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Higher frequency of regulatory T cells in the elderly and increased suppressive activity in neurodegeneration
Journal of Neuroimmunology 188 (2007) 117 – 127 www.elsevier.com/locate/jneuroim
Higher frequency of regulatory T cells in the elderly and increased suppressive activity in neurodegeneration Daniela Rosenkranz a,1 , Sascha Weyer b,1 , Eva Tolosa c,2 , Alexandra Gaenslen b , Daniela Berg b , Thomas Leyhe d , Thomas Gasser b , Lars Stoltze a,b,⁎ a Department of Cellular Neurology, Tübingen, Germany Department for Neurodegenerative Disorders, Tübingen, Germany Department of General Neurology, Hertie-Institute for Clinical Brain Research, Tübingen, Germany d Department of General Psychiatry and Psychotherapy, University Hospital, Tübingen, Germany b
c
Received 26 March 2007; received in revised form 10 May 2007; accepted 11 May 2007
Abstract The involvement of regulatory T cells (Treg) in autoimmune-disease development has been demonstrated. However, their alteration during ageing and age-related diseases has not been thoroughly investigated yet. Alzheimer (AD) and Parkinson disease (PD), are related to proteinmisfolding and are accompanied by neuroinflammation. Since, it has been hypothesized that the neuroinflammation attempts to prevent disease development, we speculated that changes in Treg might affect any relevant immune mechanism. The analysis of Treg from AD and PD patients as well as non-affected individuals, revealed that the frequency of Treg (CD4+Foxp3+) increases with age and is accompanied by intensified suppressive activity for Treg in patients. © 2007 Elsevier B.V. All rights reserved. Keywords: Immune regulation; Alzheimer's disease; Parkinson disease; Ageing; Inflammation
1. Introduction The cellular immune system is tightly regulated to maintain the balance between its ability of defending the organism against infections and avoiding autoimmunity. Regulatory T cells (Treg) are a key cell type for regulating cellular immunity and had been introduced already more than 30 years ago (Gershon and Kondo, 1971). After a period of skepticism about
Abbreviations: Aβ, amyloid-beta; AD, Alzheimer's disease; CNS, central nervous system; CSF, cerebral spinal fluid; Foxp3, forkhead transcription factor; MFI, mean flourescence intensity; PD, Parkinson disease; Treg, regulatory T cells. ⁎ Corresponding author. Department of General Neurology, Hertie-Institute for Clinical Brain Research, Tübingen, Otfried Müller Str. 27, 72076 Tübingen, Germany. Tel.: +49 7071 2981948; fax: +49 7071 294521. E-mail address: [email protected] (L. Stoltze). 1 Contributed equally to this study. 2 Current address: Institute for Neuroimmunology and Clinical MS Research, University of Hamburg, Germany. 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.05.011
their existence, Sakaguchi et al., brought them again into focus, by discovering that the CD4+CD25+ cell population has the highest regulatory function within the T cell compartment and can prevent autoimmune disease in experimental mouse models (Sakaguchi et al., 1995). Since then many more subtypes of Treg have been identified (reviewed in (Mills, 2004)) and it became clear that the marker CD25 is a useful but not exclusive marker for Treg. The most specific marker for Treg to date is the forkhead transcription factor (Foxp3) (Fontenot et al., 2003; Hori et al., 2003). The importance of Treg in regulating human cellular immunity could be demonstrated by the fact that changes in Treg activity or frequency contribute to autoimmune diseases. Functional impairment of Treg has been described for multiple scleroses (Viglietta et al., 2004), myasthenia gravis (Balandina et al., 2005) as well as other autoimmune diseases (Kriegel et al., 2004; Yamano et al., 2005) and lower numbers of Treg have been demonstrated in chronic graft-versus-host disease (Zorn et al., 2005). They also play an important role in cancer immunity (reviewed in (Zou, 2006)) but whether Treg
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change during aging or in age-related diseases remains unclear (Dejaco et al., 2006). Alzheimer's (AD) and Parkinson disease (PD) are neurodegenerative disorders of the central nervous system (CNS). Genetic risk factors have been discovered for both diseases but these account currently for less then 10% of all cases (Nussbaum and Ellis, 2003). The highest risk factor for the genetic as well as for the remaining 90% sporadic cases is age. Both disorders are associated with the accumulation of pathological protein aggregates within the CNS and neuroinflammation. Whether this inflammation is contributing to the disease pathology or is part of a defense mechanism is still unknown. It is however interesting to note that in addition to features of neuroinflammation in both diseases such as up-regulation of MHC class I and II on glial and neuronal cells, production of inflammatory cytokines and a limited T cells infiltration (Akiyama et al., 2000; Hunot and Hirsch, 2003) also autoreactive antibodies and T cells against Aβ exist in humans (Du et al., 2001; Hyman et al., 2001; Monsonego et al., 2003). Furthermore, the use of certain immunization strategies leads to the reduction of pathological features in animal models of both diseases. This has been achieved by either immunizing with the major aggregating proteins, amyloid-beta (Aβ) for AD and alpha-synuclein for PD (Masliah et al., 2005; Schenk et al., 1999) or with glatiramer acetate, a drug consisting of a peptide mixture, which mimics CNS relevant antigenic epitopes (Benner et al., 2004; Frenkel et al., 2005). For the protein immunization with Aβ, it has been shown that antibodies against the relevant protein are sufficient to obtain reduction of pathology. In contrast the effect of glatiramer acetate, which is believed to be T cell mediated, has also been observed in mice unable to produce antibodies. Combining these observations and the fact that immune cells are able to produce brain-derived neurotrophic factor (Kerschensteiner et al., 1999), increase motoneuron survival after injury (Byram et al., 2004) and may contribute to neurogenesis (Ziv et al., 2006), the possibility exists that a beneficial autoimmune response may prevent disease development within the CNS in healthy individuals. Even though this concept and its relevant effector mechanism(s) remains to be proven in humans, we hypothesized that higher activity or numbers of Treg may suppress any beneficial immune effector mechanism and influence either AD or PD. To address additionally the issue of age-dependent changes in Treg we compared not only AD and PD patients against elderly non-affected individuals but also healthy young controls.
PD patients were clinically diagnosed according to the UK Brain Bank criteria (Hughes et al., 1992a,b) and had disease stages between I–V of Hoehn and Yahr (Hoehn and Yahr, 1967) (mean 2.6 ± 1.0 SD). Of the 30 PD patients evaluated for Treg function, 26 received L-Dopa, levodopa or dopamine agonists and 4 received no medication. Patients were not treated with any immunomodulatory drugs and did not receive corticosteroids. Non-affected elderly controls were of same age as the AD and PD groups. Young controls as well as all other donors had a negative history of autoimmune diseases and did not suffer from common cold or influenza infection. The numbers, gender and age of donors examined per group are given in Table 1. 2.2. Antibodies and flow cytometry Human CD4+ CD25high and CD4+CD25− T cells were separated on a FACSAria cell sorter (BD Biosciences). PBMC were isolated by Ficoll (PAA Laboratories, Austria) gradient centrifugation and were incubated for 1 h on ice with saturating amounts of anti-CD4-CyChrome, anti-CD25-PE, and a mix of anti-CD14, anti-CD32 and anti-CD116, all FITC labeled (BD Biosciences). Control PBMC (1 × 106 cells each) were also stained with the aforementioned antibodies in combination with the relevant isotype control mouse IgGs (all purchased from BD Biosciences). Cells were washed and gated according to their forward and side scatter properties on lymphocytes, excluding large activated and all FITC labeled cells. The sort gate for the CD4+CD25high population was set to sort only cells with high CD25 expression identified by lower CD4 expression, sorting 4–8% of all CD4+ cells. Feeder cells were obtained by mixing Ficoll-purified PBMC of four different donors (two young and two elderly). The mix was stored at − 150 °C and irradiated before use at 3300 rad. For Foxp3 staining, PBMC were incubated with anti-CD4PB (DakoCytomation, Denmark), the mix of anti-CD14, CD32 and CD116-FITC, as well as in some cases anti-CD45RO-APC (BD Biosciences) and intracellular staining with anti-Foxp3-PE (E-Bioscience, USA) was performed as instructed by the manufacturer. Cells were analyzed on a CyanADP flow cytometer (Dakocytomation, Denmark) and gated on lymphocytes by forward and side scatter excluding all FITC positive cells. The relative amount of Foxp3 in Treg was determined by subtracting the mean fluorescence intensity (MFI) value of the PE-channel of CD4+ Foxp3− cells from the MFI value of CD4+ Foxp3+ cells. To account for inter-experimental variations in different experiments a standard sample of one young individual was used for all experiments and the data normalized to this person.
