ORIGINAL

ARTICLE

Vitamin D Supplementation Modulates T Cell–Mediated Immunity in Humans: Results from a Randomized Control Trial Gauree Gupta Konijeti, Pankaj Arora, Matthew R. Boylan, Yanna Song, Shi Huang, Frank Harrell, Christopher Newton-Cheh, Dillon O’Neill, Joshua Korzenik, Thomas J. Wang, and Andrew T. Chan Division of Gastroenterology (G.G.K.), Scripps Clinic, La Jolla, California; Scripps Translational Science Institute (G.G.K.), La Jolla, California 92037; Division of Gastroenterology (G.G.K., M.R.B., A.T.C.), Massachusetts General Hospital, Boston, Massachusetts; Division of Cardiology (P.A.), University of Alabama, Birmingham, Alabama 35210; Department of Biostatistics (Y.S., S.H., F.H., D.O., T.J.W.), Vanderbilt University, Nashville, Tennessee 37232; Division of Cardiology (T.J.W.), Vanderbilt University, Nashville, Tennessee 37232; Division of Cardiology (C.N.-C.), Massachusetts General Hospital, Boston, Massachusetts; Division of Gastroenterology (J.K.), Brigham and Women’s Hospital, Boston, Massachusetts 02115; and Clinical and Translational Epidemiology Unit (A.T.C.), Massachusetts General Hospital, Boston, Massachusetts 02114

Context: Although studies have linked vitamin D deficiency with immune-mediated diseases, data demonstrating a direct effect on T-cell function are sparse. Objective: Our objective was to determine whether oral vitamin D3 influences T-cell activation in humans with vitamin D deficiency. Design: This was a single-center ancillary study within Vitamin D Therapy in Individuals at High Risk of Hypertension, a double-blind, multicenter, randomized controlled trial. Setting: This study was undertaken in a single academic medical center. Participants: Adults with vitamin D deficiency and untreated pre- or early stage I hypertension were included. Intervention: In Vitamin D Therapy in Individuals at High Risk of Hypertension, participants were randomized to either low- (400 IU daily) or high- (4000 IU daily) dose oral vitamin D3 for 6 months. In this ancillary study of 38 patients, we measured CD4⫹ T-cell activation estimated by intracellular ATP release after stimulation of whole blood with plant lectin phytohemagglutinin collected at baseline (pretreatment) and 2-month follow-up. Main Outcome Measure: Determining whether ATP level changes were significantly different between treatment groups was the main outcome measure. Results: Treatment with 4000 IU of vitamin D3 decreased intracellular CD4⫹ ATP release by 95.5 ng/ml (interquartile range, ⫺219.5 to 105.8). In contrast, 400 IU of vitamin D3 decreased intracellular CD4⫹ ATP release by 0.5 ng/ml (interquartile range, ⫺69.2 to 148.5). In a proportional odds model, high-dose vitamin D3 was more likely than low-dose vitamin D3 to decrease CD4⫹ ATP release (odds ratio, 3.43; 95% confidence interval, 1.06 –1.11). Conclusions: In this ancillary study of a randomized controlled trial, we found that high-dose vitamin D3 significantly reduced CD4⫹ T-cell activation compared to low-dose vitamin D3, providing human evidence that vitamin D can influence cell-mediated immunity. (J Clin Endocrinol Metab 101: 533–538, 2016) ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2016 by the Endocrine Society Received October 2, 2015. Accepted December 8, 2015. First Published Online December 14, 2015

doi: 10.1210/jc.2015-3599

Abbreviations: 25(OH)D, 25-hydroxyvitamin D; CI, confidence interval; CMI, cell-mediated immunity; DAYLIGHT, Vitamin D Therapy in Individuals at High Risk of Hypertension; IBD, inflammatory bowel disease; IFN, interferon; IQR, interquartile range; MS, multiple sclerosis; OR, odds ratio; PHA, phytohemagglutinin; RCT, randomized controlled trial; Th1, T helper 1; Th2; T helper 2.

