Accepted Manuscript

Blocking activator protein-1 activity in donor cells reduces severity of acute GVHDthrough reciprocal regulation of Th17/regulatory T cells

Min-Jung Park, Su-Jin Moon, Sung-Hee Lee, Eun-Kyung Kim, Eun-Ji Yang, Jun-KiMin, Sung-Hwan Park, Ho-Youn Kim, Chul-Woo Yang, Mi-La Cho

PII: S1083-8791(14)00286-9

DOI: 10.1016/j.bbmt.2014.04.031

Reference: YBBMT 53455

To appear in: Biology of Blood and Marrow Transplantation

Received Date: 12 July 2013

Accepted Date: 29 April 2014

Please cite this article as: Park MJ, Moon SJ, Lee SH, Kim EK, Yang EJ, Min JK, Park SH, Kim HY,Yang CW, Cho ML, Blocking activator protein-1 activity in donor cells reduces severity of acute GVHDthrough reciprocal regulation of Th17/regulatory T cells, Biology of Blood and Marrow Transplantation(2014), doi: 10.1016/j.bbmt.2014.04.031.

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Blocking activator protein-1 activity in donor cells reduces severity of acute

GVHD through reciprocal regulation of Th17/regulatory T cells

Min-Jung Park1,4*, Su-Jin Moon2*, Sung-Hee Lee1, 4, Eun-Kyung Kim1, 4, Eun-Ji Yang1,4,

Jun-Ki Min2, Sung-Hwan Park1, Ho-Youn Kim1, Chul-Woo Yang3,4**, Mi-La Cho1,4**

1 The Rheumatism Research Center, Catholic Research Institute of Medical Science, The

Catholic University of Korea, Seoul, South Korea; 2 Division of Rheumatology, Department

of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, South

Korea; 3 Transplant Research Center, Seoul St. Mary's Hospital, College of Medicine, The

Catholic University of Korea, Seoul, South Korea; 4 Convergent Research Consortium for

Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic

University of Korea Seoul, Seoul, Republic of Korea.

Authorship note

* Min-Jung Park and Su-Jin Moon contributed equally to this work.

** Chul-Woo Yang and Mi-La Cho contributed equally to this work

Correspondence

Mi-La Cho, PhD.

Rheumatism Research Center, Catholic Institutes of Medical Science, The Catholic

University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-040, Korea (South) (Tel: 82-2-

2258-7467, Fax: 82-2-599-4287, E-mail: iammila@catholic.ac.kr)

Or

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Chul Woo Yang, M.D.

Transplant Research Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic

University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-040, Korea. (South) (Tel: 82-2-

2258-6037, Fax: 82-2-536-0323, Email: yangch@catholic.ac.kr)

Financial disclosure: See Acknowledgments on page 15.

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ABSTRACT

Acute graft-versus-host disease (aGVHD) is a major cause of mortality in allogeneic bone

marrow transplantation. Here, the diminishing effect of activator protein-1 (AP-1) blocking

with a synthetic retinoid (SR11302) on the severity of aGVHD in a murine model was

investigated. MHC-mismatched strain combinations were used in vivo: C57BL/6 (H-2kb)

donors into lethally irradiated BALB/c (H-2kd) recipients. SR11302 inhibited alloreactive T

cell response in a dose-dependent manner and negatively regulated STAT3 activation. AP-1

blocking in T cells inhibited the differentiation of Th1 and Th17. Conversely, Foxp3+

regulatory T cells (Treg) population dramatically expanded. Transfer of SR11302-treated

donor splenocytes into lethally irradiated recipients diminished the lethality and clinical

severity of aGVHD. In line with these results, AP-1 blocking in donor splenocytes exhibited

reduced Th17/Th1 population and enhanced in vivo Treg population. Beneficial Treg

expanding property of SR11302 was associated with the induction of Foxp3 and STAT5

transcription factor where the inhibiting property of Th17 was achieved by suppressing the

phosphorylated form of STAT3 and enhancing SOCS3. Conclusively, the preventive potential

of AP-1 inhibitor in aGVHD may be accomplished by altering CD4+ T cell differentiation

through modulating transcription factors.

Key words: Acute graft-versus-host disease; SR11302; STAT3; AP-1; Regulatory T cell;

Th17 cells

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INTRODUCTION

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for

the life-threatening hematological malignancies. However, acute graft-versus-host disease

(aGVHD) is the leading complication of allogeneic HSCT and causes fatal morbidities [1].

