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Immunology Letters 160 (2014) 79–88

Contents lists available at ScienceDirect

Immunology Letters

j ourna l ho me page: www.elsev ier .com/ locate / immlet

IAS3 suppresses acute graft-versus-host disease by modulatingffector T and B cell subsets through inhibition of STAT3 activation

ung-Hee Leea,b, Su-Jin Moonc, Min-Jung Parka,b, Eun-Kyung Kima,b,oung-Mee Moona,b, Mi-La Choa,b,∗

The Rheumatism Research Center, Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, South KoreaLaboratory of Immune Network, Conversant Research Consortium in Immunologic Disease, College of Medicine, The Catholic University of Korea, Seoul,outh KoreaDivision of Rheumatology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, South Korea

r t i c l e i n f o

rticle history:eceived 20 January 2014eceived in revised form 28 March 2014ccepted 28 March 2014vailable online 6 April 2014

eywords:IAS3raft-versus-host-diseaseTAT3

cells cells

a b s t r a c t

Graft-versus-host disease (GVHD) caused by transplanted donor T cells remains the major obstacle of allo-geneic bone marrow transplantation (BMT). Previous reports have suggested that IL-17-producing helperT (Th17) cells mediate the development of acute GVHD (aGVHD). Protein inhibitor of activated STAT3(PIAS) inhibits the activity of the transcription factor STAT3, which is a pivotal transcription factor forTh17 differentiation. To elucidate whether PIAS3 could inhibit the development of aGVHD, pcDNA-PIAS3or mock vector was administered in a murine model of aGVHD by intramuscular injection and subsequentelectroporation. The results demonstrated that PIAS3 overexpression by pcDNA-vector administrationsignificantly attenuated the clinical severity and histopathological severities of aGHVD involving the skin,liver, intestine, and lung. Additionally, the STAT3 activities in aGVHD target organs were suppressed byPIAS3 overexpression. Furthermore, phosphorylated (p) STAT3 activity in the spleen was profoundlyattenuated in PIAS3-overexpressing GVHD mice. Interestingly, flow cytometric analysis demonstratedthat the populations of CD21highCD23low marginal zone B cells were dramatically expanded in PIAS3-overexpressing mice. PIAS3-induced inhibition of aGVHD was largely related to the downregulationof Th1 and Th17 and the upregulation of Th2 and Treg populations. Both populations of pSTAT3Tyr705-

expressing Th17 cells and B cells were significantly reduced in the spleens of PIAS3-overexpressing mice,whereas pSTAT5 activity was increased. In addition to CD4+CD25+Foxp3+ Treg cells, the populations ofCD8+CD25+Foxp3+ Treg cells were also expanded by treatment with PIAS3. These data suggest the ther-apeutic potential of PIAS3 in the development of aGVHD through reciprocal regulation of Th17/Treglineages.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Allogeneic hematopoietic stem cell transplantation (HSCT) is thenly curative therapy with proven efficacy for the management of

any hematologic malignant diseases and bone marrow failure.llogeneic HSCT is clearly indicated for severe immunodeficien-ies that are lethal in the first few years of life. However, its wide

∗ Corresponding author at: Department of Life Science, College of Medicine, Lab-ratory of Immune Network, Rheumatism Research Center, Catholic Institutes ofedical Science, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul

37-701, South Korea. Tel.: +82 2 2258 7467; fax: +82 2 599 4287.E-mail address: iammila@catholic.ac.kr (M.-L. Cho).

ttp://dx.doi.org/10.1016/j.imlet.2014.03.014165-2478/© 2014 Elsevier B.V. All rights reserved.

application is hampered by the development of graft-versus-hostdisease (GVHD) [1]. The development of GVHD requires escalatedand prolonged immunosuppressive therapy that increases the riskof infectious complications and, ultimately, mortalities in HSCTrecipients. Despite advances in the development of prophylaxisagents, acute GVHD (aGVHD; grades II–IV) occurs in 30–60% ofpatients after allogeneic HSCT from HLA-identical sibling donors[2]. Prevention of GVHD has been the major challenge of allogeneicHSCT. Although the pathogenesis of aGVHD remains unresolvedthus far, its development is considered to be caused by mature

donor T cells that recognize genetically disparate recipient antigenson APCs, resulting in the destruction of GVHD target organs, includ-ing the skin, liver, lung, and gastrointestinal tract [3,4].aGVHDis a proinflammatory process, the pathophysiology of which is