J Clin Endocrinol Metab, February 2016, 101(2):533–538

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nvironmental determinants, particularly those that impact the immune system, appear to play a critical role in the pathogenesis and course of chronic immunemediated disorders. Vitamin D, which has recognized immunomodulatory roles, is emerging as a potentially important determinant. In vitro and in vivo animal studies have demonstrated that vitamin D affects differentiation of immune cells and modulates immune responses, which play an important role in the development and course of chronic immune-mediated disorders, such as type I diabetes, Hashimoto’s thyroiditis, inflammatory bowel disease (IBD), and multiple sclerosis (MS) (1–5). Studies have demonstrated that vitamin D deficiency is common among patients with autoimmune endocrine conditions such as Hashimoto’s thyroiditis and type I diabetes. In animal models, vitamin D appears to modulate thyroid function as well as inflammatory cytokines that typically result in immune-mediated thyroid destruction in Hashimoto’s thyroiditis (5). In type I diabetes, ␤-cell destruction is mediated by T cells, and the addition of vitamin D to insulin therapy among patients with newonset type I diabetes mellitus has been associated with a slower decline of residual ␤-cell function compared with insulin alone (6, 7). Several studies also demonstrate the association of vitamin D with inflammatory autoimmune conditions such as IBD and MS. Although this may be a consequence of disease activity, we have previously shown that low levels of vitamin D are also associated with a risk of incident IBD (1). Among patients with IBD, higher circulating levels of vitamin D are inversely associated with intestinal inflammation, as determined by measurement of fecal calprotectin (8). Further, vitamin D deficiency has been associated with clinically important outcomes, such as surgery, risk of colorectal cancer, and development of Clostridium difficile infection among IBD patients (9 –11). Likewise, vitamin D deficiency is associated with an increased risk of MS, and supplementation may influence clinical and brain magnetic resonance imaging activity in patients with established MS (12, 13). Despite these compelling animal-based and observational studies, prospective human data demonstrating a direct effect of vitamin D supplementation on immune cell function are limited. In a pilot study of four healthy individuals without IBD, Allen et al (14) showed that 15 weeks of high-dose vitamin D3 supplementation (5000 –10 000 IU daily) increased IL-10 production by non-CD4⫹ nonCD8⫹ T cells and reduced frequency of IL-17–producing CD4⫹ T cells (Th17) cells. An open-label, 12-month, randomized controlled trial (RCT) of patients with MS found abnormal T-cell reactions were suppressed in vivo by cholecalciferol at a serum 25-hydroxyvitamin D (25(OH)D)

E

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level greater than 100 nmol/L (15). In an RCT of 59 healthy adults (16), oral vitamin D3 supplementation (140,000 IU monthly) significantly increased the mean percentage of Tregs (CD4⫹, CD25-high, FoxP3⫹, and CD127-dim) cells by approximately 1.5%. To extend these data, we sought to determine whether oral vitamin D3 supplementation influences T-cell activation in individuals with vitamin D deficiency enrolled in a RCT (17).

Materials and Methods This study was an ancillary study of the Vitamin D Therapy in Individuals at High Risk of Hypertension (DAYLIGHT) trial (ClinicalTrials.gov Identifier: NCT01240512), a multicenter, double-blind, RCT conducted between December 2010 and September 2013 that evaluated the effect of vitamin D on blood pressure among 534 men and women ages 18 –50 years old with 25(OH)D lower than 25 ng/mL and untreated pre- or stage I hypertension (17). Participants were randomized to receive either low-dose (400 IU daily) or high-dose (4000 IU daily) oral vitamin D3 for 6 months. Follow-up visits were conducted every 2 months in the primary trial. Participants for the primary DAYLIGHT trial were recruited from three different geographical locations: Massachusetts, Connecticut, and Minnesota. For this substudy, we only enrolled participants from Massachusetts General Hospital in Boston, MA. Recruitment was not restricted by season, and recruitment was performed throughout the year. Potential participants were excluded from DAYLIGHT for any of the following reasons: use of an antihypertensive medication within the preceding 3 months; vitamin D supplementation (defined as vitamin D found in a multivitamin or supplement totaling ⬎400 IU/d) within the 3 months before enrollment; known cardiovascular disease (defined as prior myocardial infarction, percutaneous transluminal coronary angioplasty, coronary artery bypass, or stroke). Other exclusion criteria included subjects with history of ulcerative colitis, Crohn’s disease, celiac disease, colostomy, pancreatic enzyme deficiency, short bowel syndrome, gastric bypass, cystic fibrosis, or dumping syndrome (17). Among participants enrolled at a single center (Massachusetts General Hospital), we randomly selected a subset of 38 men and women and measured T-cell function in whole blood collected at baseline (pretreatment) and at 2 months’ follow-up using the ImmuKnow assay (ViraCor-IBT Laboratories), which quantifies intracellular CD4⫹ T-cell ATP as a measure of lymphocyte activity. ImmuKnow kits were provided by Cylex, now part of Viracor-IBT Laboratories. This assay is based on stimulation of freshly collected whole blood with plant lectin phytohemagglutinin (PHA), after which CD4⫹ T cells are identified using a magnet to select for magnetic particles coated with antihuman CD4 monoclonal antibodies. CD4⫹ T cells are then lysed to release intracellular ATP, which is measured using luciferin/luciferase and a luminometer. For statistical analyses, we compared proportions between treatment groups using the Wilcoxon signed-rank test and categorical variables using Pearson’s ␹2 test. We constructed a proportional odds model with follow-up ATP value as the dependent variable, with baseline ATP value and a dose group

