In several autoimmune diseases, including multiple sclerosis (MS), a compromised regulatory T cell (Treg) function is believed to be critically involved in the disease process. In vitro, the biologically active metabolite of vitamin D has been shown to promote Treg development. A poor vitamin D status has been linked with MS incidence and MS disease activity. In the present study, we assess a potential in vivo correlation between vitamin D status and Treg function in relapsing remitting MS (RRMS) patients.
Serum levels of 25-hydroxyvitamin D (25(OH)D) were measured in 29 RRMS patients. The number of circulating Tregs was assessed by flow-cytometry, and their functionality was tested in vitro in a CFSE-based proliferation suppression assay. Additionally, the intracellular cytokine profile of T helper cells was determined directly ex-vivo by flow-cytometry. Serum levels of 25(OH)D correlated positively with the ability of Tregs to suppress T cell proliferation (R = 0.590, P = 0.002). No correlation between 25(OH)D levels and the number of Tregs was found. The IFN-γ/IL-4 ratio (Th1/Th2-balance) was more directed towards IL-4 in patients with favourable 25(OH)D levels (R = −0.435, P = 0.023).
These results show an association of high 25(OH)D levels with an improved Treg function, and with skewing of the Th1/Th2 balance towards Th2. These findings suggest that vitamin D is an important promoter of T cell regulation in vivo in MS patients. It is tempting to speculate that our results may not only hold for MS, but also for other autoimmune diseases. Future intervention studies will show whether modulation of vitamin D status results in modulation of the T cell response and subsequent amelioration of disease activity.
Citation: Smolders J, Thewissen M, Peelen E, Menheere P, Cohen Tervaert JW, et al. (2009) Vitamin D Status Is Positively Correlated with Regulatory T Cell Function in Patients with Multiple Sclerosis. PLoS ONE 4(8): e6635. doi:10.1371/journal.pone.0006635
Editor: Derya Unutmaz, New York University School of Medicine, United States of America
Received: June 18, 2009; Accepted: July 16, 2009; Published: August 13, 2009
Copyright: © 2009 Smolders et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Multiple Sclerosis (MS) is a disabling, chronic inflammatory disease of the central nervous system (CNS). Although some patients experience progression of disability from the first symptoms onwards, the disease is at its onset mostly characterised by repetitive sub-acute deteriorations of symptoms with remissions afterwards, i.e. relapsing remitting MS (RRMS). After several years, sustained and progressive disability often occurs and the disease becomes secondary progressive .
Although the pathophysiological process in MS is not yet fully elucidated, T cell mediated inflammation within the CNS plays an important role. Auto-reactive T cells move through the blood brain barrier, and contribute to local inflammation of the CNS . Historically, these auto-reactive T cells were believed to comprise predominantly interferon gamma (IFN-γ) producing T helper 1 (Th1) cells. More recently interleukin-17 (IL-17) producing Th17 cells have been argued to be important pathogenic T cells in MS , . In a healthy T cell compartment, regulatory T cells (Treg) regulate the quantity and quality of the immune response, and prevent auto-reactive T cell responses, and hereby autoimmune diseases . Although the number of Tregs is not decreased, their capability to suppress polyclonal or antigen-specific proliferation of T cells in vitro has been shown to be compromised in MS patients –.
Experimental studies showed that the biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is able to skew the T cell compartment into a more anti-inflammatory and regulated state, with inhibition of Th1 and Th17 cells and promotion of Th2 and Treg cells , . Interestingly, in the experimental autoimmune encephalomyelitis (EAE) animal model of MS, treatment with vitamin D prevented, and even cured, the disease . These findings suggest a potential role for vitamin D in MS, as has been investigated in several epidemiological studies. Near the equator, where vitamin D photosynthesis is optimal, MS incidence is low . Furthermore, increased sun exposition and a good vitamin D status in childhood and adolescence, reflected by the serum values of 25-hydroxyvitamin D (25(OH)D) , have been associated with a decreased risk for developing MS –. An increased activity and disability of MS has also been associated with a poor vitamin D status –. Since vitamin D modulates the immune response in vitro and in animal models, it is highly interesting to assess whether this modulation can also be found in vivo in MS patients. This could be, beside regulation of the transcription of MS-associated alleles , an important driver of the associations between vitamin D status and MS. At present, however, no published studies assessed the potential relationship between serum levels of 25(OH)D and the T cell compartment in MS.
