CSIRO PUBLISHING
Reproduction, Fertility and Development, 2016, 28, 1788–1797 http://dx.doi.org/10.1071/RD14503
Adoptive transfer of transforming growth factor-b1-induced CD41CD251 regulatory T cells prevents immune response-mediated spontaneous abortion Tian Qiu A, Yincheng Teng A, Yudong Wang B and Liang Xu B,C A
Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, China. B Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated International Peace Maternity and Child Health Hospital, 910 Hengshan Road, Shanghai 200030, China. C Corresponding author. Email: [email protected]
Abstract. The effects of adoptive transfer of transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells in preventing spontaneous abortion in mice were investigated. CD4þCD25 cells were isolated from the spleens of pregnant CBA/J mice and induced into Treg cells positive for CD4, CD25 and forkhead box P3 (FOXP3) ex vivo using interleukin (IL)-2 and TGF-b1. CBA/J mice were mated with DBA/2J mice to establish a model of spontaneous abortion and, on the first day of pregnancy, mice were injected intravenously with 2 105 either freshly isolated Treg cells or those induced with TGF-b1. After 14 days, the surviving and reabsorbed fetuses in both groups were counted, and serum cytokine concentrations were measured by ELISA. Adoptive transfer of CD4þCD25þ or TGF-b1-induced Treg cells significantly reduced the fetal resorption rate, increased serum IL-10 and TGF-b1 concentrations and decreased interferon-g levels. In conclusion, the results of the present study indicate that adoptive transfer of TGF-b1-induced Treg cells prevents spontaneous abortion in mice by increasing the secretion of T helper (TH) 2 cytokines and decreasing the secretion of TH1 cytokines. Additional keywords: forkhead box P3 (FOXP3). Received 22 July 2014, accepted 17 April 2015, published online 14 May 2015
Introduction In reproductive immunology, the embryo is considered to be a semi-allograft that is not rejected by the maternal immune system in a physiological pregnancy. Thus, the maternal immune system shows a special type of immune tolerance to the fetus. However, if this immune tolerance is compromised, pathological pregnancy, including spontaneous abortion, occurs. CD4þCD25þ regulatory T (Treg) cells are a subset of T cells with potent immunosuppressive activity (Sakaguchi et al. 1995) that can inhibit effector T cell activation and maintain peripheral tolerance and immune system homeostasis. During mammalian pregnancy, levels of Treg cells increase in response to maternal immune cells encountering paternal antigens (Zenclussen et al. 2010; Clark and Chaouat 2012; Sereshki et al. 2014). Recent studies have associated a deficiency in Treg cells with a propensity for implantation rejection and miscarriage (Winger and Reed 2011; Inada et al. 2013; Lu et al. 2013), suggesting that Treg cells play a critical role in the maintenance of immune tolerance to the fetus (Zenclussen et al. 2005; Bao et al. 2011). The immunosuppressive activity of Treg cells depends on the expression of forkhead box P3 (FOXP3), which is a transcription factor and a specific biomarker of Treg cells (Hori et al. 2003; Journal compilation Ó CSIRO 2016
Hori and Sakaguchi 2004). Thus, CD4þCD25þFOXP3þ Treg cells are crucial for maintaining a normal pregnancy. Decreased levels of peripheral CD4þCD25þ T cells and FOXP3 expression are associated with the pathogenesis of unexplained recurrent spontaneous abortion (Mei et al. 2010). Therefore, modulating the number of Treg cells may represent a promising therapeutic target for preventing spontaneous abortion. A previous study indicated that adoptive transfer with Treg cells isolated from pregnant mice inhibits spontaneous abortion in abortion-prone pregnant mice (Zenclussen et al. 2005). This protection was not observed with Treg cells isolated from nonpregnant mice (Zenclussen et al. 2005). However, there are relatively few Treg cells in peripheral blood; therefore, it is difficult to obtain sufficient numbers of these cells for therapeutic application. Several experimental protocols have been developed for inducing Treg cells with tumour growth factor (TGF)-b1 in vitro (Park et al. 2004; Wahl and Chen 2005; Selvaraj and Geiger 2007; So and Croft 2007). TGF-b1-induced Treg cells are functionally stable and have potent suppressive activity. When naı¨ve CD4þCD25 T cells were costimulated with TGF-b1, anti-CD3 and anti-CD28 antibodies, Treg cell-associated markers, such as CD25, cytotoxic T lymphocyte-associated antigen www.publish.csiro.au/journals/rfd
Induced Treg cells prevent spontaneous abortion
(CTLA)-4 and FOXP3, were upregulated and the cells acquired anergic and suppressive phenotypes similar to those of thymusderived Treg cells (Park et al. 2004; Wahl and Chen 2005; Selvaraj and Geiger 2007; So and Croft 2007). However, whether treatment of abortion-prone pregnant mice with in vitro TGF-b1-induced Treg cells can prevent spontaneous abortion is not known. TGF-b1 is a key molecule contributing to peripheral tolerance. Abrogation of TGF-b1 signalling in T cells alone results in spontaneous T cell differentiation and autoimmune diseases, highlighting the essential role of TGF-b1 signalling in T cell homeostasis and the prevention of inflammatory autoimmunity (Gorelik and Flavell 2000; Lucas et al. 2000). Several studies have also demonstrated the relevance of TGF-b1 in Treg cell physiology. For example, TGF-b1 expands and amplifies the function of CD4þCD25þ Treg cells ex vivo (Yamagiwa et al. 2001) and TGF-b1 deficiency or blockade in vivo eliminates the suppressive capabilities of Treg cells (Powrie 1995; Fuss et al. 2002). These findings suggest that the immunosuppressive activity of Treg cells depends on TGF-b1. In the present study we used a mouse model of spontaneous abortion to determine whether adoptive transfer of in vitro TGFb1-induced Treg cells could prevent spontaneous abortion in abortion-prone pregnant mice. CBA/J mice were mated with DBA/2J mice to produce an in vivo model of spontaneous abortion. Adoptive transfer of TGF-b1-induced Treg cells significantly reduced fetus resorption rates; therefore, this procedure may represent a therapeutic approach for spontaneous miscarriage. Materials and methods Animals Eight- to 10-week-old female CBA/J (MHC H2 Haplotype k (h-2k)), male DBA/2J (MHC H2 Haplotype d (h-2d)), and male BALB/c (h-2d) mice weighing 20–30 g were purchased from the Experimental Animal Center of the Chinese Academy of Sciences, Shanghai; these strains were originally obtained from Japan’s National Institute of Genetics, Shizuoka, Japan. Mice were housed under specific pathogen-free conditions and were provided 1.5 g food and 2.5 mL water per 10 g bodyweight each day. The Experimental Animal Center of the Chinese Academy of Sciences bred the mice following International Genetic Standards (White and Lee 1998) to ensure the genetic quality of the strains. This study was approved by the Institutional Review Board of the Sixth People’s Hospital Affiliated to Shanghai Jiaotong University. Isolation and induction of CD41CD252 T cells Splenic cells from pregnant CBA/J mice mated with BALB/c mice (normal pregnancy model) were gently minced in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; both from Bio-Light Biotech, Shanghai, China) and single cell suspensions were prepared by passage through a 100-mm nylon cell strainer. CD4þ T cells (105–106 cells) were isolated by eliminating cells expressing CD8a, CD11b, CD45R, CD49b and Ter-119 using the CD4þCD25þ Treg isolation kit (Miltenyi Biotec, San Diego, CA, USA). CD4þCD25 T cells were then isolated by eliminating cells expressing CD25.
