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. 2011 Dec;134(4):459-68.
doi: 10.1111/j.1365-2567.2011.03507.x.

1,25-Dihydroxyvitamin D3 inhibits proliferation but not the suppressive function of regulatory T cells in the absence of antigen-presenting cells

Ai-Leng Khoo  1 , Irma Joosten, Meta Michels, Rob Woestenenk, Frank Preijers, Xue-Hui He, Mihai G Netea, André J A M van der Ven, Hans J P M Koenen
Affiliations

1,25-Dihydroxyvitamin D3 inhibits proliferation but not the suppressive function of regulatory T cells in the absence of antigen-presenting cells

Ai-Leng Khoo et al. Immunology. 2011 Dec.

Abstract

Vitamin D3 is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)2D3 inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)2D3 than conventional T cells(.) 1,25(OH)2D3 neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)2D3 on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D3). Increased serum 1,25(OH)2D3 levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3+ Treg cell population. In conclusion, 1,25(OH)2D3 directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)2D3 levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.

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Figures

Figure 1
Figure 1
Flow cytometry of vitamin D receptor (VDR) expression by regulatory T (Treg) and conventional T (Tconv) cells. (a) Histogram overlay of VDR expression in Treg and Tconv cells with isotype staining. Histograms showing VDR expression (x-axis) in anti-CD3/anti-CD28 monoclonal antibody-coated microbeads stimulated versus unstimulated (b) Treg and (c) Tconv cells on day 2. Data are representative for three independent experiments performed with cells obtained from different donors.
Figure 2
Figure 2
1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits regulatory T (Treg) and conventional T (Tconv) cell proliferation. (a) The effect of graded doses of 1,25(OH)2D3 on Treg or Tconv cell proliferation, stimulated with anti-CD3/anti-CD28 monoclonal antibody-coated microbeads and in the presence of interleukin-2 (IL-2). Cell proliferation as assayed by [3H]thymidine incorporation at day 4 of culture and expressed as mean ± SD. Data are representative for five independent experiments. (b) Percentage growth inhibition of Treg and Tconv cells upon treatment with 1,25(OH)2D3 at 1, 10 and 100 nm was calculated. Data are the cumulative results of five independent experiments using cells isolated by high purity flow cytometric cell sorting. *P< 0·05 when comparing the degree of growth inhibition between Treg and Tconv cells. Effect of 1,25(OH)2D3 (100 nm) on CD69 expression (y-axis), in (c) Treg and (d) Tconv cells labelled with carboxyfluorescein succinimidyl ester (CFSE; x-axis), 24 or 48 hr after activation. (e) Cell division of Treg and Tconv cells as analysed by CFSE dilution (x-axis) on day 5 with and without addition of 100 nm 1,25(OH)2D3. Data are representative for three independent experiments performed with cells isolated by high-purity flow cytometric cell sorting, obtained from different donors.
Figure 3
Figure 3
Effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on regulatory T (Treg) cell suppressive function and phenotypic characteristics. (a) Treg cells were expanded with anti-CD3/anti-CD28 monoclonal antibody (mAb) -coated microbeads with or without 100 nm 1,25(OH)2D3 for 7 days. The cells were harvested and rested for 2 days. Co-culture suppression assays were conducted to analyse the suppressor potential of these 1,25(OH)2D3-pre-treated cells. Cell proliferation was determined by [3H]thymidine incorporation at day 4. Results are expressed as percentage suppression = {1 – ([3H]thymidine incorporation co-culture/[3H]thymidine incorporation control mixed lymphocyte culture) × 100%} (y-axis). (b) Suppression assay performed in the absence or presence of 100 nm 1,25(OH)2D3. Percentage suppression (y-axis) was measured and calculated as mentioned above. (c) Treg cells and conventional T (Tconv) cells were expanded with anti-CD3/anti-CD28 mAb-coated microbeads with or without 100 nm 1,25(OH)2D3 for 7 days. The cells were harvested and rested for 2 days. Proliferative capacity of expanded cells upon re-stimulation with anti-CD3/anti-CD28 mAb-coated microbeads was determined in the absence or presence of interleukin-2 by [3H]thymidine incorporation (y-axis). (d) Representative histogram overlay depicting the expression of Foxp3 (y-axis) in Treg and Tconv cells on day 8, as measured by flow cytometry. Data are representative for five or six independent experiments performed with cells isolated by high-purity flow cytometric cell sorting, obtained from different donors. *P < 0·05 as compared with respective cell culture without the addition of 1,25(OH)2D3.
Figure 4
Figure 4
Effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on cytokine production. Intracellular staining for cytokines in (a) regulatory T (Treg) cells and (b) conventional T (Tconv) cells. Both Treg and Tconv cells were stimulated with anti-CD3/anti-CD28 monoclonal antibody-coated microbeads with or without the addition of 100 nm 1,25(OH)2D3. At day 8 of cultures, the cells were stimulated with PMA plus ionomycin in the presence of brefeldin A and intracellularly stained with antibodies directed against the cytokines listed: IL, interleukin; IFN, interferon; TNF, tumour necrosis factor. Data are representative for five independent experiments performed with cells isolated by high-purity flow cytometric cell sorting, obtained from different donors. *P < 0·05 as compared with respective cell culture without the addition of 1,25(OH)2D3.
Figure 5
Figure 5
Effect of cholecalciferol supplementation in vitamin D3-deficient HIV-infected patients. (a) Changes in serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] concentrations and (b) regulatory T (Treg) cell numbers over time. *P < 0·05 compared with baseline, **P < 0·05 compared with week 24. The subjects were treated with a daily dose of 2000 IU cholecalciferol during the first 12 weeks and thereafter for at least 48 weeks with a dose of 1000 IU daily. (c) Representative flow cytometry dot plot showing intracellular Ki-67 (x-axis) and Foxp3 (y-axis) staining on CD4+ gated T cells. Cumulative data showing the percentages of Ki-67-expressing cells within the (d) CD4+ Foxp3+ and (e) CD4+ Foxp3− population at week 24 and week 48 in eight subjects, where serum 25(OH)D3 levels at week 48 were reduced compared with week 24. *P < 0·05 compared with week 24.
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