The improvements in conditioning regimens and infectious prophylaxis have contributed to

improved outcomes of HSCT. However, aGVHD remains lethal and a significant obstacle. In

fact, the incidence of severe aGVHD occurs in approximately 50% of recipients who receive

a human leukocyte antigen-matched graft from an unrelated donor [2].

GVHD is a complex inflammatory process involving donor T cells that recognize major

histocompatibility complex (MHC) on host-derived antigen-presenting cells (APCs) and

dysregulation of proinflammatory cytokines release. An overwhelming immune system

attacking the recipient tissues, such as the skin and gut, by donor T cells can lead to the

destruction of tissues [3]. Th1 cells and its associated cytokine, interferon-γ (IFN-γ), have

been reported to play a critical role during aGVHD. However, recent research has shown IL-

17 producing T (Th17) cell differentiation to impact the progression of aGVHD. In vitro

polarized Th17 cells were shown to induce lethal aGVHD [4]. On the opposite sides of Th17,

there is a CD4+CD25+Foxp3+ regulatory T cells (Treg) that plays a pivotal role for the

maintenance of self tolerance in various autoimmune diseases and alloresponse [5-7]. Treg

contributes to the tolerance acquisition to donor antigen in solid organ transplantations [8]

and protects the development of fatal aGVHD in a murine model [9].

Despite clinical concerns, GVHD prophylaxis and treatment relies heavily on unspecific

immunosuppressive agents. Prolonged immunosuppressive therapy increases the risk of

various complications including severe infections, which ultimately causes mortalities in

HSCT recipients. In addition, immunosuppressive therapy has shown to reduce the effects of

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T-cell mediated graft-versus-leukemia (GVL) and subsequently increase the disease relapse

rate.

The transcription factor activator protein 1 (AP-1) consists of a variety of dimers composed

of members from the Fos and Jun families [10]. AP-1 is involved in cellular proliferation,

death, survival, and differentiation [11]. Although, AP-1 is an important transcription factor

controlling the upregulation of proinflammatory cytokines, its role in the pathogenesis of

aGVHD remains unknown. In the current study, the effects of inhibiting AP-1 activity by

SR11302 (an AP-1 blocking synthetic retinoid) on aGVHD severity or reduce mortality have

been investigated.

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MATERIALS AND METHODS

Mice

Eight to ten weeks old C57BL/6 (H-2kb, termed B6) and BALB/c (H-2kd) mice were

purchased from OrientBio (Sungnam, Korea). Foxp3-GFP knock-in mice (C57BL/6 strain)

were purchased from the Jackson Laboratory. The mice were maintained under specific

pathogen-free (SPF) conditions in an animal facility with controlled humidity (55 ± 5%),

light (12/12h light/dark), and temperature (22 ± 1°C). The air in the facility was passed

through a HEPA filter system designed to exclude bacteria and viruses. Animals were fed

mouse chow and tap water ad libitum. The protocols used in this study were approved by the

Animal Care and Use Committee of The Catholic University of Korea.

Bone marrow transplantation (BMT) model and histopathology scoring

Recipients (BALB/c mice) were injected intraveneously (i.v.) with 5 × 106 bone marrow cells

from donor mice after lethal irradiation with 800 cGy. To induce aGVHD, splenocytes were

isolated from B6. To depleted Treg cells of donor cell, GFP negative cells (non-Tregs) sorted

from Foxp3-GFP mice (B6 strain). The purity of the sorted CD4+Foxp3– cells was 98–99% as

evaluated by flow cytometry. B6 or sorted Foxp3 GFP-negative splenocytes (1 × 107 cells)

were incubated with 10 µM SR11302 or control (DMSO) for 2 h at 37°C before adoptive

transfer into recipient mice. Survival after BMT was monitored daily, and the clinical severity

of aGVHD was assessed twice a week using a scoring system changing in five clinical

parameters: weight loss, posture, activity, fur texture, and skin integrity [12]. Mice were

sacrificed 14 days after BMT for blinded histopathological analysis of GVHD targets (skin,

liver, small and large intestine). Organs were harvested, cryo-embedded, and subsequently

sectioned. Tissue sections were fixed in 10% buffered formalin and stained with hematoxylin

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and eosin (H&E) for histological examination.

