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.
ABSTRACT The use of hypocalcemic vitamin D analogs is an appealing strategy to exploit the immunomodulatory actions of active vitamin D in vivo while circumventing its calcemic side effects. The functional modulation of dendritic cells by these molecules is regarded as the key mechanism underlying their ability to regulate T cell reactivity. In this article, we demonstrate the capacity of the vitamin D analog, TX527, to target T cells directly. Microarray analysis of purified human CD3(+) T cells, cultured in the presence of TX527, revealed differential expression of genes involved in T cell activation, proliferation, differentiation, and migratory capacity. Accordingly, functional analysis showed a TX527-mediated suppression of the T cell proliferative capacity and activation status, accompanied by decreased expression of effector cytokines (IFN-γ, IL-4, and IL-17). Furthermore, TX527 triggered the emergence of CD4(+)CD25(high)CD127(low) regulatory T cells featuring elevated levels of IL-10, CTLA-4, and OX40 and the functional capacity to suppress activation and proliferation of effector T cells. Moreover, the vitamin D analog profoundly altered the homing receptor profile of T cells and their migration toward chemokine ligands. Remarkably, TX527 not only modulated skin-homing receptors as illustrated for the parent compound, but also reduced the expression of lymphoid organ-homing receptors (CD62L, CCR7, and CXCR4) and uniquely promoted surface expression of inflammatory homing receptors (CCR5, CXCR3, and CXCR6) on T cells. We conclude that TX527 directly affects human T cell function, thereby inhibiting effector T cell reactivity while inducing regulatory T cell characteristics, and imprints them with a specific homing signature favoring migration to sites of inflammation.
- SourceAvailable from: Rhiannon Kundu[Show abstract] [Hide abstract]
ABSTRACT: The active vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25[OH]2 D) potently inhibits dendritic cell (DC) priming of T-cell activation, suggesting that it mediates a homeostatic role in this context. Therefore, careful regulation of 1,25[OH]2 D levels is necessary to avoid inappropriate inhibition of T-cell activation. Cell-autonomous control of vitamin D activity can be modulated by the action of the vitamin D-activating and -inactivating hydroxylases, CYP27B1 and CYP24A1, respectively. We show that in comparison to macrophages, human monocyte-derived DCs exhibit significantly less activation of 25[OH]D to 1,25[OH]2 D, and that DCs predominantly express a truncated CYP27B1 transcript that may contribute to the deficiency in activation of vitamin D. Furthermore, in response to stimulation with 1,25[OH]2 D, up-regulation of the inactivating enzyme CYP24A1 curtailed the functional effects of vitamin D in DCs, but not macrophages. Production of 1,25[OH]2 D by macrophages was adequate to induce expression of vitamin D-responsive genes by DCs, inhibit DC maturation in response to innate immune stimulation and DC-dependent T-cell responses. Our data suggest that in comparison to macrophages, differential regulation of hydroxylases limits autocrine vitamin D activity in DCs, and that paracrine activation of vitamin D exerts a more potent mechanism for homeostatic control of DC function. This article is protected by copyright. All rights reserved.European Journal of Immunology 02/2014; · 4.97 Impact Factor
Article: The future of vitamin D analogs.[Show abstract] [Hide abstract]
ABSTRACT: The active form of vitamin D3, 1,25-dihydroxyvitamin D3, is a major regulator of bone and calcium homeostasis. In addition, this hormone also inhibits the proliferation and stimulates the differentiation of normal as well as malignant cells. Supraphysiological doses of 1,25-dihydroxyvitamin D3 are required to reduce cancer cell proliferation. However, these doses will lead in vivo to calcemic side effects such as hypercalcemia and hypercalciuria. During the last 25 years, many structural analogs of 1,25-dihydroxyvitamin D3 have been synthesized by the introduction of chemical modifications in the A-ring, central CD-ring region or side chain of 1,25-dihydroxyvitamin D3 in the hope to find molecules with a clear dissociation between the beneficial antiproliferative effects and adverse calcemic side effects. One example of such an analog with a good dissociation ratio is calcipotriol (Daivonex®), which is clinically used to treat the hyperproliferative skin disease psoriasis. Other vitamin D analogs were clinically approved for the treatment of osteoporosis or secondary hyperparathyroidism. No vitamin D analog is currently used in the clinic for the treatment of cancer although several analogs have been shown to be potent drugs in animal models of cancer. Transcriptomics studies as well as in vitro cell biological experiments unraveled basic mechanisms involved in the antineoplastic effects of vitamin D and its analogs. 1,25-dihydroxyvitamin D3 and analogs act in a cell type- and tissue-specific manner. Moreover, a blockade in the transition of the G0/1 toward S phase of the cell cycle, induction of apoptosis, inhibition of migration and invasion of tumor cells together with effects on angiogenesis and inflammation have been implicated in the pleiotropic effects of 1,25-dihydroxyvitamin D3 and its analogs. In this review we will give an overview of the action of vitamin D analogs in tumor cells and look forward how these compounds could be introduced in the clinical practice.Frontiers in Physiology 01/2014; 5:122.
- [Show abstract] [Hide abstract]
ABSTRACT: The aim of our study was to analyze the possible relationship between growing pains, vitamin D levels, and bone mineral status. We enrolled 33 children affected by growing pains. Their pain intensity was evaluated through a questionnaire using the Wong-Baker Faces Pain Rating Scale for pain assessment. Serum 25-hydroxyvitamin D (25-OH-D), parathyroid hormone (PTH), and alkaline phosphatase levels were measured as well. A quantitative ultrasound assessment (QUS) was also done, measuring both the amplitude-dependent speed of sound (AD-SOS) and the bone transmission time (BTT), correlating, respectively, with bone density and with cortical thickness. After 3 and 24 months of vitamin D supplementation, we re-evaluated pain intensity and laboratory results. After 24 months we re-assessed QUS parameters. At the beginning of the study the children reported a mean growing pain intensity of 7.5 ± 1.6 SD. The mean values of 25-OH-D and PTH levels were 15.7 ± 6.9 ng/ml and 57.3 ± 27.3 pg/ml, respectively. The AD-SOS Z score was -0.53 ± 1.19 SD, and the mean value of the BTT Z score was -0.72 ± 0.96 SD. After the first 3 months of vitamin D supplementation we observed an increase in 25-OH-D levels (34.1 ± 17.8, p < 0.001) and a reduction in both PTH levels (47.3 ± 30.6, p = 0.135) and pain intensity (2.7 ± 2.2, p < 0.001). After 24 months we observed a further significant reduction in the pain intensity (3.9 ± 3.4, p < 0.001) and in PTH levels (43.7 ± 28.5, p = 0.004) and an improvement in the QUS parameters, in particular in BTT Z scores (p = 0.014). Our study suggests an interesting relationship between growing pains, vitamin D levels and bone mineral status.Journal of Bone and Mineral Metabolism 03/2014; · 2.22 Impact Factor
The Journal of Immunology
The Vitamin D Analog, TX527, Promotes a Human
CD4+CD25highCD127lowRegulatory T Cell Profile and
Induces a Migratory Signature Specific for Homing to Sites of
Femke Baeke,*,1Hannelie Korf,*,1Lut Overbergh,* Annemieke Verstuyf,*
Lieven Thorrez,†Leentje Van Lommel,†Mark Waer,‡Frans Schuit,†Conny Gysemans,*
and Chantal Mathieu*
The use of hypocalcemic vitamin D analogs is an appealing strategy to exploit the immunomodulatory actions of active vitamin D
in vivo while circumventing its calcemic side effects. The functional modulation of dendritic cells by these molecules is regarded as
the key mechanism underlying their ability to regulate T cell reactivity. In this article, we demonstrate the capacityof the vitamin D
analog, TX527, to target T cells directly. Microarray analysis of purified human CD3+T cells, cultured in the presence of TX527,
revealed differential expression of genes involved in T cell activation, proliferation, differentiation, and migratory capacity.