2. Methods 2.1. Patients The Ethical Review Board at the University of Tübingen approved this work and all donors were enrolled upon informed consent. AD patients had disease according to NINCDSADRDA Criteria (McKhann et al., 1984) with a Mini-Mental State Examination Index score of 16–26 (mean 22.6 ± 2.4 SD).
Table 1 Numbers, gender and age of donors examined per group for this study Group
Foxp3 (n; female + male)
Functional (n; f + m)
Age in years (mean ± SD)
AD PD CO CY
29 (21 + 8) 40 (18 + 22) 33 (16 + 17) 38 (18 + 20)
23 (17 + 6) 30 (11 + 19) 29 (12 + 17) 30 (12 + 18)
62–86 (73.9 ± 6.5) 46–80 (67.5 ± 8.7) 51–87 (71.2 ± 6.5) 23–40 (28.0 ± 4.2)
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2.3. Regulatory T cell assay CD4+CD25− (responder) and CD4+CD25high (suppressor) T cells (7 × 103 cells/well) were stimulated with soluble anti-CD3 (OKT3) used at 1 μg/ml, in U-bottom 96-well plates. CD4+CD25− T cells were also co-cultured with decreasing numbers of CD4+ CD25high T cells (responder/suppressor ratios: 1:1, 1:0.3, 1:0.1, and 1:0.03). All cells were cultured in a final volume of 200 μl αMEM (Cambrex, USA) containing 5% human serum (C.C.pro, Neustadt, Germany), 2 mM glutamine (C.C.pro, Neustadt, Germany), 50 units Penicillin/Streptomycin (PAA, Austria), in the presence of 5 × 104 feeder cells/well. After 5 d of culture, 1 μCi [3H]thymidine (Hartmann Analytic, Braunschweig, Germany) was added to each well. The cells were harvested after 12 h and cpm/well were determined by scintillation counting (PerkinElmer). Thymidine incorporation reached for CD4+CD25− cells values between 20 000 and 120 000 cpm and for irradiated feeder
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cells alone less than 3% of the CD4+CD25− cell values. Experiments in which the proliferation of CD4+CD25+ cells alone reached more then 20% were excluded from evaluation because of possible contamination of CD4+CD25− cells within the CD4+CD25+ population (10 out of 122 assays). 2.4. Tau determination in CSF The amount of human tau protein within the cerebral spinal fluid (CSF) of AD patients was measured by commercially available ELISA (INNOTEST TM hTAU AG, INNOGENETICS, Gent, Belgium). 2.5. Statistics In proliferation assays the mean ± SD thymidine uptake and cytokine secretion of sixplicate cultures was calculated for each
Fig. 1. Frequency of CD4+Foxp3+ cells. PBMC from each donor where stained by flow cytometry for CD4 and Foxp3 expression. (A) Histograms of one representative case from each group gated on the lymphocyte population are shown and the numbers depicted represent the percentage of Foxp3+ cells of total CD4+ cells. (B) For each individual donor the percentage of Foxp3+ cells of total CD4+ cells and (C) the absolute number/100 000 gated lymphocytes of CD4+Foxp3+ cells and (D) of CD4+ Foxp3− cells is given. Horizontal bars represent the mean value of each group including the standard deviation. CY = diamonds (n = 38), CO = triangles (n = 33), AD = squares (n = 29), PD = circles (n = 40). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p ≤ 0.05, ns = not significant).
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experimental condition. The response of CD4+CD25− T cells was normalized to 100% to calculate the percent remaining proliferation resulting from the addition of CD4+CD25high cells to the cultures. Data from AD, PD as well as CY groups were compared by pairwise Wilcoxon test against CO as control using the JMP software (version 5.1, SAS Institute Inc). p-values were adjusted for multiple testing by the method of Bonferroni–Holm with a global significance level of 0.05. Correlations were estimated by calculating Spearmans r by linear regression analysis. 3. Results 3.1. Higher Frequency of CD4+Foxp3+ cells in the elderly For evaluation of the frequency of CD4+ Foxp3+ cells in human peripheral lymphocytes, the number of CD4+ Foxp3+
cells in young (control young = CY), elderly non-affected (control old = CO) and elderly individuals affected by neurodegeneration (AD, PD) was determined by flow cytometry. Fig. 1A shows a representative Foxp3 staining of CD4+ cells in total lymphocytes from each of the donor groups. Interestingly, the elderly group showed a significant increase in the Foxp3 + Treg frequency compared to the young individuals ( p = 0.006, Fig. 1B) without any difference between the elderly affected or non-affected groups. The mean size of the Foxp3+ population was 6% within the CD4+ cells for the elderly groups whereas the young group had an average frequency of 4.6%. The increase of the CD4+Foxp3+ frequency in the elderly was due to an increase in the number of CD4+Foxp3+ cells (Fig. 1C). Although the comparison of the young and elderly group barely missed significance ( p = 0.06) this difference was
Fig. 2. Frequency of CD4+CD25high cells. PBMC from each donor where stained by flow cytometry for CD4 and CD25 expression. The CD25high population was identified by its slightly lower CD4 expression. (A) Histograms of one representative case from each group gated on the lymphocyte population are shown and the numbers depicted represent the percentage of CD25high cells of total CD4+ cells. (B) For each individual donor the percentage of CD25high cells of total CD4+ cells and (C) the absolute number/100 000 gated lymphocytes of CD4+CD25high cells and (D) of CD4+CD25− and CD25med cells is given. Horizontal bars represent the mean value of each group including the standard deviation. CY = diamonds (n = 39), CO = triangles (n = 31), AD = squares (n = 28), PD = circles (n = 34). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p ≤ 0.05, ns = not significant).
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sufficient to result in a change of frequency, because of the constant number in CD4+Foxp3-cells (Fig. 1D). The higher tendency for PD patients in CD4+Foxp3+ cell numbers was not significant compared to the elderly control group and resulted most likely from a total increase in CD4+ cells for the PD population investigated. However, this increase in total CD4+ cells did not change the frequency of CD4+Foxp3+ cells compared to the other two elderly groups (Fig. 1B). Comparing the analyzed groups by the less specific marker for Treg of CD25high expression (Baecher-Allan et al., 2001) confirmed the Foxp3 data (Fig. 2). The frequency of CD4+ CD25high cells increased from young controls to the elderly controls and AD patients (p b 0.001, Fig. 2B), which was due to
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an increased CD4+CD25high cell number ( p b 0.001, Fig. 2C) by constant CD4+ cell numbers (Fig. 2D). The frequency of CD25high cells in PD patients differed from the results obtained for Foxp3+ cells. Even though PD patients showed also an increased number of CD4+CD25high cells (Fig. 2C), this increase was not sufficient to compensate for the increase of total CD4+ cells compared to elderly controls ( p = 0.016, Fig. 2D). However, since Foxp3 expression includes also Treg which are not CD25high and here the PD group showed the strongest increase in cell numbers (Fig. 1C), we conclude that the Foxp3 data represent more accurate the Treg frequency. Differences in total cell numbers from both experiments result from different processing of the cells for flow cytometry.
