years which is associated Rating Scale (MDS-UPDRS), Hoehn and Yahr Stage, with higher mortality [18, 24]. Infection with cyto- Addenbrooke’s Cognitive-Examination-Revised (ACE-R), megalovirus (CMV) has been proposed as the key assessment of semantic fluency (animal naming in 90 s) and pathogenic driver of this senescent CD8 subset [18], pentagon copying and the Beck Depression Inventory. with T cell receptor (TCR) restricted, CMV-specific T Levodopa equivalent daily doses were calculated [30]. Cases cells comprising a large proportion of the CD8 T cells were stratified in terms of prognosis using factors in the elderly [25, 26]. Within the CD4 population, identified in our previous CamPaIGN study: they were similar phenotypic changes occur with repeated anti- classified as high dementia risk if they carried the genic stimulation, but late differentiated CD4 cells MAPT (microtubule-associated protein tau) H1/H1 accumulate at a much lower frequency [27]. Loss of genotype and performed poorly on tests of semantic thymic T cell production can also be measured in the fluency (< 20 animals in 90 s) and/or pentagon copy- CD4 pool as a loss of CD4+ recent thymic emigrants ing (< 2/2), intermediate risk if they carried one of (RTEs), CD4+ naïve cells that co-express CD31 [19]. these factors and low risk if they carried no risk factors [31]. The functional impact of CD8+ T cell replicative senescence is an overall impairment of the adaptive Sample processing immune response, with a consequent reduction in the Up to 50 ml of venous blood was collected from each ability to fight novel infections and impairment in re- participant in a combination of lithium heparin tubes for sponses to vaccinations in the elderly [17]. The impact peripheral blood mononuclear cell (PBMC) isolation, of CD8+ cell senescence on the development and plain tubes for serum separation and ethylenediamine- progression of PD is unknown. Such changes may tetraacetic acid (EDTA) tubes for measurement of full reduce responses to disease-related antigens such as blood count and differential count. Samples for misfolded alpha synuclein, thus limiting clearance of patient-control pairs were collected on the same day (9– toxic proteins and potentially contributing to PD. 11 am) and processed in parallel to minimise inter-assay Alternatively, they may be protective through reducing variability. Plain tubes were left to clot for 15 min before the capacity of the immune system to become acti- centrifugation at 2000 rpm and removal of serum, which vated, hence limiting an important driver of central was stored at − 80 °C prior to subsequent analysis for neuroinflammation [28]. Here, we sought to answer CMV IgG using a LIAISON® CMV IgG II chemilumines- this question by investigating whether there was an as- cent immunoassay (DiaSorin, Italy). PBMCs were sepa- sociation between an immunosenescent T cell pheno- rated by centrifugation over a Ficoll gradient and type and PD and to determine the relevance of CMV washed and divided into aliquots for immunophenotyp- infection to this. ing and cell separation into T cell subsets. Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 3 of 8 PBMCs for phenotyping were blocked with 2% Table 1 Demographic and clinical characteristics of subjects mouse serum prior to labelling with two panels of Variable PD (n = 41) Controls p (n = 41) fluorochrome-conjugated monoclonal antibodies to T Age (years) 68.4 (6.3) 68.1 (5.6) 0.85 cell surface markers of interest [panel 1: CD3(FITC), CD4(PECy5), CD8(APCCy7), HLA-DR(V500), CD38(PE); Gender (% male) 68.3 68.3 1.0 panel 2: CD3(APC), CD4(V500), CD8(APCCy7), Disease duration (years) 4.3 (1.2) CD28(PECy7), CCR7(FITC), CD45RA(PECy5), CD57(PE), MDS-UPDRS motor score 34.9 (12.4) CD31(V450), BD Biosciences). Cells were fixed with 2% ACE-R 93.0 (8.3) paraformaldehyde. Flow cytometry was performed on a Levodopa equivalent daily dose (mg) 591.5 (292.9) BD Biosciences FACS CantoII with BD FACS Diva soft- CMV IgG (% positive) 46.3 61.0 0.18 ware. Isotype-matched mouse monoclonal antibodies were used where appropriate to exclude non-specific anti- Values shown are mean (SD) unless otherwise stated. Continuous variables compared using Student’s t tests and categorical variables compared using body binding. Unstained lymphocytes were also used as chi-square tests or Fisher’s exact test as appropriate negative controls. MDS-UPDRS Movement Disorder Society