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doi: 10.1210/jc.2015-3599

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Table 1. Baseline Characteristics, According to Daily Oral Vitamin D3 Dose Assignment

Female (%) Age (y), mean (SD) Race White (%) Black (%) Other (%) Baseline 25(OH)D (ng/ml), mean (SD) Days on vitamin D3, mean (SD)

Low-Dose Vitamin D3 400 IU/d (n ⴝ 18)

High-Dose Vitamin D3 4000 IU/d (n ⴝ 20)

5 (28%) 39.0 (9.7)

4 (20%) 43.9 (5.6)

5 (27.8%) 13 (72.2%) 0 (0%) 15.7 (5.1)

3 (15%) 16 (80%) 1 (5%) 16.7 (8.5)

.78

113 (49)

121 (55)

.58

Table 2. Changes in ATP Levels Before and After Vitamin D3 Treatment

P .57 .13 .42

indicator as covariates to test if ATP level changes were significantly different between treatment groups. To examine the potential for a differential association according to sex or race, we also conducted analyses in which we included cross-product terms for either sex and vitamin D dose or race and vitamin D dose. We used the Wald test to determine the statistical significance of the cross-product terms. All analyses were conducted using statistical software R, version 3.0.1.

Results Among the cohort of 38 patients for whom T-cell function was assessed, 20 participants were randomized to lowdose vitamin D and 18 participants were randomized to high-dose vitamin D (Table 1). The median age was 45 years (interquartile range, 39 – 47 years); nine were women (24%); eight (21%) were white, 29 (76%) were black, and one (3%) was of other or unknown race. Patients were treated with vitamin D for a mean of 117 days (SD, 52 days). Both groups were vitamin D– deficient, with similarly low mean baseline 25(OH)D levels (mean, 16.2 ng/mL; SD, 6.8 ng/mL). After 2 months of treatment, 25(OH)D levels significantly increased by 5.77 ng/ml (P ⬍ .01) among those assigned low-dose vitamin D and 9.77 ng/ml (P ⬍ .01) among those assigned high-dose vitamin D. Changes in CD4⫹ T cell ATP production in response to stimulation with plant lectin PHA before and after vitamin D3 supplementation are shown in Table 2. Treatment with high-dose vitamin D significantly decreased intracellular CD4⫹ ATP release (difference ⫽ 95.5 ng/ml; interquartile range [IQR], –219.5 to –105.8; P ⫽ .026). In contrast, treatment with low-dose vitamin D3 did not significantly influence intracellular CD4⫹ ATP release (difference ⫽ 0.5 ng/mL; IQR, – 69.2 to –148.5; P ⫽ .538). The difference in follow-up ATP levels at 2

535

ATP levels Baseline (ng/ml), median (IQR) Follow-up (ng/ml), median (IQR)

Low-Dose Vitamin D3 400 IU/d (n ⴝ 18)

High-Dose Vitamin D3 4000 IU/d (n ⴝ 20)

P

456 (432–556)

417 (343–516)

.37

484 (383–568)

340 (232–502)

.04

months was significantly different between the low- and high-dose vitamin D3 groups (Table 2, Figure 1). In a proportional odds model, treatment with highdose vitamin D3 was more likely to decrease ATP after antigen stimulation compared to low-dose vitamin D3 (odds ratio [OR], 3.43; 95% confidence interval [CI], 1.06 –1.11). Eleven of the 20 patients (45%) treated with high-dose vitamin D3 were considered responders with significant decreases in ATP levels. Among those treated with high-dose vitamin D3, 63.5% (7/16) of men, 25% of women (1 of 4), 52.9% (9/17) of white, and 48.1% (8/17) of black participants were responders. We considered the possibility that are results may differ according to race or sex. We did not observe a significant difference in our results according to race (pinteraction ⫽ 0.12). However, we did find a significant difference according to sex (pinteraction ⫽ 0.02). Among men, treatment with high-dose vitamin D3 was more likely to decrease ATP antigen stimulation compared to low-dose vitamin D3 (OR, 7.24; 95% CI, 1.83–28.75), whereas for women no significant association was found (OR, 0.21; 95% CI, 0.02–2.65).