Accordingly, functional analysis showed a TX527-mediated suppression of the T cell proliferative capacity and activation status,
accompanied by decreased expression of effector cytokines (IFN-g, IL-4, and IL-17). Furthermore, TX527 triggered the emer-
gence of CD4+CD25highCD127lowregulatory T cells featuring elevated levels of IL-10, CTLA-4, and OX40 and the functional
capacity to suppress activation and proliferation of effector T cells. Moreover, the vitamin D analog profoundly altered the homing
receptor profile of T cells and their migration toward chemokine ligands. Remarkably, TX527 not only modulated skin-homing
receptors as illustrated for the parent compound, but also reduced the expression of lymphoid organ-homing receptors (CD62L,
CCR7, and CXCR4) and uniquely promoted surface expression of inflammatory homing receptors (CCR5, CXCR3, and CXCR6)
on T cells. We conclude that TX527 directly affects human T cell function, thereby inhibiting effector T cell reactivity while
inducing regulatory T cell characteristics, and imprints them with a specific homing signature favoring migration to sites of
inflammation.The Journal of Immunology, 2011, 186: 132–142.
immune effects observed in vitro and in vivo (1, 2). Most reports
itamin D, in particular its active metabolite 1,25-
dihydroxyvitamin D3[1,25(OH)2D3], has been identified
as a potentially interesting immunomodulator based on
on 1,25(OH)2D3 put forward its actions on APCs, especially
dendritic cells (DCs), as the key feature underlying the immuno-
modulatory properties of this compound (3, 4). Active vitamin D
alters the phenotype and functionality of DCs, locking them in
a nonmature state, with features of tolerogenic DCs (3). The al-
tered DCs are CD14+and characterized by reduced expression of
costimulatory molecules (e.g., CD40, CD80, and CD86), as well
as reduced CD1a, CD83, and MHC class II expression (5, 6).
These 1,25(OH)2D3-treated DCs are able to alter the behavior of
T lymphocytes, leading to effector T cell hyporesponsiveness and,
more importantly, the generation of regulatory T cells (Tregs) (5–
7). It has been assumed that the effects of 1,25(OH)2D3on T cells
are mainly indirect and mediated via modulation of DCs (4).
Nevertheless, the presence of vitamin D receptors (VDRs) in ac-
tivated T cells suggests possible direct effects on T lymphocytes
and thus the existence of additional mechanisms for 1,25(OH)2D3
to shape T cell responses (8). Some older reports on direct effects
of 1,25(OH)2D3 on Ag- and lectin-stimulated proliferation of
human and murine T cells and their cytokine responses exist (9–
13). More recently, it was reported that 1,25(OH)2D3treatment
of purified human CD4+T cells effectively inhibits production
of proinflammatory cytokines including IFN-g, IL-17, and IL-21
while promoting the emergence of Tregs (14), expressing CTLA-4
and FOXP3, strengthening the concept of direct 1,25(OH)2D3-
mediated modulation of T cell responses.
In vivo, exploitation of these interesting immunoregulatory ef-
fects is limited by the actions of 1,25(OH)2D3on calcium and bone
metabolism, as the doses needed for achieving the immune effects
*Laboratory of Experimental Medicine and Endocrinology,†Gene Expression Unit,
Department of Molecular Cell Biology, and‡Laboratory of Experimental Transplan-
tation, Catholic University of Leuven, Leuven, Belgium
1F.B. and H.K. contributed equally to this article.
Received for publication March 8, 2010. Accepted for publication October 31, 2010.
Thisworkwassupported bygrants from theFlemishResearch Foundation(FondsVoor
lowship to C.G. and H.K., and a clinical fellowship to C.M.), the Belgium Program on
Interuniversity Poles of Attraction initiated by the Belgian State (P5/17 and P6/40), the
Katholieke Universiteit Leuven (GOA 2004/10, GOA 2009/10, and SymBioSys CoE
EF/05/007), the 6th Framework Program of the European Union with SAVEBETA as
well as of the 7th Framework Program of the European Union with Natural Immuno-
modulators as Novel Immunotherapies for Type 1 Diabetes, and the Juvenile Diabetes
Research Foundation Center for b Cell Therapy in Diabetes (Grant 4-2005-1327).
The microarray data presented in this article have been submitted to the Gene Ex-
pression Omnibus under accession number GSE23984.
Address correspondence and reprint requests to Dr. Chantal Mathieu, Campus Gasthuis-
berg O&N1, Laboratory of Experimental Medicine and Endocrinology, Herestraat 49
bus 902, 3000 Leuven, Belgium. E-mail address: Chantal.Mathieu@med.kuleuven.be
The online version of this article contains supplemental material.
Abbreviations used in this paper: 7-AAD, 7-aminoactinomycin D; CLA, cutaneous
lymphocyte-associated Ag; DC, dendritic cell; GADD45A, growth arrest and DNA
damage-inducible a; GITR, glucocorticoid-induced TNFR-related protein; KEGG,
Kyoto Encyclopedia of Genes and Genomes; ND, not detected; nTreg, naturally
occurring regulatory T cell; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; Treg, regula-
tory T cell; VDR, vitamin D receptor.
greatly exceed the levels seen in physiological circumstances. One
solution could be topical application of 1,25(OH)2D3, but even
this administration route leads to hypercalcemia and bone de-
calcification (15). Only the use of synthetic analogs of 1,25
(OH)2D3, in which calcemic and immunomodulatory actions have
been dissociated, can lead to a possible in vivo application, topically
or systemically. In this study, we explored the ability of TX527
[19-nor-14,20-bisepi-23-yne-1,25(OH)2D3], a hypocalcemic vita-
min D analog with established immunoregulatory properties (16),
to directly interfere with T cell phenotype and functionality. By
using a synchronized system of T cell activation, we demonstrate
direct effects of TX527 on human T lymphocytes, both at the
phenotypic and functional level. Analysis of surface markers
suggests the promotion of a Treg population (CD4+CD25high
CD127low), with differential alterations in cytokine production in
CD4+and CD8+T cells. Finally, TX527 alters the chemokine re-
ceptor profile of T lymphocytes, inducing a specific homing sig-
nature, which would direct migration to skin (CCR4 and CCR10)
and, in particular, to sites of inflammation (CCR5, CXCR3, and
Materials and Methods
RPMI 1640 medium with Glutamax-I, FCS, and antibiotics (penicillin and
streptomycin) were purchased from Invitrogen (Merelbeke, Belgium).
Purified anti-CD3 mAb (clone UCHT1) and anti-CD28 mAb (clone 37407)
were obtained from R&D Systems (Minneapolis, MN). Human rIL-2 was
purchased from PeproTech (London, U.K.). Human rCCL27, rCXCL12,
and rCXCL16 were obtained from R&D Systems. The vitamin D ana-
log TX527 [19-nor-14,20-bisepi-23-yne-1,25(OH)2D3] was synthesized by
M. Vandewalle and P. De Clercq (University of Ghent, Ghent, Belgium)
and obtained from The ´ramex (Monaco, France).
T cell isolation and culture
were isolated by Ficoll-gradient centrifugation (Axis-Shield Poc, Oslo,
Norway). CD3+T cells were further purified by a negative selection
method using the Pan T cell Isolation Kit II (Miltenyi Biotec, Bergisch
Gladbach, Germany) according to manufacturer’s recommendations (pu-
rity routinely .96%). CD3+T cells (0.7 3 106cells/ml) were seeded in
24-well culture plates coated with anti-CD3 (1 mg/ml) and anti-CD28 (1
mg/ml) in RPMI 1640 medium, supplemented with Glutamax-I, 10% heat-
inactivated FCS, penicillin (100 IU/ml), and streptomycin (100 IU/ml).