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CD4þCD25þ T cells (103–104 cells) were isolated by positive selection using the CD4þCD25þ Treg isolation kit (Miltenyi Biotec). The CD4þCD25þ T cells were pooled before transfer into pregnant mice. For in vitro TGF-b1 induction, approximately 1 106 CD4þCD25 T cells were stimulated with 5 mg mL1 platebound anti-CD3 (BD Biosciences, San Diego, CA, USA), 0.5 mg mL1 soluble anti-CD28 (BD Biosciences), 5 ng mL1 TGF-b1 (R&D Systems, Minneapolis, MN, USA) and 10 U mL1 interleukin (IL)-2 (PeproTech, Rocky Hill, NJ, USA) for 5 days. The Treg cells were then harvested and analysed by flow cytometry. Suppression assay To determine the suppressive activity of TGF-b1-induced Treg cells isolated from pregnant CBA/J mice with normal pregnancies (Day 14) on CD4þ T cell proliferation, carboxyfluorescein succinimidyl ester (CFSE)-labelled CD4þ T cells were cocultured with TGF-b1-induced Treg cells, as described previously (Park et al. 2004). Briefly, freshly isolated splenic CD4þ T cells from CBA/J mice were washed and resuspended at a concentration of 1.0 107 cells mL1 in phosphate-buffered saline (PBS). CFSE (Invitrogen, Carlsbad, CA, USA) was added at a final concentration of 5 mM and samples were incubated for 10 min at room temperature. The reaction was stopped by the addition of 500 mL cold FBS, and the cells were washed three times with PBS. Then, 1.0 106 CFSE-labelled CD4þ T cells were mixed with 1.0 105 TGF-b1-induced Treg cells, CD4þCD25þFOXP3þ Treg cells or CD4þCD25 cells in a volume of 500 mL per well in 48-well plates containing 1 mg mL1 immobilised anti-CD3 and 0.2 mg mL1 soluble anti-CD28 (both from BD Biosciences). After 72 h coculture, the fluorescence intensity of live CFSE-labelled cells was determined by flow cytometry (see below). Animal groups Male BALB/c or DBA/2 mice were mated with female CBA/J mice. After pregnancy had been confirmed by the presence of a vaginal plug (Day 0), which was assessed twice daily at 0800 and 1600 hours, mice were separated into the following five groups: (1) CBA/J mice mated with BALB/c mice (normal pregnancy model); (2) CBA/J mice mated with DBA/2 mice (abortion model without treatment); (3) CBA/J mice mated with DBA/2 mice and injected intravenously with 2 105 CD4þCD25þFOXP3þ Treg cells on Day 1 of pregnancy; (4) CBA/J mice mated with DBA/2 mice and injected intravenously with 2 105 TGF-b1-induced Treg cells on Day 1 of pregnancy; and (5) CBA/J mice mated with DBA/2 mice and injected intravenously with 2 105 CD4þCD25 T cells on Day 1 of pregnancy. There were no significant differences in the age or weight of the mice randomly assigned to the five groups, either within or between groups. On Day 14 of pregnancy, pregnant mice were killed and their uteri removed to evaluate implantation sites. Compared with normal embryos and placentas, the abortion sites were identified by their relatively small sizes and necrotic and haemorrhagic appearance, as described previously (Zhao and Bao 1999).
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The resorption rate was calculated as: (no. total abortions/no. total implantations) 100%. For the flow cytometry assays, spleen samples were cut into small pieces and collected in Hanks’ balanced salt solution without Ca2þ or Mg2þ. The uterine horns were opened longitudinally and the feto-placental unit was separated from the uterine implantation sites, and decidual tissues were isolated. For RNA extraction and western blot analysis, decidual tissues were washed thoroughly with cold sterile PBS, pH 7.40, snap frozen and stored at 808C until further use. Because tissue from resorption sites is necrotic, which makes RNA isolation from these sites extremely difficult, we harvested tissues from healthy implantation sites for RNA extraction, as described by Zenclussen et al. (2005).
buffer (Amresco, Solon, OH, USA). Proteins were transferred to 0.45 mm Optitran nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA) in Tris-glycine buffer (Amresco). Membranes were blocked in PBS containing 5% non-fat dry milk and incubated overnight at 48C with anti-FOXP3 (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-glyceraldehyde-3-phosphate dehydrogenase (1 : 20 000 dilution; Research Diagnostics, Flanders, NJ, USA) antibodies. The membranes were washed several times with PBS containing 0.1% Tween 20 (PBS-T) and incubated with peroxidase-conjugated IgG (1 : 2000 dilution) in blocking solution. After the membranes had been washed several times in PBS-T, they were developed with enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA) and exposed to film.
Determination of cytokine levels On Day 14 of pregnancy, serum samples were collected from anaesthetised pregnant mice (ketamine, 10 mg/100 g body weight, intraperitoneal injection) via retro-orbital bleeding. Serum concentrations of total TGF-b1, interferon (IFN)-g and IL-10 were determined using commercially available ELISA kits (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions. For analysis of IL-10 and TGF-b1 levels produced by CD4þCD25 T cells and CD4þCD25þFOXP3þ or TGF-b1induced Treg cells, 5 103cells per well were cultured in 96-well microplates for 2 h (RPMI1640 with 2 mmol L1 L- glutamine,1105 U L1 penicillin,100 g L1 streptomycin,50 mmol L1 2-mercaptoethanol and 100 mL L1 fetal calf serum). The microplates were cultured at 378C and 5% CO2.), after which the cell culture supernatant was collected and 0.5 mL aliquots were used for ELISA according to the manufacturer’s instructions.
Real-time polymerase chain reaction Total RNA was extracted from placental tissue using TRIzol reagent (Invitrogen). First-strand cDNA was synthesised using random hexamers and Superscript II reverse transcriptase (Invitrogen) in a final volume of 40 mL using 2 mg RNA. Amplification was performed in an ABI 7900 Sequence Detector (Applied Biosystems, Foster City, CA, USA) using the Takara SYBR Premix Ex Taq kit (Takara, Shiga, Japan) in a final reaction volume of 10 mL, containing 0.5 mL cDNA, 10 pmol each primer and 5 mL Sybr Green premix. The cycling parameters were as follows: 2 min at 508C, 10 s at 958C, 40 cycles of 5 s at 958C and 30 s at 608C, and then 10 min at 708C. The following Foxp3-specific primers were designed using Primer Express Software v2.0 (ABI) based on sequences from the National Center for Biotechnology Information (NCBI): sense, 50 -CCAGACGGCGGCCTGTTTGC-30 ; antisense, 50 -GCTCTCCACT CGCACAAAGC-30 .