Unified Parkinson’s Disease Rating Scale, ACE-R Addenbrooke’s Cognitive Examination-Revised Data analysis Immunophenotyping data was analysed using Flowjo. Lym- PD and 20 controls revealed lower total lymphocyte phocytes were gated using forward scatter and side scatter, counts in PD cases than controls (1.4 (0.4) versus 1.9 and single cells were identified using forward-scatter area (0.6) × 10 /L; p = 0.01) with no significant differences in versus width. CD4 and CD8 subsets were identified by gat- monocyte or neutrophil numbers. Flow cytometric data ing on CD3-high CD4-high and CD3-high CD8-high cells from the full cohort indicated no difference in the respectively. Naïve, central memory, effector memory CD4:CD8 ratio between PD cases and controls [3.8 (2.3) and terminally differentiated memory CD45RA+ cells versus 3.3 (3.0); p > 0.05]. (TEMRA) were identified based on CCR7 and CD45RA However, there was a reduction in the number and expression [19]. Senescent ‘late differentiated’ cells were proportion of CD28 CD57 CD8+ T cells in individuals lo hi defined as CD28-low CD57-high cells within the CD8 with PD compared to controls, along with a marginally population. RTEs were identified as CD31+ cells within significant reduction in CD8+ TEMRA cells and accom- the CD4 naïve (CCR7+CD45RA+) population. Expres- panying small increase in CD8+ central memory cells sion of markers of activation (CD28, CD38, HLA-DR) (Table 2 and Fig. 1a, b). Expression of the activation and senescence (CD57) were quantified within CD4+ markers CD38 and HLA-DR on CD8+ T cells was not and CD8+ populations as the ratio of median fluores- different between patients and controls, but expression cence intensity in labelled versus unlabelled lympho- of CD57 was reduced and expression of CD28 was in- cytes (MFI ratio), to allow for variations in voltage creased in PD patients (Table 3; Fig. 1c), in keeping with between samples. the CD8+ subset data (Table 3; Fig. 1c). No differences The primary statistical analyses involved comparison of T Table 2 T lymphocyte subsets cell subsets (as a percentage of lymphocytes) and MFI ratios for relevant markers in PD cases versus matched Subset (% of lymphocytes) PD Control p controls using paired t tests. CMV positivity in patients CD8 Naïve 3.4 (2.3) 3.1 (2.4) 0.511 versus controls was compared using chi-square tests, and Central memory 1.5 (1.2) 1.1 (0.9) 0.015 analyses of variance (ANOVA), including age as a covariate, Effector memory 6.6 (3.6) 7.2 (5.0) 0.516 were used for patient-control comparisons of T cell markers TEMRA 6.2 (6.3) 9.3 (7.9) 0.042 in CMV-positive and CMV-negative subgroups. Relation- CD28 57 5.3 (4.5) 9.0 (8.0) 0.007* lo hi ships between relevant markers and clinical measures of motor and cognitive functions were explored using Pear- CD4 Naïve 17.5 (9.0) 16.0 (8.2) 0.406 son’s correlations. Statistical analysis was performed using Central memory 18.0 (6.7) 15.8 (6.7) 0.052 GraphPad Prism version 6.0 and SPSS version 25 (IBM). Effector memory 14.2 (6.5) 12.8 (6.4) 0.245 Results TEMRA 2.1 (2.4) 2.0 (2.3) 0.747 Forty-one patients with PD and 41 age/gender-matched RTE 2.2 (3.3) 2.1 (2.2) 0.827 controls were recruited. Demographic and clinical char- T lymphocyte subsets are expressed as a percentage of the acteristics of the subjects and CMV status are shown in lymphocyte population. TEMRA terminally differentiated effector memory CD45RA+ve cells, RTE recent Table 1. Nine PD cases were designated high dementia thymic emigrants risk, 18 were low risk and 14 were intermediate risk. *p value (from paired t test) which remains < 0.05 following Bonferroni Analysis of full blood and differential counts in n =20 correction for multiple testing Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 4 of 8 Fig. 1 CD8 immunophenotyping in PD cases (n = 41) versus age-matched controls (n = 41). a Following gating of the lymphocyte and singlet populations, CD8 cells were gated on the basis of high CD3 and CD8 staining, and the CD28 CD57 subset was identified as a discrete lo hi population of CD8+ cells using 5% contour plots. Quadrant gates were used to divide the CD8 population into naïve, central memory, effector memory and TEMRA (terminally differentiated effector memory CD45RA+ve cells) subsets on pseudocolour dot plots. b Individual-level data for CD8 cell subsets (shown as percentage of lymphocytes). c Individual-level data for surface marker expression