Where indicated, naturally occurring Tregs (nTregs) were depleted from
the starting CD3+T cell fraction, using CD25-microbeads II (Miltenyi
Biotec) according to the protocol provided by the manufacturer. Every
second day, cells were split and supplemented with fresh medium con-
taining human rIL-2 (12.5 ng/ml). The cells were treated with TX527 (1028
M) or vehicle (ethanol) every other day starting at day 2. This treatment
schedule is chosen based on previous results showing an optimal induction
of VDR expression levels in human T cells on day 2 following activation
(17). In some experiments, CD4+T cells were further separated from the
total CD3+T cell fraction after the 10-d culture period by negative selection
(CD4+T cell Isolation Kit II, Miltenyi Biotec, purity .95%). Also, the
CD4-depleted cell fraction, which contained .85% CD8+T cells, was
cells from vehicle-treated and TX527-treated T cell populations were iso-
lated by flow cytometric cell sorting on a FACSVantage (BD Biosciences,
San Jose, CA).
RNA samples were prepared from five independent experiments using the
was verified on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa
Clara, CA). Total RNA (500 ng) was reverse-transcribed into cDNA using
the IVT express kit (Affymetrix, Santa Clara, CA) with oligo(dT) primers
containing a T7 RNA polymerase promoter site. cDNAwas further in vitro
transcribed to cRNA and biotin labeled, according to Affymetrix user
manual 702646Rev.8 (Affymetrix). Biotinylated cRNA was purified and
fragmented.Thequalityof labeledand fragmented cRNA, respectively,was
assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies).
Fragmented biotinylated cRNA (10 mg) was hybridized overnight to the
Human Genome U133 Plus 2.0 Array (Affymetrix). The arrays were
washed and stained with streptavidin-PE on an automated Affymetrix
Fluidics Station and scanned using the Affymetrix 3000 GeneScanner
(Affymetrix). The image files were analyzed using Affymetrix GCOS
Software (Affymetrix), and the RMA algorithm was used to calculate the
signal intensities for each probe cell. The microarray datasets are deposited
into the public database Gene Expression Omnibus (http://www.ncbi.nlm.
nih.gov/geo/) with accession number GSE23984. Gene probes displaying
a fold change of .1.5 combined with p , 0.01 were considered as dif-
ferentially expressed. The biological function analysis tool of the Ingenuity
Pathway Analysis Software (Ingenuity Systems, Redwood City, CA; http://
www.ingenuity.com) was used to identify the principal biological pro-
cesses that were most significantly associated with the set of differentially
expressed genes. Heat maps were constructed by hierarchical clustering
with Functional Treeview (18) using log2transformed expression values
centered around zero.
RNA isolation and real-time RT-PCR
Total RNA was routinely isolated using the High Pure RNA Isolation Kit
(Roche) or the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) in the
case of sorted cells. The RNA samples (0.5–1 mg) were reverse transcribed
using 100 U Superscript II Reverse Transcriptase (Invitrogen) and 5 mM
oligo(dT)16at 42˚C for 80 min. Real time RT-PCR conditions were as
previously reported (19, 20) using the MyIQ-Cycler (Bio-Rad, Hercules,
CA) and TaqMan systems (Applied Biosystems, Foster City, CA). Primer
and probe sets for b-actin, VDR, 24-hydroxylase, IFN-g, IL-4, IL-10, and
IL-17 were the same as reported previously (17, 19, 21, 22). cDNA
plasmid standards were used to quantify mRNA expression levels (19). For
normalization, RT-PCR mRNA target copy numbers were divided by
b-actin copy numbers, as a stably expressed housekeeping gene among all
On day 8 of the cultures, CD3+T cells were labeled with 2.5 mM CFSE
(Molecular Probes, Leiden, The Netherlands) and replated in 24-well
plates (0.7 3 106/well). Cells were replenished with fresh medium sup-
plemented with IL-2 (12.5 ng/ml) with or without TX527 (1028M) and
cultured for an additional 48 h (until day 10). For analysis, cells were
labeled with mAbs against CD4 and CD8, and the percentage of dividing
cells (determined by a decrease of CFSE-signal) in each T cell subset was
determined by flow cytometry. Dead cells were excluded from analysis by
7-aminoactinomycin D (7-AAD) staining (eBioscience, San Diego, CA).
Flow cytometric analysis of T cell phenotype
For analysis of surface protein expression, CD3+T cells were harvested
at the end of the 10-d culture term and stained with directly conjugated
mAbs against CD4, CD8, CD25, CD69, CCR5 (eBioscience), cutaneous
lymphocyte-associated Ag (CLA), integrin b7,glucocorticoid-induced TNFR-
related protein (GITR; Biolegend, San Diego, CA), CCR4, CCR6, CCR9,
CCR10, CXCR6 (R&D Systems), CXCR3, CXCR4, CCR7, L-selectin
(CD62L), CD40L, IL-7Ra (CD127), OX40 (CD134/TNFRSF4), ICOS
(CD278) (BD Biosciences), or matching isotype controls. Dead cells were
excluded from analysis using 7-AAD (eBioscience). Intracellular staining
isotype controls was performed using the human FOXP3 Buffer Set (BD
Biosciences), according to the manufacturer’s instructions. Samples were
acquired on an FACSCanto instrument (BD Biosciences), and the analysis
was performed using the FACSDiva software (BD Biosciences). Overlay
graphs were constructed using the FlowJo software (Tree Star, Ashland,
Autologous CD4+CD252responder T cells, isolated from PBMCs, were
labeled with eFluor 670 Proliferation Dye (eBioscience). PBMCs, depleted
from CD3+T cells (CD3-microbeads, Miltenyi Biotec) and mitomycin C
treated, were used as accessory cells. Suppression assays were performed
in round-bottom 96-well plates containing 5 3 104responder T cells, 5 3
104accessory cells, and soluble anti-CD3 (0.5 mg/ml, OKT3, eBioscience).
CD4+CD25+T cells, isolated from 10-d TX527-treated or vehicle-treated
T cell cultures, using the human CD4+CD25+Regulatory T cell Isolation
Kit (Miltenyi Biotec), were implemented as putative suppressor cells at
different concentrations (responder-to-suppressor ratios 1:2, 1:1, and
1:0.25). After 12 h, CD69 expression of the responder T cell fraction was
measured. In addition, responder T cell proliferation was determined after
72 h by analysis of eFluor 670 Proliferation Dye dilution. To control for
The Journal of Immunology133
crowding in proliferation assays, equal numbers of unlabeled responder
T cells were added to the cultures.
T cell migration was evaluated using 3-mm pore polycarbonate filters in 24-
well transwell chambers (Corning, Lowell, MA). Control and TX527-
treated CD3+T cells (5 3 105cells in 100 ml RPMI 1640 + 0.5% BSA)
were added to the upper chamber and allowed to migrate toward the lower
chambers, containing 600 ml RPMI + 0.5% BSA supplemented with hu-
man rCXCL12 (100 ng/ml), rCXCL16 (150 ng/ml), or rCCL27 (2 mg/ml).
A control condition, without chemokines, was included to determine
background migration. Following incubation for 2 h at 37˚C, cells in the
lower compartment were collected and counted using Countbright Abso-
lute Counting Beads (Molecular Probes). The percentage of migrating cells
was calculated as a ratio between the number of migrating cells and the
initial number of cells. Specific migration was calculated by subtracting
the mean number of spontaneously migrating cells (migration to medium
only) from the mean number of cells that migrated in response to the
Functional enrichment scores were calculated by the Benjamini-Hochberg
corrected Fischer’s exact test. All other statistical analysis was performed
using the Student t test. Significance was defined as p , 0.05.
