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The Vitamin D Analog, TX527, Promotes a Human CD4+CD25highCD127low Regulatory T Cell Profile and Induces a Migratory Signature Specific for Homing to Sites of Inflammation

Femke Baeke, Hannelie Korf, Lut Overbergh, Annemieke Verstuyf, Lieven Thorrez, Leentje Van Lommel, Mark Waer, Frans Schuit, Conny Gysemans and Chantal Mathieu
J Immunol January 1, 2011, 186 (1) 132-142; DOI: https://doi.org/10.4049/jimmunol.1000695
Femke Baeke
*Laboratory of Experimental Medicine and Endocrinology,
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Hannelie Korf
*Laboratory of Experimental Medicine and Endocrinology,
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Lut Overbergh
*Laboratory of Experimental Medicine and Endocrinology,
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Annemieke Verstuyf
*Laboratory of Experimental Medicine and Endocrinology,
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Lieven Thorrez
†Gene Expression Unit, Department of Molecular Cell Biology, and
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Leentje Van Lommel
†Gene Expression Unit, Department of Molecular Cell Biology, and
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Mark Waer
‡Laboratory of Experimental Transplantation, Catholic University of Leuven, Leuven, Belgium
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Frans Schuit
†Gene Expression Unit, Department of Molecular Cell Biology, and
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Conny Gysemans
*Laboratory of Experimental Medicine and Endocrinology,
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Chantal Mathieu
*Laboratory of Experimental Medicine and Endocrinology,
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  • FIGURE 1.
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    FIGURE 1.

    TX527 alters the transcriptional program of human T cells. Purified human CD3+ T cells, isolated from whole blood samples of healthy donors, were activated by plate-bound anti-CD3/anti-CD28 as described in the Materials and Methods section. From day 2 onwards, the cells were repeatedly treated with vehicle (ctr) or TX527 (10−8 M) every second day until day 10. A, At the end of the culture term, CD3+ T cells were further separated into a CD4+ and a CD8-enriched T cell population, and mRNA-expression levels of 24-hydroxylase and VDR in the different T cell fractions were analyzed by quantitative real-time RT-PCR. The results shown represent the mean values ± SEM from one out of four independent experiments. B, The biological function analysis tool of the Ingenuity Pathway Analysis Software identified the most significant biological processes that were associated with the set of TX527-regulated genes, obtained by microarray analysis. The x-axis of the bar plot depicts the significance score of the top five of functional categories regulated by TX527. The statistical threshold is indicated by the dashed line. C, Pie chart displaying the number of TX527-regulated genes for each of the top five biological processes most significantly affected by the analog. D, Heat map showing the gene expression profiles of TX527-regulated genes (n = 140) that are involved in cellular movement. As indicated, the expression levels are mapped to a color gradient from low (green) to high expression (red), and the individual columns represent expression profiles of five independent donors. *p < 0.05; **p < 0.001 versus ctr.

  • FIGURE 2.
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    FIGURE 2.

    TX527 reduces the proliferative capacity of CD4+ and CD8+ T cells. Purified human CD3+ T cells were activated with anti-CD3/anti-CD28 and repeatedly treated with vehicle (ctr) or TX527 (10−8 M) every second day as described before. On day 8, cells were harvested, CFSE-labeled, and seeded out again in the presence or absence of TX527 (10−8 M). The cultures were supplemented with IL-2 (12.5 ng/ml) on the usual 2-d intervals to maintain T cell growth. On day 10, T cells were harvested and stained with mAbs directed against CD4 and CD8 before flow cytometric analysis was performed. The proliferating cell fraction was identified by a decrease of CFSE signal. Dead cells were excluded from analysis using 7-AAD staining. Mean values ± SEM of the percentages of proliferating cell fractions from one representative experiment are shown. A total of three independent donors were tested. *p < 0.05; **p < 0.005 versus ctr.

  • FIGURE 3.
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    FIGURE 3.

    Cytokine responses in CD4+ and CD8-enriched T cell fractions are differentially regulated by TX527. Human CD3+ T cells, activated by anti-CD3/anti-CD28 and treated with vehicle (ctr) or TX527 (10−8 M) as described earlier, were further separated into a CD4+ and a CD8-enriched T cell populations following the 10-d culture period. The relative mRNA levels of IFN-γ, IL-17, IL-4, and IL-10 in the different T cell fractions were analyzed by quantitative real-time RT-PCR. The values depicted are the mean ± SEM of one representative experiment out of two to four independently tested donors. *p < 0.05 versus ctr. ND, not detected.