Fig. 3. Increasing frequency of Foxp3+CD45RO+ cells with age. (A) PBMC from randomly selected donors of each group where stained by flow cytometry for CD4, Foxp3 and CD45RO expression. Histograms represent one representative case of each group gated on CD4+Foxp3+ cells. Numbers represent the percentage of CD45RO+ in the CD4+Foxp3+ population. (B) Gating on CD4+Foxp3− cells revealed a constant CD45RO+ population with age but (C) gating on CD4+Foxp3+ cells showed an increased CD45RO+ population in the elderly. The percentage of CD45RO+ cells is shown for each individual donor. Horizontal bars represent the mean value of each group including the standard deviation. CY = diamonds (n = 13), CO = triangles (n = 15), AD = (n = 14), PD = (n = 8). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p ≤ 0.05, ns = not significant).
122 D. Rosenkranz et al. / Journal of Neuroimmunology 188 (2007) 117–127 Fig. 4. Treg suppressive activity increases with neurodegeneration. CD4+CD25− and CD4+CD25+ cells of each donor were cultured in the presence of irradiated allogenic feeder cells, alone and together with decreasing amounts of CD4+CD25+ cells. All cells were stimulated with soluble anti-CD3 antibody (1 μg/ml). (A) Proliferation of cells was measured by thymidine incorporation after 6 days of culture and (B) expressed as percentage of proliferation, after normalizing the proliferation of CD4+CD25− cells alone to 100%. (C) All assays resulted in a linear relationship when plotted as % proliferation against the ratio of CD25−/CD25+ expressed as log scale. A–C show one representative assay of one donor. (D) Background proliferation values from CD4+CD25+ cells alone showed no difference between groups, (E) whereas the suppressive activity of Treg at the ratio 1:1 increased with age and was further enhanced by a neurodegenerative phenotype. For true suppression values the background proliferation of CD4+CD25+ cells alone, shown in D was subtracted from the proliferation at the ration CD25−/CD25+ of 1:1. Horizontal bars represent the mean value of each group including the standard deviation. CY =(n =30), CO=(n=29), AD =(n=23), PD=(n =30). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p≤ 0.05, ns=not significant).
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3.2. CD4+Foxp3+ cells are antigen-experienced, and this memory phenotype increases with age
the young and elderly groups (p = 0.0006) but again no difference between elderly and disease-affected groups was observed.
To address the question of the phenotypic development for CD4+ cells in the elderly, we stained cells of randomly selected individuals from each group for CD45RO, which is a marker for antigen experienced memory cells (Fig. 3A). The CD4+Foxp3population did not show significant age-dependent increase in CD45RO staining, which was expressed on 46% of the cells in the young group and up to 58% for the PD group (Fig. 3B). This result is in line with previous published data, showing that CD4+CD45RO+ cells increase strongly until the age of 30 but not thereafter (Cossarizza et al., 1996). Because Treg are mainly antigen experienced cells, we were not surprised that the CD4+Foxp3+ cell population showed a higher memory percentage with an average of 80% CD4+ Foxp3+CD45RO+ cells for the young group (Fig. 3C). Higher age leads to a further pronouncement of this phenotype reaching up to 90% for the elderly control and AD group. This increase of CD4+Foxp3+CD45RO+ cells was highly significant comparing
3.3. Increased regulatory T cell activity in AD and PD To assess whether any functional differences exist between Treg from young and elderly non-affected and affected individuals, CD4+CD25high cells of PBMC from each donor were sorted by flow cytometry (standard purity ≥ 96%) and tested in a T cell proliferation assay for their suppressive activity. Each assay included the evaluation of four different ratios of CD4+CD25− to CD4+CD25+ cells (Fig. 4A) and the results were expressed as % proliferation relative to CD4+CD25− cells alone (Fig. 4B). In all assays selected for analysis, the suppressive activity exponentially declined with decreasing ratios of CD4+CD25−/CD4+CD25+ cells, resulting in a straight line when the ratio was expressed on a logarithmic scale against % proliferation (Fig. 4C). To compare the functional activity of CD4+CD25+ cells between the groups, only the values at a ratio of 1:1 were used.
Fig. 5. Foxp3 expression correlates with tau in CSF of AD patients. (A) The relative amount of Foxp3 in CD4+Foxp3+ cells was determined as mean fluorescence intensity by flow cytometry for each donor. Horizontal bars represent the mean value of each group including the standard deviation. CY = (n = 38), CO = (n = 33), AD = (n = 29), PD = (n = 40). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (ns = not significant). (B) The amount of Foxp3 in CD4+Foxp3+ cells for all donors does not correlate with the suppressive activity of Treg from the same donor at the ratio 1:1, (C) but a relationship exists between the amount of Foxp3 in CD4+Foxp3+ cells of AD patients and their amount of total tau protein in the CSF (r2 = 0.34, p = .0025). Each data point represents one individual donor.
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This was the case because we realized that the true suppressive value is only obtained if the background proliferation of the CD4+CD25+-only sample is subtracted from the value at the ratio 1:1. We were not able to do this for all ratios, because of missing background values for lower CD4+CD25+ cell numbers in early assays. Interestingly, even though the background values of CD4+CD25+ cell proliferation showed no difference between any group (Fig. 4D), we observed an increased suppressive activity in CD4+CD25+ cells from the elderly group compared to the young group, reflected by the reduced proliferation of CD4+ CD25− cells at the cell ratio 1:1 (Fig. 4E). This increase of suppressive activity did not reach significance for the elderly controls but was highly significant for the AD and PD group (AD p = 0.0059, PD p = 0.0019). However, the suppressive activity of CD4+ CD25+ cells from AD and PD was not significantly higher compared to the non-affected elderly control group (AD p = 0.10, PD p = 0.03). Because the PD group only missed significance because of adjustment for multiple testing we wondered whether the L-Dopa or a dopamine related medication from most of the PD patients investigated in this study influenced our results. This was of special interest, because dopamine has been shown to lower Treg function (Kipnis et al., 2004), which means that treated PD patients could have in reality even stronger increased Treg activity. The comparison of patients with and without medication showed however no significant difference in Treg activity (3.9%, mean proliferation SD 3.2, at ratio 1:1 for dopamine medication (n = 26), compared to 2.3%, SD 0,9 for no medication (n = 4), p = 0.67). This implies that peripheral administration of dopamine related medication in concentrations used for PD treatment does not significantly alter the activity of Treg. 3.4. Foxp3 expression reflects not suppressive activity but correlates with tau levels in the CSF of AD patients Because of the importance of Foxp3 expression in Treg function, which is sufficient to drive CD4+ T cells into the Treg linage (Hori et al., 2003), we assessed whether the amount of Foxp3 expression correlates with suppressive activity. Therefore, the relative amount of Foxp3 expressed in Treg was determined by flow cytometry for each donor at a single cell level. The mean fluorescence intensity for Foxp3 in CD4+ cells was compared in all four groups, but no differences were observed (Fig. 5A). Additionally, did the amount of Foxp3 neither correlate within the subgroups nor in all groups combined with suppressive activity (Fig. 5B). Since the increase in Treg suppression observed in our patient population suggests a relationship between suppression and neurodegeneration, we evaluated whether our observation has any direct relationship with clinical markers. Disease severity correlated with no Treg data but surprisingly, when the amount of Foxp3 expression from AD patients was compared with the amount of tau protein within the CSF, we found a moderate correlation (r2 = 0.34, p = 0.0025, Fig. 5C). This observation remains however difficult to interpret because of lacking comparable CSF data from the other groups.