Alloreactive T cell prolifereation in vitro and viability studies

Splenic APC derived from B6 mice were used as allogeneic stimulators and splenic T cells

from BALB/c mice were used as responder cells in mixed lymphocyte reaction (MLR). T cell

proliferation in this assay is used as a parameter of alloreactivity. Red blood cells were

removed using ammonium-chloride-potassium lysis buffer, washed, and resuspended in

complete culture medium (RPMI 1640 medium supplemented with 10% heat-inactivated fetal

calf serum, 1mM sodium pyruvate, 5 × 105 M 2-ME, 20 mM HEPES, and antibiotics [100

U/ml penicillin, 100 µg/ml streptomycin]). Aliquots of 2 × 105 CD4+ T cells (responder) were

cultured with 2 × 105 irradiated (2500 cGy) APC in 96-well plates containing 200 µL of

complete medium at 37 °C in a humidified 5% CO2 atmosphere, pulsed with 1 µCi of [3H]

TdR (NEN life Science Products Inc., Boston, MA, USA) 18 h before harvesting, and

counted using an automated harvester (PHD Cell Harvester; Cambridge Technology, Inc.,

Cambridge, MA, USA). Splenic T cells were cultured with syngenic splenic APC or allogenic

splenic APC in the absence or presence of SR11302. Results are expressed as the mean

counts per minute (cpm) of triplicate samples ± standard deviation (SD). The stimulation

index was calculated by comparing the anti-stimulators response with the anti-self response.

The effect of SR11302 on the viability of cultured murine splenocytes were determined using

a MTT assay (Pierce, Rockford, IL).

Western blot

Protein samples were separated by SDS gel electrophoresis and transferred to a nitrocellulose

membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were stained

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with primary antibodies to c-Jun, c-Fos, phosphorylated signal transducer and activator of

transcription (STAT)3 on tyrosine 705 (pSTAT3Tyr705) or on serine 727 (pSTAT3Ser727), STAT3

(all from Cell Signaling, Danvers, MA) and β-actin. The HRP-conjugated secondary antibody

was then added.

Flow cytometry

Mononuclear cells were immunostained with various combinations of the following

fluorescence-conjugated antibodies: CD4, CD25, Foxp3, IFN-γ, IL-4 and IL-17. These cells

were also intracellularly stained with the following antibodies: IL-4 (BD Biosciences), IL-10

(Biolegend), IL-17, and Foxp3 (eBioscience). Before intracellular staining, the cells were

restimulated for 4 h with 25 ng/mL PMA and 250 ng/mL ionomycin in the presence of

GolgiSTOP (BD Biosciences). Intracellular staining was conducted using an intracellular

staining kit (eBioscience) according to the manufacturer’s protocol. Harvested cells were

stained with surface mAb, fixed, permeabilized and stained for anti-cytokine mAb. Flow

cytometric analysis was performed on a FACS Calibur cytometer (BD Biosciences).

Splenocytes isolated from B6 mice were stimulated with (1 µg/ml) anti-CD3 mAb and

(1µg/ml) of anti-CD28 mAb (BD PharMingen, CA, USA) for 72 h (Th0 condition). Cells

were stained first with mAbs to CD4, CD25, inducible costimulator ((ICOS), glucocorticoid

induced TNF related (GITR), and programmed death-1 (PD-1), follow by mAbs to cytotoxic

T lymphocyte antigen 4 (CTLA-4) and Foxp3 using the regulatory T Cell Staining Kit

(eBioscience, San Diego, CA). All analyses were based on control cells incubated with

appropriate isotype control to adjust background fluorescence.

Measurement of cytokines

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The concentrations of cytokines in culture supernatants were measured using a sandwich

enzyme-linked immunosorbent assay (ELISA) kit (Duoset; R&D Systems, Lille, France) as

described by the manufacturer.

Reverse transcription–polymerase chain reaction analysis

Total RNA (2 µg) was reverse transcribed into cDNA using a transcription kit (TaqMan

Reverse Transcription Reagents; Applied Biosystems, Darmstadt, Germany). The resulting

cDNA was amplified by PCR using Foxp3 sense (5'- GGC CCT TCT CCA GGA CAG A-3')

and antisense (5'- GCT GAT CAT GGC TGG GTT GT-3') primers, and using SOCS3 sense

(5'- CGC CTC AAG ACC TTC AGC TC-3') and antisense (5'- CTG ATC CAG GAA CTC

CCG AA-3') primers. PCR products were separated on a 1.5% agarose gel and stained with

ethidium bromide.