in CD8 T cells (median fluorescence intensity (MFI) ratio versus unstained lymphocytes). Horizontal lines denote means; *p < 0.05, **p < 0.01 in paired t tests were identified in the CD4+ T cell pool between patients and controls. For cell subsets/markers reaching significance (p < 0.05), ANOVA were performed to assess the effect of dementia risk group on the observed case-control differences (with Table 3 T cell surface marker expression ‘case-control status’ and ‘risk subgroup’ included as fixed Marker PD Control p factors and age and gender as covariates). Main effects of CD8 CD28 188.6 (102.5) 131.5 (88.3) 0.005* ‘case-control status’ were confirmed for the markers previ- CD38 1.9 (1.2) 2.3 (1.6) 0.117 ously identified, but there was no interaction with risk subgroup. Amongst the PD cases, no significant correla- HLA-DR 1.8 (0.4) 2.0 (0.6) 0.055 tions were found between T cell subset percentages, or CD57 38.2 (69.9) 161.0 (261.7) 0.005* surface markers of activation and senescence, and either clinical measures of motor and cognitive function or CD4 CD28 334.6 (97.3) 299.8 (89.1) 0.067 equivalent daily levodopa dose. CD38 6.1 (3.0) 7.1 (4.0) 0.057 CMV IgG seropositivity was not significantly different HLA-DR 1.1 (0.2) 1.2 (0.2) 0.413 between PD cases (19/41) and controls (25/41) (p =0.18). CD57 1.8 (1.5) 2.0 (1.3) 0.493 Nonetheless, given the previously described association Values shown are mean (SD) ratio of median fluorescent intensity versus between CMV exposure and CD8 immunosenescence, unstained lymphocytes (MFI ratio) we explored this relationship further. As anticipated, CD8+ *p value which remains < 0.05 following Bonferroni correction for multiple testing senescence markers were elevated in CMV-positive versus Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 5 of 8 CMV-negative subjects overall, including CD57 expression [14, 34], as well as more widespread genomic associations (ANOVA with age as covariate, F = 4.66, p = 0.03), in loci affecting T cell regulation and function [35, 36]. CD28 CD57 cells (% of lymphocytes, F = 18.75, p < 0.001) Furthermore, specific T cell responses to epitopes of alpha lo hi and TEMRA cells (% of lymphocytes, F =12.71, p = 0.001). synuclein have recently been described at a higher fre- However, this effect was more apparent for controls than quency in PD patients than controls by Sulzer and col- for PD patients, with significantly higher CD57 ex- leagues [11] and were closely associated with possession pression (p = 0.017) and CD28 CD57 cells (% of of known PD risk alleles at the HLA locus. In addition, lo hi lymphocytes, p = 0.028) in controls versus PD cases in alpha synuclein peptides have been shown to bind to these the CMV-positive group (Fig. 2). HLA ‘risk variant’ molecules in vitro, thus revealing a potential functional mechanism through which the Discussion well-described HLA association may contribute to the This study demonstrates for the first time that the per- development of PD. Whilst the authors of this work ipheral immune profile in PD is distinctly atypical for a highlighted the importance of the Th2-type CD4+ more elderly population, with a lack of the CD8+ T cell cell-mediated response observed in their experiments, replicative senescence which characterises normal age- they also found evidence of a significant CD8+-mediated ing. Although other authors have reported phenotypic response when stimulating cells with shorter alpha synu- alterations in the T lymphocyte population in PD, these clein peptides, and in fact, the strongest association be- alterations have mainly been restricted to CD4+ cell sub- tween alpha synuclein-specific T cell responses and HLA sets with conflicting findings [9, 32, 33] and we found was seen for the MHC class I allele HLA A*11.02, thus no changes in the CD4+ T cell pool in this study. No underlining the relevance of the CD8 (MHC class 1 re- previous studies have specifically explored markers of T stricted) response [11]. The latter would be in keeping cell replicative senescence in PD. CMV infection has with our observations that PD-associated immunophe- been strongly implicated in CD8+ replicative senescence notypic changes in our cohort were within the CD8+ [18], but this does not appear to be confounding our ob- population. Other authors have similarly shown an servations, with similar levels of CMV IgG positivity in elevated CD8+ T cell response to alpha synuclein