1. Improved Vitamin D3 status is associated with increases in circulating T-regulatory cells during pregnancy
Jennifer K. Mulligan, PhD
Dept of Otolaryngology, Division of Rhinology & Sinus Surgery
Dept of Pediatrics, Division of Neonatology
Medical University of South Carolina

2. Role of the immune system in pregnancy
Arck & Hecher, Nature Medicine, 2013

3. T-regulatory cells (T-regs) help to maintain immune balance during pregnancy
↑ Pregnancy loss
↑ Preeclampsia
↑ Preterm labor
↑ Infections X
Cytotoxic T-cells
Dendritic cell
↑ Pre-term labor
↑ Preeclampsia
↑ Pregnancy-induced hypertension
T-reg
Macrophage
Helper T-cell
↑ Pregnancy loss
↑ Preeclampsia
↑ Pregnancy loss
Natural Killer cell
↑ Pregnancy loss
↑ Preeclampsia

4. So how do we generate more T-regulatory cells?
Vitamin D3

5. Study Questions
Does supplementation with 4,400 IU vitamin D3 daily during pregnancy result in increased numbers of circulating T-regulatory cells as compared to 400 IU daily?
Does increasing circulating T-regulatory cell numbers result in reduced pregnancy complications?

6. Study Design
Peripheral blood was collected from woman participating in the “Preventing Health Disparities During Pregnancy Through Vitamin D Supplementation” clinical trial
1st trimester = 8-12 weeks (study entry)
2nd trimester = weeks
3rd trimester = weeks
Control subjects = gestation matched individuals, that had no pregnancy complications and had a term delivery
Maternal peripheral blood immune cell profile was determined by flow cytometry (cell phenotype)
T-regulatory cells (T-regs) were defined as CD4+CD25+FoxP3+
T-reg precursors as CD4+CD25+FoxP3-
Dosing arm stratification
18 patients received 400 IU/daily vitamin D3
22 patients received 4,400 IU/daily vitamin D3

7. 25(OH)D3 levels by trimester & dosing arm

8. T-regulatory cells & T-regulatory cell precursors increase throughout pregnancy

9. Circulating T-regulatory cells are increased in woman receiving 4,400 IU/day of vitamin D3

10. What 25(OH)D3 value should we use to define vitamin D3 sufficiency?
No differences in T-regulatory cells or precursors when
stratified 20, 32 or 50 ng/ml of 25(OH)D3

11. 25(OH)D3 >40 ng/ml is associated with increased T-regulatory cell precursors in the 1st trimester

12. Does increasing circulating T-regulatory cell and/or 25(OH)D3 levels result in improved pregnancy outcomes?

13. Woman that miscarry prior to 22 weeks have lower circulating T-regs and 25(OH)D3 than healthy controls
Study entry; 8 – 12 weeks gestation

14. Woman who develop preeclampsia have lower circulating T-regs and 25(OH)D3 than pregnant controls
Study entry; 8 – 12 weeks gestation
Differences in T-regulatory cells were only observed in the 1st trimester

15. Summary
During pregnancy higher circulating 25(OH)D3 is associated with increased circulating T-regulatory cells and T-regulatory cell precursors.
Reduced numbers of circulating T-regulatory cells and their precursors were observed in woman who miscarried vs those who went on to have healthy, full term pregnancies.
Reduced numbers of circulating T-regulatory cells in the 1st trimester were observed in woman who developed preeclampsia vs those with healthy pregnancies.
Update!!!!

16. Take Home Message
Higher intake of vitamin D3 may improve pregnancy-related outcomes through activation of immune modulatory mediators such as T-regulatory cells.
Should higher intake of vitamin D3 be a part of preconception counseling?

17. Acknowledgements W.K. Kellogg Foundation Mulligan Laboratory
Aman Sumal
Carol Wagner, MD
Hal Harmon
Sarah Smith
Bruce Hollis, PhD
Whitney Nagel
Kellogg Study Team
Renee Washington
Judy Shary

18. Thank you for your attention! Questions?

19. Increased 25(OH)D3 levels correlates with elevated circulating T-regulatory cells in the 2nd trimester

20. What level of 25(OH)D3 constitutes VD3 “sufficiency” in pregnancy?
Kinetic Reaction Graph of 25(OH)D and 1,25(OH)2D
25(OH)D had direct influence on 1,25(OH)2D levels throughout pregnancy (p<0.0001)
DOES NOT OCCUR DURING ANY OTHER TIME DURING THE LIFESPAN
First Order becoming Zero Order Kinetics Saturation Curve:
Inflection point at 40 ng/mL (100 nmol/L) 25(OH)D3
Level required to optimize 1,25(OH)2D production
Courtesy of Carol Wagner, MD

21. Correlation between 25(OH)D3 vs plasma cytokines
25(OH)D3 levels correlate with increases in factors associated with T-reg induction & suppressive function
IL-2
IL-10
TGF-β1
Naïve T-cell/
T-reg precursor
T-regulatory
cell
Effector cell
Correlation between 25(OH)D3 vs plasma cytokines
1st Trimester
2nd Trimester
3rd Trimester p r
IL-2
0.339
0.265
0.005
0.646
0.002
0.741
IL-10
0.953
0.006
0.991
.008
0.016
0.664
TGF-β1
<0.0001
0.4821
0.058
0.091
0.702
0.004

22. Circulating immune cells
Table I: Association between 25(OH)D3 Levels vs circulating maternal immune cells & mediators
1st Trimester
2nd Trimester
3rd Trimester
 Circulating immune cells p r
Total T-cells (CD3+)
0.561
0.095
0.259
0.185
0.659
0.072
Helper T-cell (CD4+)
0.121
0.249
0.273
0.180
0.425
0.130
Cytotoxic T-cell (CD8+)
0.641
-0.076
0.927
0.015
0.573
0.092
Monocyte-derived dendritic cells (MHC+CD14+CD209+)
0.706
-0.062
0.645
0.271
0.178
Mature dendritic cell (CD83+)
0.932
0.014
0.175
0.222
0.979
-0.004
B-cell (CD19+)
0.342
-0.154
0.753
0.052
0.683
-0.067
Monocyte (CD14+)
0.631
-0.078
0.429
0.206
0.204
T-regulatory cell (CD4+CD25+FoxP3+)
0.542
-0.099
0.032
0.345
0.297
-0.169
T-regulatory cell precursor (CD4+CD25+ FoxP3-)
0.086
0.275
0.001
0.513
0.689
-0.065
Macrophage (CD68+)
0.965
0.007
0.789
0.044
0.240
-0.190
Activated macrophage (CD163+)
0.873
-0.026
-0.156
0.358
-0.149
Activated antigen presenting cells (CD86+)
0.453
-0.124
0.400
-0.137
NK cells (CD56+)
0.562
0.514
-0.108
0.667
-0.070
Plasma Cytokines
IL-2
0.339
0.265
0.005
0.646
0.002
0.741
IL-10
0.606
0.084
0.826
0.036
0.122
TGF-β1
0.504
0.109
0.348
0.256
0.184
Statistics shown are the results of a Pearson correlation matrix analysis. Values in red indicate statistical significance (p<0.05) and their accompanying r value.