Flow cytometry Cells were isolated from the spleen by disaggregating and filtering the tissue through a sterile net. Erythrocytes were then lysed with a solution of NHCl4–NaCl. Cells were collected, washed and incubated with antibodies for specific surface markers for 10 min at 48C in the dark before being fixed with a 1% paraformaldehyde solution overnight at 48C. After permeabilising the cells with 0.2% saponin, intracellular antibodies were added and samples were incubated for 20 min at 48C in the dark. Then, cells were washed with cold sterile PBS, pH 7.40, and analysed using a FACScan flow cytometer (BD Bioscience). The following monoclonal antibodies (mAbs) were used: fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD4, allophycocyanin (APC)-conjugated rat anti-mouse CD25 and phycoerythrin (PE)-labelled anti-mouse FOXP3 and CD69. FITC-, APC- and PE-conjugated rat IgG1 or Ig2b were used as negative controls in separate tubes (all from eBioscience, San Diego, CA, USA). Western blot analysis In western blot assays, 20 ng protein was separated on a 10% Tris-glycine sodium dodecyl sulfate (SDS)–polyacrylamide gel (Invitrogen) in Tris-Glycine SDS Running Buffer (TG-SDS)
Statistical analysis Normally distributed data are summarised as the mean s.d. and were compared by one-way analysis of variance (ANOVA) with post hoc Bonferroni correction. Skewed data are expressed as median values with the interquartile range (IQR) in parentheses and were compared using the Kruskal–Wallis test, with the Mann–Whitney test as a post hoc test. Statistical analyses were performed using SAS software version 9.2 (SAS Institute, Cary, NC, USA). Two-tailed P , 0.05 was considered significant. Results TGF-b1 induction of CD25 and FOXP3 expression by CD41CD252 T cells The purity of the CD4þCD25 T cells after isolation was 99.05 9.73%, with only 3.4 1.3% of cells expressing FOXP3. As shown in Fig. 1a, b, the purified CD4þCD25 T cell fraction contained ,1.0% CD25þ cells. Specifically, flow cytometric analysis of the purified cells showed that the proportion of CD4þCD25 cells to CD4þ cells in six independent experiments was 99.40%, 99.80%, 99.30%, 98.70%, 99.10% and 99.20%. The proportion of CD25þ cells increased significantly from 0.92 0.28% to 79.46 7.08% (P , 0.001; Table 1) after stimulation with anti-CD3 and anti-CD28 antibodies for 5 days in the presence of TGF-b1 in fresh medium
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Fig. 1. Stimulation with transforming growth factor (TGF)-b1 induces CD25 and forkhead box P3 (FOXP3) expression by CD4þCD25 T cells. Representative expression of (a, b) CD25 and (c, d) Foxp3 by CD4þCD25 T-cells (a, c) and those stimulated with anti-CD3 and anti-CD28 antibodies for 5 days in the presence of TGF-b1 and interleukin-2 (b, d ).
Table 1. Stimulation with transforming growth factor-b1 induces CD25 and forkhead box P3 (FOXP3) expression by CD41CD252 T cells Data are the mean s.d. Before, before stimulation with transforming growth factor-b1; After, after stimulation with transforming growth factor-b1
CD25 (%) Foxp3 (%)
Before
After
P-value
0.92 0.28 3.13 1.63
79.46 7.08 66.23 8.55
,0.001 ,0.001
containing exogenous IL-2. In addition, the presence of TGF-b1 during polyclonal stimulation of CD4þCD25 T cells enhanced the expression of FOXP3 from 3.13 1.63% to 66.23 8.55% (P , 0.001; Fig. 1c, d; Table 1). CD4þCD25þ T cells (103–104 cells) were isolated by positive selection using the CD4þCD25þ Treg isolation kit, and their purity ranged between 75% and 85%.
Suppression of CD41 T cell proliferation by TGF-b1induced Treg cells A functional hallmark of Treg cells is their ability to suppress the development and expansion of effector T cells. We therefore investigated whether TGF-b1-induced Treg cells were able to suppress the proliferation of CD4þ T cells using coculture assays. As indicated in Fig. 2 and Table 2, coculture with TGF-b1-induced Treg cells or CD4þCD25þFOXP3þ Treg cells significantly increased the number of non-dividing CD4þ T cells compared with untreated cells (P , 0.05). Moreover, the suppressive activity of TGF-b1-induced Treg cells was similar to that of CD4þCD25þFOXP3þ Treg cells. Effects of adoptive transfer of CD41CD251FOXP31 and TGF-b-induced Treg cells in mice The proportion of CD4þCD25þFOXP3þ T cells in the spleen and decidua was significantly lower in the untreated group, as well as in mice treated with CD4þCD25 T cells,
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CD4⫹ T cells alone
No stimulation
21.4%
93.2%
Table 2. Suppression of CD41 T cell proliferation by transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells Data are the mean s.d. (n ¼ 10 per group). Significant differences were found by post hoc analysis with Bonferroni correction. *P , 0.05 compared with untreated cells; †P , 0.05 compared with CD4þ T cells alone; z P , 0.05 compared with CD4þ T cells treated with CD4þCD25þFOXP3þ Treg cells; yP , 0.05 compared with CD4þ T cells treated with TGF-b1induced Treg cells. FOXP3, forkhead box P3; CFSE, carboxyfluorescein succinimidyl ester CFSE-labelled cells (%)
CFSE CD4⫹ T cells ⫹ TGF-β1-induced CD4⫹CD25⫹Treg cells
CD4⫹ T cells ⫹ CD4⫹CD25⫺ T cells
58.1%
Count
18.9%
CFSE
CD4⫹ T cells ⫹ ⫹ ⫹
CD4 CD25 FOXP3⫹ Treg cells 56.7%
No stimulation CD4þ T cells alone CD4þ T cells treated with CD4þCD25þFOXP3þ Treg cells CD4þ T cells treated with TGF-b1-induced Treg cells CD4þ T cells treated with CD4þCD25 T cells P-value between the five groups
92.68 3.47 21.44 2.82* 57.07 5.94*† 58.64 5.26*† 18.92 2.96*zy ,0.001
with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (P , 0.05 for both; Table 3). Using CD69 as a marker of activated T cells, we next evaluated the proportion of CD4þCD69þ T cells to CD4þ T cells in the spleen and decidua in the different groups and found no significant differences (P . 0.05 for all; Table 3). Finally, treatment of mice with CD4þCD25þFOXP3þ Treg cells significantly increased the proportion of CD4þCD25þFoxp3þ T cells : CD4þCD69þ T cells in the spleen compared with untreated control mice and mice treated with CD4þCD25 T cells (P , 0.001 for both). The proportion of CD4þCD25þFoxp3þ T cells to CD4þCD69þ T cells in the decidua was significantly lower in mice treated with CD4þCD25 T cells than in the normal pregnancy group (P ¼ 0.001; Table 3). Foxp3 mRNA and protein levels were higher in deciduas of mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells than in mice treated with CD4þCD25 T cells or untreated mice (P , 0.001 for both). However, Foxp3 expression remained slightly lower in deciduas of Treg cell-treated mice than mice with normal pregnancies (Table 3).