4. Discussion The aim of this study was to quantitatively and qualitatively analyze peripheral Treg of young and elderly non-affected donors as well as AD and PD patients. By using Foxp3 which is the most specific marker for Treg our analysis differs from previously published data in which controversial results have been obtained when CD25 was used for quantitative analysis of Treg in elderly individuals (Gregg et al., 2005; Tsaknaridis et al., 2003), AD (Speciale et al., in press) or PD patients (Baba et al., 2005; Bas et al., 2001). Quantifying Treg by Foxp3 reveals that the frequency of Treg within the CD4+ pool increases with age, which is in line with data obtained in mice using Foxp3 as marker (Sharma et al., 2006). This is due to increasing Treg numbers and not a decrease of the CD4+ T cell number and shows that Treg undergo immunological senescence. Using CD25 expression as Treg marker mainly confirmed our Foxp3 data. However, differences in Treg frequency for the PD group with this two markers argues for the use of the most accurate Treg marker, which is Foxp3. Increased frequency of Treg may be due to clonal expansion of Treg or accumulation of memory type Treg because with advanced age the frequency of CD45RO+ Treg rises from 80% in the young group to 90% in the elderly. Hence, this accumulation goes in parallel to the previously published decline of so-called naïve Treg with age, which are Foxp3+ and classified by being CD4+CD25+CD45RA+ (Valmori et al., 2005). This increase of Treg frequency is not influenced by a neurodegenerative phenotype but may contribute to the observation of weaker immune responses in the elderly against infections such as influenza (Thompson et al., 2003). Evaluating Treg reactivity in a functional suppression assay showed that Treg activity increases with a neurodegenerative phenotype. Even though it is generally believed that aged T cells have a lower proliferating capacity, we observed no difference in the mean proliferation of sorted CD4+CD25− cells between the groups, which did not change when the proliferation of CD4+CD25− and CD4+CD25+ cells were combined (data not shown). This could be due to the stimulus used, as the extend of the alloreaction against the mixed feeder cells may vary among donors, but one should also consider that in cases of lower T cell proliferation in the elderly, only bulk CD4+ cells have been used and the aspect of increasing Treg number with age has not yet been considered. Taking further into account that the accuracy of AD/PD diagnosis is about 80% (Cummings, 2004; Hughes et al., 1992b) and without knowing whether some of the elderly control individuals with high Treg suppressive activity may develop AD/PD in the near future, we cannot rule out that the difference between the elderly affected and non-affected groups would in reality become significant. The fact that some young donors show high Treg suppressive activity does not disagree with this interpretation because this may represent a risk factor to develop disease. However, this would have to be confirmed by a larger longitudinal study. It is also to consider that we analyzed only the total pool of peripheral Treg. In the case of Treg specific for CNS-relevant antigens, the differences between the groups analyzed may be
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larger. In this context it is of interest to note that in Leishmaniainfected mice antigen-specific Treg act locally (Suffia et al., 2006). Consequently, it will be of special interest to analyze Treg specific for antigens relevant to AD or PD, such as Aβ or alpha-synuclein. That the observed effect of higher Treg activity is specific for Treg and not due to a change in APC function can be excluded because of the fact, that we used the same pool of irradiated feeder cells for all donors, consisting of a mix of four different donors (two elderly, two young individuals). Alternatively the effect could be explained by higher sensitivity of the CD4+CD25− cells to Treg. Even though we did not perform crossover experiments, others have done this under various conditions and the defect was always accounted to Treg (Viglietta et al., 2004; Wing et al., 2003). Furthermore, by the inclusion of an internal standard in the flow cytometry experiments as well as the realization that CD4+CD25+ cells contribute in suppression assays to background proliferation, we believe to have used a very sensitive experimental set-up. Nonetheless, our sort strategy allowed only the analysis of CD4+ CD25high Treg. This population may only partially include the naïve CD4+CD25+CD45RA+ Treg, which show a slightly lower CD25 expression. However, because no difference in suppressive activity has been demonstrated between naïve or standard Treg (Valmori et al., 2005), we would not expect that the full inclusion of this Treg population would change our results. Furthermore it has been shown that the CD4+CD25high Treg population can be subdivided into CD45RO− and CD45RO+ cells (Taams et al., 2001; Yagi et al., 2004). Only the CD45RO + population showed thereby suppressive activity (Yagi et al., 2004). Even though we normalized the amount of Treg to 7000 cells in our functional assays, we may speculate that due to the increase of CD45RO+ Treg with age, the proportion of suppressive CD4+CD25+ CD45RO+ Treg increases in our assay. This may explain the non-significant difference in suppressive Treg activity between the healthy young and elderly group. However, this does not explain the further increase for the AD/PD groups, because we observed no difference between the Foxp3+CD45RO+ population of elderly-controls versus AD and for PD this population was even slightly (but not significantly) smaller. In search for an explanation of our observed Treg differences, the most obvious candidate was Foxp3, because it has the highest impact on development and function of Treg. Hence, we speculated whether the amount of Foxp3 expressed in Treg could influence their suppressive activity. But in contrast to their suppressive activity, all groups showed similar Foxp3 expression and even though the expression of Foxp3 in mouse CD4+CD25− T cells seems to be sufficient to drive CD4+ T cells into a regulatory phenotype (Hori et al., 2003), the amount of Foxp3 in our experiments does not correlate with their suppressive function and shows that further experiments are required to understand why Treg from AD and PD patients are more active. The relevance of peripheral Treg changes for a CNS-based disease might be questioned, however one should consider that these T cells may be present within the CNS during neurodegeneration, since a modest T cell infiltration into the CNS has been demonstrated for AD (Itagaki et al., 1988; Togo et al., 2002) and
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PD patients (McGeer et al., 1987) as well as in animal models of AD (Stalder et al., 2005) and PD (Kurkowska-Jastrzebska et al., 1999). Even though this represents a rather minor T cell infiltration in comparison to classical autoimmune diseases like multiple sclerosis, a low number of Treg may contribute to a chain of events, which affect protein misfolding, and thus neurodegeneration. Especially in the case of localized events within the CNS, like the loss of dopaminergic neurons in PD, which results in reduced dopamine and may result in higher Treg function (Kipnis et al., 2004) or the induction of Treg by activated neurons (Liu et al., 2006). Additionally, other changes due to neurodegeneration, for instance elevated CSF tau levels may affect Treg function. The observation that Treg frequency increases with age and that Treg have a higher reactivity in the presence of a neurodegenerative phenotype leads to the question how Treg and neurodegeneration affect each other? Since other regulatory T cell subpopulations such as CD8+CD28− T cells are also affected by a neurodegenerative phenotype (Speciale et al., in press) it is possible that imbalanced immune regulation may affect protective immune mechanisms in neurodegenerative pathology. The influence of Treg in acute CNS-injury models has been shown (Kipnis et al., 2002) and Treg function may further influence beneficial effects of auto-reactive T cells (Moalem et al., 1999) or the auto-reactive antibody response (Du et al., 2001). Thus, changes in immune regulation may disturb the balance of beneficial clearance mechanisms and protein aggregation and thus indirectly increase the pathology mediated by protein aggregation. This is not contradicted by the fact that higher reactivity of Aβ-specific T cells exists in elderly and AD (Monsonego et al., 2003). An increase in reactivity tested by in vitro assays could be due to a higher frequency of Aβ-specific T cells, which may outnumber the effect of increasing Treg activity. Since the function of Aβ-specific T cells in vivo is not known, it will be interesting to see whether their increase and the increase of Treg have synergistic or antagonistic effects. Thus, it will be highly interesting to see whether a change in Treg activity affects any mechanism involved in clearance of protein aggregates in either AD or PD. Acknowledgments The authors wish to thank Cornelia Grimmel, Andreas Böhmler and Hans-Jörg Bühring for cell sorting, Birgitt Schönfisch for statistical advice and Michael Calhoun and Mathias Jucker for the critical reading of the manuscript. This work was supported by the FORTÜNE Program of the University of Tübingen. References Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., Finch, C.E., Frautschy, S., Griffin, W.S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I.R., McGeer, P.L., O'Banion, M.K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F.L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G., Wyss-Coray, T., 2000. Inflammation and Alzheimer's disease. Neurobiol. Aging 21, 383–421.