TX527 regulates transcription of immune-related genes in
purified human CD3+T cells
Affymetrix GeneChip Arrays (Affymetrix) were performed on
peripheral blood CD3+T cells that were cultured as described in
Materials and Methods for 10 d in the presence or absence of
TX527. Of ∼54,000 probe sets analyzed, 936 (including 846 with
an assigned gene identifier) were differentially expressed in TX527-
treated T cells compared with vehicle-treated T cells (.1.5-fold
regulated; p , 0.01). Within this group, 473 (50.5%) showed an
increase in expression, and 463 (49.5%) showed a decrease in ex-
To provide evidence that our experimental system generated
reliable data, we first verified differential expression of VDR- and
known vitamin D-regulated genes among the two experimental
conditions. Asexpected, upregulationof VDRand direct vitaminD
target genes, such as 24-hydroxylase (known as CYP24A1) and
also carbonic anhydrase II (CA2), demonstrate a direct regulation
by ligand (TX527)-bound VDR in T cells (Supplemental Fig. 1A,
Table I). Differential gene expression of VDR and 24-hydroxylase
was confirmed by real-time RT-PCR (Supplemental Fig. 1B).
Separation of CD3+T cells into CD4+and CD8-enriched T cell
fractions prior to real-time RT-PCR analysis revealed a compara-
ble degree of TX527-mediated regulation of these genes in both
T cell subsets, suggesting that CD4+and CD8+T cells are equally
responsive to the analog (Fig. 1A). These results indicate that
this hypocalcemic vitamin D analog shares important metabolic-
related actions with the parent compound.
More importantly, TX527 also affected transcription of pre-
viously described immune-related vitamin D targets, such as IFN-
g, CCR10, and growth arrest and DNA damage-inducible a (Table
I), suggesting direct immunomodulatory actions on T cells and
adaptive immune responses. Ingenuity Pathway Analysis (Ingenu-
ity Systems, http://www.ingenuity.com), a literature-based online
annotation tool, was used to identify the interactions and bi-
ological significance of the affected genes. The data revealed a
preferential regulation of genes involved in cellular growth and
proliferation, cell death, cellular development, cellular movement,
and cell-to-cell signaling and interaction. These groups of genes
represented the top five biological networks most significantly
affected by the vitamin D analog (Fig. 1B, 1C, Supplemental Table
I). First, we identified targets involved in cell growth, prolifera-
tion, and cell death, already described for the parent compound,
1,25(OH)2D3. In addition to these described pathways, TX527
uniquely regulated the expression of a large set of genes (n = 140)
involved in cellular movement (Fig. 1D) and cell-to-cell signaling
of T cells.
TX527 inhibits proliferation of CD4+and CD8+T cells
We next investigated whether the transcriptional regulation of
genes involved in cell growth and proliferation by TX527 cor-
Table I. Microarray analysis reveals TX527-mediated gene regulation in purified human CD3+T cells
Gene Symbol Gene Title
by TX527 UniGene ID
Vitamin D target genes
Carbonic anhydrase II +2.9
Cytochrome P450, family 24, subfamily A, polypeptide 1
Vitamin D (1,25-dihydroxyvitamin D3) receptor
Growth arrest and DNA damage-inducible, a
Cytotoxic T-lymphocyte–associated protein 4
TNFR superfamily, member 4
Chemokine (C-C motif) receptor 10
Chemokine (C-X-C motif) receptor 3
Chemokine (C-X-C motif) receptor 6
Fucosyltransferase 7 [a (1, 3) fucosyltransferase]
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 a.
134TX527 MODULATES T CELL FUNCTION AND HOMING PROPERTIES
related with the functional ability to inhibit T cell proliferation
upon activation. The data indicated that, although CD4+and CD8+
T cells did proliferate at the end 10-d culture term, cell division of
both T cell populations was significantly inhibited by TX527 (Fig.
2). Importantly, there was no significant difference in the viability
of T cells cultured with TX527 compared with vehicle-treated
cells, indicating that the compound is not inherently toxic to
T cells at this concentration (data not shown).
TX527 differentially modulates cytokine profiles in CD4+and
CD8-enriched T cell subsets
As microarray analysis revealed a TX527-mediated regulation of
genes involved in cellular development, we analyzed the direct
effects of TX527 on T cell differentiation by measuring the cy-
tokine profile of activated human T cells postexposure to the vi-
tamin D analog. For this purpose, CD3+T cells were cultured as
described above, but further separated into a CD4+T cell frac-
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 (1028M) 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 6 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.
TX527 alters the transcriptional program of human T cells. Purified human CD3+T cells, isolated from whole blood samples of healthy
The Journal of Immunology 135
tion and a CD4-depleted fraction (consisting out of .85% CD8+
T cells) at the end of the culture term, allowing a specific analysis
of the cytokine profiles of the distinct T cell fractions.
Exposure of the cells to TX527 almost completely abrogated the
Th1 cytokine function of CD4+T cells, as demonstrated by the
near basal levels of IFN-g mRNA (Fig. 3A). In contrast to IFN-g,
CD4+control cells showed rather low levels of the Th2 and Th17
cytokines, IL-4, and IL-17, respectively. Nevertheless, a similar
TX527-mediated inhibition of IL-4 and IL-17 could be observed
(Fig. 3A). Importantly, TX527 did not completely inhibit T cell
cytokine production altogether because a specific upregulation of
the immunoregulatory cytokine IL-10 was observed within the
CD4+T cell subset (Fig. 3A), whereas expression of IL-2 and
TGF-b remained unchanged (data not shown). Analysis of CD8-
enriched T cell fractions revealed similar results for all cytokines
analyzed, except for IL-10, as TX527 treatment failed to upreg-
ulate the expression of this cytokine (Fig. 3B), indicating that the
IL-10–inducing capacity of this synthetic VDR-agonist is re-
stricted to CD4+T cells.
TX527 alters the activation status of CD4+and CD8+T cells
and promotes a Treg phenotype
We next investigated whether the TX527-mediated inhibition of
T cell proliferation and cytokine responses is in agreement with
a decreased activation status of the cells, because the microarray
data revealed decreased mRNA expression levels of the T cell
activation markers CD69 and CD40L in CD3+T cells exposed to
TX527 (Table I). To verify these inhibitory effects of TX527 on
T cell activation, surface expression levels of CD69 and CD40L
were measured by flow cytometry. By gating on the CD4+T cell
subset, the data revealed that, despite the 10-d time lapse since the
initial T cell activating signal, CD4+T cells from vehicle-treated
cell cultures still featured a substantial number of CD69+(36.1%)
and CD40L+T cells (26.8%) (Fig. 4A). In accordance with the
reduced proliferative capacity, TX527 prevented CD4+T cells to
become fully activated, because only a minimal proportion of the
cells stained positive for CD69 (21.0%; p , 0.0005) and CD40L
(7.5%; p , 0.05). Similar results for CD69 were obtained on the
gated CD8+T cell population (p , 0.05), whereas CD40L was
almost not expressed by this population (Fig. 4A).
Remarkably, microarray analysis revealed that TX527 enhanced
the expression of IL-2RA by human CD3+T cells, also known as
T cells. Purified human CD3+T cells were activated with anti-CD3/anti-
CD28 and repeatedly treated with vehicle (ctr) or TX527 (1028M) 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 (1028
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 cyto-
metric analysis was performed. The proliferating cell fraction was iden-
tified by a decrease of CFSE signal. Dead cells were excluded from
analysis using 7-AAD staining. Mean values 6 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
TX527 reduces the proliferative capacity of CD4+and CD8+
anti-CD3/anti-CD28 and treated with vehicle (ctr) or TX527 (1028M) 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-g, 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 6 SEM of one representative experiment out of two to four independently tested donors.
*p , 0.05 versus ctr. ND, not detected.