CFSE Fig. 2. Suppression of responder cell proliferation by transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells. Purified CD4þCD25 T cells from normal pregnant CBA/J mice were stimulated with 5 ng mL1 TGF-b1 for 5 days. After extensive washing, 1.0 105 TGF-b1-induced Treg cells were mixed with 1.0 106 carboxyfluorescein succinimidyl ester (CFSE)-labelled splenic T cells from CBA/J mice and stimulated with immobilised anti-CD3 and soluble anti-CD28 antibodies. After gating for CD4þ T cells, the fluorescence intensity of live CFSE-labelled cells after 72 h culture is shown as a histogram. The percentages in the figure represent undivided CFSEþ cells. These results are representative of 10 independent experiments. FOXP3, forkhead box P3.
compared with the normal pregnancy control group (P , 0.05; Table 3). Furthermore, compared with untreated mice, the proportion of CD4þCD25þFOXP3þ T cells in the spleen and decidua was significantly higher in mice treated
Effects of adoptive transfer of CD41CD251FOXP31 and TGF-b-induced Treg cells on abortion rate In the spontaneous abortion model, the embryo resorption rate was significantly higher in untreated mice (median 19.1%) compared with the normal pregnancy model (median 0%; P , 0.001; Table 4). The embryo resorption rates of mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (10.8% and 11.8%, respectively) were significantly lower than in untreated mice and did not differ significantly from that observed in the normal pregnancy model. The embryo resorption rate for mice treated with CD4þCD25 T cells (20.2%) did not differ significantly from that of untreated mice and was significantly higher the rates in mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (Table 4). There were no significant differences in fetal weights, placental
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Table 3. Phenotypic characterisation of splenic CD41 cells following T cell transfer and forkhead box P3 (Foxp3) protein and mRNA expression in deciduas of mice Data are the mean s.d. (n ¼ 10 per group). Significant differences were found by post hoc analysis with Bonferroni correction. zP , 0.05 compared with Group 1; yP , 0.05 compared with Group 2; *P , 0.05 compared with Groups 3, †P , 0.05 compared with Groups 4. Group 1, normal pregnancy model (CBA/ J BALB/c); Group 2, untreated abortion model; Group 3, abortion model treated with CD4þCD25þFOXP3þ Treg cells; Group 4, abortion model treated with transforming growth factor-b1-induced Treg cells; Group 5, abortion model treated with CD4þCD25 T cells Abortion models (CBA/J DBA/2)
Group 1 CD4þCD25þFoxp3þ T cells/CD4þ T cells (%) Spleen 7.25 1.11 Decidua 29.71 5.44 Splenic CD4þ cells CD25/CD4 (%) 7.64 1.98 Foxp3/CD4 (%) 7.25 1.11 Foxp3 expression Protein 0.805 0.110 mRNA 0.555 0.084 CD4þCD69þ T cells/CD4þ T cells (%) Spleen 4.77 1.25 Decidua 3.48 1.26 CD4þCD25þFoxp3þT cell/CD4þCD69þT cells (%) Spleen 1.64 0.57 Decidua 10.39 6.08 A
Group 5
P-valueA
7.47 0.58y* 21.67 3.62zy
3.46 0.48z† 13.58 3.02z†
,0.001 ,0.001
12.57 3.78zy 9.50 0.69zy
10.58 3.08y 7.47 0.58y*
4.88 1.73† 3.46 0.48z†
,0.001 ,0.001
0.638 0.058zy 0.481 0.052zy
0.556 0.045zy 0.384 0.032zy*
0.285 0.039z† 0.064 0.020z†
,0.001 ,0.001
Group 2
Group 3
3.3 0.48z 15.74 2.96z
9.5 0.69zy 23.86 4.06zy
5.01 1.88 3.30 0.48z 0.244 0.056z 0.075 0.020z
Group 4
4.45 1.30 3.52 1.05
5.38 1.95 3.66 1.10
5.14 1.44 3.34 1.01
4.92 1.25 3.84 0.96
0.82 0.35 4.83 1.49
1.99 0.75y 7.16 2.72
1.59 0.58 7.18 2.95
0.76 0.28† 3.74 1.15z
0.674 0.866 ,0.001 0.001
Determined by ANOVA.
Table 4. Comparisons of embryo resorption rates in pregnant CBA/J mice Data are presented as the mean s.d. (with P-values determined by ANOVA) or as median values with the interquartile range in parentheses (and P-values determined by the Kruskal–Wallis test). The number of embryos from each mouse ranged between 6 and 11. *P , 0.05 compared with Group 1; †P , 0.05 compared with Group 2, zP , 0.05 compared with Groups 3 and 4. Group 1, normal pregnancy model (CBA/J BALB/c); Group 2, untreated abortion model; Group 3, abortion model treated with CD4þCD25þFOXP3þ Treg cells; Group 4, abortion model treated with transforming growth factor-b1-induced Treg cells; Group 5, abortion model treated with CD4þCD25 T cells; FOXP3, forkhead box P3 Group 1
No. mice No. embryos No. resorptions Resorption rateA (%) A
10 8.7 1.5 0.0 (0.0, 1.0) 0.0 (0.0, 9.1)
Abortion model
P-value
Group 2
Group 3
Group 4
Group 5
10 9.3 1.1 2.0 (1.0, 2.0)* 19.1 (11.1, 22.2)*
10 8.6 1.7 1.0 (0.0, 1.0)† 10.8 (0.0, 14.3)†
10 8.3 1.6 1.0 (0.0, 1.0)† 11.8 (0.0, 14.3)†
10 8.8 1.5 2.0 (1.0, 3.0)*z 20.2 (11.1, 33.3)*z
0.658 ,0.001 ,0.001
The resorption rate was calculated as (no. resorptions/no. embryos) 100.
weights or their ratio (P . 0.05 for all; see Table S1 available as Supplementary Material to this paper). Effects of adoptive transfer of CD41CD251FOXP31 and TGF-b-induced Treg cells on TH1 and TH2 responses Analysis of serum cytokines indicated that concentrations of serum IFN-g were significantly higher in untreated mice or mice treated with CD4þCD25 T cells (P , 0.001 for all; Fig. 3a). However, there was no significant difference in serum IFN-g levels among mice in the normal pregnancy group and those treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (P . 0.05; Fig. 3a). In addition, serum concentrations of TGF-b1 and IL-10 in mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells were significantly higher than in the other three groups (P , 0.001 for all; Fig. 3b, c).
Analysis of IL-10 and TGF-b1 levels produced by CD4þCD25 T cells and CD4þCD25þFOXP3þ or TGF-b1induced Treg cells revealed that IL-10 and TGF-b1 secretion by CD4þCD25 T cells was significantly lower than that of either CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (P , 0.001 for all; see Table S2). Discussion Although adoptive transfer of CD4þCD25þ Treg cells from pregnant mice prevents spontaneous abortion (Zenclussen et al. 2005), their relatively low yield may limit their therapeutic potential. Because Treg cells isolated from non-pregnant mice can be induced by anti-CD3 and anti-CD28 antibodies and IL-2 into therapeutic Treg cells (Yin et al. 2012), the aim of the present study was to investigate whether T cells induced with anti-CD3
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(b)
(a) 80
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160
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∗
40
120
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Group 4
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Abortion models Fig. 3. Adoptive transfer of CD4þCD25þFOXP3þ and transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells modulated T helper (TH) 1 and TH2 responses towards TH2. FOXP3, forkhead box P3. Peripheral blood samples were obtained from individual mice on Day 14 of pregnancy (n ¼ 10 per group) and serum concentrations of (a) interferon (IFN)-g, (b) interleukin (IL)-10, and (c) TGF-b1 were determined using ELISA. Group 1, normal pregnancy model; Group 2, untreated abortion model; Group 3, abortion model treated with CD4þCD25þFOXP3þ Treg cells; Group 4, abortion model treated with TGF-b1-induced Treg cells; Group 5, abortion model treated with CD4þCD25 T cells. Data are the mean s.d. Significant differences were determined by post hoc analysis with Bonferroni correction. *P , 0.05 compared with Group 1; †P , 0.05 compared with Group 2; zP , 0.05 compared with Group 3; yP , 0.05 compared with Group 4.