In the present study, we assess whether vitamin D status is correlated with the in vivo composition and function of the T cell compartment in MS patients. Since in RRMS patients with a short disease duration disease pathophysiology has been argued to be predominantly inflammatory  and Treg function has been shown to be particularly compromised , we assess a potential anti-inflammatory and in particular Treg-promoting effect of vitamin D, as being related to vitamin D status, in these patients.
Materials and Methods
We included in this study 29 Caucasian subjects, by approaching consecutive patients matching the inclusion criteria, who visited the outpatient clinic of our MS centre. All subjects had RRMS according to the revised McDonald criteria , with a disease duration of <5 years since the first to MS attributable symptoms. Both patients treated with interferon beta 1a or 1b, and patients untreated with immune modulation were included. Exclusion criteria were: no clinically definite phenotype, use of other immune modulation than interferon beta 1a or 1b, occurrence of a relapse <6 weeks before blood collection, recent start (<1/2 year) of vitamin D supplementation. The characteristics of the patients are described in Table 1.
Table 1. Patient characteristics.doi:10.1371/journal.pone.0006635.t001
Written informed consent was acquired from all subjects participating in this study, according to the declaration of Helsinki. The study was reviewed and approved by both the regional ethical committee on human research ‘Atrium-Orbis-Zuyd’, and the institutional ethical committee ‘Local Advisory board on Scientific Research’.
Vitamin D assays
Blood withdrawal was performed in the period from September 2008 until February 2009. The cells for the cellular assays and the serum for vitamin D measurement were retrieved at the same time, and cellular assays were performed on the day of blood collection. The collected serum was immediately shielded from direct light and stored at −20°C. At the end of the study, all samples were analysed simultaneously. The serum values of 25(OH)D were measured with a commercially available radioimmuno assay (Immunodiagnostic Systems, Boldon, UK) .
Cell culture reagents
Cells were cultured in RPMI glutamax medium (Gibco Invitrogen, Breda, The Netherlands) supplemented with 10% Foetal Calf Serum (Greiner Bio-One, Alphen a/d Rijn, The Netherlands), 1% non-essential amino acids (Gibco Invitrogen), 1% sodium pyruvate (Gibco Invitrogen) and 2% penicillin-streptomycin (Gibco Invitrogen) in U-bottom 96-wells plates.
Cell isolation was performed as described before, with minor modifications . PBMC were isolated by Ficoll gradient centrifugation (Histopaque; Sigma Aldrich, Zwijndrecht, The Netherlands). CD4+ T cells were selectively isolated with RosetteSep (Stem Cell Technologies, Grenoble, France). Approximately 20–30×106 CD4+ T cells were incubated at 4°C for 30 minutes with anti-CD4-APC (BD Biosciences, Breda, The Netherlands), anti-CD25-PE (BD Biosciences) and anti-CD127-FITC (BD Biosciences). Human CD4+CD25+CD127− Tregs ,  and CD4+CD25− responder T cells (Tresps) were sorted on a FacsAriaTM (BD Biosciences) cell sorter (Figure 1A). The mean purity of the sorted populations was 97.51% (SD 0.81) for the Tregs and 97.45% (SD 1.65) for the Tresps. Accessory cells were obtained by irradiating autologous PBMC with 66.2 gray.
Figure 1. Results proliferation suppression assay.