Discussion Vitamin D, which has recognized immunomodulatory roles, is emerging as a potentially important determinant of the etiopathogenesis of chronic immune-mediated disorders. Although studies have linked vitamin D deficiency with the onset and course of conditions such as type I diabetes, IBD, and MS, prospective data demonstrating a direct effect of vitamin D supplementation on immune cell function in humans are limited. In this ancillary study of an RCT, we found that treatment with high-dose vitamin D3 reduces CD4⫹ T-cell activation, providing direct human data that vitamin D may influence cell-mediated immunity (CMI). Prior experimental studies have shown that vitamin D regulates CD4⫹ T-cell responses by promoting T helper 2 (Th2) cells and suppressing T helper 1 (Th1) cells, thereby

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J Clin Endocrinol Metab, February 2016, 101(2):533–538

Figure 1. Median ATP change over time.

limiting Th1-mediated inflammatory responses and tissue damage while enhancing Th2-mediated anti-inflammatory responses (18, 19). In a mouse model using CD4⫹ T cells in culture, Boonstra et al demonstrated decreased Th1 cytokine (interferon [IFN]-␥) and increased Th2 cytokine (IL-4, IL-5, and IL-10) production after treatment with vitamin D3 (18). Recent studies have shown that CD4⫹ T cells have the capacity to convert inactive 25(OH)D to the active 1,25(OH)2D, which enhances vitamin D receptor expression and decreases vitamin D receptor proteasome degradation, both of which enable gene activation that results in CD4⫹ T-cell differentiation, T-cell antigen receptor signaling, and cytokine production (18, 20, 21). Despite this compelling preclinical evidence, comparable data in humans are limited. In a pilot study of four healthy adults with vitamin D deficiency (mean baseline 25(OH)D 38 nmol/L, or 15.2 ng/mL), Allen et al (14) showed that 15 weeks of vitamin D supplementation (5000 –10 000 IU daily) increased IL-10 production and decreased the frequency of Th17 cells in parallel with expected increases in serum 25(OH)D. Bock et al (16) compared vitamin D3 supplementation (140 000 IU monthly) vs placebo in 59 healthy adults with baseline vitamin D insufficiency [25(OH)D mean 25–26 ng/ml]. They found a 1.5% increase in Treg cells with vitamin D treatment

compared to placebo. Our data significantly extend these findings by offering additional mechanistic support for an effect of vitamin D supplementation on T-cell function. This evidence of a direct effect of vitamin D on immune function provides a compelling biological rationale for additional studies examining the role of vitamin D as a therapeutic adjunct for patients with established IBD. To date, such studies are limited. Jorgensen et al randomized mostly immunocompetent CD patients to receive either 1200 IU vitamin D3 daily or placebo for 12 months (22). They found a modest yet statistically nonsignificant reduction in risk of disease relapse (13% vs 29%, respectively; P ⫽ .06). These findings suggest the possibility that higher doses of vitamin D, such as the 4000 IU used in this study, may be more effective. The High Dose Vitamin D in Patient’s With Crohn’s Disease trial (ClinicalTrials.gov Identifier: NCT02208310), an ongoing multicenter trial comparing high-dose (10,000 IU daily) with low-dose (400 IU daily) vitamin D3 supplementation in CD patients with vitamin D deficiency, will hopefully provide additional data regarding the clinical relevance of vitamin D supplementation in patients with CD. The Vitamin D Supplementation in Multiple Sclerosis trial (ClinicalTrials.gov Identifier: NCT01490502) is an ongoing multicenter trial in the United States comparing moderate-dose (5000 IU daily) with low-dose (600 IU