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elieved to be a multistep process. Initially, host tissues are dam-ged by preparative chemotherapy or radiotherapy. GVHD occurss a result of T cell activation followed by alloreactive T cell expan-ion and differentiation [5]. It is generally accepted that aGVHDs driven by mainly T helper 1 (Th1) cells, which produce IFN-�nd IL-2 [6]. Recent evidence has suggested that the mechanismf GVHD is more complex and might involve a new lineage ofD4+ effector T cells, identified as IL-17-producing helper T (Th17)ells [7]. Th17 cells have been implicated in several inflamma-ory diseases [8]. Regarding GVHD pathobiology, Th17 cells play

direct role in the development of GVHD [9]. Adoptive transfer ofn vitro-differentiated Th17 cells can induce lethal aGVHD [10]. IL-7 stimulates the production of other inflammatory cytokines, suchs IL-6, IL-8, TNF-�, and vascular endothelial growth factor, sup-orting the proinflammatory milieu [11,12]. The signal transducernd activator of transcription (STAT)3 and ROR�t are key transcrip-ion factors for Th17 differentiation from naïve CD4+ T cells [13].rolonged activation of STAT3 is a dominant event during the devel-pment of aGVHD in target organs [14] and also plays a critical rolen T-cell alloactivation [15]. Furthermore, it was reported that theTAT3 signaling pathway negatively contributes to an enhancedopulation of CD4+CD25+Foxp3+ Treg cells in the development ofVHD, whereas it does not affect Th1 differentiation [16]. Previ-us results have suggested the identification of STAT3 protein as aotential target of treatment to prevent or inhibit aGVHD.

Protein inhibitor of activated STAT3 (PIAS3) was initially identi-ed as an endogenous molecule that inhibits DNA binding of STAT317]. PIAS3 is known to inhibit gene induction through activatedTAT3 by blocking the DNA binding activity of the transcriptionactor. In addition, PIAS3 has also been linked to the inhibition ofhosphorylation of STAT3 [18]. The in vivo function of PIAS3 as a keyolecule in suppressing the microphthalamia transcription fac-

or (MITF)-induced transcriptional activity has been demonstrated19,20]. PIAS3 activates TGF-�/Smad transcriptional responses byorming a complex with Smad and increases SUMO-E3 ligase activ-ty [21,22].

Therefore, we hypothesized that a PIAS3-enhancing strategyould suppress the development of aGVHD in an experimentalurine model. To elucidate the mechanism by which PIAS3 exerts

therapeutic effect, we investigated whether PIAS3 affects T-cellubset populations, particularly the Th17/Treg imbalance, and B cellubsets. In the present study, we identified the preventive potentialf PIAS3 in the development of aGVHD in a murine model throughodulation of T- and B-cell subpopulations.

. Materials and methods

.1. Animals

Female BALB/c (BALB/c, H2d) and C57BL/6 (B6, H2b) mice wereurchased from The Jackson Laboratory (Bar Harbor, ME). Miceere between 8 and 10 weeks of age at the start of the exper-

ments. The mice were maintained under specific pathogen-freeonditions in an animal facility with controlled humidity (55 ± 5%),ight (12/12 h light/dark), and temperature (22 ± 1 ◦C). The air inhe facility was passed through a HEPA filtration system designedo exclude bacteria and viruses. Animals were fed mouse chow andap water ad libitum. All experimentation was carried out in strictccordance with the guidelines of the Animal Care and Use Commit-

ee of The Catholic University of Korea. The protocol was approvedy the Animal Research Ethics Committee of the Catholic Univer-ity of Korea (permit number: 2012-0155-01), which conforms toll National Institutes of Health of the USA guidelines.

tters 160 (2014) 79–88

2.2. Hematopoietic stem cell transplantation procedures

The bone marrow transplantation (BMT) procedure was per-formed as described previously. BALB/c (H2d) mice were used asrecipients. Recipient mice received total body irradiation (400 cGy32 split dose, total 800 cGy). Animals were followed by the infu-sion of 5 × 106 total bone marrow cells intravenously with 1 × 107

splenocytes (as a source of allogeneic T cells) from allogeneic donorC57BL/6 (B6, H2b). All experiments were conducted with 12 miceper group. The recipient mice were randomly divided into the con-trol group and therapy group. One days after GVHD induction,the mice were injected intramuscular injection of 50 �g of thepCMV-PIAS3 vector in the both (left and right) leg with electri-cal stimulation (electroporation) using a 31-gauge needle insulinsyringe. Recipients were monitored twice a day for survival, weightloss, and the degree of clinical GVHD was assessed during the 30 dayobservation period using a scoring system that summed changes infive clinical parameters: weight loss, posture, activity, fir texture,and skin integrity. Survival of mice was monitored by daily obser-vation, and the day of death was recorded as the day the mousespontaneously died. Animals were humanely sacrificed when theyexhibited the euthanasia GVHD criteria (greater than 20% weightloss or animals that received a score of. 6.5 or higher).

2.3. Immunohistochemistry and histopathologic analysis ofGVHD target organs

The small intestine and skin from GVHD control and PIAS3vector-treated mice of the BMT model were fixed in 10% formalinand embedded in paraffin. Small intestine and skin tissues werethen sectioned at 7-�m thickness, deparaffinated using xylene,dehydrated through a gradient of alcohols, and then stained withH&E. GvHD was scored by two trained pathologists blinded tothe treatment groups as previously published histopathology sco-ring system [23,24]. Endogenous peroxidase activity was quenchedwith 3% H2O2 in methanol. Immunohistochemistry was performedusing the Vectastain ABC kit (Vector Laboratories, Burlingame, CA,USA). Tissues were incubated with the first primary anti-mouseantibody STAT3 overnight at 4 ◦C. The primary antibodies weredetected using a biotinylated secondary linking antibody, followedby incubation with streptavidin–peroxidase complex for 1 h. Thefinal color product was developed using the DAB chromogen (Dako,Carpinteria, CA, USA). Positive cells were counted, and the resultswere expressed as means ± SD.