and anti-CD28 antibodies , IL-2 and TGF-b1 could also confer protective effects. After abortion-prone mice had been injected with TGF-b1-induced Treg cells, the rate of embryo loss was reduced from 19.1% to 11.8%, and was similar to that of abortionprone mice treated with CD4þCD25þFOXP3þ Treg cells. Thus, adoptive transfer of either CD4þCD25þFOXP3þ or TGF-b1induced Treg cells could prevent fetal loss. However, there was no significant difference in the rate of embryo loss between abortion-prone mice treated with CD4þCD25 T cells and the untreated controls. After the adoptive transfer of CD4þCD25þFOXP3þ or TGF-b1-induced CD4þCD25þ Treg cells, triple staining analysis revealed that the proportion of CD4þCD25þFOXP3þ cells in the spleen and levels of FOXP3 protein and mRNA in deciduas of abortion-prone mice increased markedly, but there was no such change in FOXP3 expression in
abortion-prone mice that were treated with CD4þCD25 T cells. The adoptive transfer of CD4þCD25þFOXP3þ or TGF-b1induced CD4þCD25þFOXP3þ T cells could protect fetuses from spontaneous abortion, possibly by increasing the secretion of TH2 cytokines and decreasing the secretion of TH1 cytokines. Thus, the autologous transfer of in vitro TGF-b1-induced Treg cells in the early stage of pregnancy may represent a novel therapeutic strategy for women with unexplained recurrent spontaneous abortions. In cases of human miscarriage, the proportion of FOXP3þCD4þ T cells is reduced compared with normally progressing pregnancies (Inada et al. 2013). In the present study, the decidual expression of FOXP3 was significantly higher in mice receiving the adoptive transfer of CD4þCD25þFOXP3þ and TGF-b1-induced cells. A transcription factor, FOXP3 is
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expressed in Treg cells but not in other types of T cells (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003). Mice deficient in FOXP3 exhibit serious defects in their Treg cells and develop severe autoimmune diseases, and CD4þCD25 T cells ectopically expressing FOXP3 acquire a regulatory phenotype (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003). Furthermore, there was a significant association between polymorphisms in the Foxp3 gene and unexplained recurrent spontaneous abortion in Chinese Han patients (Wu et al. 2012). Thus, FOXP3 appears to be a master gene governing the immunosuppressive activity of CD4þCD25þ Treg cells. TGF-b1 converts naı¨ve CD4þCD25 cells into a population that is indistinguishable from CD4þCD25þFOXP3þ Treg cells in phenotype and suppressive activity (Sakaguchi et al. 2001; Zheng et al. 2002; Chen et al. 2003; Park et al. 2004; Fantini et al. 2004; Piccirillo and Shevach 2004; Wahl and Chen 2005; Selvaraj and Geiger 2007; So and Croft 2007). When naı¨ve CD4þCD25 T cells were activated with TGF-b1, the expression of Treg cell-associated markers, such as CD25, CTLA-4 and FOXP3, was upregulated. These TGF-b1-induced changes occur in CD25þ Treg cell-depleted cultures, confirming that TGF-b1 actually transforms CD4þCD25 cells, as opposed to merely expanding CD4þCD25þ cells migrating from the thymus (Park et al. 2004). TGF-b1 induced CD4þCD25þ Treg cells had a contact-dependent mechanism of action that was not affected by anti-TGF-b1 or anti-IL-10 antibodies and they were shown to be potent inhibitors of CD8þ T cell activation (Piccirillo and Shevach 2001; Yamagiwa et al. 2001). This mechanism of action distinguishes TGF-b1-converted Treg cells from TH3 and Type 1 regulatory T (Tr1) cells, the activities of which are solely dependent on soluble TGF-b1 and IL-10, respectively, and are contact independent. After stimulation with anti-CD3 and anti-CD28 antibodies, TGF-b1 and IL-2 for 5 days in the present study, the proportion of cells expressing CD25 and FOXP3 increased significantly. In addition, the TGF-b1-induced CD4þCD25þ T cells significantly inhibited the proliferation of responder T cells, confirming their immunosuppressive function that was indistinguishable from CD4þCD25þFOXP3þ Treg cells. TGF-b1-induced Treg cells are potentially vital for clinical immunosuppression. In addition to reducing fetus resorption rates in abortion-prone mice (Zenclussen et al. 2005; Yin et al. 2012), Zheng et al. (2006) showed that ex vivo induction of Treg cells with TGF-b1 can act like a vaccine that generates host suppressor cells with the potential to protect major histocompatibility complex (MHC)-mismatched organ grafts from rejection. Weber et al. (2006) showed that adaptive TGF-b1-induced Treg cells may control diabetes in part by impairing the survival of islet-specific TH1 cells, and thereby inhibiting the localisation and response of autoaggressive T cells in the pancreatic islets. In addition, data from Fantini et al. (2006) suggest that Treg cells expanded by TGF-b1 emerge as a class of regulatory T cells with therapeutic potential in T cell-mediated chronic intestinal inflammation. These studies led us to postulate that adoptive transfer of TGF-b1-induced CD4þCD25þ Treg cells may represent a therapeutic solution for maternal–fetal immune tolerance and prevent fetal loss in abortion-prone mice with the added advantage of the possibility of one-to-one adaptive transfer given the high yield of cells.
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The study of Saini et al. (2011) revealed that an imbalance in the TH1/TH2 response is associated with the development of unexplained recurrent spontaneous abortion, and that modulation of TH1 and TH2 responses towards TH2 can suppress maternal rejection of the fetus. In the present study, we found that serum concentrations of IFN-g were significantly higher in mice with abortions than in mice with normal pregnancies, supporting the notion that strong TH1 responses contribute to the development of spontaneous abortion. Adoptive transfer of CD4þCD25þFOXP3þ or TGF-b-induced Treg cells markedly decreased the serum concentrations of IFN-g and increased IL-10 and TGF-b1 levels, which coincided with reduced rates of spontaneous abortion. These data indicate that modulation of pro- and anti-inflammatory cytokine responses in abortionprone mice inhibits spontaneous abortion, and that adoptive transfer of CD4þCD25þFOXP3þ or TGF-b-induced Treg cells tends to maintain immune tolerance during pregnancy. These findings are clinically relevant because Treg cells from pregnant women can be collected, induced and transferred back early in pregnancy. Given the possible role of IL-6 transduction signalling in blocking Treg cell expansion (Arruvito et al. 2009), further studies will assess the effects of adaptive Treg cell transfer on this signalling pathway. The present study is limited in that in vivo models do not always represent the pathophysiology observed in patients. In addition, the mechanism underlying the protective effects was not examined, but will be determined in future studies. Furthermore, the transfer of Treg cells to the pregnancy tissues was not determined and needs to be assessed using cell tracker experiments in future studies. Finally, although we added anti-CD3 and anti-CD28 antibodies, TGF-b1 and IL-2 into the culture medium to induce cell phenotype switching based on previous studies (Park et al. 2004), we did not examine Ki67 expression in the TGF-b1-induced cells to differentiate them from expanding cells; again, this requires investigation in future studies. In conclusion, we found that isolated Treg cells can be induced by anti-CD3 and anti-CD28 antibodies, IL-2 and TGF-b1 in vitro, and that the adoptive transfer of these cells could prevent spontaneous abortion in mice by increasing the secretion of TH2 cytokines and decreasing TH1 cytokines. These studies are clinically relevant in that Treg cell isolation from pregnant women, in vitro expansion and autologous transfer may represent a novel therapeutic strategy for women with unexplained recurrent spontaneous abortions. Acknowledgement This study was supported by a grant from the National Natural Science Foundation of China (No. 81100474).