Confocal staining

Spleen tissue was obtained 14 days after BMT and was snap-frozen in liquid nitrogen and

stored at -80 °C. Tissue cryosections (7 µm thick) were fixed with acetone and stained using

PE-labeled anti-IFN-γ, IL-4, IL-17 or Foxp3 Ab (eBioscience, San Diego, CA), FITC-labeled

anti anti-pSTAT3Tyr705, pSTAT3Ser727, pSTAT5, PD-1 or GITR (eBioscience, San Diego, CA),

PerCP–labeled anti-CD4 Ab (Biolegend, San Diego, CA), APC-labeled anti-CD25 Ab. In all

experiments, background staining was established using isotype-matched PE -, or FITC-

conjugated isotype control. After incubation overnight at 4 °C, stained sections were

analyzed using a confocal microscopy system (LSM 510 Meta. Zeiss, Gottingen, Germany).

Statistical analysis

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Data was presented as mean ± SD. Data comparison between more than two groups and two

groups were performed with Kruskal-Wallis test and Mann–Whitney U-test or Student t test,

respectively. To assess the Gaussian distribution and the equality of variance, the Shapiro–

Wilk test and Leven test were used, respectively. The overall survival rate in aGVHD was

evaluated using the Kaplan-Meier estimate and compared using the log-rank test. Weight data

were analyzed at each time point. Statistical analysis was performed using the SPSS

statistical software package (standard version 16.0; SPSS, Chicago, IL, USA). P values less

than 0.05 (two-tailed) were considered significant.

RESULTS

AP-1blocking modulates alloreactive T cell responses in vitro

Prior to the in vitro experiment, cell viability with increasing treatment dose of SR11302 was

assessed using the MTT colorimetric assay. The results showed that SR11302 treatment up to

10 µM did not show any significant toxicity to murine splenocytes (Figure 1A). To determine

the impact of AP-1 activity on the proliferative capacity of donor CD4+ T cells in response to

alloantigens, T cell alloreactivity with treatment of SR11302 was measured by incorporating

[3H]-thymidine. Proliferative responses to B6 splenic APC (allogeneic stimulator) were

measured in BALB/c splenic T cells (responder cells) and were compared with those of

BALB/c APC (syngeneic stimulator). BALB/c T cells that were incubated with B6 splenic

APC in the presence of SR11302 displayed decreased alloreactivity when compared to those

in the absence of SR11302 in a dose-dependent manner (Figure 1B). The concentrations of

IFN-γ, IL-17 and IL-10 in culture supernatants were measured using ELISA (Figure 1C).

Treatment with SR11302 markedly reduced IFN-γ and IL-17 levels and reciprocally

increased the level of IL-10 in a dose-dependent manner (Figure 1D). To determine the

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inhibition effect of the AP-1 activity was due to the treatment of SR11302, AP-1 proteins, c-

Jun and c-Fos were analyzed using western blot. c-Jun and c-Fos expression were effectively

diminished with SR11302 treatment (Figure 1D). Total STAT3 and pSTAT3 expressions were

analyzed to examine if SR11302 negatively regulates STAT3 activation in alloreactive T cells

(Figure 1E). STAT3 and pSTAT3 (pSTAT3Tyr705 and pSTAT3Ser727) activity decreased

following SR11302 treatment, although the changes of pSTAT3 expressions were more

significant than that of total STAT3 activity.

Altered differentiation of T cells in vitro by AP-1 blocking

IL-17 producing cells (mainly Th17) and IFN-γ producing cells (mainly Th1) increased in

responder T cells stimulated with allogeneic APC for 4 days (Figure 2A). Th17 and Th1 cells

decreased with SR11302 treatment in a dose-dependent manner. Conversely, SR11302

increased approximately two-fold in CD4+Foxp+ Treg cells (Figure 2A). BALB/c splenic T

cells were incubated with anti-CD3 and anti-CD28 for 72 hours in the presence or absence of

SR11302 (10 µM). CD25+Foxp3+-double positive T cells dramatically increased (Figure 2B).

The suppressive function of Treg on conventional T cells was gained through the cell-cell

contact-dependent mechanism, such as CTLA-4 [13]. The proportion of CTLA-4, ICOS, PD-

1, or GITR-expressing Treg were analyzed using flow cytometry. The CTLA-4, and ICOS-

positive Treg population were increased by SR11302 compared to control (Figure 2C).

Conclusively, AP-1 signaling blocking led to the attenuation of the alloreactive T cell

response and Th17 differentiation via the interruption of STAT3 activity. Furthermore, results

demonstrated treatment with SR11302 in vitro can expand Treg and upregulate CTLA-4 and

ICOS molecules, thus showing enhanced suppressor activity of Treg cells.

