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![Page 1: Hypothesis: Human Umbilical Cord Blood-Derived Stromal Cells Regulate the Foxp3 Expression of Regulatory T Cells Through the TGF-β1/Smad3 Pathway](https://reader030.fdocument.org/reader030/viewer/2022020603/5750702b1a28ab0f07d3b707/html5/thumbnails/1.jpg)
ORIGINAL PAPER
Hypothesis: Human Umbilical Cord Blood-Derived Stromal CellsRegulate the Foxp3 Expression of Regulatory T Cells Throughthe TGF-b1/Smad3 Pathway
Cheng Zhang • Xi Zhang • Xing-Hua Chen
Published online: 13 December 2011
� Springer Science+Business Media, LLC 2011
Abstract Despite the improvements in transplant immu-
nology and clinical and supportive care, graft-versus-host
disease (GVHD) is still among the most common causes of
overall mortality and morbidity after allogeneic hemato-
poietic cell transplantation. The development and severity
of GVHD are strongly related with post-transplant out-
comes. New strategies should be explored to overcome
GVHD. Regulatory T cells (Treg cells), as dedicated sup-
pressors of diverse immune responses and inflammation
and important gatekeepers of immune homeostasis, con-
tribute to the prevention of graft rejection and induce
transplantation tolerance. Foxp3, a transcription factor, is
predominantly expressed in Treg cells and is a master
regulator of the development and function of Treg cells.
Foxp3 mutations and Foxp3 deficiency lead to lethal
autoimmune lymphoproliferative disease, which results
from a defect in Treg cells. TGF-b1 is required to maintain
Foxp3 expression in Treg cells. We isolated a novel pop-
ulation from among CD34? cells in our laboratory, referred
to as human umbilical cord blood-derived stromal cells
(hUCBDSCs), which exert an immunosuppressive effect
and can notably increase Foxp3 expression in Treg cells.
Our previous study also revealed that hUCBDSCs con-
stantly secrete TGF-b1. Based on the literature searchings
and our experimental findings, we hypothesize that
hUCBDSCs, which secrete a high level of TGF-b1,
modulate the Foxp3 expression of Treg cells through the
TGF-b1/Smad3 pathway to regulate GVHD.
Keywords Human umbilical cord blood-derived stromal
cells � Foxp3 regulatory T cells � TGF-b1/Smad3
Abbreviations
allo-HSCT Allogeneic hematopoietic cell transplantation
GVHD Graft-versus-host disease
hUCBDSCs Human umbilical cord blood-derived
stromal cells
Treg cells Regulatory T cells
IPEX syndrome Immune dysfunction/polyendocrinopathy/
enteropathy/x-linked syndrome
Role of Treg Cells in Transplantation Tolerance
It has been shown that Regulatory T cells (Treg cells) play a
pivotal role in transplantation tolerance [1]. Several studies
have implicated Treg cells in the maintenance of trans-
plantation tolerance to donor antigens [2–6]. The removal of
Treg cells from normal mice enhanced graft rejection (i.e.,
reduced the survival of the grafts) [7]. Conversely, when
Treg cells enriched from normal syngeneic mice were
introduced, together with naive T cells, into syngeneic T
cell-deficient mice with allografts, graft survival was sig-
nificantly prolonged [8]. In the absence of the Treg cell
subset, various treatments fail to induce allograft tolerance
and the suppressive effects mediated by tolerant lympho-
cytes in adoptive transfer systems are neutralized [2, 5].
Role of Foxp3 in Treg Cells
Foxp3, a transcription factor, is predominantly expressed in
CD4?CD25? Treg cells and is a master regulator of the
development and function of Treg cells [9–11]. Continued
C. Zhang � X. Zhang � X.-H. Chen (&)
Department of Hematology, Xinqiao Hospital,
The Third Military Medical University,
Chongqing 400037, People’s Republic of China
e-mail: [email protected]
123
Cell Biochem Biophys (2012) 62:463–466
DOI 10.1007/s12013-011-9328-8
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Foxp3 expression in mature Treg cells is required to
maintain the transcriptional and functional program
established during Treg cell development [12]. Foxp3-
mutant scurfy mice and Foxp3-deficient mice display lethal
autoimmune lymphoproliferative disease, which results
from a defect in CD4?CD25? Treg cells [10, 11]. Muta-
tions of the FOXP3 gene in humans are responsible for a
severe autoimmune disease, called IPEX syndrome (Immune
dysfunction/Polyendocrinopathy/Enteropathy/X-linked syn-
drome) [13, 14]. Dramatically, when Foxp3 was ectopically
expressed via retroviral gene transfer, non-Treg cells in mice
and humans acquired a Treg cell phenotype similar to that of
naturally occurring Treg cells [15].
Stable Foxp3 expression is clearly a prerequisite for the
maintenance of the transcriptional and functional program
established during Treg cell development. As the result of
the deletion of Foxp3, immature Treg cells lost their sup-
pressive function in vivo [12]. Foxp3 amplifies and fixes
pre-established molecular features of Treg cells and
solidifies Treg cell lineage stability [16].
Regulation of Foxp3 Expression of Treg Cells
Given the importance of Foxp3 in the development and
function of Treg cells, it is essential to understand how
Foxp3 expression is controlled. Recently, many studies
have focused on the factors that control the Foxp3
expression and attempted to explain the mechanisms
underlying the transcriptional regulation of the Foxp3
locus. Foxp3 expression is regulated at the molecular level
by factors such as NFAT, Smad, and STAT. However, the
TGF-b/Smad pathway is the most important pathway [17].