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Higher frequency of regulatory T cells in the elderly and increased suppressive activity in neurodegeneration Daniela Rosenkranz a,1 , Sascha Weyer b,1 , Eva Tolosa c,2 , Alexandra Gaenslen b , Daniela Berg b , Thomas Leyhe d , Thomas Gasser b , Lars Stoltze a,b,⁎ a Department of Cellular Neurology, Tübingen, Germany Department for Neurodegenerative Disorders, Tübingen, Germany Department of General Neurology, Hertie-Institute for Clinical Brain Research, Tübingen, Germany d Department of General Psychiatry and Psychotherapy, University Hospital, Tübingen, Germany b
c
Received 26 March 2007; received in revised form 10 May 2007; accepted 11 May 2007
Abstract The involvement of regulatory T cells (Treg) in autoimmune-disease development has been demonstrated. However, their alteration during ageing and age-related diseases has not been thoroughly investigated yet. Alzheimer (AD) and Parkinson disease (PD), are related to proteinmisfolding and are accompanied by neuroinflammation. Since, it has been hypothesized that the neuroinflammation attempts to prevent disease development, we speculated that changes in Treg might affect any relevant immune mechanism. The analysis of Treg from AD and PD patients as well as non-affected individuals, revealed that the frequency of Treg (CD4+Foxp3+) increases with age and is accompanied by intensified suppressive activity for Treg in patients. © 2007 Elsevier B.V. All rights reserved. Keywords: Immune regulation; Alzheimer's disease; Parkinson disease; Ageing; Inflammation
1. Introduction The cellular immune system is tightly regulated to maintain the balance between its ability of defending the organism against infections and avoiding autoimmunity. Regulatory T cells (Treg) are a key cell type for regulating cellular immunity and had been introduced already more than 30 years ago (Gershon and Kondo, 1971). After a period of skepticism about
Abbreviations: Aβ, amyloid-beta; AD, Alzheimer's disease; CNS, central nervous system; CSF, cerebral spinal fluid; Foxp3, forkhead transcription factor; MFI, mean flourescence intensity; PD, Parkinson disease; Treg, regulatory T cells. ⁎ Corresponding author. Department of General Neurology, Hertie-Institute for Clinical Brain Research, Tübingen, Otfried Müller Str. 27, 72076 Tübingen, Germany. Tel.: +49 7071 2981948; fax: +49 7071 294521. E-mail address: [email protected] (L. Stoltze). 1 Contributed equally to this study. 2 Current address: Institute for Neuroimmunology and Clinical MS Research, University of Hamburg, Germany. 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.05.011
their existence, Sakaguchi et al., brought them again into focus, by discovering that the CD4+CD25+ cell population has the highest regulatory function within the T cell compartment and can prevent autoimmune disease in experimental mouse models (Sakaguchi et al., 1995). Since then many more subtypes of Treg have been identified (reviewed in (Mills, 2004)) and it became clear that the marker CD25 is a useful but not exclusive marker for Treg. The most specific marker for Treg to date is the forkhead transcription factor (Foxp3) (Fontenot et al., 2003; Hori et al., 2003). The importance of Treg in regulating human cellular immunity could be demonstrated by the fact that changes in Treg activity or frequency contribute to autoimmune diseases. Functional impairment of Treg has been described for multiple scleroses (Viglietta et al., 2004), myasthenia gravis (Balandina et al., 2005) as well as other autoimmune diseases (Kriegel et al., 2004; Yamano et al., 2005) and lower numbers of Treg have been demonstrated in chronic graft-versus-host disease (Zorn et al., 2005). They also play an important role in cancer immunity (reviewed in (Zou, 2006)) but whether Treg
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change during aging or in age-related diseases remains unclear (Dejaco et al., 2006). Alzheimer's (AD) and Parkinson disease (PD) are neurodegenerative disorders of the central nervous system (CNS). Genetic risk factors have been discovered for both diseases but these account currently for less then 10% of all cases (Nussbaum and Ellis, 2003). The highest risk factor for the genetic as well as for the remaining 90% sporadic cases is age. Both disorders are associated with the accumulation of pathological protein aggregates within the CNS and neuroinflammation. Whether this inflammation is contributing to the disease pathology or is part of a defense mechanism is still unknown. It is however interesting to note that in addition to features of neuroinflammation in both diseases such as up-regulation of MHC class I and II on glial and neuronal cells, production of inflammatory cytokines and a limited T cells infiltration (Akiyama et al., 2000; Hunot and Hirsch, 2003) also autoreactive antibodies and T cells against Aβ exist in humans (Du et al., 2001; Hyman et al., 2001; Monsonego et al., 2003). Furthermore, the use of certain immunization strategies leads to the reduction of pathological features in animal models of both diseases. This has been achieved by either immunizing with the major aggregating proteins, amyloid-beta (Aβ) for AD and alpha-synuclein for PD (Masliah et al., 2005; Schenk et al., 1999) or with glatiramer acetate, a drug consisting of a peptide mixture, which mimics CNS relevant antigenic epitopes (Benner et al., 2004; Frenkel et al., 2005). For the protein immunization with Aβ, it has been shown that antibodies against the relevant protein are sufficient to obtain reduction of pathology. In contrast the effect of glatiramer acetate, which is believed to be T cell mediated, has also been observed in mice unable to produce antibodies. Combining these observations and the fact that immune cells are able to produce brain-derived neurotrophic factor (Kerschensteiner et al., 1999), increase motoneuron survival after injury (Byram et al., 2004) and may contribute to neurogenesis (Ziv et al., 2006), the possibility exists that a beneficial autoimmune response may prevent disease development within the CNS in healthy individuals. Even though this concept and its relevant effector mechanism(s) remains to be proven in humans, we hypothesized that higher activity or numbers of Treg may suppress any beneficial immune effector mechanism and influence either AD or PD. To address additionally the issue of age-dependent changes in Treg we compared not only AD and PD patients against elderly non-affected individuals but also healthy young controls.
PD patients were clinically diagnosed according to the UK Brain Bank criteria (Hughes et al., 1992a,b) and had disease stages between I–V of Hoehn and Yahr (Hoehn and Yahr, 1967) (mean 2.6 ± 1.0 SD). Of the 30 PD patients evaluated for Treg function, 26 received L-Dopa, levodopa or dopamine agonists and 4 received no medication. Patients were not treated with any immunomodulatory drugs and did not receive corticosteroids. Non-affected elderly controls were of same age as the AD and PD groups. Young controls as well as all other donors had a negative history of autoimmune diseases and did not suffer from common cold or influenza infection. The numbers, gender and age of donors examined per group are given in Table 1. 2.2. Antibodies and flow cytometry Human CD4+ CD25high and CD4+CD25− T cells were separated on a FACSAria cell sorter (BD Biosciences). PBMC were isolated by Ficoll (PAA Laboratories, Austria) gradient centrifugation and were incubated for 1 h on ice with saturating amounts of anti-CD4-CyChrome, anti-CD25-PE, and a mix of anti-CD14, anti-CD32 and anti-CD116, all FITC labeled (BD Biosciences). Control PBMC (1 × 106 cells each) were also stained with the aforementioned antibodies in combination with the relevant isotype control mouse IgGs (all purchased from BD Biosciences). Cells were washed and gated according to their forward and side scatter properties on lymphocytes, excluding large activated and all FITC labeled cells. The sort gate for the CD4+CD25high population was set to sort only cells with high CD25 expression identified by lower CD4 expression, sorting 4–8% of all CD4+ cells. Feeder cells were obtained by mixing Ficoll-purified PBMC of four different donors (two young and two elderly). The mix was stored at − 150 °C and irradiated before use at 3300 rad. For Foxp3 staining, PBMC were incubated with anti-CD4PB (DakoCytomation, Denmark), the mix of anti-CD14, CD32 and CD116-FITC, as well as in some cases anti-CD45RO-APC (BD Biosciences) and intracellular staining with anti-Foxp3-PE (E-Bioscience, USA) was performed as instructed by the manufacturer. Cells were analyzed on a CyanADP flow cytometer (Dakocytomation, Denmark) and gated on lymphocytes by forward and side scatter excluding all FITC positive cells. The relative amount of Foxp3 in Treg was determined by subtracting the mean fluorescence intensity (MFI) value of the PE-channel of CD4+ Foxp3− cells from the MFI value of CD4+ Foxp3+ cells. To account for inter-experimental variations in different experiments a standard sample of one young individual was used for all experiments and the data normalized to this person.