Cytokine responses in CD4+and CD8-enriched T cell fractions are differentially regulated by TX527. Human CD3+T cells, activated by
136 TX527 MODULATES T CELL FUNCTION AND HOMING PROPERTIES
CD25, another surface marker implicated in T cell activation
(Table I). Importantly, CD25 is, next to its status as an activation
marker, also a marker for the identification of human nTregs,
which were originally defined as CD4+CD25highT cells. Fur-
thermore, the gene expression profile of TX527-treated T cells
showed a decreased expression level of IL-7R, also known as
CD127 (Table I), a recently proposed surface marker that further
discriminates effector T cells and Tregs in the CD4+CD25high
T cell fraction, defining human nTregs as CD4+CD25high
CD127low. To investigate whether TX527 would increase the
emergence of such Tregs, surface expression of CD25 and CD127
on CD4+T cells was measured by flow cytometry. In addition to
the microarray data, TX527 significantly enhanced CD25 protein
expression (p , 0.00001), while reducing CD127 levels on CD4+
T cells p , 0.0005) (Fig. 4B, upper panel). Overall, a strong in-
crease in the percentage of CD4+CD25highCD127lowT cells after
chronic exposure to TX527 was observed (4.8% in vehicle-treated
versus 20.9% in TX527-treated cells; p , 0.005) (Fig. 4B, lower
panel). Together, these data indicate that TX527 diminishes T cell
activation while favoring the emergence of T cells with a regula-
TX527 promotes Treg conversion from naive T cells with
functional suppressive capacity
To assess whether the increased abundance of CD4+CD25high
CD127lowT cells upon TX527 treatment resulted from an ex-
pansion of pre-existing nTregs or, instead, Treg conversion of
naive CD4+T cells was promoted by the analog, the starting
population was depleted of nTregs prior to exposure to the analog
(Fig. 5A). TX527 treatment of nTreg-depleted T cells induced the
appearance of the characteristic CD4+CD25highCD127lowpop-
ulation at the end of the culture period, suggesting that the analog
induces a Treg phenotype rather than expanding nTregs already
present in the starting culture (Fig. 5A).
To verify whether the TX527-induced Tregs exhibit functional
capacity to suppress T cell responses, we tested their ability to
CD4+CD25+T cell fractions, isolated from 10-d cultured vehicle-
or TX527-treated T cells, were cocultured with Cell Proliferation
Dye-labeled CD4+CD252responder T cells in a standard sup-
pression assay. T cell-depleted PBMCs and anti-CD3 provided the
costimulatory and TCR stimuli, respectively. Of note, in line with
the ability of TX527 to promote a Treg phenotype from naive
T cells, clear elevatedyields ofCD4+CD25+T cells were recovered
from TX527-treated cultures (7.3% from vehicle-treated versus
TX527-conditioned CD4+CD25+T cells effectively prevented full
activation of effector T cells as evidenced by the reduced expres-
sion of early activation marker, CD69, on labeled responder T cells
(72.1% on activated responder T cells alone versus 60.4% in the
presence of TX527 suppressors; p , 0.05) (Fig. 5B). Corroborating
these findings, activation-induced responder T cell proliferation
was inhibitedwhencultured inthe presenceofTX527 CD4+CD25+
T cells (72.8% proliferation of responder T cells alone versus
25.2% in the presence of TX527 suppressors; p , 0.05) (Fig. 5C).
However, although the vitamin D analog promoted elevated Treg
numbers displaying functional activity, TX527-conditioned T cells
did not exhibit a superior suppressive function, because equal cell
numbers of control CD4+CD25+T cells showed a comparable
ability to reduce expression of early activation markers and pro-
liferation of responder T cells (Fig. 5B, 5C, lower panels).
In an attempt to unravel the mechanism by which TX527-
induced Tregs are able to suppress effector T cell responses, we
first tested their ability to produce secreted regulatory mediators.
As IL-10 represents an important hallmark cytokine of various
Treg subsets, we investigated whether the TX527-induced CD4+
CD25highCD127lowfraction was responsible for the increased IL-
10 mRNA levels observed in the total CD4+T cell fraction upon
exposure to the analog (Fig. 3A). CD4+T cells derived from
vehicle-treated and TX527-treated cell cultures were sorted into
a CD4+CD25highCD127lowfraction (Tregfraction) and a remaining
(non-Treg) fraction. FACS-sorted Treg fractions of TX527-treated
T cells displayed elevated IL-10 mRNA levels compared with
control Treg counterparts (Fig. 5D). However, also the TX527-
treated non-Treg fractions featured increased IL-10 copy num-
bers compared with the control non-Tregs (data not shown).
Thus, the ability of TX527 to trigger IL-10 production could not
be exclusively linked to the observed CD4+CD25highCD127lowcell
fraction. In the same line of investigation, we verified TX527-
mediated regulation of other Treg-associated molecules, includ-
ing OX40, ICOS, GITR, FOXP3, and CTLA-4, as possible con-
tributors to the suppressive activity of the cells. Flow cytometric
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
CD25highand CD127lowon the CD4+T cell population. The results are
representative of three to eight independent replicates performed on dif-
TX527 modulates surface expression of T cell activation
The Journal of Immunology 137
analysis of the CD4+T cell population revealed that continuous
exposure to TX527 resulted in the inhibition of GITR expression,
whereas FOXP3 and ICOS levels remained unaffected by the
treatment. In contrast, the clear elevated expression of CTLA-4 and
OX40 by CD4+T cells upon exposure to the analog may implicate
them as possible mediators in the suppressive function of TX527
Tregs (Fig. 5E).
TX527 induces a migratory signature in T cells specific for
homing to skin and sites of inflammation
Next to the effects of TX527 on T cell activation, proliferation, and
differentiation, a significant overrepresentation of genes involved
in cellular movement (n = 140; Fig. 1D) was found among the set
of differentially expressed genes. Remarkably, this group of cel-
lular movement genes comprised 17 genes directly acting in the
chemokine signaling pathway (Kyoto Encyclopedia of Genes and
Genomes [KEGG]: 04062) (Fig. 6A). Furthermore, also various
genes participating in other aspects of cell motility were found to
be affected by TX527, including members of the pathways regu-
lating focal adhesion (n = 11; KEGG: 04510), cell adhesion
molecules (n = 10; KEGG: 04514), and actin cytoskeleton (n = 9;
Togain insightinto the roleof TX527as apotentialmodulator of
T cell movement, we first analyzed the surface expression levels of
a broad range of homing receptors. Our search comprised a se-
lection of those receptors affected by TX527 at the mRNA level
(Fig. 6A, Table I), as well as a large set of other tissue- or envi-
homing receptors (CCR4, CCR6, CCR10, and CLA), receptors
regulating T cell trafficking to the gut (integrin b7and CCR9),
homing receptors for secondary lymphoid organs (L-selectin/
CD62L, CCR7, and CXCR4), and receptors directing T cells to
sites of inflammation (CCR5, CXCR3, and CXCR6) (Fig. 6B). In
accordance with data reported for 1,25(OH)2D3, TX527 condi-
tioning of the cells induced expression of CCR10 on CD4+(p ,
0.001) and CD8+T cells (p , 0.005), whereas expression of CLA
showed a decreasing trend, which was most pronounced in CD8+
T cells (CD4+: NS; CD8+: p , 0.05). Our data revealed another
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+CD25highCD127lowTregs 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+CD252
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+CD252responders 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+CD25highCD127lowT cell population. Data shown
illustrate relative IL-10 mRNA levels (mean 6 SEM) of FACS-sorted CD4+CD25highCD127lowTX527-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.
TX527 drives de novo conversion of naive T cells into functional Tregs. A, TX527-mediated induction of CD4+CD25highCD127lowTregs
138TX527 MODULATES T CELL FUNCTION AND HOMING PROPERTIES
TX527-responsive skin-homing receptor, as the analog strongly
elevated the number of cells expressing CCR4 (p , 0.05), espe-
cially within the CD8+T cell compartment, whereas expression of
CCR6, another receptor proposed to be involved in skin homing,
was not altered by TX527. Gut-homing receptors CCR9 and
integrin b7were not affected by the analog. Of interest, TX527
impaired the expression of receptors involved in T cell homing
to secondary lymphoid organs within CD4+and CD8+T cell pop-
ulations, including CD62L (CD4+: p , 0.0005; CD8+: p , 0.005),
CCR7 (CD4+: p , 0.02; CD8+: p = 0.05), and CXCR4 (CD4+: p ,
0.01; CD8+: NS). Instead, TX527 uniquely triggered elevated ex-
pression of multiple chemokine receptors guiding T cells to sites of
inflammation in both T cell subsets, including CCR5 (CD4+: p ,
0.01; CD8+: p , 0.05), CXCR3 (CD4+: p , 0.001; CD8+: p ,
0.05), and CXCR6 (CD4+: p , 0.005; CD8+: p , 0.05).