2.4. CD4+ T cell isolation and differentiation

CD4+ T cells were isolated from spleen using CD4+ T cell isolationkits (Miltenyi Biotec) according to the manufacturer’s instructions.The purity of the isolated CD4+ T cells was >95%. Isolated CD4+

T cells were stimulated with plate-bound anti-CD3 (0.5 �g/ml);and soluble anti-CD28 (0.5 �g/ml) for 72 h in 24-well plates. Th17cell differentiation was induced by treatment with, anti-IFN-�(4 �g/ml), anti-IL-4 (4 �g/ml), TGF-� (2 ng/ml), and IL-6 (20 ng/ml),for 72 h.

2.5. Transfection and expression of PIAS3 and mockoverexpression vectors

To generate PIAS3 overexpression vector, PIAS3 cDNA was pur-

chased from Korea Human Gene Bank, Medical Genomics ResearchCenter, KRIBB, Korea and subcloned into the Kpn1 and Xho1 sitesof pcDNA3.1+ (Invitrogen). The mock, PIAS3 vector constructs weretransfected using an Amaxa 4D-Nucleofector X unit according to

S.-H. Lee et al. / Immunology Letters 160 (2014) 79–88 81

Fig. 1. PIAS3 overexpression ameliorates acute GVHD severity. (A) Recipient BALB/c mice received 5 × 106 total bone marrow cells and 1 × 107 splenocytes from B6 mice,and mock or PIAS overexpresssion vector was administrated on 1 day post BMT, using electroporation method. Mice were monitored for weight and clinical signs of acuteGVHD. Data combined from two independent experiments (n = 12 per group) are shown. Improvement of the clinical GVHD score after bone marrow transplantation (BMT)w ity. **a his seh ix mic

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as evaluated in terms of weight loss, posture, activity, fur texture, and skin integrnd lung after BMT (n = 12 per group) from one of two independent experiments. Tistopathology score of each tissue group. Results are expressed as means ± SD of s

he manufacturer’s recommendations with the program DN-100Lonza).

.6. PCR

PCR amplification of cDNA aliquots was performed by adding.5 mM dNTPs, 2.5 U of Taq DNA polymerase (Takara), and 0.25 �Mf sense and antisense primers. The reaction was performed in PCRuffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3)) in aotal volume of 25 �l. The following sense and antisense primersor each molecule were used: IL-17 sense, 5′-CCT CAA AGC TCA GCGGT CC-3′; IL-17 antisense, 5′-GAG CTC ACT TTT GCG CCA AG-3′; IL-

sense, 5′-TCA ACC CCC AGC TAG TTG TC-3′; IL-4 antisense, 5′-TGTCT TCG TTG CTG TGA GG-3′; IFN-r sense, 5′-GAA AAT CCT GCA GAGCA GA-3′; IFN-r antisense, 5′-TGA GCT CAT TGA ATG CTT GG-3′;oxp3 sense, 5′-GGC CCT TCT CCA GGA CAG A-3′; Foxp3 antisense,′-GCT GAT CAT GGC TGG GTT GT-3′; b-actin sense, 5′-GAA ATC GTGGT GAC ATC AAA G-3′; b-actin antisense, 5′-TGT AGT TTC ATG GATCC ACA G-3′.

Reactions were processed in a DNA thermal cycler (Perkin-Elmeretus, Norwalk, CT, USA) through cycles of 30 s of denaturation at4 ◦C, 1 min of annealing at 56–64 ◦C for IL-4 and IL-17, IFN-r, andoxp3 followed by 1 min of elongation at 72 ◦C. PCR cycles wereepeated for 25–33 cycles each for IL-, IL-17, IFN-r, and Foxp3, andhe cycle number was determined to be within the exponentialhase of amplification for each molecule. The level of mRNA expres-ion was presented as the ratio of the expression of the IL-17 PCRroduct to that of the b-actin product.

.7. Intracellular staining

Lymphoid organs from GVHD mice were removed androcessed into a single cell suspension. Absolute numbersor each organ were determined using a hemocytometer. Forytokine staining, cells were stimulated in vitro with 25 ng/mL

*P < 0.001, **P < 0.01, *P < 0.05. (B) Histopathology of the skin, liver, small intestine,ction was stained with H&E (original magnification, 200×). Bar graph indicates thee. *P < 0.05, **P < 0.01.