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AP-1 signaling is involved in aGVHD-induced lethalty and target organ damage

T-cell depleted (TCD) BMT model was used as a negative control. All of the control mice

died from aGVHD, whereas recipients receiving SR11302-treated splenocytes survived

longer, where 50% survived beyond 60 days after BMT (Figure 3A). Weight loss was fairly

mitigated in SR11302-treated aGVHD animals. SR11302-treated aGVHD animals showed

less severe clinical scores compared to the control group (Figure 3A). Skin, liver and intestine

are primary target sites of aGVHD. Histopathology results on day 14 post-BMT after GVHD

induction demonstrated significantly improved pathologic cutaneous lesions in SR11302-

treated recipients. Inflammatory cell infiltration and structural destruction of intestinal

mucosa that were shown in control recipients were also attenuated in SR11302-treated

aGVHD mice (Figure 3B). Furthermore, to ascertain whether the protective function of

SR11302 work by inhibiting the function of AP-1, the expression of its components, c-Fos

and c-Jun, in target organs were assessed by confocal and immunohistochemical staining.

Expression of both proteins in spleen, liver, and epithelial tissues of small intestine was

significantly reduced in SR11302-treatd aGVHD animals (Figure 3C and D).

The preventive effect of SR11302 were accompanied by altered T cell population in vivo

IFN-γ-producing CD4+ cell population (mainly Th1) and IL-17-producing CD4+ T cells

(mainly Th17) were dramatically reduced in SR11302-treated GHVD recipients (Figure 4A).

Interestingly, IL-4-expressing CD4+ T cells (mainly Th2) slightly expanded, suggesting the

differentiation toward Th2 subtype following SR11302 treatment. SOCS3 has been shown to

inhibit STAT4 signaling, leading the immune response to skew toward Th2 [14]. In the fate of

Th17, SOCS3 blocked STAT3 signaling, leading to the inhibition to generate Th17

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population [15]. Foxp3 is essential for differentiation of Treg cells [16]. Splenocytes isolated

from aGVHD mice that were transferred SR11302-treated splenocytes showed enhanced

mRNA expression of SOCS3 and Foxp3 compared to the control group, suggesting the

mechanisms accounting for the aGVHD attenuation by SR11302 (Figure 4B). In line with

this, spleens isolated on day 14 after BMT demonstrated that Th1 and Th17 dramatically

decreased in SR11302-treated recipients compared to the vehicle-treated animals. Conversely,

IL-4+CD4+ (mainly Th2) and Foxp3+ CD4+ T (mainly Treg) cells were significantly enhanced

in SR11302-treated GVHD mice (Figure 4C). Those results suggest that AP-1 blocking in

donor splenocytes effectively attenuates aGVHD severity by altering the differentiation of

CD4+ T cells during the alloreactive T cell responses.

Regulation of STAT3 and STAT5 activity via AP-1 signal blocking in vivo

The expression of pSTAT3Tyr705 and pSTAT3Ser727 in spleens isolated from aGVHD receiving

SR11302-treated splenocytes displayed reduced pSTAT3 among CD4+ T cells. Conversely,

the expression of pSTAT5 demonstrated in spleen dramatically increased in SR11302-treated

mice compared to control (Figure 5A). Regarding the suppressive property of Treg, GITR

and PD-1-expressing Treg portions in SR11302-treated group seemed to increase when

compared to the control group (Figure 5B). Thus, AP-1 signal blocking by SR11302 could

prevent lethal aGVHD by inhibiting STAT3 biologic activity and reciprocal induction of

STAT5.

The substantial potential of Treg cells to attenuate the severity of aGVHD

As shown in Figure 6A, treatment with SR11302 on Treg cells-depleted donor splenocytes

did not exert a significant benefit to weight loss and survival gain. All recipient mice failed to

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survive beyond 20 days after BMT. The ex-vivo results demonstrated that SR11302 treatment

on Treg cells-depleted donor splenocytes failed to regulate the differentiation of CD4+ T cells

in recipient mice (Figure 6B). Taken together, donor Treg cells may exert substantial direct

effects on the attenuated severity of aGVHD following SR11302 treatment.

DISCUSSION

We demonstrated that SR11302, an AP-1 inhibition-specific synthetic retinoid, can inhibit the

expansion of Th1/Th17 through attenuation of STAT3 activity and SOCS3 induction. In

addition, SR11302 can induce Treg by enhanced phosphorylation of STAT5. Ultimately, AP-1

blocking in donor lymphocytes led to reduced mortality in aGVHD recipients and attenuated

the clinical severity of aGVHD.