Next, the functional relevance of the unique chemokine receptor
profile was assessed by measuring the chemotactic response of
TX527-conditioned CD3+T cells to three chemokines: CXCL12,
mokineligands.Tcells,activated byanti-CD3/anti-CD28andculturedin thepresenceofTX527orvehicle,werescreenedforalteredexpressionofgenesand
from low (green) to high expression(red), of thosegenes actingin 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+subsetsof vehicle-treated (solidlines) versus TX527-treated (dashedlines) T cells depict surface expression ofreceptors directingT cells to
skin (CCR4, CCR6, CCR10, and CLA), gut (CCR9 and integrin b7), secondary lymphoid organs (CD62L, CCR7, and CXCR4), or to inflammatory sites
(CCR5,CXCR3,and CXCR6). 7-AADwasused to exclude deadcells from analysis.The resultsare representativeof threeto eight independent experiments.
(150 ng/ml), and CCL27 (2 mg/ml) was investigated using a transwell tissue culture system (with a 3-mm 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.
TX527 imprints T cells with a homing receptor signature specific for migration to inflammatory sites and alters their responsiveness to che-
The Journal of Immunology139
CXCL16, and CCL27, being the ligands for CXCR4 (secondary
lymphoid organ homing), CXCR6 (homing to inflammatory sites),
and CCR10 (skin-homing), respectively. Cells that had been pre-
exposed to TX527 exhibited a substantial decrease in their mi-
gratory capacity to the chemokine CXCL12, whereas chemotaxis
toward CXCL16 and CCL27 was enhanced compared with
vehicle-treated T cells (Fig. 6C). Overall, these data demonstrate
that TX527 dramatically alters the migratory behavior of human
T cells in response to chemokine ligands by modulating their
homing receptor signature, as well as by targeting important sig-
naling pathways underlying cellular movement processes.
Analogs of vitamin D have been developed to allow in vivo ex-
ploitation of the anticancer and immunomodulatory effects of
vitamin D with reduced calcemic side effects (23). Vitamin D
affects the immune system in a wide variety of ways, and in an-
imal models, the therapeutic potential of analogs in the modula-
tion of graft survival or autoimmunity has been demonstrated (2).
The main target for vitamin D and its analogs is thought to be the
DC, thereby indirectly altering T cell responses, but effects in-
volving direct modulation of T cell responses by these compounds
remain less well characterized. In this report, we investigated the
ability of a hypocalcemic vitamin D analog, TX527, to directly
interfere with proximal pathways involved in human T cell acti-
vation at a transcriptional, phenotypical, as well as functional level.
We demonstrated that the synthetic VDR agonist exerted inhibitory
actions on T cell proliferation and effector cytokine production,
functions that are shared with the parent compound. However,
TX527 also exclusively triggered the induction of a functional
CD4+CD25highCD127lowTreg population in the absence of any
additional immunoregulatory compounds and imprinted T cells
with a migratory signature, which would target them to sites of
In alignment with the functional ability of VDR agonists to
inhibit the proliferative capacity of human CD4+and CD8+T cells,
as shown in this study with the hypocalcemic vitamin D analog
TX527 and by others using the parent compound (9, 24–26), the
genome-wide screening revealed that 199 genes involved in T cell
growth and survival were regulated by TX527. In addition, ex-
posure of T cells to TX527 decreased their activation status, as
evidenced by TX527-mediated suppression of common activation
markers such as CD69 and CD40L. Furthermore, the synthetic
agonist also effectively inhibited Th1, Th17, as well as Th2 re-
activity of the cells. Up to now, varying results regarding the direct
effectsofactivevitaminD metaboliteson T cell cytokineresponses
have been reported, with a majority of studies showing inhibitory
effects on Th1 (IFN-g) and Th17 responses (IL-17), whereas Th2
(IL-4) was mostly shown to be stable or increased under un-
polarized conditions in studies using mouse T cells (11–13) or
human T cells (14, 26–29). However, in our hands, triggering of
VDR signaling by the hypocalcemic vitamin D analog TX527
resulted in an overall inhibition of T effector cytokine responses,
including Th2 cytokines.
Importantly, TX527 did not paralyze overall T cell function, as
evidenced by a specific elevation of the regulatory cytokine IL-10
within the CD4+T cell subset. The potent and broad-spectrum
tolerogenic functions of this cytokine have unequivocally been
established in various models of infection, inflammation, and
cancer (30), and modulation of IL-10 might therefore contribute to
the immunomodulatory effects of VDR agonists. Previous in vitro
studies revealed that 1,25(OH)2D3enhanced IL-10 secretion by
DCs (5), as well as CD4+T cells as supported in this study and by
others (11, 14, 31). Also in vivo, IL-10 signaling was identified as
an essential component for 1,25(OH)2D3-mediated inhibition of
experimental autoimmune encephalomyelitis (32). Interestingly,
in an experimental setup comparable to ours, a combination of
1,25(OH)2D3and dexamethasone was shown to induce IL-10–
producing CD4+Tregs in vitro, which were able to suppress au-
toimmune demyelination invivo in an Ag-specific way (27, 31). In
accordance with our data, these induced Tregs did not produce
IFN-g and IL-4, nor did they express Foxp3 (27, 33).
The concept of VDR agonists favoring the development of
T cells with regulatory properties is further strengthened by the
presence of CD4+CD25highCD127lowT cells upon TX527 treat-
ment. Our data suggest the de novo conversion of naive CD4+
T cells into CD4+CD25highCD127lowT cells rather than expansion
of existing Tregs, because depletion of CD4+CD25+cells from the
starting culture still reproduced the characteristic Treg phenotype
following TX527 treatment. More importantly, TX527-induced
Tregs exhibited the functional capacity to suppress activation as
well as proliferation of effector T cells. However, although the
analog clearly triggered increased Treg numbers, their suppressive
functions remained comparable to that of CD4+CD25+T cells
isolated from control T cell cultures. Nevertheless, TX527-induced
Tregs featured elevated levels of regulatory mediators such as
IL-10 as well CTLA-4 and OX40, which might be relevant for
increased suppressive function in more relevant in vivo settings.
Different groups have previously reported increased numbers of
CD4+CD25+and/or CD4+CD25+Foxp3+Tregs upon 1,25(OH)2D3
or analog treatment in vivo (34, 35). Until now, this effect is pro-
posed to rely on the ability of VDR agonists to induce myeloid DCs
with tolerogenic properties, because in vitro 1,25(OH)2D3-treated
DCs favor the emergence of CD4+FOXP3+Tregs (7). The results
presented in this study strongly indicate that TX527 can directly
modulate T cell signaling pathways to promote a CD4+CD25high
CD127lowTreg phenotype, underlining the capacity of this agonist
to affect T cell responses in the absence of APCs or APC-derived
factors. In support of our data, others have reported upregulation of
CD25 on human T cells when simultaneously exposed to IL-2 and
1,25(OH)2D3(36). In addition, a recent study demonstrated the
ability of 1,25(OH)2D3, acting in synergism with IL-2, to induce
IL-10–producing CD4+FOXP3+CTLA-4+Tregs from human pu-
rified CD4+peripheral blood T cells in vitro (14). The fact that we
did not observe FOXP3 induction at day 10 could possibly be
linked to its transient expression pattern, peaking at 4 d and de-
creasing thereafter, as shown in the presence of 1,25(OH)2D3(14).