PMA (Sigma–Aldrich, St. Louis, MO, USA), 250 ng/mL ionomycin(Sigma–Aldrich), and 500 ng/mL of Golgi Plug (BD Biosciences,Franklin Lakes, NJ, USA) in a 48-well plate, and then were incubatedat 37 ◦C for 4–5 h before staining. Surface staining was performedfor 5 min in FcR block with the corresponding cocktail of antibodies.Cells were washed and resuspended in fixation/permeabilizationsolution (BD Cytofix/Cytoperm kit; BD Biosciences), and intracel-lular staining was performed per the manufacturer’s protocol. Absagainst mouse CD4, CD25, CD8a, Foxp3, CD21, CD23, B220, CD19,CD1d, CD5, IgM, IgD, CD138, IFN-r, IL-17, IL-4, IL-10, and Foxp3 wereobtained from BioLegend (San Diego, CA, USA), BD Biosciences,eBioscience (San Diego, CA, USA), or Invitrogen (Carlsbad, CA, USA).Gates were determined by isotype staining performed on the sameorgan and time point.

2.8. ELISA

The concentrations of IFN-�, IL-17, TGF-�, and IL-10 in cell cul-ture supernatants and serum were measured by sandwich ELISA(Duoset; R&D Systems, Lille, France). Serum levels of IgG antibod-ies were measured using a commercially available ELISA kit (BethylLaboratories, Montgomery, TX).

2.9. Western blotting for STAT3, p-STAT3 705, p-STAT5, and STAT5

Spleen tissues were collected from GVHD control and PIAS3vector-treated mice of the BMT model. Samples were separatedby SDS gel electrophoresis and transferred to a nitrocellulosemembrane (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Membranes were stained with primary antibodies against STAT3,p-STAT3 705, p-STAT5, and STAT5 (all from Cell Signaling, Dan-vers, MA) and b-actin. The appropriate HRP-conjugated secondaryantibody was then added.

82 S.-H. Lee et al. / Immunology Letters 160 (2014) 79–88

Fig. 2. Blocking of the STAT3 pathway by PIAS3 suppresses acute GVHD. Recipient BALB/c mice received 5 × 106 total bone marrow cells and 1 × 107 splenocytes from B6mice, and mock or PIAS overexpresssion vector was administrated on 1 day post BMT, using electroporation method. Mice were monitored for weight and clinical signs ofacute GVHD. (A) Twelve days after BMT, immunohistochemistry was performed for STAT3 expression in representative skin, small intestine, liver, and lung biopsies fromB ocytesf pSTATv e mic

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MT mice and PIAS3 vector-treated mice of the BMT model. (B) The lysates of splenor STAT3, pSTATTyr705, and �-actin expression levels. *P < 0.05, **P < 0.01. (C), (D)

isualized by confocal microscopy on day 15 after BMT. Bars represent the SD of fiv

.10. Staining for confocal microscopy analysis

Spleen tissues were obtained 7 weeks after primary immu-ization. The tissues were stained using anti-Foxp3, anti-IL-17,nti-IL-4, anti-IFN-r, anti-CD25, anti-p-STAT3 705, anti-p-STAT327, anti-STAT3, anti-p-STAT5, anti-STAT5, and anti-CD4 in PBS.tained tissues were analyzed by confocal microscopy (LSM 510eta; Carl Zeiss, Thornwood, NY, USA).

.11. Statistical analysis

Results were calculated using the GraphPad Prism 4.0 softwarend are presented as the means ± SD of at least three experiments.

P value of <0.05 was considered to indicate statistical signif-cance. Log-rank test was used for survival analysis. Data wereompared by two-factor ANOVA with Bonferroni’s post-test or byhe Mann–Whitney U-test, as appropriate.

. Results

.1. PIAS3 suppresses the development of acute GVHD

PIAS3 can specifically inhibit STAT3 activity through binding tohe STAT3 DNA binding domain [17]. To determine whether PIAS3verexpression modulates the development of aGVHD in vivo, thecDNA-PIAS3 vector was administered by intramuscular infusionnd subsequent electroporation as described previously (Section, Fig. 1a). The results demonstrated that aGVHD mice treatedith the PIAS3-overexpressing vector showed attenuated weight

oss, less severe clinical scores of aGVHD, and significantly delayedGVHD-induced lethality in recipient mice compared with theock vector-treated group (Fig. 1A). The skin, liver, small intestine,

nd lung are the target organs of aGVHD. Mice from each treatmentroup were sacrificed on day 12 post-BMT after GVHD induction.o determine the protective effects of PIAS3 activity on the devel-pment of aGVHD, the tissue pathologies of the skin, liver, small

from both groups were separated by SDS-PAGE and subjected to Western blottingTyr705 levels were determined in splenic CD4+ T cells and B220+ B cells and were

e per group. ***P < 0.001, *P < 0.05.

intestine, and lung were evaluated. As shown in Fig. 1c, pcDNA-PIAS3 vector-treated mice showed less severe aGVHD pathologyscores in the skin, liver, small intestine, and lung, whereas mockvector-treated recipient mice demonstrated moderate-to-severeaGVHD in those organs. The histopathologic changes of the nucleusand cytoplasm, as well as the extent of infiltrating inflammatorycells found in the aGVHD-target organs, were dramatically reducedin PIAS3-treated mice (Fig. 1B). Thus, PIAS3 protein overexpress-ion suppressed the development of aGVHD in a murine model ofallogeneic BMT.