(A) Representative dot-plot of the sorting protocol. CD25+CD127− cells within the CD4+ lymphogate were sorted as Tregs, CD25− cells were sorted as Tresps. (B) The percentages of suppression which were achieved for the respective Treg/Tresp ratios of the individual patients are shown. The Treg/Tresp ratio at which 50% inhibition of proliferation was achieved is defined as ED50 of suppression. The distribution of ED50 is shown in the rightmost column. The lines show the median values. The closed dots represent the Beta Interferon-treated patients, the open dots the untreated patients.doi:10.1371/journal.pone.0006635.g001
The CFSE-based proliferation suppression assay was adapted from Venken et al. . In short, the freshly isolated CD4+CD25− Tresps were labelled with carboxy fluorescein diacetate succinimidyl ester (CFSE; Molecular Probes Invitrogen, Breda, The Netherlands). In a U-bottom 96-wells plate, 2×105 Tresps were stimulated with soluble anti-CD3 (2.0 pg/mL, WT32 IgG2a monoclonal antibody, kindly provided by dr. W Tax, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) in the presence of 2×105 irradiated accessory cells. Tresps were co-cultured for 5 days with varying amounts of Tregs (Treg/Tresp ratios 0/1, 0.25/1, 0.5/1, 1/1 or 2/1). All conditions were performed in triplo in a final volume of 200 µL. After culture, the cells were stained with anti-CD4-APC and 7AAD (BD Biosciences), and the CFSE signal of cells in the CD4+7AAD− lymphogate was analysed by Flow Cytometry on a FacsCaliburTM flowcytometer (BD Biosciences). Data were analysed with CellQuest software (BD Biosciences). The amount of proliferation achieved in the 0/1 ratio was set at 0% suppression. The mean relative amount of suppression in the other ratios was calculated. By linear interpolation, the Treg/Tresp ratio at which 50% suppression of proliferation was achieved (ED50) was calculated.
T cell phenotyping
Tregs were defined as CD25+FoxP3+ CD4+ T cells ,  and as CD25+CD127− CD4+T cells , . For determining the proportion of Tregs, PBMC were stained with anti-CD3-PerCP (BD Biosciences), anti-CD4-APC, anti-CD4-FITC (BD Biosciences), anti-CD25-PE, anti-CD127-FITC or anti-FoxP3-APC (E-Bioscience, San Diego, USA), and analysed on a FacsCaliburTM flowcytometer . The absolute number of lymphocytes was determined with a haematological cell counter (Beckman Coulter, Woerden, The Netherlands).
T helper cell subsets were determined by assessing the intracellular cytokine pattern of CD3+CD8− lymphocytes, which are further referred to as CD4+ T cells, by flowcytometry . PBMC were stimulated for 5 hours with calcium ionomycine (Sigma Aldrich), phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich) and monensin (BD Biosciences). Cells were stained intra-cellularly with anti-IL-4-PE (BD Biosciences), anti-IFN-γ-FITC (BD Biosciences), anti-IL-17-PE (E-Bioscience) and anti-IL-10-PE (E-Bioscience), and extra-cellularly with anti-CD3-PerCP and anti-CD8-APC.
Statistical analysis was conducted with Statistical Package for Social Sciences version 15.0 software (SPSS inc., Chicago IL, USA) and figures were constructed with GraphPad Prism 5 software (GraphPad Software inc., La Jolla CA, USA). Of continuous variables, the median value and corresponding range (min–max) are provided. When normally distributed (Shapiro-Wilk test P>0.05), a linear relationship between two continuous variables was tested with the Pearson correlation coefficient (Pearson R). Abnormally distributed variables were Log-transformed. In case of persistent abnormal distribution, the Spearman correlation coefficient (Spearman R) was determined. A P value<0.05 was considered statistically significant.
Serum 25(OH)D levels
We determined the serum levels of 25(OH)D, the vitamin D metabolite which is accepted to reflect vitamin D status best . The median serum 25(OH)D level was 54 nmol/L (19–133).