Also important to note here is that the Tregs induced by 1,25
(OH)2D3and IL-2 that were implemented for demonstration of
suppressive function were originally activated in the presence of
monocytes at the start of the culture (14). This is in sharp contrast
with TX527-conditioned Tregs, which were never in contact with
APCs during the initial 10-d culture term. Considering the im-
portance of costimulatory signals for Treg homeostasis and func-
tion (37), these crucial differences in experimental setup in the
above-mentioned study and the current one may explain the fail-
ure of the analog to promote FOXP3 induction and superior sup-
Besides the ability to induce functional Tregs, TX527 also
preferentially regulated 140 genes involved in cellular movement,
representing one of the top five biological processes most signifi-
cantly affected by TX527. Several of them encode chemokine
receptors that are not uniquely involved in the regulation of T cell
homing tocutaneoussites, stronglyarguingforamuchbroader role
agreement with previous studies (38, 39), our results demonstrated
a similar TX527-mediated regulation of CCR10 and CLA. In
addition, we identified the skin-homing receptor CCR4 as an
140TX527 MODULATES T CELL FUNCTION AND HOMING PROPERTIES
important target for TX527, especially in CD8+T cells. Recently,
CCR4+CD8+T cells were identified as an immature memory
T cell subset in the differentiation pathway of human CD8+
T cells, which have the ability to migrate to inflammatory sites in
the skin, where they are involved in cutaneous immunity (40).
TX527 uniquely impaired the expression of receptors involved in
T cell homing to secondary lymphoid organs, including CD62L,
CCR7, and CXCR4. In support of our data, Topilski et al. (41)
demonstrated a reduced chemotactic capacity of 1,25(OH)2D3-
treated CD4+T cells toward CXCL12 and CCL21, the ligands of
CXCR4 and CCR7, respectively. In accordance, transfer of CD4+
T cells to 1,25(OH)2D3-treated mice resulted in a reduced lymph
node homing capacity of these cells as compared with control
mice, revealing a mechanism by which this hormone prevents
T cell recruitment and thus subsequent activation in the lymph
nodes. Taken together, our data suggest that the homing receptor
profile, induced by TX527 after T cell activation, favors T cell
egress from secondary lymphoid organs while promoting their
migration to inflammatory sites (42), as demonstrated by the el-
evated levels of inflammation-homing receptors such as CCR5,
CXCR3, and CXCR6, as well as by altered chemotactic responses
toward CXCL12 (the ligand for the lymph node-homing receptor
CXCR4) and CXCL16 (the ligand for the inflammation homing
receptor CXCR6). The data obtained in this study provide im-
portant additional insights into the immunomodulatory actions of
TX527. Nevertheless, in vivo, environmental signals might further
shape the homing receptor profiles induced by VDR agonists. For
example, induction of CCR6+CCR10+Th22 cells was observed by
1,25(OH)2D3, but only when applied in combination with TNF-a
and IL-6 or with plasmacytoid DCs (43).
In summary, our data show that the hypocalcemic vitamin D
analog TX527 directly targets T lymphocytes, thereby inhibiting
effector T cell functions while inducing increased numbers of
functional CD4+CD25highCD127lowT cells. In addition, TX527
imprints T cells with a unique homing signature, favoring their
migration to sites of inflammation. Migrationofsuppressorcellsto
without affecting the ability of the host to combat systemic infec-
tions, is one of the major challenges in combating diseases with
unwanted or uncontrolled immune responses. Considering the
TX527-induced alterationsinhumanT cell functionandphenotype
described in this paper, exploitation of the immunomodulatory
effects of this potent vitamin D analog may open therapeutic pos-
sibilities for many inflammatory and autoimmune disorders.
We thank Wim Cockx, Jos Depovere, Nursen Gol, Ame ´lie Fossoul, Eefje
Verdrengh, and Dirk Valckx for excellent technical assistance.
The authors have no financial conflicts of interest.
1. Adams, J. S., and M. Hewison. 2008. Unexpected actions of vitamin D: new
perspectives on the regulation of innate and adaptive immunity. Nat. Clin. Pract.
Endocrinol. Metab. 4: 80–90.
2. Baeke, F., E. van Etten, C. Gysemans, L. Overbergh, and C. Mathieu. 2008.
Vitamin D signaling in immune-mediated disorders: Evolving insights and
therapeutic opportunities. Mol. Aspects Med. 29: 376–387.
3. Adorini, L., and G. Penna. 2009. Dendritic cell tolerogenicity: a key mechanism
in immunomodulation by vitamin D receptor agonists. Hum. Immunol. 70: 345–
4. Adorini, L., G. Penna, N. Giarratana, A. Roncari, S. Amuchastegui, K. C. Daniel,
and M. Uskokovic. 2004. Dendritic cells as key targets for immunomodulation
by Vitamin D receptor ligands. J. Steroid Biochem. Mol. Biol. 89-90: 437–441.
5. Penna, G., and L. Adorini. 2000. 1 Alpha,25-dihydroxyvitamin D3 inhibits
differentiation, maturation, activation, and survival of dendritic cells leading to
impaired alloreactive T cell activation. J. Immunol. 164: 2405–2411.
6. van Halteren, A. G., E. van Etten, E. C. de Jong, R. Bouillon, B. O. Roep, and
C. Mathieu. 2002. Redirection of human autoreactive T-cells Upon interaction
with dendritic cells modulated by TX527, an analog of 1,25 dihydroxyvitamin D
(3). Diabetes 51: 2119–2125.
7. Penna, G., A. Roncari, S. Amuchastegui, K. C. Daniel, E. Berti, M. Colonna, and
L. Adorini. 2005. Expression of the inhibitory receptor ILT3 on dendritic cells
is dispensable for induction of CD4+Foxp3+ regulatory T cells by 1,25-
dihydroxyvitamin D3. Blood 106: 3490–3497.
8. Veldman, C. M., M. T. Cantorna, and H. F. DeLuca. 2000. Expression of 1,25-
dihydroxyvitamin D(3) receptor in the immune system. Arch. Biochem. Biophys.
9. Rigby, W. F., T. Stacy, and M. W. Fanger. 1984. Inhibition of T lymphocyte
mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol). J. Clin. Invest. 74: 1451–
10. Bhalla, A. K., E. P. Amento, B. Serog, and L. H. Glimcher. 1984. 1,25-Dihy-
droxyvitamin D3 inhibits antigen-induced T cell activation. J. Immunol. 133:
11. Boonstra, A., F. J. Barrat, C. Crain, V. L. Heath, H. F. Savelkoul, and A. O’Garra.
2001. 1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(+)
T cells to enhance the development of Th2 cells. J. Immunol. 167: 4974–4980.
12. Staeva-Vieira, T. P., and L. P. Freedman. 2002. 1,25-dihydroxyvitamin D3
inhibits IFN-gamma and IL-4 levels during in vitro polarization of primary
murine CD4+ T cells. J. Immunol. 168: 1181–1189.
13. Mahon, B. D., A. Wittke, V. Weaver, and M. T. Cantorna. 2003. The targets of
vitamin D depend on the differentiation and activation status of CD4 positive
T cells. J. Cell. Biochem. 89: 922–932.
14. Jeffery, L. E., F. Burke, M. Mura, Y. Zheng, O. S. Qureshi, M. Hewison,
L. S. Walker, D. A. Lammas, K. Raza, and D. M. Sansom. 2009. 1,25-Dihy-
droxyvitamin D3 and IL-2 combine to inhibit T cell production of inflammatory
cytokines and promote development of regulatory T cells expressing CTLA-4
and FoxP3. J. Immunol. 183: 5458–5467.
15. Mortensen, J. T., J. Lichtenberg, and L. Binderup. 1996. Toxicity of 1,25-
dihydroxyvitamin D3, tacalcitol, and calcipotriol after topical treatment in rats.
