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: xhchen888@yahoo.com.cn

123

Cell Biochem Biophys (2012) 62:463–466

DOI 10.1007/s12013-011-9328-8

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

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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