In this study, the effects of high dose vitamin D3 supplementation on safety and T cell parameters in patients with RRMS were assessed. We observed that supplementation of 20 000 IU/d vitamin D3 for 12 weeks was without negative side-effects and without decompensation of calcium metabolism (i.e. hypercalcaemia and hypercalciuria). The number of Treg remained unaffected, as well as the numbers of Treg memory and naïve subtypes. Results regarding Treg suppressive capacity were inconclusive. However, a shift was observed in the cytokine profiles of CD4+T cells, with an increased circulating proportion of IL-10+ CD4+ T cells, and a decreased ratio of Th1/Th2 cells (IFN-γ+/IL-4+ CD4+ T cell ratio). These results further add to the notion that vitamin D is a physiological immune modulator in vivo. Monitoring the cytokine profile of CD4+ T cells could be a valuable tool to assess the immunological efficacy of vitamin D3 in randomized controlled clinical trials on vitamin D supplementation in patients with MS.
This study confirmed that supplementation of high doses vitamin D3
does not induce a decompensation of calcium metabolism in patients with MS 
. Without a dose-escalation supplementation scheme, we did not detect hypercalcaemia or hypercalciuria. The slight non-significant increase in serum calcium and phosphorus most likely reflects a correction of vitamin D insufficiency in this cohort. A gradual increase in serum creatinine levels was observed during vitamin D3
supplementation, which however remained below 110 µmol/L in all patients. The dramatic complication of a hypercalcaemia associated decrease of glomerular filtration rate (GFR) is unlikely, since neither hypercalcaemia nor hypercalciuria was detected. Earlier dose-escalation trials supplementing up to 280 000 IU/w for 28 weeks and up to 40 000 IU for 52 weeks did not report either significant elevations of serum creatinine levels, or calcifications in the kidneys 
. Interestingly, however, earlier reports described a rise in serum creatinine which was not accompanied by a decreased GFR in patients treated with 1,25(OH)2
D or 1α(OH)D 
. Physiologically, 25(OH)D is filtrated in complex with vitamin D binding protein (DBP) by the glomeruli and re-absorbed by proximal tubular epithelial cells 
. Interestingly, creatinine is being secreted in the proximal renal tubuli as well. We postulate that a massive increase of 25(OH)D-DBP complex reabsorbtion competes with tubular creatinine excretion and hereby increases serum creatinine levels. Although we believe that the increased serum creatinine levels do not reflect a decreased GFR, future studies on massive dose vitamin D supplementation should be cautious towards patients with an impaired kidney function. On the whole, biochemical data show the absence of vitamin D3
toxicity in participants of this study.
We found no effect of vitamin D supplementation on the number of Treg in the circulation. In MS, the function rather than the number of Treg has been reported to be impaired 
, and to relate with serum 25(OH)D levels 
. Additionally, other immune modulating therapies also have been shown to improve Treg function rather than Treg numbers 
. An expansion of naïve Treg has been described in Glatiramer Acetate treatment 
, and a decrease of memory Treg cells in IFN-β treatment 
. Therefore, we also assessed these Treg subsets, but found no effect of vitamin D3
supplementation on the proportions of naïve and memory Treg.
Previously, we observed a correlation of Treg suppressive capacity with serum 25(OH)D levels 
. Although Treg suppressive function was improved in several patients after 12 weeks of vitamin D3
supplementation, this improvement was not statistically significant. The size of the cohort assessed is most likely too small to detect a possible effect in this complex assay. Alternatively, one could speculate from our previous work that only in the patients with the poorest vitamin D statuses, an improved suppression can be expected 
. Although vitamin D statuses at week 0 were lower in the 9 improving patients when compared to the 4 non-improving patients, the difference was not significant. Additionally, Treg suppressive function is not impaired in all RRMS patients, and shows a substantial overlap with healthy individuals 
. Therefore, it can be speculated that an adequate Treg function cannot be improved further on vitamin D3
supplementation. Indeed, the patients of whom Treg suppressive function improved tended to have a poorer suppression at week 0. Lastly, the genetic background of an individual may affect the immunological response to vitamin D3
. Altogether, the data collected in this study regarding Treg suppressive function remain inconclusive.
A significant effect on the composition of the T helper cell compartment was detected. The ratio of IFN-γ+
T cells represents the balance between pro-inflammatory Th1 and anti-inflammatory Th2 cells, and is regarded an important variable in autoimmune diseases 
. During supplementation, this ratio changed towards a less pro-inflammatory profile. The effect on absolute proportions of IFN-γ+
T cells may be too small to be detected in this small cohort. This is in accordance with previous cross-sectional findings 
. Interestingly, we detected also more IL-10+
T cells after supplementation. The source of IL10+
cells in the CD4+
T cell compartment may be the T regulatory cell type 1 (Tr1) 
. Alternatively, IL-10 has also been proposed to be a typical Th2 cytokine. However, we measured no double producers of IL-10 and the (other) typical Th2 cytokine IL-4 (data not shown). Therefore, we speculate that the rise in IL-10+
T cells might reflect expansion of inducible regulatory Tr1 cells. The finding of an altered profile of pro- and anti-inflammatory CD4+
T cells conforms to observations in experimental studies 
and further supports the assessment of vitamin D3
as a natural immune modulator in MS 
Although the present study did not comprise a placebo-group, it delivered valuable new information. We earlier performed a cross-sectional study, in which serum 25(OH)D levels correlated with peripheral T cell homeostasis 
. However, the cross-sectional design did not allow statements on causality, since physical exercise and UV exposure per se could be the real underlying mediators 
. Our current study revealed 25(OH)D as a good causal candidate. Ultimately, however, placebo controlled studies should demonstrate that vitamin D3
is an immune modulator in vivo
. Regarding these upcoming trials, it is also of interest that we included patients treated with immune modulating drugs (IFN-β). Interestingly, therapy with 1,25(OH)2
D showed synergistic effects with IFN-β in an EAE model of MS 
. Upcoming clinical trials will most likely assess add-on effects of vitamin D3
on current immune modulating drugs. Results from the present study suggest that it is worthwhile to combine immune-modulating drugs like IFN-β with vitamin D3
in order to further modulate the immune system in a for MS beneficial way.
In conclusion, we showed in a cohort of RRMS patients that supplementation of high doses vitamin D3 did not result in a decompensated calcium metabolism. Additionally, vitamin D3 appeared to skew the CD4+ T cell compartment to a more pronounced anti-inflammatory state. Herewith, we confirmed that the peripheral CD4+ T cell compartment cytokine profile is a potential outcome measure in upcoming randomized controlled clinical trials. Ultimately, these trials should demonstrate whether vitamin D3 is an immune and disease modulating compound in MS.