Although a recent study suggested that GVHD can be elicited in the absence of host APCs

[17], progression of aGVHD is traditionally summarized in three sequencial phases: (1)

activation of host APCs by the intensive conditioning regimen, (2) activation, and

proliferation of alloreactive donor T cells, and (3) subsequent tissue destruction [1]. Activated

donor CD4+ T cells that interact with host APCs can proliferate and differentiate into several

types of Th cells: Th1, Th2 and Th17 cells, depending on the milieu. aGVHD has been

traditionally proposed to be Th1-dominant condition. But, donor cells isolated from mice

lacking IFN-γ, a key cytokine of Th1, led to an accelerated mortality due to a GVHD [18].

Recently, Th17 cells have been reported to play a pivotal role in some autoimmune diseases,

including rheumatoid arthritis [19] and autoimmune uveitis [20]. Yi et al. showed that Th1

and Th17 cells play a different role under the progression of GVHD [21]. In their study, gut

GVHD heavily depends on Th1, whereas skin GVHD is Th17-dominant and lung GVHD is

Th2 dependent. On the opposing side of Th1 and Th17, Treg plays a pivotal role in

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maintaining tolerance to alloantigens where Foxp3 was discovered to be a key transcription

factor contributing to new insights into Treg biology. Regarding Treg induction, STAT5 has

been considered to have nonredundant roles. STAT5 can upregulate Foxp3 expression by

directly binding to the Foxp3 gene [22]. Removal of Treg from the graft can accelerate

aGVHD-induced mortality in animal models [9, 23]. However, Foxp3+ Treg cells can lose

Foxp3 expression in vivo and take on an effector memory T cell phenotype [24, 25].

Activated Treg cells with low expression of Foxp3 have been described to produce more IL-

17 than any other CD4+ T cell fraction [26]. The plasticity of Treg as well as the scarcity of

circulating Treg population suggests a limitation of adoptive transfer of Treg in various

immune diseases, including GVHD. However, preclinical experiments have documented the

preventive effect of Treg [27] and GVHD-inducing effect of Th17 cells [4], which give rise

for the need of novel strategies that can induce Treg and to inhibit the differentiation and

proliferation of Th1/ Th17 lineages.

The prevention of aGVHD could be achieved by the depletion or functional inactivation of

donor immune cells within the context of disease pathophysiology [28]. However, that

approach could be a double-edged sword due to the reduced T-cell induced GVL effect in

recipients. Treg cells protect the host from autoimmune diseases by suppressing self-reactive

cells, blocking antitumor immune responses. Furthermore, Treg cells found in solid cancer

sucah as lung cancer and ovarian cancer were shown to secret TGF-β, contributing to

immune dysfunction in patients with cancer [29]. Curiel et al. demonstrated that Treg cells in

ovarian cancer may lead to the failure of host antitumor immunity, therefore favoring tumor

development or growth and influencing poor survival [30]. However, it has not yet been

discovered whether elimination of Treg cells in hematologic malignancies also exhibits anti-

tumor immunity. A growing number of studies have suggested that Treg cells can reduce the

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clinical severity of GVHD after allogeneic BMT in animal models as well as in human [31-

33]. One decade ago, Edinger et al. reported that Treg cells could be the potent

immunoregulatory T cells that inhibit lethal aGVHD while preserving GVL effect in a murine

model [34]. Furthermore, one recent study demonstrated that adoptive transfer of Treg leads

to the preservation of thymic and peripheral lymph node architecture as well as abrogation of

GVHD [35]. Based on these findings, expanding Treg population is a rational therapeutic

strategy to reduce clinical severity of aGVHD while preserving GVT effects induced by

conventional T cells following allogeneic BMT. Therefore, the reciprocal regulation of

Th1/Th17 and Treg seems to be a rational approach to inhibit fatal aGVHD as well as the

preservation of the GVL effect, in the aspect of prevention of disease relapse. But, the

probability that enhanced Treg and inhibited Th17 populations in BMT recipients could lead

to attenuated GVL effects should be addressed. Our study did not investigate whether GVL

effect or other immune function in BMT recipients was affected by SR11302 treatment.