Suppressive capacity of Tregs correlates positively with 25(OH)D levels
To evaluate Treg suppressive capacity, we conducted an in vitro CFSE-based proliferation suppression assay (Figure 1B). Of 4 patients, not enough cells were retrieved to start the assay. In all other individual patients, Tregs suppressed proliferation of T responder cells in a dose-dependent manner. There was no apparent difference in Treg suppression between Beta Interferon treated and untreated patients (Figure 1B). The median Treg/Tresp ratio at 50% suppression of proliferation (ED50) was 0.82 (0.16 - 1.96). Serum 25(OH)D levels correlated negatively with ED50 (Spearman R = −0.590, P = 0.002) (Figure 2A). This correlation was reflected by the positive correlation between vitamin D status and the amount of suppression in the individual Treg/Tresp ratios (Figure 2B–E).
Figure 2. Correlation between Treg suppressive function and serum 25(OH)D levels.
(A) The correlation between ED50 and serum 25(OH)D levels. (B–E) Correlation between inhibition of suppression and 25(OH)D levels for the individual Treg/Tresp ratios. The Spearman correlation coefficient is shown. The regression line illustrates the direction of the association. The closed dots represent the Beta Interferon-treated patients, the open dots the untreated patients.doi:10.1371/journal.pone.0006635.g002
Number of circulating Tregs does not correlate with 25(OH)D levels
Next, we evaluated the absolute and relative number of Tregs in the circulation (Figure 3). Regulatory cells within the CD4+ T cell compartment were defined as CD25+CD127− and CD25+FoxP3+ cells. The median percentage of CD25+FoxP3+ Tregs in the CD4+ T cell compartment was 5.63% (2.67–15.81) (absolute number 9.61×107 c/L (4.65–25.41)). The median percentage of CD25+CD127− Tregs was 6.56% (2.93–12.32) (absolute 10.32×107 c/L (4.44–24.14)). A strong positive correlation was observed between the numbers of CD25+CD27− and CD25+FoxP3+ CD4+Tregs (R = 0.826, P<0.001). Serum levels of 25(OH)D were not correlated with the relative and absolute number of Tregs (Figure 4).
Figure 3. Relative and absolute number of circulating Tregs.
Within the CD3+CD4+ lymphogate, Tregs were defined as either being (A) CD25+FoxP3+ or being (B) CD25+CD127−. (C) The percentages of Tregs within the CD4+ T cell compartment. (D) Absolute number of circulating Tregs. The lines indicate the median values.doi:10.1371/journal.pone.0006635.g003
Figure 4. Correlation of the number of circulating Tregs with serum 25(OH)D levels.
(A–D) Correlation of relative and absolute number of Tregs with 25(OH)D levels. The Pearson correlation coefficient and corresponding regression line are shown.doi:10.1371/journal.pone.0006635.g004
The Th1/Th2 balance correlates negatively with 25(OH)D levels
CD4+ T cells were classified as Th1, Th2 or Th17 by assessing their intracellular cytokine profile (IFN- γ, IL-4 and IL-17, resp.) (Figure 5A). We also assessed the percentage of CD4+ T cells producing the regulatory cytokine IL-10 . The median percentage of IFN-γ+ cells within the CD4+ T cell compartment was 12.11% (2.60 - 26.18), of IL-4+ cells 2.07% (0.90–4.93), of IL-17+ cells 0.92% (0.31–2.35), and of IL-10+ cells 0.83% (0.43–1.45). No significant correlations between serum 25(OH)D levels and the individual percentages of T helper cell subsets were found, although there was a trend towards a negative correlation with the percentage of IFN-γ+CD4+ T cells (Pearson R = −0.336 P = 0.060). The balance between Th1 and Th2 is used to indicate a more pro- or anti-inflammatory composition of the T cell compartment . This balance is described by the ratio of IFN-γ+ and IL-4+ CD4+ T cells. The IFN-γ/IL-4 ratio correlated negatively with serum 25(OH)D levels (Pearson R = −0.435 P = 0.023) (Figure 5B).
Figure 5. Correlation of T helper cell subsets with serum 25(OH)D levels.