2. Methods 2.1. Patients The Ethical Review Board at the University of Tübingen approved this work and all donors were enrolled upon informed consent. AD patients had disease according to NINCDSADRDA Criteria (McKhann et al., 1984) with a Mini-Mental State Examination Index score of 16–26 (mean 22.6 ± 2.4 SD).
Table 1 Numbers, gender and age of donors examined per group for this study Group
Foxp3 (n; female + male)
Functional (n; f + m)
Age in years (mean ± SD)
AD PD CO CY
29 (21 + 8) 40 (18 + 22) 33 (16 + 17) 38 (18 + 20)
23 (17 + 6) 30 (11 + 19) 29 (12 + 17) 30 (12 + 18)
62–86 (73.9 ± 6.5) 46–80 (67.5 ± 8.7) 51–87 (71.2 ± 6.5) 23–40 (28.0 ± 4.2)
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2.3. Regulatory T cell assay CD4+CD25− (responder) and CD4+CD25high (suppressor) T cells (7 × 103 cells/well) were stimulated with soluble anti-CD3 (OKT3) used at 1 μg/ml, in U-bottom 96-well plates. CD4+CD25− T cells were also co-cultured with decreasing numbers of CD4+ CD25high T cells (responder/suppressor ratios: 1:1, 1:0.3, 1:0.1, and 1:0.03). All cells were cultured in a final volume of 200 μl αMEM (Cambrex, USA) containing 5% human serum (C.C.pro, Neustadt, Germany), 2 mM glutamine (C.C.pro, Neustadt, Germany), 50 units Penicillin/Streptomycin (PAA, Austria), in the presence of 5 × 104 feeder cells/well. After 5 d of culture, 1 μCi [3H]thymidine (Hartmann Analytic, Braunschweig, Germany) was added to each well. The cells were harvested after 12 h and cpm/well were determined by scintillation counting (PerkinElmer). Thymidine incorporation reached for CD4+CD25− cells values between 20 000 and 120 000 cpm and for irradiated feeder
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cells alone less than 3% of the CD4+CD25− cell values. Experiments in which the proliferation of CD4+CD25+ cells alone reached more then 20% were excluded from evaluation because of possible contamination of CD4+CD25− cells within the CD4+CD25+ population (10 out of 122 assays). 2.4. Tau determination in CSF The amount of human tau protein within the cerebral spinal fluid (CSF) of AD patients was measured by commercially available ELISA (INNOTEST TM hTAU AG, INNOGENETICS, Gent, Belgium). 2.5. Statistics In proliferation assays the mean ± SD thymidine uptake and cytokine secretion of sixplicate cultures was calculated for each
Fig. 1. Frequency of CD4+Foxp3+ cells. PBMC from each donor where stained by flow cytometry for CD4 and Foxp3 expression. (A) Histograms of one representative case from each group gated on the lymphocyte population are shown and the numbers depicted represent the percentage of Foxp3+ cells of total CD4+ cells. (B) For each individual donor the percentage of Foxp3+ cells of total CD4+ cells and (C) the absolute number/100 000 gated lymphocytes of CD4+Foxp3+ cells and (D) of CD4+ Foxp3− cells is given. Horizontal bars represent the mean value of each group including the standard deviation. CY = diamonds (n = 38), CO = triangles (n = 33), AD = squares (n = 29), PD = circles (n = 40). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p ≤ 0.05, ns = not significant).
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experimental condition. The response of CD4+CD25− T cells was normalized to 100% to calculate the percent remaining proliferation resulting from the addition of CD4+CD25high cells to the cultures. Data from AD, PD as well as CY groups were compared by pairwise Wilcoxon test against CO as control using the JMP software (version 5.1, SAS Institute Inc). p-values were adjusted for multiple testing by the method of Bonferroni–Holm with a global significance level of 0.05. Correlations were estimated by calculating Spearmans r by linear regression analysis. 3. Results 3.1. Higher Frequency of CD4+Foxp3+ cells in the elderly For evaluation of the frequency of CD4+ Foxp3+ cells in human peripheral lymphocytes, the number of CD4+ Foxp3+
cells in young (control young = CY), elderly non-affected (control old = CO) and elderly individuals affected by neurodegeneration (AD, PD) was determined by flow cytometry. Fig. 1A shows a representative Foxp3 staining of CD4+ cells in total lymphocytes from each of the donor groups. Interestingly, the elderly group showed a significant increase in the Foxp3 + Treg frequency compared to the young individuals ( p = 0.006, Fig. 1B) without any difference between the elderly affected or non-affected groups. The mean size of the Foxp3+ population was 6% within the CD4+ cells for the elderly groups whereas the young group had an average frequency of 4.6%. The increase of the CD4+Foxp3+ frequency in the elderly was due to an increase in the number of CD4+Foxp3+ cells (Fig. 1C). Although the comparison of the young and elderly group barely missed significance ( p = 0.06) this difference was
Fig. 2. Frequency of CD4+CD25high cells. PBMC from each donor where stained by flow cytometry for CD4 and CD25 expression. The CD25high population was identified by its slightly lower CD4 expression. (A) Histograms of one representative case from each group gated on the lymphocyte population are shown and the numbers depicted represent the percentage of CD25high cells of total CD4+ cells. (B) For each individual donor the percentage of CD25high cells of total CD4+ cells and (C) the absolute number/100 000 gated lymphocytes of CD4+CD25high cells and (D) of CD4+CD25− and CD25med cells is given. Horizontal bars represent the mean value of each group including the standard deviation. CY = diamonds (n = 39), CO = triangles (n = 31), AD = squares (n = 28), PD = circles (n = 34). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p ≤ 0.05, ns = not significant).
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sufficient to result in a change of frequency, because of the constant number in CD4+Foxp3-cells (Fig. 1D). The higher tendency for PD patients in CD4+Foxp3+ cell numbers was not significant compared to the elderly control group and resulted most likely from a total increase in CD4+ cells for the PD population investigated. However, this increase in total CD4+ cells did not change the frequency of CD4+Foxp3+ cells compared to the other two elderly groups (Fig. 1B). Comparing the analyzed groups by the less specific marker for Treg of CD25high expression (Baecher-Allan et al., 2001) confirmed the Foxp3 data (Fig. 2). The frequency of CD4+ CD25high cells increased from young controls to the elderly controls and AD patients (p b 0.001, Fig. 2B), which was due to
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an increased CD4+CD25high cell number ( p b 0.001, Fig. 2C) by constant CD4+ cell numbers (Fig. 2D). The frequency of CD25high cells in PD patients differed from the results obtained for Foxp3+ cells. Even though PD patients showed also an increased number of CD4+CD25high cells (Fig. 2C), this increase was not sufficient to compensate for the increase of total CD4+ cells compared to elderly controls ( p = 0.016, Fig. 2D). However, since Foxp3 expression includes also Treg which are not CD25high and here the PD group showed the strongest increase in cell numbers (Fig. 1C), we conclude that the Foxp3 data represent more accurate the Treg frequency. Differences in total cell numbers from both experiments result from different processing of the cells for flow cytometry.