both patient and control groups. Instead, the senescent peptides and herpes simplex virus peptides in PD shift typically induced by chronic latent CMV infection versus controls [37]. is not observed in this PD cohort. This is an intriguing The mechanism by which alterations in peripheral finding, raising the possibility that there are inherent CD8+ T cell function and CNS pathology interact in PD differences in the adaptive immune response to chronic is still not fully elucidated. However, it is now clear that viral infections in PD. We hypothesise that these differ- there is an abundance of T lymphocytes within the men- ences may contribute to a more active T cell-mediated inges which keep the brain under immune surveillance immune response to PD-relevant antigens such as alpha and which drain, together with CNS antigens, via the synuclein, leading in turn to an exaggerated neuroin- CNS lymphatic system to the local lymph nodes [38]. flammatory response which may contribute to the These meningeal T cells may be activated by ‘alarm neuropathology of PD. signals’ released into the CSF by degenerating CNS neu- In keeping with this hypothesis, there is well-established rones [39] and in turn produce inflammatory cytokines evidence from genetic studies of an immune-mediated which can diffuse into the brain parenchyma and lead to susceptibility to PD, with association between the HLA further activation of already ‘primed’ microglia [40]and/ locus, which is critical in antigen recognition, and PD risk or impact directly on neuronal function [41]. Fig. 2 CD8+ senescence markers in CMV-positive versus CMV-negative subjects. The figure shows CD57 expression on CD8+ lymphocytes (median fluorescence intensity (MFI) ratio versus unstained lymphocytes), CD8+ CD28 CD57 cells (percentage of lymphocytes) and TEMRA cells lo hi (percentage of lymphocytes) in PD versus age-matched controls stratified by CMV seropositivity. Horizontal lines denote means; *p < 0.05 and ***p ≤ 0.001 Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 6 of 8 Furthermore, the presence of CD8+ cells within the age-related neurodegenerative disorders such as PD, brain parenchyma in postmortem tissue from PD pa- through limiting immune activation to aberrant peptides tients raises the possibility of direct interaction with associated with these diseases. On a practical level, this neurones presenting relevant antigen in the context of work suggests that the CD8+ CD28 CD57 immunophe- lo hi MHC class 1, with consequent cytotoxic effects [12]. notype warrants further evaluation as a biomarker in PD, In this study, we adopted a hypothesis-driven ap- which could be relevant for patient stratification for clin- proach and so restricted our T cell phenotyping to ical trials of immunomodulating therapies. Furthermore, specifically explore cell subsets and markers relevant to whilst immunomodulatory strategies targeting the CD4 activation and replicative senescence. A consequent limi- population are being developed [43], the CD8 population tation of the study is that we were not able to fully char- may also warrant consideration as a target for immune acterise the T cell population in our cohort, nor assess therapies in PD. changes in T regulatory cells, which have been reported Abbreviations to be reduced in both number, and functional ability to ACE-R: Addenbrooke’s Cognitive Examination-Revised; ANOVA: Analysis of suppress effector T cells in PD patients [9]. Another po- variance; CMV: Cytomegalovirus; CSF: Cerebrospinal fluid; EDTA: Ethylenediaminetetraacetic acid; HLA: Human leucocyte antigen; tential limitation of our study is that the majority of our MAPT: Microtubule-associated protein tau; MDS-UPDRS: Movement patients were taking dopaminergic medication, which Disorder Society Unified Parkinson’s Disease Rating Scale; MFI: Median has been implicated as a potential confounding factor in fluorescence intensity; PBMC: Peripheral blood mononuclear cell; PD: Parkinson’s disease; RTE: Recent thymic emigrant; SD: Standard deviation; TEMRA: Terminally alterations in lymphocyte populations in PD cohorts differentiated effector memory CD45RA+ cells [33]. In particular, in vitro data indicates that dopamine is an activator of effector T cells and an inhibitor of Acknowledgements We gratefully acknowledge the participation of all our patient volunteers and regulatory T cells [42]. However, studies specifically NIHR