(A) The percentages of IFN-γ+, IL-4+, IL-17+ and IL-10+ CD4+ T cells. The lines show the median values. (B) The correlation between IFN-γ/IL-4 ratio and serum 25(OH)D levels. The Pearson correlation coefficient and corresponding regression line are shown.doi:10.1371/journal.pone.0006635.g005
No association between MS disease activity and vitamin D status or T cell parameters
Neither vitamin D status, nor T cell parameters as Treg function or T cell phenotype did correlate with measures of MS disease activity as described in table 1 (data not shown).
Vitamin D status has been linked with MS either in large epidemiological studies or in experimental in vitro and animal studies. This is the first study to investigate the correlation between vitamin D status and the composition of the T cell compartment and in particular Treg function in vivo in MS patients. Our data shows that serum 25(OH)D levels correlate positively with the ability of CD4+CD25+CD127− Tregs to suppress proliferation of Tresp cells. The absolute and relative Treg numbers in the circulation were not correlated with vitamin D status. Although we did not observe a significant association between 25(OH)D levels and the individual percentages of CD4+ T helper cell subsets, the decreasing IFN-γ/IL-4 ratio reflected a skewing of the Th1/Th2-balance towards a more Th2 phenotype in patients with higher serum 25(OH)D levels. Altogether, this data indicates that high serum 25(OH)D levels are associated with a less inflammatory and better regulated T cell compartment in MS.
In our data, we found no association between vitamin D status and disease severity and activity of MS. This is in contrast with earlier observations –. However, since we measured patients in the period from September until February, the seasonal fluctuation of vitamin D status is likely to confound these associations . In contrast to the MS activity variables, this seems not critical for the correlation with the T cell tests: the data obtained reflects the status of the T cell compartment at the moment of blood collection. Hereby, several studies have shown a seasonal fluctuation of immunological parameters in MS patients comparable to the fluctuation of serum 25(OH)D levels , .
In a previous study, CD4+CD25high Tregs of RRMS patients achieved only about 40% suppression in a 1:1 Treg/Tresp ratio as measured in a 3H-Thymidine assay . A later study confirmed a mean suppression of 40% in untreated RRMS patients, and of 50% in Beta interferon users . Our RRMS patients reached in the 1:1 ratio a mean suppression of 60% (20–90). Beta Interferon treatment has been shown to promote Treg function . Therefore, the fact that most of our patients were treated with Beta Interferon might have contributed to this substantially more favourable suppression. Additionally, we included the α-chain of the IL-7 receptor (CD127), which has been shown to be specifically absent on CD4+CD25+FoxP3+ Tregs, in our sorting protocol to define Tregs , . Therefore, the sorted population of CD4+CD25+CD127− Tregs may comprise a more pure Treg fraction, compared to CD4+CD25high sorted T cells alone .
Our data shows that in RRMS patients with a short disease duration, in which Treg function has been shown to be particularly compromised , Treg suppressor function correlates positively with serum 25(OH)D levels. The relationship between Treg function and 25(OH)D levels is likely to contribute to the inter-individual variation of Treg suppressive function in MS cohorts in other studies –. However, we could not find an association of serum vitamin D values with the number of CD4+CD25+FoxP3+ or CD4+CD25+CD127− Tregs in the circulation. This discrepancy between the impaired number and function of Tregs has been shown in other studies on Tregs in MS and other autoimmune diseases . However, the underlying mechanism is not know. Interestingly, a recent publication showed a negative correlation between the percentage of FoxP3+ Tregs within the T cell compartment of MS patients and their vitamin D status . However, the heterogeneous study population considering race and disease duration, and the use of linear regression models to analyse non-equally distributed parameters in a limited sample size, require careful interpretation of these results. The discrepancy with our data not showing a comparable correlation, might be attributable to these methodological differences.