STAT3 has been revealed to be activated via tyrosine phosphorylation of Tyr705 in response

to various factors such as IL-6 [36]. Despite of the dominant role of Tyr705 phosphorylation

in STAT3 activation, Ser727 phosphorylation was reported to be involved in maximal activity

[37]. However, Ser727 phosphorylation could suppress STAT3 activity in another

circumstance [38]. Despite the elusive role of Ser727 in STAT3, we showed that the

inhibitory effects of SR11301 in an aGVHD model were associated with decreased

phosphorylation of both Ser727 and Tyr705.

Conclusively, the present study demonstrated that AP-1 blocking in donor lymphocytes

ameliorated lethal aGVHD through the expansion of the Treg population as well as the

inhibition of Th17 differentiation and proliferation. AP-1 blocking in transplanted donor T

cells seems to be a reasonable strategy to prevent mortality and to decrease the severity

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scores of aGVHD in allogeneic HSCT recipients.

ACKNOWLEDGMENT

Financial disclosure: This study was supported by a grant of the Korea Healthcare

Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI09C1555).

This research was supported by the Bio & Medical Technology Development Program of the

National Research Foundation (NRF) funded by the Korean government (MEST) (No.

2012M3A9C6049783).

Conflict of Interest Statement: There are no conflicts of interest to report.

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Figure legends

Figure 1. SR11302 inhibits alloreactive T cell proliferation in vitro through down-regulation

of AP-1/STAT3 signaling. (A) The cellular viability to SR11302 treatment in alloresponse

condition in vitro was determined by MTT assay. A total of 2 × 105 RBC-lysed BALB/c

splenic T cells (responders) were incubated with 2 × 105 irradiated RBC-lysed BALB/c

splenic APC (syngenic stimulator) or C57BL/6 splenic APC (allogeneic stimulator) for 4

days. (B) In MLR assay, a total of 2 × 105 RBC-lysed BALB/c splenic T cells (responders)

were incubated with 2 × 105 irradiated RBC-lysed BALB/c splenic APC (syngenic

stimulator) or C57BL/6 (B6) splenic APC (allogeneic stimulator) for 4 days. Responder cells

were cultured in the absence or presence of SR11302 (2.5 µM and 10 µM). SR11302 was

added on day 0, and T cell proliferation was measured by [3H]-thymidine incorporation in

each group. (C) The supernatants were collected, and ELISA was performed to determine

the IFN-γ, IL-17 and IL-10 levels. Data represent means ± SD of triplicates (*P < 0.05 vs.

Alloresponse). (D) A total of 105 RBC-lysed BALB/c splenic T cells (responders) were

incubated with 105 irradiated RBC-lysed BALB/c splenic APC (syngenic stimulator) or B6

splenic APC (allogenic stimulators). SR11302 (2.5 µM) was added on day 0 and cells were

harvested on day 2. And then, western blotting analysis of AP-1 (D) and STAT3

phosphorylation associated protein expression (E) was performed in each sample.

(1:syngenic stimulator, 2: allogenic stimulator in the absence of SR11302, 3:allogenic

stimulator in the presence of SR11302).

Figure 2. Altered T cell differentiation following SR11302 treatment in donor allogeneic T

cells is associated with reduced alloreactivity. (A) A total of 105 RBC-lysed BALB/c splenic

T cells (responders) were incubated with 105 irradiated RBC-lysed BALB/c splenic APC

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(syngenic stimulator) or C57BL/6 splenic APC (allogeneic stimulator) for 4 days.

Responder cells were cultured in the absence or presence of SR11302 (2.5 µM and 10 µM).

SR11302 was added on day 0 and cells were harvested on day 4. Then, intracellular staining

for IFN-γ, IL-17, and Foxp3 in isolated CD4+ T cells was performed and analyzed by flow

cytometric analysis. Right panel shows the absolute number of CD4+IFN-γ+, CD4+IL-17+,

CD4+CD25+Foxp3+ T cells. (B) Splenic T cells were incubated for 72 h with plate-bound

anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) antibody in the absence or presence of 10 µM

SR11302. CD4+CD25+Foxp3+ Treg cell proportion were analyzed by flow cytometry. The

data are expressed as the mean ± SD from four independent experiments. (C) The mean

fluorescence intensity (MFI) of various Treg markers, including CTLA-4, ICOS, PD-1, and

GITR, were analyzed by flow cytometry. The data are expressed as mean ± SD for four

mice per group. *P < .05, ** P < .01, and *** P < .001, compared with the vehicle (control)-

treated alloresponse.

