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. 2022 May 26;13:893702. doi: 10.3389/fimmu.2022.893702

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Copyright © 2022 Koetzier, van Langelaar, Wierenga-Wolf, Melief, Pol, Musters, Lubberts, Dik, Smolders and van Luijn

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The sensitivity of MDR1^low Th17 and MDR1^high Th17.1 cells to 1,25(OH)₂D_3. (A) FACS plot showing representative gating for CCR6⁺ memory T helper (CD4⁺CD45RA^-CD25^low) subsets Th17 (CCR4⁺CXCR3^-) and Th17.1 (CCR4^-/dim). (B) Relative ABCB1 expression for healthy donor Th17 and Th17.1 cells as determined by qPCR (n = 16 per group). (C) Relative IL-17A, IFNG and CSF2 expression for healthy donor PMAionomycin and anti-CD3/CD28-stimulated Th17 and Th17.1 cells before and after 1,25(OH)₂D₃ exposure as determined by qPCR (n = 7 per group). (D) Relative VDR expression for healthy donor non-activated Th17 and Th17.1 cells as determined by qPCR (n = 7 per group). (E) Relative VDR expression for healthy donor non-activated versus anti-CD3/CD28 activated Th17 and Th17.1 cells, including a comparison for these activated Th17 and Th17.1 cells (F), as determined by qPCR (n = 8 per group). (G) Relative CYP24A1 expression for healthy donor PMAionomycin and anti-CD3/CD28-stimulated Th17 and Th17.1 cells after 1,25(OH)₂D₃ exposure as determined by qPCR (n = 7 per group). (H) Amount (pg/ml) of IFN-γ, GM-CSF, TNF-α, LT, granzyme B, and IL-10 measured in the supernatants of healthy donor anti-CD3/CD28-stimulated Th17.1 cells before and after 1,25(OH)₂D₃ exposure as determined by Luminex (n = 8 per group). (I) MDR1 surface expression (median fluorescent intensity) on healthy donor anti-CD3/CD28-stimulated Th17.1 cells before and after 1,25(OH)₂D₃ exposure (n = 13 per group). (J) Graphical model displaying the 1,25(OH)₂D₃ sensitivity of non-activated versus activated Th17.1 cells. When TCR-activated, Th17.1 cells increase their VDR expression resulting in 1,25(OH)₂D₃-signaling and a decrease in their pro-inflammatory (IFN-γ⁺GM-CSF⁺) and MDR1-expressing phenotype. Lines represent paired observations for cells from the same donors. Data were compared using either Wilcoxon rank-sum or (E) Mann-Whitney U tests. *p < 0.05, **p < 0.01 and ***p < 0.001. “D₃ = 1,25(OH)₂D₃”, “MDR1”, multidrug resistance protein 1; “MFI, median fluorescence intensity”; “1,25(OH)₂D₃, calcitriol”, “TCR”, T-cell receptor and “VDR, vitamin D receptor”.