Additionally, we found a negative correlation between serum 25(OH)D levels and the IFN-γ/IL-4 ratio. This ratio is commonly used to describe the balance in the immune system between pro-inflammatory IFN-γ+ Th1 cells and anti-inflammatory IL-4+ Th2 cells . Our data suggests that, in patients with relatively high 25(OH)D-levels, the balance between Th1 and Th2 is more skewed in the direction of Th2. Interestingly, in vitro research also suggested that vitamin D skews the T cell compartment from a Th1 towards a Th2 phenotype , . Therefore, high 25(OH)D D levels appear to be associated with a less-pro-inflammatory T-cell compartment in MS patients.
In conclusion, we have shown that serum 25(OH)D levels are associated with the suppressive function of Tregs and the composition of the T cell compartment in MS patients. These findings further support the hypothesis that vitamin D is an important promoter of T cell regulation in MS. Since in RRMS patients the Treg compartment has been shown to be compromised, our findings provide additional arguments to maintain a healthy vitamin D status in these patients. Also other autoimmune diseases have been associated with both low vitamin D levels, and a reduced regulatory T cell function , . Whether a poor vitamin D status correlates in healthy populations with a reduced Treg function and potentially a subsequently increased risk for developing autoimmune diseases, remains to be established. Altogether, it is tempting to speculate about the potential therapeutic effect of vitamin D supplementation in autoimmune diseases. Previously, it was demonstrated that therapy with Beta Interferon in MS results in a promotion of regulatory T cell function  and a decrease of relapse rate . Since we observed an additional effect of 25(OH)D levels in Beta Interferon users, this might provide a rationale for add-on therapy with vitamin D during Beta Interferon therapy in MS. Randomized clinical trials should assess an additional role of vitamin D treatment on the T cell compartment and more importantly on disease activity of MS.
The authors would like to thank MS-nurses Tiny Kempkens, Judith Poeth and Ingrid Mevissen for their help with patient inclusion and blood collection, and Jozien Jaspers-Spits and Dennis Meesters for their skilled technical assistance with the laboratory procedures.
Conceived and designed the experiments: JS MT PM JWCT JD RH. Performed the experiments: JS MT EP. Analyzed the data: JS MT. Contributed reagents/materials/analysis tools: PM JWCT JD RH. Wrote the paper: JS MT PM JWCT JD RH.
- 1. Compston A, Coles A (2008) Multiple Sclerosis. Lancet 372: 1502–1517.
- 2. Bar-Or A (2008) The immunology of multiple sclerosis. Semin Neurol 28: 29–45.
- 3. Kebir H, Kreymborg K, Ifergan I, et al. (2007) Human Th17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat Med 13: 1173–1175.
- 4. Steinman L (2007) A brief history of T(h)17, the first major revision in the T(h)1/T(h)2 hypothesis of T cell-mediated tissue damage. Nat Med 13: 139–145.
- 5. Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133: 775–787.
- 6. Haas J, Hug A, Viehöver A, et al. (2005) Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis. Eur J Immunol 35: 3343–3352.
- 7. Venken K, Hellings N, Hensen K, et al. (2006) Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression. J Neurosci Res 83: 1432–1446.
- 8. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA (2004) Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 199: 971–979.
- 9. Correale J, Ysrraelit MC, Gaitán MI (2009) Immunomodulatory effects of vitamin D in multiple sclerosis. Brain 132: 1146–1160.
- 10. Smolders J, Damoiseaux J, Menheere P, Hupperts R (2008) Vitamin D as an immune modulator in multiple sclerosis, a review. J Neuroimmunol 194: 7–17.
- 11. Cantorna MT, Hayes CE, DeLuca HF (1996) 1,25-dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc Natl Acad Sci U S A 93: 7861–7864.
- 12. Kurtzke JF (1967) On the fine structure of the distribution of multiple sclerosis. Acta Neurol Scand 43: 257–282.
- 13. Hollis BW (1996) Assessment of vitamin D nutritional and hormonal status: what to measure and how to do it. Calcif Tissue Int 58: 4–5.
- 14. Islam T, Gauderman WJ, Cozen W, Mack TM (2007) Childhood sun exposure influences risk of multiple sclerosis in monozygotic twins. Neurology 69: 381–388.