J. Investig. Dermatol. Symp. Proc. 1: 60–63.
16. Van Etten, E., B. Decallonne, L. Verlinden, A. Verstuyf, R. Bouillon, and
C. Mathieu. 2003. Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent
immunomodulators. J. Cell. Biochem. 88: 223–226.
17. Baeke, F., H. Korf, L. Overbergh, E. van Etten, A. Verstuyf, C. Gysemans, and
C. Mathieu. 2010. Human T lymphocytes are direct targets of 1,25-dihydrox-
yvitamin D3 in the immune system. J. Steroid Biochem. Mol. Biol. 121: 221–
18. Freudenberg, J. M., V. K. Joshi, Z. Hu, and M. Medvedovic. 2009. CLEAN:
CLustering Enrichment ANalysis. BMC Bioinformatics 10: 234–249.
19. Overbergh, L., A. Giulietti, D. Valckx, R. Decallonne, R. Bouillon, and
C. Mathieu. 2003. The use of real-time reverse transcriptase PCR for the
quantification of cytokine gene expression. J. Biomol. Tech. 14: 33–43.
20. Giulietti, A., L. Overbergh, D. Valckx, B. Decallonne, R. Bouillon, and
C. Mathieu. 2001. An overview of real-time quantitative PCR: applications to
quantify cytokine gene expression. Methods 25: 386–401.
21. Stoffels, K., L. Overbergh, A. Giulietti, L. Verlinden, R. Bouillon, and C. Mathieu.
2006. Immune regulation of 25-hydroxyvitamin-D3-1alpha-hydroxylase in human
monocytes. J. Bone Miner. Res. 21: 37–47.
22. Bullens, D. M., E. Truyen, L. Coteur, E. Dilissen, P. W. Hellings, L. J. Dupont,
and J. L. Ceuppens. 2006. IL-17 mRNA in sputum of asthmatic patients: linking
T cell driven inflammation and granulocytic influx? Respir. Res. 7: 135–144.
23. Bouillon, R., A. Verstuyf, L. Verlinden, G. Eelen, and C. Mathieu. 2003. Pros-
pects for vitamin D receptor modulators as candidate drugs for cancer and (auto)
immune diseases. Recent Results Cancer Res. 164: 353–356.
24. Rigby, W. F., B. Yirinec, R. L. Oldershaw, and M. W. Fanger. 1987. Comparison
of the effects of 1,25-dihydroxyvitamin D3 on T lymphocyte subpopulations.
Eur. J. Immunol. 17: 563–566.
25. Lemire, J. M., J. S. Adams, V. Kermani-Arab, A. C. Bakke, R. Sakai, and
S. C. Jordan. 1985. 1,25-Dihydroxyvitamin D3 suppresses human T helper/
inducer lymphocyte activity in vitro. J. Immunol. 134: 3032–3035.
26. Correale, J., M. C. Ysrraelit, and M. I. Gaita ´n. 2009. Immunomodulatory effects
of Vitamin D in multiple sclerosis. Brain 132: 1146–1160.
27. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul,
R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, and A. O’Garra. 2002.
In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is
induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)-
and Th2-inducing cytokines. J. Exp. Med. 195: 603–616.
28. Borgogni, E., E. Sarchielli, M. Sottili, V. Santarlasci, L. Cosmi, S. Gelmini,
A. Lombardi, G. Cantini, G. Perigli, M. Luconi, et al. 2008. Elocalcitol inhibits
inflammatory responses in human thyroid cells and T cells. Endocrinology 149:
29. Bartels, L. E., S. P. Jørgensen, J. Agnholt, J. Kelsen, C. L. Hvas, and
J. F. Dahlerup. 2007. 1,25-dihydroxyvitamin D3 and dexamethasone increase
interleukin-10 production in CD4+ T cells from patients with Crohn’s disease.
Int. Immunopharmacol. 7: 1755–1764.
30. Mosser, D. M., and X. Zhang. 2008. Interleukin-10: new perspectives on an old
cytokine. Immunol. Rev. 226: 205–218.
31. Urry, Z., E. Xystrakis, D. F. Richards, J. McDonald, Z. Sattar, D. J. Cousins,
C. J. Corrigan, E. Hickman, Z. Brown, and C. M. Hawrylowicz. 2009. Ligation of
The Journal of Immunology 141
TLR9 induced on human IL-10-secreting Tregs by 1alpha,25-dihydroxyvitamin D3
abrogates regulatory function. J. Clin. Invest. 119: 387–398.
32. Spach, K. M., F. E. Nashold, B. N. Dittel, and C. E. Hayes. 2006. IL-10 signaling
is essential for 1,25-dihydroxyvitamin D3-mediated inhibition of experimental
autoimmune encephalomyelitis. J. Immunol. 177: 6030–6037.
33. Vieira, P. L., J. R. Christensen, S. Minaee, E. J. O’Neill, F. J. Barrat, A. Boonstra,
T. Barthlott, B. Stockinger, D. C. Wraith, and A. O’Garra. 2004. IL-10-secreting
regulatory T cells do not express Foxp3 but have comparable regulatory function
to naturally occurring CD4+CD25+ regulatory T cells. J. Immunol. 172: 5986–
34. Gregori, S., N. Giarratana, S. Smiroldo, M. Uskokovic, and L. Adorini. 2002. A
1alpha,25-dihydroxyvitamin D(3) analog enhances regulatory T-cells and arrests
autoimmune diabetes in NOD mice. Diabetes 51: 1367–1374.
35. Ghoreishi, M., P. Bach, J. Obst, M. Komba, J. C. Fleet, and J. P. Dutz. 2009.
Expansion of antigen-specific regulatory T cells with the topical vitamin
d analog calcipotriol. J. Immunol. 182: 6071–6078.
36. Rigby, W. F., B. J. Hamilton, and M. G. Waugh. 1990. 1,25-Dihydroxyvitamin
D3 modulates the effects of interleukin 2 independent of IL-2 receptor binding.
Cell. Immunol. 125: 396–414.
37. Qu, Y., and Y. Zhao. 2007. Regulatory CD4(+)CD25(+) T-cells are controlled by
multiple pathways at multiple levels. Int. Rev. Immunol. 26: 145–160.
38. Sigmundsdottir, H., J. Pan, G. F. Debes, C. Alt, A. Habtezion, D. Soler, and
E. C. Butcher. 2007. DCs metabolize sunlight-induced vitamin D3 to ‘program’
T cell attraction to the epidermal chemokine CCL27. Nat. Immunol. 8: 285–293.
39. Yamanaka, K., C. J. Dimitroff, R. C. Fuhlbrigge, M. Kakeda, I. Kurokawa,
H. Mizutani, and T. S. Kupper. 2008. Vitamins A and D are potent inhibitors of
cutaneous lymphocyte-associated antigen expression. J. Allergy Clin. Immunol.
121: 148–157, e3.
40. Kondo, T., and M. Takiguchi. 2009. Human memory CCR4+CD8+ T cell subset
has the ability to produce multiple cytokines. Int. Immunol. 21: 523–532.
41. Topilski, I., L. Flaishon, Y. Naveh, A. Harmelin, Y. Levo, and I. Shachar. 2004.
The anti-inflammatory effects of 1,25-dihydroxyvitamin D3 on Th2 cells in vivo
are due in part to the control of integrin-mediated T lymphocyte homing. Eur. J.
Immunol. 34: 1068–1076.
42. D’Ambrosio, D., P. Panina-Bordignon, and F. Sinigaglia. 2003. Chemokine
receptors in inflammation: an overview. J. Immunol. Methods 273: 3–13.
43. Duhen, T., R. Geiger, D. Jarrossay, A. Lanzavecchia, and F. Sallusto. 2009.
Production of interleukin 22 but not interleukin 17 by a subset of human skin-
homing memory T cells. Nat. Immunol. 10: 857–863.
142TX527 MODULATES T CELL FUNCTION AND HOMING PROPERTIES