Cambridge BioResource volunteers. We thank the Cambridge BioResource comparing medicated and unmedicated PD patients have staff for their help with volunteer recruitment. found no relationship between levodopa treatment and We thank members of the Cambridge BioResource SAB and Management Committee for their support of our study. We also acknowledge the support changes in T cell subsets [32]. Similarly, we found no as- of the Cambridge NIHR BRC Cell Phenotyping Hub. sociations between T cell phenotypes or surface marker expression and equivalent levodopa dose in our patient Funding Grant funding from the Academy of Medical Sciences, UK, the Rosetrees cohort suggesting that medication was not a significant Trust, the Stevenage Biosciences Catalyst and Addenbrooke’s Charitable Trust factor driving the observed reduction in markers of CD8 contributed to this work. The research was also supported by the NIHR replicative senescence. Cambridge Biomedical Research Centre (Cambridge University Hospitals NHS Trust/University of Cambridge). C. H. Williams-Gray is supported by a fellowship Although we found clear differences in T cell senes- from the Medical Research Council. R. S. Wijeyekoon was supported by a cence markers between patient and control groups, we fellowship from Addenbrooke’sCharitableTrust.K.M. Scott andJ.L. were not able to demonstrate a relationship between Jones are supported by fellowships from the Wellcome Trust. R. A. Barker is supported by the Wellcome-MRC Cambridge Stem Cell Institute. these markers and dementia risk status or clinical mea- The funding bodies had no role in the design of the study; the collection, sures of disease severity within the PD group. This may analysis and interpretation of data; or the writing of the manuscript. be because the observed immunophenotypic differences Availability of data and materials are most relevant to the earliest stages of the disease, Datasets used during the current study are available from the corresponding impacting on susceptibility but not on the rate of pro- author on reasonable request. Access to Cambridge BioResource volunteers gression. However, the power of our study to detect and their data and samples is governed by the Cambridge BioResource SAB. Documents describing access arrangements and contact details are available at differences between dementia risk subgroups was likely http://www.cambridgebioresource.org.uk/. suboptimal given the size of our cohort. In terms of measures of disease severity, the spread across the group Authors’ contributions CHW-G contributed to the study design, acquisition of data, analysis and was relatively minimal (Table 1) which consequently lim- interpretation of data and drafting of the manuscript. RW contributed to the ited our ability to explore correlations with disease stage. acquisition of data and critical revision of the manuscript. KMS contributed Further studies exploring these markers both across a to the acquisition of data and critical revision of the manuscript. SH contributed to the acquisition of data and critical revision of the manuscript. range of disease stages, and longitudinally in individual RAB contributed to the study design and supervision and critical revision of patients, would be of interest. the manuscript. JLJ contributed to the supervision of study, study design, interpretation of data and critical revision of the manuscript. All authors read and approved the final manuscript. Conclusions Our data reveal that there is a reduction in age-associated Ethics approval and consent to participate replicative senescence of CD8+ T cells in PD patients This study was approved by the East of England—Cambridge Central compared with strictly age-matched controls. Hence, Research Ethics Committee (ref 03/303). All participants provided written informed consent. whilst immunosenescence may be detrimental in terms of increased susceptibility to infections and cancers with in- Competing interests creasing age, it may provide a degree of protection against The authors declare that they have no competing interests. Williams-Gray et al. Journal of Neuroinflammation (2018) 15:166 Page 7 of 8 Publisher’sNote 17. Chou JP, Effros RB. T cell replicative senescence in human aging. 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Journal of NeuroinflammationSpringer Journals

Published: May 28, 2018

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