- 15. Kampman MT, Wilsgaard T, Mellgren SI (2007) Outdoor activities and diet in childhood and adolescence relate to MS risk above the Arctic circle. J Neurol 254: 471–477.
- 16. Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A (2006) Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA 296: 2832–2838.
- 17. Kragt J, van Amerongen B, Killestein J, et al. (2009) Higher levels of 25-hydroxyvitamin D are associated with a lower incidence of multiple sclerosis only in women. Mult Scler 15: 9–15.
- 18. Smolders J, Menheere P, Kessels A, Damoiseaux J, Hupperts R (2008) Association of vitamin D metabolite levels with relapse rate and disability in multiple sclerosis. Mult Scler 14: 1220–1224.
- 19. Van der Mei IA, Ponsonby AL, Dwyer T, et al. (2007) Vitamin D levels in people with multiple sclerosis and community controls in Tasmania, Australia. J Neurol 254: 581–590.
- 20. Ramagopalan SV, Maugeri NJ, Handunnetthi L, et al. (2009) Expression of the multiple sclerosis-associated MHC class II allele HLA-DRB1*1501 is regulated by vitamin D. PLoS Genet 5: e1000369.
- 21. Polman CH, Reingold SC, Edan G, et al. (2005) Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria”. Ann Neurol 58: 840–846.
- 22. Venken K, Thewissen M, Hellings N, et al. (2007) A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses. J Immunol Methods 322: 1–11.
- 23. Liu W, Putnam AL, Xu-Yu Z, et al. (2006) CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ Treg cells. J Exp Med 203: 1701–1711.
- 24. Seddiki N, Santner-Nanan B, Martinson J, et al. (2006) Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 203: 1693–1700.
- 25. Fontenot JD, Gavin MA, Rudensky AY (2003) FoxP3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4: 330–336.
- 26. Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by transcription factor FoxP3. Science 299: 1057–1061.
- 27. Ordonez L, Bernard I, L'faqihi-Olive FE, et al. (2009) CD45RC isoform expression identifies functionally distinct T cell subsets differentially distributed between healthy individuals and AAV patients. PLoS ONE 4: e5287.
- 28. Kimlin MG (2008) Geographic location and vitamin D synthesis. Mol Aspects Med 29: 453–461.
- 29. Stewart N, Taylor B, Ponsonby AL, et al. (2007) The effect of season on cytokine expression in multiple sclerosis and healthy subjects. J Neuroimmunol 188: 181–186.
- 30. Balashov KE, Olek MJ, Smith DR, Khoury SJ, Weiner HL (1998) Seasonal variation of interferon-gamma production in progressive multiple sclerosis. Ann Neurol 44: 824–828.
- 31. Venken K, Hellings N, Thewissen M, et al. (2008) Compromised CD4+CD25high regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FoxP3-positive cells and reduced FoxP3-expression at the single-cell level. Immunology 123: 79–89.
- 32. Michel L, Berthelot L, Pettré S, et al. (2008) Patients with relapsing-remitting multiple sclerosis have normal Treg function when cells expressing IL-7 receptor alpha-chain are excluded from the analysis. J Clin Invest 118: 3411–3419.
- 33. Costantino CM, Baecher-Allan CM, Hafler DA (2008) Human regulatory T cells and autoimmunity. Eur J Immunol 38: 921–924.
- 34. Royal W3rd, Mia Y, Li H, Naunton K (2009) Peripheral blood regulatory T cell measurements correlate with serum vitamin D levels in patients with multiple sclerosis. J Neuroimmunol. doi:10.1016/j.jneuroim.2009.05.012.
- 35. Arnson Y, Amital H, Shoenfeld Y (2007) Vitamin D and autoimmunity: new aetiological and therapeutic considerations. Ann Rheum Dis 66: 1137–1142.
- 36. (1995) Interferon Beta 1b in the treatment of multiple sclerosis: final outcome of the randomised controlled trial. The IFNB multiple sclerosis study group and the University of British Columbia MS/MRI analysis group. Neurology 45: 1277–1285.