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doi: 10.1210/jc.2015-3599

daily) oral vitamin D3 on the activity of established MS. A prior 96-week RCT examining the effect of vitamin D3 on bone mineral density in participants with MS found no significant difference relapse rates, disability, MS functional components, grip strength, or fatigue (23). In contrast, another study found that the addition of vitamin D3 to treatment with IFN-␤-1b significantly reduced magnetic resonance imaging disease activity (T1 enhancing lesions) in MS compared to patients receiving IFN-␤ alone (13). The strengths of this study include a well-characterized cohort nested within a carefully conducted RCT in which vitamin D treatment was assigned and participants were closely followed for adherence and collection of blood for measurement of T-cell activation at uniform time points. Our study has several limitations. First, we conducted the study only among a limited subset of the trial population because of the substantial cost of the assay and the requirement for freshly collected blood. Nonetheless, despite our relatively small sample size, we did detect a significant difference in T-cell activation that should be corroborated in larger studies. Second, we estimated T-cell activation based on measurement of intracellular ATP production in PHA-stimulated CD4⫹ T cells (Immuknow assay). This assay provides information regarding ATP release from peripheral CD4⫹ T cells as a biomarker for T-cell activation as reflected by its clinical use in the solid organ transplant population to assess CMI However, the assay does not distinguish among CD4⫹ T-cell subsets or correlate with overall leukocyte levels (24). Whether it is the optimal marker of CMI in a healthy population remains to be validated. Finally, our study was limited to individuals with vitamin D deficiency (defined as 25(OH)D ⱕ25 ng/mL). It is unclear if vitamin D supplementation may have a similar effect on T-cell activation in a population that is vitamin D sufficient. In summary, we found that high-dose vitamin D3 decreased CD4⫹ T-cell activation, providing direct human evidence of the potential role of vitamin D supplementation on CMI. These findings offer a mechanistic correlate for the potential influence of vitamin D on the course of immune-mediated disorders.

Acknowledgments Address all correspondence and requests for reprints to: Andrew T. Chan, MD, MPH, Massachusetts General Hospital, Division of Gastroenterology, 55 Fruit Street, GRJ 825C, Boston, MA 02114. E-mail: [email protected]. The Vitamin D Therapy in Individuals at High Risk of Hypertension study was supported by a research grant from Diasorin. Assay support was provided by LabCorp. The ancillary

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study on T-cell activation was funded by K24 DK098311 (to A.T.C.). Supported was provided by T32 DK 007191 and National Center for Advancing Translational Sciences (NCATS)/ National Institutes of Health (NIH) grant KL2 TR001112-03 (to G.G.K.). ImmuKnow kits were provided by Cylex, now part of Viracor-IBT Laboratories. Author Contributions: G.G.K. undertook study concept and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, and statistical analysis. P.A. undertook study concept and design, analysis and interpretation of data, critical revision of the manuscript for important intellectual content, funding, and study supervision. M.R.B. undertook collection of data, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. Y.S. undertook analysis and interpretation of the data, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. S.H. undertook analysis and interpretation of the data, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. F.H. undertook analysis and interpretation of the data, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. C.N.-C. undertook study concept and design, analysis and interpretation of data, critical revision of the manuscript for important intellectual content, funding, and study supervision. D.O. undertook critical revision of the manuscript, and administrative, technical, or material support. J.K. undertook study concept and design and analysis and interpretation of the data. T.W. undertook study concept and design, analysis and interpretation of data, critical revision of the manuscript for important intellectual content, funding, and study supervision. A.T.C. undertook study concept and design, analysis and interpretation of data, critical revision of the manuscript for important intellectual content, funding, and study supervision. Disclosure Summary: The authors have nothing to disclose.

References 1. Ananthakrishnan AN, Khalili H, Higuchi LM, et al. Higher predicted vitamin D status is associated with reduced risk of Crohn’s disease. Gastroenterology. 2012;142:482– 489. 2. Hayes CE, Hubler SL, Moore JR, Barta LE, Praska CE, Nashold FE. Vitamin D actions on CD4(⫹) T cells in autoimmune disease. Front Immunol. 2015;6:100. 3. White JH. Vitamin D metabolism and signaling in the immune system. Rev Endocrine Metab Disorders. 2012;13:21–29. 4. Yang CY, Leung PS, Adamopoulos IE, Gershwin ME. The implication of vitamin D and autoimmunity: a comprehensive review. Clin Rev AllergyI Immunol. 2013;45:217–226. 5. Muscogiuri G, Tirabassi G, Bizzaro G, et al. Vitamin D and thyroid disease: to D or not to D? Eur J Clin Nutr. 2015;69:291–296. 6. Herold KC, Vignali DA, Cooke A, Bluestone JA. Type 1 diabetes: translating mechanistic observations into effective clinical outcomes. Nat Rev Immunol. 2013;13:243–256. 7. Gabbay MA, Sato MN, Finazzo C, Duarte AJ, Dib SA. Effect of cholecalciferol as adjunctive therapy with insulin on protective immunologic profile and decline of residual beta-cell function in newonset type 1 diabetes mellitus. Arch Pediatr Adolesc Med. 2012; 166:601– 607.