3.2. PIAS3 controls the development of acute GVHD via STAT3modulation in a murine model of allogeneic BMT

Th17 cells and their effector molecules are involved in the patho-genesis of various autoimmune and inflammatory diseases [25–27].Additionally, Th17 cells and the STAT3 pathway were revealed toplay critical roles in the pathogenesis of GVHD [16]. Therefore,we next investigated whether PIAS3 could prevent the develop-ment of aGVHD by affecting STAT3 activity. Immunohistochemicalstaining showed that STAT3 activities were dramatically attenu-ated by treatment with the PIAS3-overexpressing vector (Fig. 2A).To quantify STAT3 activity in both groups of mice, Western blot-ting was used. The results showed that STAT3 and pSTAT3Tyr705

activities in splenocytes isolated from each group of mice weresignificantly decreased by PIAS3 treatment (Fig. 2B). Conversely,pSTAT3Ser727 expression was not affected by PIAS3 treatment (datanot shown). In addition, the numbers of CD4+IL-17+ pSTAT3Tyr705+

T cells in spleens isolated from each group were counted usingconfocal staining to investigate the in vivo mechanism of PIAS3treatment in a murine model of aGVHD. The numbers of CD4+IL-17+pSTAT3Tyr705+ T cells were decreased in the PIAS3-treated group

compared with the mock vector-treated group (Fig. 2C). Similarly,the numbers of pSTAT3Tyr705+B220+ B cells were also significantlydecreased by PIAS3 treatment (Fig. 2D). Taken together, the findingsshow that decreased STAT3 activities, particularly pSTAT3Tyr705, in

S.-H. Lee et al. / Immunology Letters 160 (2014) 79–88 83

Fig. 3. Analysis of T helper cell subtype in PIAS3-treated GVHD mice. Recipient BALB/c mice received 5 × 106 total bone marrow cells and 1 × 107 splenocytes from B6 mice,and mock or PIAS overexpresssion vector was administrated on 1 day post BMT, using electroporation method. Mice were monitored for weight and clinical signs of acuteGVHD. (A) Serum levels of IFN-�, IL-17, and IL-10 in both groups were measured by ELISA. Data are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Twelvedays after BMT, the mRNA levels of IL-17, IL-4, Treg, and IFN-� in the control and PIAS3 vector-treated GVHD mice were analyzed by quantitative PCR. **P < 0.01. (C) Twelved els ofw cytes ia sualiz

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ays after BMT, splenocytes and lymph nodes were isolated, and the expression levas performed once using five mice per group. (D) Twelve days after BMT, spleno

nti-IL-17, anti-IL-4, anti-IFN-� antibodies, and then the expression patterns were vi

arget organs (the skin, small intestine, liver, and lung) and spleno-ytes (both T and B cells) mediate the attenuated development ofGVHD.

.3. PIAS3 overexpression modulates effector T cell subsets andnflammatory cytokine production in vivo

We next examined whether PIAS3 overexpression could affect cell subsets in the murine model of aGVHD. First, the serumFN-� and IL-17 levels tended to decrease. However, the IL-10evel was significantly increased by treatment with the PIAS3-verexpressing vector (Fig. 3A) The mRNA levels of IFN-�, IL-4,L-17, and Foxp3 were determined in spleen T cells that were iso-ated from each group of mice. The mRNA levels of IFN-� and IL-17

ere decreased in the PIAS3-treated group. Foxp3 is a key trans-riptional factor for Treg cell differentiation. Foxp3 mRNA levels inhe spleens of PIAS3-treated mice were increased compared withhose in the mock vector-treated group (Fig. 3B). Flow cytometricnalysis demonstrated that enhanced expression of PIAS3 signif-

cantly decreased IL-17 (Th17) and IFN-�-expressing T cell (Th1)opulations in spleens and lymph nodes, whereas IL-4-expressingTh2) and Foxp3-expressing (Treg) T cells were increased (Fig. 3C).onfocal staining of the spleens isolated from each group of mice

IL-4, IL-17, IFN-�, and Foxp3 were determined by flow cytometry. The experimentsolated from each group were stained with anti-CD4, followed by anti-Foxp3 anded by confocal microscopy. Bars represent the SD of five mice per group. ***P < 0.001.

on day 12 after BMT was conducted. The results also demonstratedthat the Th1 and Th17 populations in spleens were decreasedin PIAS3-treated mice. By contrast, Th2 and Treg subsets wereprofoundly expanded by PIAS3 overexpression in recipient mice(Fig. 3D). To summarize, PIAS3 overexpression could act as a recip-rocal regulator on Th1/Th17 and Th2/Treg subsets in a murinemodel of aGVHD transplanted with allogeneic BMT, thereby exert-ing a suppressive property on aGVHD development.