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Reproduction, Fertility and Development, 2016, 28, 1788–1797 http://dx.doi.org/10.1071/RD14503
Adoptive transfer of transforming growth factor-b1-induced CD41CD251 regulatory T cells prevents immune response-mediated spontaneous abortion Tian Qiu A, Yincheng Teng A, Yudong Wang B and Liang Xu B,C A
Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, China. B Department of Obstetrics and Gynecology, Shanghai Jiao Tong University Affiliated International Peace Maternity and Child Health Hospital, 910 Hengshan Road, Shanghai 200030, China. C Corresponding author. Email: [email protected]
Abstract. The effects of adoptive transfer of transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells in preventing spontaneous abortion in mice were investigated. CD4þCD25 cells were isolated from the spleens of pregnant CBA/J mice and induced into Treg cells positive for CD4, CD25 and forkhead box P3 (FOXP3) ex vivo using interleukin (IL)-2 and TGF-b1. CBA/J mice were mated with DBA/2J mice to establish a model of spontaneous abortion and, on the first day of pregnancy, mice were injected intravenously with 2 105 either freshly isolated Treg cells or those induced with TGF-b1. After 14 days, the surviving and reabsorbed fetuses in both groups were counted, and serum cytokine concentrations were measured by ELISA. Adoptive transfer of CD4þCD25þ or TGF-b1-induced Treg cells significantly reduced the fetal resorption rate, increased serum IL-10 and TGF-b1 concentrations and decreased interferon-g levels. In conclusion, the results of the present study indicate that adoptive transfer of TGF-b1-induced Treg cells prevents spontaneous abortion in mice by increasing the secretion of T helper (TH) 2 cytokines and decreasing the secretion of TH1 cytokines. Additional keywords: forkhead box P3 (FOXP3). Received 22 July 2014, accepted 17 April 2015, published online 14 May 2015
Introduction In reproductive immunology, the embryo is considered to be a semi-allograft that is not rejected by the maternal immune system in a physiological pregnancy. Thus, the maternal immune system shows a special type of immune tolerance to the fetus. However, if this immune tolerance is compromised, pathological pregnancy, including spontaneous abortion, occurs. CD4þCD25þ regulatory T (Treg) cells are a subset of T cells with potent immunosuppressive activity (Sakaguchi et al. 1995) that can inhibit effector T cell activation and maintain peripheral tolerance and immune system homeostasis. During mammalian pregnancy, levels of Treg cells increase in response to maternal immune cells encountering paternal antigens (Zenclussen et al. 2010; Clark and Chaouat 2012; Sereshki et al. 2014). Recent studies have associated a deficiency in Treg cells with a propensity for implantation rejection and miscarriage (Winger and Reed 2011; Inada et al. 2013; Lu et al. 2013), suggesting that Treg cells play a critical role in the maintenance of immune tolerance to the fetus (Zenclussen et al. 2005; Bao et al. 2011). The immunosuppressive activity of Treg cells depends on the expression of forkhead box P3 (FOXP3), which is a transcription factor and a specific biomarker of Treg cells (Hori et al. 2003; Journal compilation Ó CSIRO 2016
Hori and Sakaguchi 2004). Thus, CD4þCD25þFOXP3þ Treg cells are crucial for maintaining a normal pregnancy. Decreased levels of peripheral CD4þCD25þ T cells and FOXP3 expression are associated with the pathogenesis of unexplained recurrent spontaneous abortion (Mei et al. 2010). Therefore, modulating the number of Treg cells may represent a promising therapeutic target for preventing spontaneous abortion. A previous study indicated that adoptive transfer with Treg cells isolated from pregnant mice inhibits spontaneous abortion in abortion-prone pregnant mice (Zenclussen et al. 2005). This protection was not observed with Treg cells isolated from nonpregnant mice (Zenclussen et al. 2005). However, there are relatively few Treg cells in peripheral blood; therefore, it is difficult to obtain sufficient numbers of these cells for therapeutic application. Several experimental protocols have been developed for inducing Treg cells with tumour growth factor (TGF)-b1 in vitro (Park et al. 2004; Wahl and Chen 2005; Selvaraj and Geiger 2007; So and Croft 2007). TGF-b1-induced Treg cells are functionally stable and have potent suppressive activity. When naı¨ve CD4þCD25 T cells were costimulated with TGF-b1, anti-CD3 and anti-CD28 antibodies, Treg cell-associated markers, such as CD25, cytotoxic T lymphocyte-associated antigen www.publish.csiro.au/journals/rfd
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(CTLA)-4 and FOXP3, were upregulated and the cells acquired anergic and suppressive phenotypes similar to those of thymusderived Treg cells (Park et al. 2004; Wahl and Chen 2005; Selvaraj and Geiger 2007; So and Croft 2007). However, whether treatment of abortion-prone pregnant mice with in vitro TGF-b1-induced Treg cells can prevent spontaneous abortion is not known. TGF-b1 is a key molecule contributing to peripheral tolerance. Abrogation of TGF-b1 signalling in T cells alone results in spontaneous T cell differentiation and autoimmune diseases, highlighting the essential role of TGF-b1 signalling in T cell homeostasis and the prevention of inflammatory autoimmunity (Gorelik and Flavell 2000; Lucas et al. 2000). Several studies have also demonstrated the relevance of TGF-b1 in Treg cell physiology. For example, TGF-b1 expands and amplifies the function of CD4þCD25þ Treg cells ex vivo (Yamagiwa et al. 2001) and TGF-b1 deficiency or blockade in vivo eliminates the suppressive capabilities of Treg cells (Powrie 1995; Fuss et al. 2002). These findings suggest that the immunosuppressive activity of Treg cells depends on TGF-b1. In the present study we used a mouse model of spontaneous abortion to determine whether adoptive transfer of in vitro TGFb1-induced Treg cells could prevent spontaneous abortion in abortion-prone pregnant mice. CBA/J mice were mated with DBA/2J mice to produce an in vivo model of spontaneous abortion. Adoptive transfer of TGF-b1-induced Treg cells significantly reduced fetus resorption rates; therefore, this procedure may represent a therapeutic approach for spontaneous miscarriage. Materials and methods Animals Eight- to 10-week-old female CBA/J (MHC H2 Haplotype k (h-2k)), male DBA/2J (MHC H2 Haplotype d (h-2d)), and male BALB/c (h-2d) mice weighing 20–30 g were purchased from the Experimental Animal Center of the Chinese Academy of Sciences, Shanghai; these strains were originally obtained from Japan’s National Institute of Genetics, Shizuoka, Japan. Mice were housed under specific pathogen-free conditions and were provided 1.5 g food and 2.5 mL water per 10 g bodyweight each day. The Experimental Animal Center of the Chinese Academy of Sciences bred the mice following International Genetic Standards (White and Lee 1998) to ensure the genetic quality of the strains. This study was approved by the Institutional Review Board of the Sixth People’s Hospital Affiliated to Shanghai Jiaotong University. Isolation and induction of CD41CD252 T cells Splenic cells from pregnant CBA/J mice mated with BALB/c mice (normal pregnancy model) were gently minced in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; both from Bio-Light Biotech, Shanghai, China) and single cell suspensions were prepared by passage through a 100-mm nylon cell strainer. CD4þ T cells (105–106 cells) were isolated by eliminating cells expressing CD8a, CD11b, CD45R, CD49b and Ter-119 using the CD4þCD25þ Treg isolation kit (Miltenyi Biotec, San Diego, CA, USA). CD4þCD25 T cells were then isolated by eliminating cells expressing CD25.