Figure 3. SR11302 suppresses the development of acute GVHD via blocking of c-Fos/c-Jun

signaling. (A) C57BL/6 (B6) splenocytes (1 × 107 cells) were incubated with 10 µM of

SR11302 or DMSO (control) for 2 h at 37 °C before adoptive transfer into lethally irradiated

(800 cGy) BALB/c (recipient) mice. Recipients also received 5 × 106 BM cells from B6

mice and were monitored for weight loss, survival and clinical scores of aGVHD. Lethal

aGVHD shown in BALB/c recipients was inhibited after administration of donor B6

splenocytes treated with SR11302. Recipient mice receiving TCD-BM cells showed no signs

of GVHD. Data were combined from three independent experiments (n = 15 per group) are

shown. (B) Histopathology of skin, small and large intestine after BMT (n = 6 per group),

which is one representative of two independent experiments. Tissues isolated on day 14 after

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BMT were stained with H&E (original magnification, 200×) (left panel) and histological

scores of each organ were higher in vehicle–treated animals, compared with those of

SR11302–treated group (right pane). The data are expressed as the mean ± SD from three

independent experiments for five mice per group. *P < .05, *** P < .001, compared to the

vehicle-treated GVHD group. (C) Fourteen days after BMT, splenocytes isolated from each

group were stained with DAPI and anti-c-Fos or c-Jun antibodies, components of AP-1, and

visualized by confocal microscopy. Representative examples of confocal staining for c-Fos

and c-Jun in spleen from GVHD mice. (D) Representative examples of

immunohistochemical staining for c-Fos and c-Jun, components of AP-1, in liver and small

intestine tissue from GVHD mice. Positive immunoreactivity appears as a brown color and

is counterstained with blue (original magnification, 400×).

Figure 4. Analysis of CD4+ T helper cells in SR11302-treated GVHD mice. (A) C57BL/6

(B6) splenocytes (1 × 107 cells) were incubated with SR11302 (10 µM) or vehicle (DMSO)

for 2 h at 37°C before adoptive transfer into lethally irradiated (800 cGy) BALB/c mice.

Recipient BALB/c mice also received 5 × 106 BM cells from B6 mice. Intracellular

cytokines were determined in the CD4+ T cells. Splenocytes of each group and were

analyzed by flow cytometry on day 14 after BMT. Results are shown as mean ± SD (n = 5

per group). *P < .05, compared to the vehicle-treated GVHD group. (B) Fourteen days after

BMT, the expression of Foxp3 and SOCS3 mRNA was determined by RT-PCR analysis

from whole splenocytes isolated from each group. (C) Spleens isolated on day 14 after BMT

in each group were analyzed by confocal microscopy for the expression of IFN-γ, IL-4, IL-

17, and Foxp3 among CD4+ cells. The proportions of IFN-γ, IL-4, IL-17, or Foxp3

expressing CD4+ cells in spleen were represented by bar graph (right panel) (mean ± SD).

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The experiment was performed once with six mice per group. *P < .05, compared to the

vehicle-treated GVHD group.

Figure 5. SR11302 regulate signaling of STAT3 and STAT5. (A) C57BL/6 (B6) splenocytes

(1 × 107 cells) were incubated with SR11302 (10 µM) or vehicle (DMSO) for 2 h at 37 °C

before adoptive transfer into lethally irradiated (800 cGy) BALB/c mice. Recipient BALB/c

mice also received 5 × 106 BM cells from B6 mice. The signal transducers STAT3 and

STAT5 in the spleens were analyzed by confocal microscopy on day 14 after BMT in each

group. Data are representative of three independent experiments. (B) Fourteen days after

BMT, isolated spleens from each group were analyzed by confocal microscopy for the

expression of GITR and PD-1 among CD4+CD25+Foxp3+ regulatory T cells. The

experiment was performed once with six mice per group. Bars represent the mean ± SD of

six mice per group.

Figure 6. SR11302 plays a critical role in Tregs-mediated inhibition of aGVHD severity. (A)

Treg-depleted splenocytes (1 × 107 cells) from B6 mice were incubated with 10 µM of

SR11302 or DMSO (control) for 2 h at 37 °C before adoptive transfer into lethally irradiated

(800 cGy) BALB/c (recipient) mice. Recipients also received 5 × 106 BM cells from B6 mice

and were monitored for weight loss and survival. (B) Spleens were isolated on day 10 after

BMT in each group. Then intracellular staining for IFN-γ, IL-17, CD25 and Foxp3 in isolated

CD4+ T cells was performed and analyzed by flow cytometric analysis.

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