3.4. PIAS3 overexpression induces the expansion of CD4+ Tregand CD8+ Treg cells in vivo

In addition to CD4+CD25+Foxp3+ Treg cells, CD8+Foxp3+ Tregcells have been reported to play a critical role in the maintenanceof tolerance after allogeneic BMT [28,29]. STAT5 signaling is essen-tial to maintain homeostasis and preserve the immunoregulatoryproperties of Treg cells [30]. Confocal microscopy showed thatthe CD4+Foxp3+pSTAT5+ Treg population was increased in pcDNA-PIAS3 vector-treated recipient mice compared with the control

group. Interestingly, CD8+Foxp3+pSTAT5+ Treg (CD8+ Treg) cellswere also increased by PIAS3 overexpression (Fig. 4A). Next, flowcytometric analysis was performed. As shown in Fig. 4B, comparedwith control mice, both CD4+ and CD8+ Treg populations were

84 S.-H. Lee et al. / Immunology Letters 160 (2014) 79–88

Fig. 4. PIAS3 overexpression induces CD4 and CD8+ Treg in GVHD. Recipient BALB/c mice received 5 × 106 total bone marrow cells and 1 × 107 splenocytes from B6 mice, andmock or PIAS overexpresssion vector was administrated on 1 day post BMT, using electroporation method. Mice were monitored for weight and clinical signs of acute GVHD.(A) Twelve days after BMT, splenocytes isolated from each group were stained with anti-CD4 and anti-CD8 antibodies, followed by anti-Foxp3 antibodies, and visualized byc .001 (W are repfl are e

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onfocal microscopy. Bars represent the SD of five mice per group. **P < 0.01, ***P < 0estern blotting for STAT5, phospho-STAT5 and �-actin expression levels. The data

ow cytometric contour plots of CD4+ or CD8+ Foxp3+ Treg cells in the spleen. Data

ncreased in the spleens of PIAS3-treated mice (1.44 versus 1.76% inhe CD4+ Treg cell population, respectively; 0.95 versus 2.05% in theD8+ Treg cell population, respectively). Western blotting showedhat STAT5 and pSTAT5 activities in isolated spleen T cells werencreased dramatically in PIAS3-overexpressing mice (Fig. 4C).

.5. In vitro regulatory effects of PIAS3 on T cell subsets

Next, we determined the immunoregulatory property of PIAS3rotein in the differentiation of T-cell subsets. CD4+ T cells thatere isolated from the spleens of normal C57BL/6 mice were

ransfected with pcDNA-PIAS3 or mock vector. The cells werehen cultured with anti-CD3 and anti-CD28 mAb. After 3 days oftimulation, flow cytometric analysis showed that Th17 cell differ-ntiation was inhibited by PIAS3 overexpression, whereas Treg cellifferentiation was augmented (Fig. 5A). Next, IFN-� and IL-17 con-entrations in the culture supernatants were measured by ELISA.n line with the in vivo results, IFN-� and IL-17 levels were signif-

cantly suppressed by PIAS3 overexpression (Fig. 5B). By contrast,IAS3-transfected CD4+ T cells under Treg cell-polarizing condi-ions increased more significantly the production of TGF-� thanontrol CD4+ T cells (Fig. 5C). The mRNA levels of TGF-�, Foxp3 and

B) Fifteen days after BMT, splenocytes of each group were isolated and subjected toresentative of at least three independent experiments. **P < 0.01 (C) Representative

xpressed as means ± SD. *P < 0.05, **P < 0.01.

IL-10 were then measured in the cells. As shown in Fig. 6d, Foxp3and IL-10 mRNA levels were increased significantly by PIAS3 over-expression. Additionally, PIAS3 activity did not affect TGF-� mRNAlevels (Fig. 5D).

3.6. PIAS3 protein modulates B cell subsets via STAT3 activity

STAT3 protein can modulate the activities of B cells, APCs,and T cells. Similar to T cells, B cells are involved in theimmunopathophysiology of autoimmunity and inflammatory con-ditions, including GVHD [31,32]. Several recent studies havereported B cell development and its activation are regulated bySTAT3 activity [33–35]. STAT3 signaling plays a critical role inthe stage of plasma cell development in B cell lines rather thanin naïve, immature, mature, and memory B cell populations [36].Therefore, we examined the effects of PIAS3 on B-cell subpop-ulations. Serum total IgG levels were significantly decreased inPIAS3-overexpressing mice (Fig. 6A). Flow cytometric analysis

demonstrated that the immature and mature B cell populationswere not affected by PIAS3 treatment (Fig. 6B). Interestingly, themarginal zone B cell (CD21highCD23low) population was signifi-cantly increased in PIAS3-overexpressing recipient mice, whereas