Fig. 3. Increasing frequency of Foxp3+CD45RO+ cells with age. (A) PBMC from randomly selected donors of each group where stained by flow cytometry for CD4, Foxp3 and CD45RO expression. Histograms represent one representative case of each group gated on CD4+Foxp3+ cells. Numbers represent the percentage of CD45RO+ in the CD4+Foxp3+ population. (B) Gating on CD4+Foxp3− cells revealed a constant CD45RO+ population with age but (C) gating on CD4+Foxp3+ cells showed an increased CD45RO+ population in the elderly. The percentage of CD45RO+ cells is shown for each individual donor. Horizontal bars represent the mean value of each group including the standard deviation. CY = diamonds (n = 13), CO = triangles (n = 15), AD = (n = 14), PD = (n = 8). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p ≤ 0.05, ns = not significant).
122 D. Rosenkranz et al. / Journal of Neuroimmunology 188 (2007) 117–127 Fig. 4. Treg suppressive activity increases with neurodegeneration. CD4+CD25− and CD4+CD25+ cells of each donor were cultured in the presence of irradiated allogenic feeder cells, alone and together with decreasing amounts of CD4+CD25+ cells. All cells were stimulated with soluble anti-CD3 antibody (1 μg/ml). (A) Proliferation of cells was measured by thymidine incorporation after 6 days of culture and (B) expressed as percentage of proliferation, after normalizing the proliferation of CD4+CD25− cells alone to 100%. (C) All assays resulted in a linear relationship when plotted as % proliferation against the ratio of CD25−/CD25+ expressed as log scale. A–C show one representative assay of one donor. (D) Background proliferation values from CD4+CD25+ cells alone showed no difference between groups, (E) whereas the suppressive activity of Treg at the ratio 1:1 increased with age and was further enhanced by a neurodegenerative phenotype. For true suppression values the background proliferation of CD4+CD25+ cells alone, shown in D was subtracted from the proliferation at the ration CD25−/CD25+ of 1:1. Horizontal bars represent the mean value of each group including the standard deviation. CY =(n =30), CO=(n=29), AD =(n=23), PD=(n =30). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (⁎p≤ 0.05, ns=not significant).
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3.2. CD4+Foxp3+ cells are antigen-experienced, and this memory phenotype increases with age
the young and elderly groups (p = 0.0006) but again no difference between elderly and disease-affected groups was observed.
To address the question of the phenotypic development for CD4+ cells in the elderly, we stained cells of randomly selected individuals from each group for CD45RO, which is a marker for antigen experienced memory cells (Fig. 3A). The CD4+Foxp3population did not show significant age-dependent increase in CD45RO staining, which was expressed on 46% of the cells in the young group and up to 58% for the PD group (Fig. 3B). This result is in line with previous published data, showing that CD4+CD45RO+ cells increase strongly until the age of 30 but not thereafter (Cossarizza et al., 1996). Because Treg are mainly antigen experienced cells, we were not surprised that the CD4+Foxp3+ cell population showed a higher memory percentage with an average of 80% CD4+ Foxp3+CD45RO+ cells for the young group (Fig. 3C). Higher age leads to a further pronouncement of this phenotype reaching up to 90% for the elderly control and AD group. This increase of CD4+Foxp3+CD45RO+ cells was highly significant comparing
3.3. Increased regulatory T cell activity in AD and PD To assess whether any functional differences exist between Treg from young and elderly non-affected and affected individuals, CD4+CD25high cells of PBMC from each donor were sorted by flow cytometry (standard purity ≥ 96%) and tested in a T cell proliferation assay for their suppressive activity. Each assay included the evaluation of four different ratios of CD4+CD25− to CD4+CD25+ cells (Fig. 4A) and the results were expressed as % proliferation relative to CD4+CD25− cells alone (Fig. 4B). In all assays selected for analysis, the suppressive activity exponentially declined with decreasing ratios of CD4+CD25−/CD4+CD25+ cells, resulting in a straight line when the ratio was expressed on a logarithmic scale against % proliferation (Fig. 4C). To compare the functional activity of CD4+CD25+ cells between the groups, only the values at a ratio of 1:1 were used.
Fig. 5. Foxp3 expression correlates with tau in CSF of AD patients. (A) The relative amount of Foxp3 in CD4+Foxp3+ cells was determined as mean fluorescence intensity by flow cytometry for each donor. Horizontal bars represent the mean value of each group including the standard deviation. CY = (n = 38), CO = (n = 33), AD = (n = 29), PD = (n = 40). Bonferroni–Holm corrected p-values of pairwise Wilcoxon tests (ns = not significant). (B) The amount of Foxp3 in CD4+Foxp3+ cells for all donors does not correlate with the suppressive activity of Treg from the same donor at the ratio 1:1, (C) but a relationship exists between the amount of Foxp3 in CD4+Foxp3+ cells of AD patients and their amount of total tau protein in the CSF (r2 = 0.34, p = .0025). Each data point represents one individual donor.
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This was the case because we realized that the true suppressive value is only obtained if the background proliferation of the CD4+CD25+-only sample is subtracted from the value at the ratio 1:1. We were not able to do this for all ratios, because of missing background values for lower CD4+CD25+ cell numbers in early assays. Interestingly, even though the background values of CD4+CD25+ cell proliferation showed no difference between any group (Fig. 4D), we observed an increased suppressive activity in CD4+CD25+ cells from the elderly group compared to the young group, reflected by the reduced proliferation of CD4+ CD25− cells at the cell ratio 1:1 (Fig. 4E). This increase of suppressive activity did not reach significance for the elderly controls but was highly significant for the AD and PD group (AD p = 0.0059, PD p = 0.0019). However, the suppressive activity of CD4+ CD25+ cells from AD and PD was not significantly higher compared to the non-affected elderly control group (AD p = 0.10, PD p = 0.03). Because the PD group only missed significance because of adjustment for multiple testing we wondered whether the L-Dopa or a dopamine related medication from most of the PD patients investigated in this study influenced our results. This was of special interest, because dopamine has been shown to lower Treg function (Kipnis et al., 2004), which means that treated PD patients could have in reality even stronger increased Treg activity. The comparison of patients with and without medication showed however no significant difference in Treg activity (3.9%, mean proliferation SD 3.2, at ratio 1:1 for dopamine medication (n = 26), compared to 2.3%, SD 0,9 for no medication (n = 4), p = 0.67). This implies that peripheral administration of dopamine related medication in concentrations used for PD treatment does not significantly alter the activity of Treg. 3.4. Foxp3 expression reflects not suppressive activity but correlates with tau levels in the CSF of AD patients Because of the importance of Foxp3 expression in Treg function, which is sufficient to drive CD4+ T cells into the Treg linage (Hori et al., 2003), we assessed whether the amount of Foxp3 expression correlates with suppressive activity. Therefore, the relative amount of Foxp3 expressed in Treg was determined by flow cytometry for each donor at a single cell level. The mean fluorescence intensity for Foxp3 in CD4+ cells was compared in all four groups, but no differences were observed (Fig. 5A). Additionally, did the amount of Foxp3 neither correlate within the subgroups nor in all groups combined with suppressive activity (Fig. 5B). Since the increase in Treg suppression observed in our patient population suggests a relationship between suppression and neurodegeneration, we evaluated whether our observation has any direct relationship with clinical markers. Disease severity correlated with no Treg data but surprisingly, when the amount of Foxp3 expression from AD patients was compared with the amount of tau protein within the CSF, we found a moderate correlation (r2 = 0.34, p = 0.0025, Fig. 5C). This observation remains however difficult to interpret because of lacking comparable CSF data from the other groups.