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CD4þCD25þ T cells (103–104 cells) were isolated by positive selection using the CD4þCD25þ Treg isolation kit (Miltenyi Biotec). The CD4þCD25þ T cells were pooled before transfer into pregnant mice. For in vitro TGF-b1 induction, approximately 1 106 CD4þCD25 T cells were stimulated with 5 mg mL1 platebound anti-CD3 (BD Biosciences, San Diego, CA, USA), 0.5 mg mL1 soluble anti-CD28 (BD Biosciences), 5 ng mL1 TGF-b1 (R&D Systems, Minneapolis, MN, USA) and 10 U mL1 interleukin (IL)-2 (PeproTech, Rocky Hill, NJ, USA) for 5 days. The Treg cells were then harvested and analysed by flow cytometry. Suppression assay To determine the suppressive activity of TGF-b1-induced Treg cells isolated from pregnant CBA/J mice with normal pregnancies (Day 14) on CD4þ T cell proliferation, carboxyfluorescein succinimidyl ester (CFSE)-labelled CD4þ T cells were cocultured with TGF-b1-induced Treg cells, as described previously (Park et al. 2004). Briefly, freshly isolated splenic CD4þ T cells from CBA/J mice were washed and resuspended at a concentration of 1.0 107 cells mL1 in phosphate-buffered saline (PBS). CFSE (Invitrogen, Carlsbad, CA, USA) was added at a final concentration of 5 mM and samples were incubated for 10 min at room temperature. The reaction was stopped by the addition of 500 mL cold FBS, and the cells were washed three times with PBS. Then, 1.0 106 CFSE-labelled CD4þ T cells were mixed with 1.0 105 TGF-b1-induced Treg cells, CD4þCD25þFOXP3þ Treg cells or CD4þCD25 cells in a volume of 500 mL per well in 48-well plates containing 1 mg mL1 immobilised anti-CD3 and 0.2 mg mL1 soluble anti-CD28 (both from BD Biosciences). After 72 h coculture, the fluorescence intensity of live CFSE-labelled cells was determined by flow cytometry (see below). Animal groups Male BALB/c or DBA/2 mice were mated with female CBA/J mice. After pregnancy had been confirmed by the presence of a vaginal plug (Day 0), which was assessed twice daily at 0800 and 1600 hours, mice were separated into the following five groups: (1) CBA/J mice mated with BALB/c mice (normal pregnancy model); (2) CBA/J mice mated with DBA/2 mice (abortion model without treatment); (3) CBA/J mice mated with DBA/2 mice and injected intravenously with 2 105 CD4þCD25þFOXP3þ Treg cells on Day 1 of pregnancy; (4) CBA/J mice mated with DBA/2 mice and injected intravenously with 2 105 TGF-b1-induced Treg cells on Day 1 of pregnancy; and (5) CBA/J mice mated with DBA/2 mice and injected intravenously with 2 105 CD4þCD25 T cells on Day 1 of pregnancy. There were no significant differences in the age or weight of the mice randomly assigned to the five groups, either within or between groups. On Day 14 of pregnancy, pregnant mice were killed and their uteri removed to evaluate implantation sites. Compared with normal embryos and placentas, the abortion sites were identified by their relatively small sizes and necrotic and haemorrhagic appearance, as described previously (Zhao and Bao 1999).
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The resorption rate was calculated as: (no. total abortions/no. total implantations) 100%. For the flow cytometry assays, spleen samples were cut into small pieces and collected in Hanks’ balanced salt solution without Ca2þ or Mg2þ. The uterine horns were opened longitudinally and the feto-placental unit was separated from the uterine implantation sites, and decidual tissues were isolated. For RNA extraction and western blot analysis, decidual tissues were washed thoroughly with cold sterile PBS, pH 7.40, snap frozen and stored at 808C until further use. Because tissue from resorption sites is necrotic, which makes RNA isolation from these sites extremely difficult, we harvested tissues from healthy implantation sites for RNA extraction, as described by Zenclussen et al. (2005).
buffer (Amresco, Solon, OH, USA). Proteins were transferred to 0.45 mm Optitran nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA) in Tris-glycine buffer (Amresco). Membranes were blocked in PBS containing 5% non-fat dry milk and incubated overnight at 48C with anti-FOXP3 (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-glyceraldehyde-3-phosphate dehydrogenase (1 : 20 000 dilution; Research Diagnostics, Flanders, NJ, USA) antibodies. The membranes were washed several times with PBS containing 0.1% Tween 20 (PBS-T) and incubated with peroxidase-conjugated IgG (1 : 2000 dilution) in blocking solution. After the membranes had been washed several times in PBS-T, they were developed with enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA) and exposed to film.
Determination of cytokine levels On Day 14 of pregnancy, serum samples were collected from anaesthetised pregnant mice (ketamine, 10 mg/100 g body weight, intraperitoneal injection) via retro-orbital bleeding. Serum concentrations of total TGF-b1, interferon (IFN)-g and IL-10 were determined using commercially available ELISA kits (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions. For analysis of IL-10 and TGF-b1 levels produced by CD4þCD25 T cells and CD4þCD25þFOXP3þ or TGF-b1induced Treg cells, 5 103cells per well were cultured in 96-well microplates for 2 h (RPMI1640 with 2 mmol L1 L- glutamine,1105 U L1 penicillin,100 g L1 streptomycin,50 mmol L1 2-mercaptoethanol and 100 mL L1 fetal calf serum). The microplates were cultured at 378C and 5% CO2.), after which the cell culture supernatant was collected and 0.5 mL aliquots were used for ELISA according to the manufacturer’s instructions.
Real-time polymerase chain reaction Total RNA was extracted from placental tissue using TRIzol reagent (Invitrogen). First-strand cDNA was synthesised using random hexamers and Superscript II reverse transcriptase (Invitrogen) in a final volume of 40 mL using 2 mg RNA. Amplification was performed in an ABI 7900 Sequence Detector (Applied Biosystems, Foster City, CA, USA) using the Takara SYBR Premix Ex Taq kit (Takara, Shiga, Japan) in a final reaction volume of 10 mL, containing 0.5 mL cDNA, 10 pmol each primer and 5 mL Sybr Green premix. The cycling parameters were as follows: 2 min at 508C, 10 s at 958C, 40 cycles of 5 s at 958C and 30 s at 608C, and then 10 min at 708C. The following Foxp3-specific primers were designed using Primer Express Software v2.0 (ABI) based on sequences from the National Center for Biotechnology Information (NCBI): sense, 50 -CCAGACGGCGGCCTGTTTGC-30 ; antisense, 50 -GCTCTCCACT CGCACAAAGC-30 .