S.-H. Lee et al. / Immunology Letters 160 (2014) 79–88 85

Fig. 5. PIAS3 controls the in vitro T cell response. Splenic CD4+ T cells sorted from C57BL/6 mice transfected with mock or PIAS3 vector. Mock or PIAS3 transfected T cellsactivated by stimulation with anti-CD3/CD28 for 3 days. (A) The Th17 and CD4+CD25+Foxp3+ Treg cells were analyzed by flow cytometry. Data are expressed as means ± SD ofthree independent experiments. *P < 0.05, **P < 0.01 versus mock. (B) IL-17 and IFN-r concentrations in culture supernatants were determined by ELISA. ***P < 0.001 (C) Mock-or PIAS3-transfected T cells were cultured under Treg-polarizing conditions. The differentiated Treg cell culture supernatants were analyzed for TGF-� expression levels byE by re*

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LISA. ***P < 0.001 (D) The mRNA levels of IL-17, TNF-�, and IFN-r were determined*P < 0.05, ***P < 0.001 versus mock.

he follicular (CD21intCD23high) B cell subset was decreased slightlyFig. 6C). Interestingly, the plasma B cell population (CD138+ B cells)as dramatically decreased by PIAS3 overexpression (Fig. 6D).

L-10 production is the hallmark of regulatory B cells, which neg-tively regulate inflammation and autoimmune disease [37–39].D19+CD5+CD1d+IL-10+ regulatory B cells have been revealed touppress autoimmunity, inflammatory responses, and cancers innimal models [40,41]. Furthermore, regulatory B cells can func-ion as a negative regulator of the development of GVHD [42].ur study demonstrated that the IL-10-expressing B cell (mainly

egulatory B cells) population was profoundly increased in recip-ent mice treated with the pcDNA-PIAS3 vector compared with

ock vector-treated mice (Fig. 3e; 3.95% versus 12.2%, respectively).hese results suggested that decreased STAT3 activity via PIAS3verexpression could induce regulatory B cells subsets and recip-ocally inhibit antibody-producing B cells, thereby suppressing theevelopment of aGVHD.

. Discussion

In the present study, we demonstrated that the PIAS3 enhancingtrategy using a pcDNA-PIAS3 vector could attenuate the clinicalnd histopathological severity of aGVHD development. The under-ying preventive function of PIAS3 in the development of aGVHDs to modulate T and B cell subsets through regulatory effects onTAT proteins (STAT3 and STAT5). This is the first study to inves-

igate the in vivo and in vitro effects of PIAS3 protein on aGVHDegarding Th17/Treg balance.

Despite the widespread use of HSCT, major obstacles to con-entional HSCT include conditioning regimen-related toxicities,

al-time PCR. Data are expressed as means ± SD of three independent experiments.

immunological aspects of GVHD and graft rejection, and limita-tions in the number of HLA-identical donors [3,4]. Nevertheless,current GVHD prophylaxis and treatment are based on non-specific immunosuppressive drug therapy, including FK506 andcyclosporine A [43]. Prolonged use of those immunosuppressivedrugs increases the risk of various complications, including seriousinfections, which lead to lethality in HSCT recipients.

Although the pathophysiology of aGVHD is complex, recentstudies have discovered the possible role of Treg/Th17 imbalancein the development of GVHD [44]. However, previous studies usingIL-17-deficient T cells have suggested that IL-17 is not essential forall manifestations of aGVHD but exerts a pathogenic role in organ-specific GVHD [45,46]. And, Th17 cells polarized ex vivo do notalways maintain their cytokine profiles in vivo after transfer. There-fore, targeted disruption of the transcription factors that are criticalfor Th17 and Treg cell differentiation would be a better strategythan direct modulation of Th17 population.

The STAT3 signaling pathway that is activated in GVHD mod-els promotes in vivo expansion of donor-derived CD4+ T cells andtheir trafficking to epithelial organs. Additionally, in the setting ofallogeneic HSCT, STAT3 signaling hinders CD4+ T cell differenti-ation into Treg cells [16]. Abrogation of STAT3 activity exerted amodulatory effect on the development of chronic GVHD after allo-geneic HSCT [16]. Among the molecules implicated in JAK-STATpathway, one key regulator is the recently discovered PIAS fam-ily. In vivo studies have identified specific PIAS-STAT interactions.

After cytokine stimulation, PIAS3 specifically interacts with STAT3[17]. And, PIAS3 can directly interact with STAT5, thereby enhanc-ing STAT5 DNA-binding activity [47]. In line with that finding,our present study also found that PIAS3 enhancement significantly

86 S.-H. Lee et al. / Immunology Letters 160 (2014) 79–88

Fig. 6. Analysis of B-cell subsets in PIAS3-treated GVHD mice. Recipient BALB/c mice received 5 × 106 total bone marrow cells and 1 × 107 splenocytes from B6 mice, andmock or PIAS overexpresssion vector was administrated on 1 day post BMT, using electroporation method. Mice were monitored for weight and clinical signs of acute GVHD.(A) On day 12 after BMT, mean serum IgG concentrations determined by ELISA were lower in PIAS3 vector-treated mice than those in the BMT group. Values are shown asmeans ± SD (n = 3 animals per group). **P < 0.01. (B–D) On day 12 after BMT, B-cell subsets were analyzed. Splenocytes isolated from control or PIAS3 vector-treated GVHDmice were stained for B220, IgM, IgD, B220, CD21, CD23, and Ig-secreting plasma cells (B220+CD138+) and then analyzed by flow cytometry. Cells shown were gated on B220.B cells),