  • FIGURE 4.
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    FIGURE 4.

    TX527 modulates surface expression of T cell activation and Treg markers. Expression of activation and Treg markers by human T cells, activated by anti-CD3/anti-CD28 and cultured for 10 d in the presence of the vehicle (solid lines) or TX527 (dashed lines), was analyzed by flow cytometry. A, Histogram overlays, gated on CD4+ or CD8+ T cell subsets, represent surface expression of T cell activation markers CD69 and CD40L. B, The upper panel shows histogram overlays, gated on CD4+ T cells, depicting surface expression of Treg markers CD25 and CD127. The lower panel shows contour graphs, depicting coexpression of CD25high and CD127low on the CD4+ T cell population. The results are representative of three to eight independent replicates performed on different donors.

  • FIGURE 5.
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    FIGURE 5.

    TX527 drives de novo conversion of naive T cells into functional Tregs. A, TX527-mediated induction of CD4+CD25highCD127low Tregs from nTreg-depleted CD3+ T cells. The upper panel depicts the composition of the starting T cell population as determined by FACS, either depleted (right panel) or not (left panel) for nTregs. The lower panel shows the percentage of end-stage CD4+CD25highCD127low Tregs induced following anti-CD3/anti-CD28 stimulation and treatment with TX527 or vehicle within the corresponding starting populations. B and C, The functional ability of TX527-induced Tregs to counteract activation and proliferation of autologous CD4+CD25- T responder cells was analyzed. For this, CD4+CD25+ T cells, isolated from 10-d cultured TX527-treated (TX527 suppressors) or vehicle-treated T cells (ctr suppressors), were cocultured with Cell Proliferation Dye labeled-CD4+CD25− responder T cells in the presence of accessory cells and soluble anti-CD3. Activation and proliferation of responder T cells were measured by surface CD69 expression (12 h) (B) and by Cell Proliferation Dye dilution (72 h) (C), respectively. Different responder-to-suppressor ratios were implemented (1:2, 1:1, and 1:0.25). To control for crowding, a condition with identical numbers of unlabeled CD4+CD25− responders instead of suppressors was included. The upper panels (B, C) depict histogram plots of the above-mentioned parameters for responder T cells alone or cocultured with TX527 suppressors. The lower panels show the percent suppression of responder T cell activation and proliferation for all the experimental conditions tested (B, C). The data represent one experiment out of three with identical outcome. D, TX527 triggers the induction of IL-10 within the CD4+CD25highCD127low T cell population. Data shown illustrate relative IL-10 mRNA levels (mean ± SEM) of FACS-sorted CD4+CD25highCD127low TX527-treated (TX527-Treg) and vehicle-treated (ctr-Treg) cells. E, Expression of Treg-associated markers by human TX527-treated T cells. CD3+ T cells, activated by anti-CD3/anti-CD28 and cultured in the presence of the vehicle (solid lines) or TX527 (dashed lines), were analyzed by FACS for surface expression of OX40, CTLA-4, GITR, FOXP3, and ICOS. The data are shown by histogram overlays, gated on CD4+ T cells, of one representative experiment out of four independent donors tested.

  • FIGURE 6.
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    FIGURE 6.

    TX527 imprints T cells with a homing receptor signature specific for migration to inflammatory sites and alters their responsiveness to chemokine ligands. T cells, activated by anti-CD3/anti-CD28 and cultured in the presence of TX527 or vehicle, were screened for altered expression of genes and proteins involved in cellular movement and altered migratory capacity. A, Heat map showing the microarray gene expression levels, mapped to a color gradient from low (green) to high expression (red), of those genes acting in the chemokine signaling pathway (KEGG: 04062) and regulated by TX527 (n = 17). Genes marked with an asterisk were further analyzed at the protein level. B, T cell homing receptor expression was measured by flow cytometry. Histogram overlays of CD4+ or CD8+ subsets of vehicle-treated (solid lines) versus TX527-treated (dashed lines) T cells depict surface expression of receptors directing T cells to skin (CCR4, CCR6, CCR10, and CLA), gut (CCR9 and integrin β7), secondary lymphoid organs (CD62L, CCR7, and CXCR4), or to inflammatory sites (CCR5, CXCR3, and CXCR6). 7-AAD was used to exclude dead cells from analysis. The results are representative of three to eight independent experiments. C, The functional ability of CD3+ T cells, conditioned with TX527 (10−8 M) or vehicle (ctr) to migrate toward the chemokines CXCL12 (100 ng/ml), CXCL16 (150 ng/ml), and CCL27 (2 μg/ml) was investigated using a transwell tissue culture system (with a 3-μm pore size). The results shown are representative of four independent experiments and depict the net migration after subtraction of background migration. *p < 0.005 versus control.