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538

Konijeti et al

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8. Raftery T, Merrick M, Healy M, et al. Vitamin D status is associated with intestinal inflammation as measured by fecal calprotectin in Crohn’s disease in clinical remission. Dig Dis Sci. 2015;60:2427– 2435. 9. Ananthakrishnan AN, Cagan A, Gainer VS, et al. Normalization of plasma 25-hydroxy vitamin D is associated with reduced risk of surgery in Crohn’s disease. Inflamm Bowel Dise. 2013;19:1921– 1927. 10. Ananthakrishnan AN, Cagan A, Gainer VS, et al. Higher plasma vitamin D is associated with reduced risk of Clostridium difficile infection in patients with inflammatory bowel diseases. Alim Pharmacol Therapeutics. 2014;39:1136 –1142. 11. Ananthakrishnan AN, Cheng SC, Cai T, et al. Association between reduced plasma 25-hydroxy vitamin D and increased risk of cancer in patients with inflammatory bowel diseases. Clin Gastroenterol Hepatol. 2014;12:821– 827. 12. Mokry LE, Ross S, Ahmad OS, et al. Vitamin D and risk of multiple sclerosis: a Mendelian randomization study. PLoS Med. 2015;12: e1001866. 13. Soilu-Hanninen M, Aivo J, Lindstrom BM, et al. A randomised, double blind, placebo controlled trial with vitamin D3 as an add on treatment to interferon beta-1b in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry. 2012;83:565–571. 14. Allen AC, Kelly S, Basdeo SA, et al. A pilot study of the immunological effects of high-dose vitamin D in healthy volunteers. Multiple Scler. 2012;18:1797–1800. 15. Kimball S, Vieth R, Dosch HM, et al. Cholecalciferol plus calcium suppresses abnormal PBMC reactivity in patients with multiple sclerosis. J Clin Endocrinol Metab. 2011;96:2826 –2834. 16. Bock G, Prietl B, Mader JK, et al. The effect of vitamin D supple-

J Clin Endocrinol Metab, February 2016, 101(2):533–538

17.

18.

19.

20.

21.

22.

23.

24.

mentation on peripheral regulatory T cells and beta cell function in healthy humans: a randomized controlled trial. Diabetes Metab Res Rev. 2011;27:942–945. Arora P, Song Y, Dusek J, et al. Vitamin D therapy in individuals with prehypertension or hypertension: the DAYLIGHT trial. Circulation. 2015;131:254 –262. Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, O’Garra A. 1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(⫹) T cells to enhance the development of Th2 cells. J Immunol. 2001;167:4974 – 4980. Mora JR, Iwata M, von Andrian UH. Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol. 2008;8:685– 698. Kongsbak M, von Essen MR, Boding L, et al. Vitamin D up-regulates the vitamin D receptor by protecting it from proteasomal degradation in human CD4⫹ T cells. PloS One. 2014;9:e96695. von Essen MR, Kongsbak M, Schjerling P, Olgaard K, Odum N, Geisler C. Vitamin D controls T cell antigen receptor signaling and activation of human T cells. NatI Immunol. 2010;11:344 –349. Jorgensen SP, Agnholt J, Glerup H, et al. Clinical trial: vitamin D3 treatment in Crohn’s disease - a randomized double-blind placebocontrolled study. Alim Pharmacol Therapeutics. 2010;32:377–383. Kampman MT, Steffensen LH, Mellgren SI, Jorgensen L. Effect of vitamin D3 supplementation on relapses, disease progression, and measures of function in persons with multiple sclerosis: exploratory outcomes from a double-blind randomised controlled trial. Multiple Scler. 2012;18:1144 –1151. Sanchez-Velasco P, Rodrigo E, Valero R, et al. Intracellular ATP concentrations of CD4 cells in kidney transplant patients with and without infection. Clin Transplant. 2008;22:55– 60.

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