TGF-b is a member of a family of dimeric polypeptide
growth factors that functions through Smad-dependent and
Smad-independent pathways [18]. Treg cells suppress the
proliferation of CD4?CD25- T cells; however, such sup-
pression is abolished by the presence of anti-TGF-b. In
addition, Treg cells suppress B cell immunoglobulin pro-
duction and anti-TGF-b again abolishes such suppression.
Stimulated Treg cells but not CD4?CD25- T cells express
high and persistent levels of TGF-b1 on the cell surface.
This expression pattern strongly suggests that Treg cells
exert immunosuppression via a cell–cell interaction
involving cell surface TGF-b1 [19]. Cell membrane-bound
TGF-b can deliver a regulatory signal to target cells via a
contact-dependent process [20]. Not only do Treg cells
express latent TGF-b [21], but these cells also bear TGF-bin its active configuration on the cell surface. TCR
engagement augments the expression of both TGF-b and
TbRII, enhancing this self-inflicted anergy. Suppressor
cell-derived TGF-b can bind to activated responder cells
and transduce contact-dependent suppressive signals
through a heteromeric complex consisting of type I (RI)
and type II (RII) receptor components [20].
It has been demonstrated that TGF-b induces Foxp3
expression in naı̈ve CD4?CD25- T cells from TCR
transgenic mice with a RAG-/- background (which lack
Foxp3? T cells) [22]. The notion that TGF-b signaling is
required for the induction of Foxp3 expression is also
supported by the fact that CD4?CD25- T cells deficient in
TGF-b signaling could not be converted into Foxp3? Treg
cells in vitro or in vivo [23]. In humans, FOXP3 expression
could also be induced in CD4?CD25- T cells by TGF-b[24, 25].
It should be noted that Smad3 binds to the intronic
enhancer 1 of Foxp3 locus and that this binding is required
for histone acetylation and the function of the enhancer [26].
Concurrently, a conserved Smad-binding element in the
promoter of the Foxp3 gene, located -85 bp upstream of the
transcriptional start site, was identified [27]. It has shown
that Smad binds to enhancer 1 first and then dissociates from
the enhancer, binding at the promoter site after stimulation
[28]. Interestingly, the number of Foxp3? T cells induced by
TGF-b in vitro was lower in Smad3-/- mice [29]. By ana-
lyzing the Foxp3 expression in Smad3-deficient mice, it has
been demonstrated that Smad3 is essential for the TGF-
b-mediated induction of Foxp3? T cells [30]. These studies
collectively suggest that TGF-b may induce Foxp3 expres-
sion through a Smad-dependent pathway [17].
Hypothesis
We isolated a novel population from CD34? cells in our
laboratory, referred to as human umbilical cord blood-
derived stromal cells (hUCBDSCs) and showed that these
cells exert an immunosuppressive effect [31, 32]. Our
previous study in the graft-versus-host disease (GVHD)
mouse model showed that hUCBDSCs significantly
decreased the mortality and prolonged the survival time of
mice, and the degree of aGVHD was much lower in the
hUCBDSC group than in the groups without hUCBDSCs.
The number of Foxp3? Treg cells was much higher in the
hUCBDSC group than that in the groups without
hUCBDSCs [33].
The level of Foxp3 was significantly related to the
seriousness of GVHD in allogeneic hematopoietic cell
transplantation (allo-HSCT) patients. In patients who have
undergone allo-HSCT, the level of Foxp3 mRNA was
found to be negatively correlated with the severity of
GVHD [34]. In allo-HSCT patients with the unrelated
donors, the frequency of GVHD was significantly higher
than that for patients with related donors, which was caused
by the low level of Foxp3 expression. Interestingly,
increasing the level of Foxp3 expression lead to a sharp
decrease in the incidence rate of GVHD. Thus, we propose
464 Cell Biochem Biophys (2012) 62:463–466
123
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that the ability of hUCBDSCs to reduce the severity of
GVHD may be related to the Foxp3 expression of Treg
cells induced by hUCBDSCs. Our in vitro experiment
showed that hUCBDSCs could induce an increase in the
number of Foxp3? Treg cells when cocultured with T cells
[35]. We also observed that hUCBDSCs could secrete
TGF-b1 as detected by ELISA. The regulation of Treg cells
by TGF-b through secreted free TGF-b and membrane
TGF-b. The production of surface-bound TGF-b was
associated with the secretion of modest amounts of TGF-b[21]. TGF-b on the cell surface binds to a TGF-bR, which
produced a signal way.
Based on literature searches and our experimental
findings, we hypothesize that hUCBDSCs, which secrete a
high level of TGF-b1, modulate the Foxp3 expression of
Treg cells through the TGF-b1/Smad3 pathway to regulate
the development of GVHD.
Future Practice
The cytokines related to the TGF-b1/Smad3 pathway and
the level of Foxp3 expression should be investigated in
vitro using co-cultures of hUCBDSCs and Treg cells. The
relationship between TGF-b1 secreted by hUCBDSCs and
the Foxp3 expression of Treg cells requires further inves-
tigation with RNAi and molecular cloning. The model of
allo-HCST constructed with the bone marrow of Smad3
nude mice combined with hUCBDSCs was used to observe
the role of TGF-b1/Smad3 pathway in the regulation of
Foxp3 expression of Treg cells in vivo.
Acknowledgments This study was funded by Grants from the
National Natural Science Foundation (No. 81170529), the Natural
Science Foundation Project of CQ ‘‘CSTC’’ (CSTC, 2010BA5178),
the Key Discipline of Medical Science of Chongqing, and the special
foundation for the ‘‘1520 project’’ of Xinqiao Hospital of Third
Military Medical University.
Conflict of interest None declared.
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