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    Table I. Microarray analysis reveals TX527-mediated gene regulation in purified human CD3+ T cells
    Gene SymbolGene TitleFold Change by TX527UniGene ID
    Vitamin D target genes
     CA2Carbonic anhydrase II+2.9Hs.155097
     CYP24A1 (24-hydroxylase)Cytochrome P450, family 24, subfamily A, polypeptide 1+17.4Hs.89663
     VDRVitamin D (1,25-dihydroxyvitamin D3) receptor+2.0Hs.524368
    Cell cycle
     GADD45AGrowth arrest and DNA damage-inducible, α+4.9Hs.80409
    Activation markers
     CTLA4Cytotoxic T-lymphocyte–associated protein 4+3.2Hs.247824
     IL2RA (CD25)IL-2R, α+2.4Hs.231367
     TNFRSF4 (OX40)TNFR superfamily, member 4+2.4Hs.129780
     CD40LGCD40L−2.2Hs.592244
     CD69CD69 molecule−2.0Hs.208854
     IL7R (CD127)IL-7R−1.8Hs.635723
    Chemokine/homing receptors
     CCR10Chemokine (C-C motif) receptor 10+5.5Hs.278446
     CXCR3Chemokine (C-X-C motif) receptor 3+1.6Hs.198252
     CXCR6Chemokine (C-X-C motif) receptor 6+4.2Hs.34526
     FUT7Fucosyltransferase 7 [α (1, 3) fucosyltransferase]−2.0Hs.457
    Cytokines
     IFNGIFN, γ−6.0Hs.856
     IL4IL-4−1.6Hs.73917
    • Gene expression profiles of human CD3+ T cells, treated with vehicle or TX527, were monitored by microarray analysis. Gene probes displaying a fold change >1.5 combined with p < 0.01 were considered as differentially expressed. A selection of genes of interest is listed. When multiple probe sets of a gene were differentially regulated, the average fold change is displayed.

    • GADD45A, growth arrest and DNA damage-inducible α.

Additional Files

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    Files in this Data Supplement:

    • Supplemental Figure 1 (PDF, 36.2 Kb) - TX527 triggers VDR-dependent signaling pathways in human activated T cells.
    • Supplemental Table (XLS, 126 Kb) - List of differentially expressed genes in the category "Cell-To-Cell Signaling and Interaction", as assigned by Ingenuity Pathway Analysis...
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The Journal of Immunology: 186 (1)
The Journal of Immunology
Vol. 186, Issue 1
1 Jan 2011
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The Vitamin D Analog, TX527, Promotes a Human CD4+CD25highCD127low Regulatory T Cell Profile and Induces a Migratory Signature Specific for Homing to Sites of Inflammation
Femke Baeke, Hannelie Korf, Lut Overbergh, Annemieke Verstuyf, Lieven Thorrez, Leentje Van Lommel, Mark Waer, Frans Schuit, Conny Gysemans, Chantal Mathieu
The Journal of Immunology January 1, 2011, 186 (1) 132-142; DOI: 10.4049/jimmunol.1000695

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The Vitamin D Analog, TX527, Promotes a Human CD4+CD25highCD127low Regulatory T Cell Profile and Induces a Migratory Signature Specific for Homing to Sites of Inflammation
Femke Baeke, Hannelie Korf, Lut Overbergh, Annemieke Verstuyf, Lieven Thorrez, Leentje Van Lommel, Mark Waer, Frans Schuit, Conny Gysemans, Chantal Mathieu
The Journal of Immunology January 1, 2011, 186 (1) 132-142; DOI: 10.4049/jimmunol.1000695
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