* ls (CD*

is

TrrGaiosai[mTt

pokRtCkpc

220+ B cells included IgMhighIgDlow (immature B cells), IgMhighIgDhigh (mature B

P < 0.05, **P < 0.001. (E) The IL-10 cytokine level within the proportion of B10 celP < 0.05.

nhibited STAT3 activity and reciprocally enhanced STAT5 expres-ion in vivo, therefore suppressed aGVHD development.

PIAS3 can modulate effector B- and T-cell populations. Although cells have classically been considered the main underlying cause,ecent studies have suggested that B cells also play fundamentaloles in GVHD development [31,48]. B cell-induced aggravation ofVHD is identified to induce the clonal expansion, differentiation,nd survival of pathogenic CD4+ T cells [49]. As in T cells, STAT3s also involved in B-cell development, particularly in the stagef terminal differentiation of B cells into plasma cells [34]. In ourtudy, IL-10-producing regulatory B cells (B10) were significantlyugmented by PIAS3 treatment. Regulatory B cells may regulatemmune responses through their production of the cytokine IL-1050]. IL-10 can act on the suppressive function of Treg cells through

aintaining Foxp3 expression in a murine model of colitis [51].herefore, IL-10 might play a critical role in PIAS3-induced inhibi-ion during the development of aGVHD.

Our study showed that Foxp3+CD8+ and Foxp3+CD4+ T-cellopulations were increased in mice treated with the PIAS3-verexpressing vector. CD8+Foxp3+ regulatory T cells are nownown to be important for restoration of immune balance.egarding GVHD development, recent research has shown consis-ent results regarding the regulatory function of CD8+ Treg cells.

D8+ Treg cells can abrogate GVHD severity in vivo [52]. To ournowledge, our study is the first to show the immune modulatoryroperties of PIAS3 in terms of induction of CD8+CD25+Foxp3+ Tregells.

CD21lowCD23high (follicular B cells), and CD21highCD23low (marginal zone B cells).19highCD1dhighCD5high) in the spleen increased in the PIAS3 vector-treated group.

Nonspecific immunosuppressive agents have been used asGVHD prophylaxis strategy. However, immunosuppressive ther-apy has shown to reduce the T-cell mediated graft-versus-leukemia(GVL) effects. As GVL effect of allogeneic T cells is due largely torecognition by donor T cells of host alloantigens that are expressedon malignant cells, strongest GVL effect are observed in the set-ting of donor-host MHC disparity. Therefore, it has been consideredthat GVL effect induced by donor T cells occurs parallel to GVHD.But, there are some exceptions in that general concept regardingthe link between GVHD and GVL effects. One is NKT cells [53] andthe other is Treg cells. GVHD modulating property of Treg cells hasbeen identified to be separated from GVL effects [54]. About onedecade ago, Edinger et al. demonstrated that Treg cells attenuateGVHD activity while permitting GVL effects mediated by donor Tcells [55]. After that study, there have been many animal studiesthat determined the GVHD inhibiting effects of Treg cells withoutabrogating GVL effects [54]. In addition, HSCT patients receivinglow numbers of Treg showed a significantly increased incidence ofaGVHD when compared with individuals receiving high numbersof Treg cells [56]. In the present study, the changes of GVL effects bySTAT3 inhibition were not elucidated. Surely, additional studies arewarranted to determine whether or not STAT3 inhibition by PIAS3affects GVL effects derived by donor T cells.

In summary, the present report showed that PIAS3 inhibitedT-cell responses and IFN-r and IL-17 production in vitro. ThePIAS3-enhancing strategy using the pcDNA-PIAS3 vector atten-uated aGVHD severity and shifted responses from Th1, Th17,

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nd terminal differentiation of B cells into plasma cells in vivo.dditionally, PIAS3 treatment in recipient mice transplanted withllogeneic bone marrow resulted in the expansion of CD4 as wells CD8+ Treg cells in vivo. PIAS3 regulation of the immune bal-nce between CD4+/CD8+ Treg and Th17 cells by modulating pro-r anti-inflammatory cytokines and specific transcription factorsay be a promising treatment choice for the development of

GVHD.

onflicts of interest

The authors declare no financial or commercial conflicts of inter-st.

cknowledgments

This research was supported by the Bio & Medical Tech-ology Development Program of the National Research Foun-ation (NRF) funded by the Korean government (MEST) (No.012M3A9C6049783). This study was supported by a grant of theorean Health Technology R&D Project, Ministry for Health & Wel-

are, Republic of Korea (HI09C1555).

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