4. Discussion The aim of this study was to quantitatively and qualitatively analyze peripheral Treg of young and elderly non-affected donors as well as AD and PD patients. By using Foxp3 which is the most specific marker for Treg our analysis differs from previously published data in which controversial results have been obtained when CD25 was used for quantitative analysis of Treg in elderly individuals (Gregg et al., 2005; Tsaknaridis et al., 2003), AD (Speciale et al., in press) or PD patients (Baba et al., 2005; Bas et al., 2001). Quantifying Treg by Foxp3 reveals that the frequency of Treg within the CD4+ pool increases with age, which is in line with data obtained in mice using Foxp3 as marker (Sharma et al., 2006). This is due to increasing Treg numbers and not a decrease of the CD4+ T cell number and shows that Treg undergo immunological senescence. Using CD25 expression as Treg marker mainly confirmed our Foxp3 data. However, differences in Treg frequency for the PD group with this two markers argues for the use of the most accurate Treg marker, which is Foxp3. Increased frequency of Treg may be due to clonal expansion of Treg or accumulation of memory type Treg because with advanced age the frequency of CD45RO+ Treg rises from 80% in the young group to 90% in the elderly. Hence, this accumulation goes in parallel to the previously published decline of so-called naïve Treg with age, which are Foxp3+ and classified by being CD4+CD25+CD45RA+ (Valmori et al., 2005). This increase of Treg frequency is not influenced by a neurodegenerative phenotype but may contribute to the observation of weaker immune responses in the elderly against infections such as influenza (Thompson et al., 2003). Evaluating Treg reactivity in a functional suppression assay showed that Treg activity increases with a neurodegenerative phenotype. Even though it is generally believed that aged T cells have a lower proliferating capacity, we observed no difference in the mean proliferation of sorted CD4+CD25− cells between the groups, which did not change when the proliferation of CD4+CD25− and CD4+CD25+ cells were combined (data not shown). This could be due to the stimulus used, as the extend of the alloreaction against the mixed feeder cells may vary among donors, but one should also consider that in cases of lower T cell proliferation in the elderly, only bulk CD4+ cells have been used and the aspect of increasing Treg number with age has not yet been considered. Taking further into account that the accuracy of AD/PD diagnosis is about 80% (Cummings, 2004; Hughes et al., 1992b) and without knowing whether some of the elderly control individuals with high Treg suppressive activity may develop AD/PD in the near future, we cannot rule out that the difference between the elderly affected and non-affected groups would in reality become significant. The fact that some young donors show high Treg suppressive activity does not disagree with this interpretation because this may represent a risk factor to develop disease. However, this would have to be confirmed by a larger longitudinal study. It is also to consider that we analyzed only the total pool of peripheral Treg. In the case of Treg specific for CNS-relevant antigens, the differences between the groups analyzed may be
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larger. In this context it is of interest to note that in Leishmaniainfected mice antigen-specific Treg act locally (Suffia et al., 2006). Consequently, it will be of special interest to analyze Treg specific for antigens relevant to AD or PD, such as Aβ or alpha-synuclein. That the observed effect of higher Treg activity is specific for Treg and not due to a change in APC function can be excluded because of the fact, that we used the same pool of irradiated feeder cells for all donors, consisting of a mix of four different donors (two elderly, two young individuals). Alternatively the effect could be explained by higher sensitivity of the CD4+CD25− cells to Treg. Even though we did not perform crossover experiments, others have done this under various conditions and the defect was always accounted to Treg (Viglietta et al., 2004; Wing et al., 2003). Furthermore, by the inclusion of an internal standard in the flow cytometry experiments as well as the realization that CD4+CD25+ cells contribute in suppression assays to background proliferation, we believe to have used a very sensitive experimental set-up. Nonetheless, our sort strategy allowed only the analysis of CD4+ CD25high Treg. This population may only partially include the naïve CD4+CD25+CD45RA+ Treg, which show a slightly lower CD25 expression. However, because no difference in suppressive activity has been demonstrated between naïve or standard Treg (Valmori et al., 2005), we would not expect that the full inclusion of this Treg population would change our results. Furthermore it has been shown that the CD4+CD25high Treg population can be subdivided into CD45RO− and CD45RO+ cells (Taams et al., 2001; Yagi et al., 2004). Only the CD45RO + population showed thereby suppressive activity (Yagi et al., 2004). Even though we normalized the amount of Treg to 7000 cells in our functional assays, we may speculate that due to the increase of CD45RO+ Treg with age, the proportion of suppressive CD4+CD25+ CD45RO+ Treg increases in our assay. This may explain the non-significant difference in suppressive Treg activity between the healthy young and elderly group. However, this does not explain the further increase for the AD/PD groups, because we observed no difference between the Foxp3+CD45RO+ population of elderly-controls versus AD and for PD this population was even slightly (but not significantly) smaller. In search for an explanation of our observed Treg differences, the most obvious candidate was Foxp3, because it has the highest impact on development and function of Treg. Hence, we speculated whether the amount of Foxp3 expressed in Treg could influence their suppressive activity. But in contrast to their suppressive activity, all groups showed similar Foxp3 expression and even though the expression of Foxp3 in mouse CD4+CD25− T cells seems to be sufficient to drive CD4+ T cells into a regulatory phenotype (Hori et al., 2003), the amount of Foxp3 in our experiments does not correlate with their suppressive function and shows that further experiments are required to understand why Treg from AD and PD patients are more active. The relevance of peripheral Treg changes for a CNS-based disease might be questioned, however one should consider that these T cells may be present within the CNS during neurodegeneration, since a modest T cell infiltration into the CNS has been demonstrated for AD (Itagaki et al., 1988; Togo et al., 2002) and
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PD patients (McGeer et al., 1987) as well as in animal models of AD (Stalder et al., 2005) and PD (Kurkowska-Jastrzebska et al., 1999). Even though this represents a rather minor T cell infiltration in comparison to classical autoimmune diseases like multiple sclerosis, a low number of Treg may contribute to a chain of events, which affect protein misfolding, and thus neurodegeneration. Especially in the case of localized events within the CNS, like the loss of dopaminergic neurons in PD, which results in reduced dopamine and may result in higher Treg function (Kipnis et al., 2004) or the induction of Treg by activated neurons (Liu et al., 2006). Additionally, other changes due to neurodegeneration, for instance elevated CSF tau levels may affect Treg function. The observation that Treg frequency increases with age and that Treg have a higher reactivity in the presence of a neurodegenerative phenotype leads to the question how Treg and neurodegeneration affect each other? Since other regulatory T cell subpopulations such as CD8+CD28− T cells are also affected by a neurodegenerative phenotype (Speciale et al., in press) it is possible that imbalanced immune regulation may affect protective immune mechanisms in neurodegenerative pathology. The influence of Treg in acute CNS-injury models has been shown (Kipnis et al., 2002) and Treg function may further influence beneficial effects of auto-reactive T cells (Moalem et al., 1999) or the auto-reactive antibody response (Du et al., 2001). Thus, changes in immune regulation may disturb the balance of beneficial clearance mechanisms and protein aggregation and thus indirectly increase the pathology mediated by protein aggregation. This is not contradicted by the fact that higher reactivity of Aβ-specific T cells exists in elderly and AD (Monsonego et al., 2003). An increase in reactivity tested by in vitro assays could be due to a higher frequency of Aβ-specific T cells, which may outnumber the effect of increasing Treg activity. Since the function of Aβ-specific T cells in vivo is not known, it will be interesting to see whether their increase and the increase of Treg have synergistic or antagonistic effects. Thus, it will be highly interesting to see whether a change in Treg activity affects any mechanism involved in clearance of protein aggregates in either AD or PD. Acknowledgments The authors wish to thank Cornelia Grimmel, Andreas Böhmler and Hans-Jörg Bühring for cell sorting, Birgitt Schönfisch for statistical advice and Michael Calhoun and Mathias Jucker for the critical reading of the manuscript. This work was supported by the FORTÜNE Program of the University of Tübingen. References Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., Finch, C.E., Frautschy, S., Griffin, W.S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I.R., McGeer, P.L., O'Banion, M.K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F.L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G., Wyss-Coray, T., 2000. Inflammation and Alzheimer's disease. Neurobiol. Aging 21, 383–421.
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