Flow cytometry Cells were isolated from the spleen by disaggregating and filtering the tissue through a sterile net. Erythrocytes were then lysed with a solution of NHCl4–NaCl. Cells were collected, washed and incubated with antibodies for specific surface markers for 10 min at 48C in the dark before being fixed with a 1% paraformaldehyde solution overnight at 48C. After permeabilising the cells with 0.2% saponin, intracellular antibodies were added and samples were incubated for 20 min at 48C in the dark. Then, cells were washed with cold sterile PBS, pH 7.40, and analysed using a FACScan flow cytometer (BD Bioscience). The following monoclonal antibodies (mAbs) were used: fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD4, allophycocyanin (APC)-conjugated rat anti-mouse CD25 and phycoerythrin (PE)-labelled anti-mouse FOXP3 and CD69. FITC-, APC- and PE-conjugated rat IgG1 or Ig2b were used as negative controls in separate tubes (all from eBioscience, San Diego, CA, USA). Western blot analysis In western blot assays, 20 ng protein was separated on a 10% Tris-glycine sodium dodecyl sulfate (SDS)–polyacrylamide gel (Invitrogen) in Tris-Glycine SDS Running Buffer (TG-SDS)
Statistical analysis Normally distributed data are summarised as the mean s.d. and were compared by one-way analysis of variance (ANOVA) with post hoc Bonferroni correction. Skewed data are expressed as median values with the interquartile range (IQR) in parentheses and were compared using the Kruskal–Wallis test, with the Mann–Whitney test as a post hoc test. Statistical analyses were performed using SAS software version 9.2 (SAS Institute, Cary, NC, USA). Two-tailed P , 0.05 was considered significant. Results TGF-b1 induction of CD25 and FOXP3 expression by CD41CD252 T cells The purity of the CD4þCD25 T cells after isolation was 99.05 9.73%, with only 3.4 1.3% of cells expressing FOXP3. As shown in Fig. 1a, b, the purified CD4þCD25 T cell fraction contained ,1.0% CD25þ cells. Specifically, flow cytometric analysis of the purified cells showed that the proportion of CD4þCD25 cells to CD4þ cells in six independent experiments was 99.40%, 99.80%, 99.30%, 98.70%, 99.10% and 99.20%. The proportion of CD25þ cells increased significantly from 0.92 0.28% to 79.46 7.08% (P , 0.001; Table 1) after stimulation with anti-CD3 and anti-CD28 antibodies for 5 days in the presence of TGF-b1 in fresh medium
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(a) 104
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M1 10
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0 100
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FOXP3
Fig. 1. Stimulation with transforming growth factor (TGF)-b1 induces CD25 and forkhead box P3 (FOXP3) expression by CD4þCD25 T cells. Representative expression of (a, b) CD25 and (c, d) Foxp3 by CD4þCD25 T-cells (a, c) and those stimulated with anti-CD3 and anti-CD28 antibodies for 5 days in the presence of TGF-b1 and interleukin-2 (b, d ).
Table 1. Stimulation with transforming growth factor-b1 induces CD25 and forkhead box P3 (FOXP3) expression by CD41CD252 T cells Data are the mean s.d. Before, before stimulation with transforming growth factor-b1; After, after stimulation with transforming growth factor-b1
CD25 (%) Foxp3 (%)
Before
After
P-value
0.92 0.28 3.13 1.63
79.46 7.08 66.23 8.55
,0.001 ,0.001
containing exogenous IL-2. In addition, the presence of TGF-b1 during polyclonal stimulation of CD4þCD25 T cells enhanced the expression of FOXP3 from 3.13 1.63% to 66.23 8.55% (P , 0.001; Fig. 1c, d; Table 1). CD4þCD25þ T cells (103–104 cells) were isolated by positive selection using the CD4þCD25þ Treg isolation kit, and their purity ranged between 75% and 85%.
Suppression of CD41 T cell proliferation by TGF-b1induced Treg cells A functional hallmark of Treg cells is their ability to suppress the development and expansion of effector T cells. We therefore investigated whether TGF-b1-induced Treg cells were able to suppress the proliferation of CD4þ T cells using coculture assays. As indicated in Fig. 2 and Table 2, coculture with TGF-b1-induced Treg cells or CD4þCD25þFOXP3þ Treg cells significantly increased the number of non-dividing CD4þ T cells compared with untreated cells (P , 0.05). Moreover, the suppressive activity of TGF-b1-induced Treg cells was similar to that of CD4þCD25þFOXP3þ Treg cells. Effects of adoptive transfer of CD41CD251FOXP31 and TGF-b-induced Treg cells in mice The proportion of CD4þCD25þFOXP3þ T cells in the spleen and decidua was significantly lower in the untreated group, as well as in mice treated with CD4þCD25 T cells,
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CD4⫹ T cells alone
No stimulation
21.4%
93.2%
Table 2. Suppression of CD41 T cell proliferation by transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells Data are the mean s.d. (n ¼ 10 per group). Significant differences were found by post hoc analysis with Bonferroni correction. *P , 0.05 compared with untreated cells; †P , 0.05 compared with CD4þ T cells alone; z P , 0.05 compared with CD4þ T cells treated with CD4þCD25þFOXP3þ Treg cells; yP , 0.05 compared with CD4þ T cells treated with TGF-b1induced Treg cells. FOXP3, forkhead box P3; CFSE, carboxyfluorescein succinimidyl ester CFSE-labelled cells (%)
CFSE CD4⫹ T cells ⫹ TGF-β1-induced CD4⫹CD25⫹Treg cells
CD4⫹ T cells ⫹ CD4⫹CD25⫺ T cells
58.1%
Count
18.9%
CFSE
CD4⫹ T cells ⫹ ⫹ ⫹
CD4 CD25 FOXP3⫹ Treg cells 56.7%
No stimulation CD4þ T cells alone CD4þ T cells treated with CD4þCD25þFOXP3þ Treg cells CD4þ T cells treated with TGF-b1-induced Treg cells CD4þ T cells treated with CD4þCD25 T cells P-value between the five groups
92.68 3.47 21.44 2.82* 57.07 5.94*† 58.64 5.26*† 18.92 2.96*zy ,0.001
with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (P , 0.05 for both; Table 3). Using CD69 as a marker of activated T cells, we next evaluated the proportion of CD4þCD69þ T cells to CD4þ T cells in the spleen and decidua in the different groups and found no significant differences (P . 0.05 for all; Table 3). Finally, treatment of mice with CD4þCD25þFOXP3þ Treg cells significantly increased the proportion of CD4þCD25þFoxp3þ T cells : CD4þCD69þ T cells in the spleen compared with untreated control mice and mice treated with CD4þCD25 T cells (P , 0.001 for both). The proportion of CD4þCD25þFoxp3þ T cells to CD4þCD69þ T cells in the decidua was significantly lower in mice treated with CD4þCD25 T cells than in the normal pregnancy group (P ¼ 0.001; Table 3). Foxp3 mRNA and protein levels were higher in deciduas of mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells than in mice treated with CD4þCD25 T cells or untreated mice (P , 0.001 for both). However, Foxp3 expression remained slightly lower in deciduas of Treg cell-treated mice than mice with normal pregnancies (Table 3).
CFSE Fig. 2. Suppression of responder cell proliferation by transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells. Purified CD4þCD25 T cells from normal pregnant CBA/J mice were stimulated with 5 ng mL1 TGF-b1 for 5 days. After extensive washing, 1.0 105 TGF-b1-induced Treg cells were mixed with 1.0 106 carboxyfluorescein succinimidyl ester (CFSE)-labelled splenic T cells from CBA/J mice and stimulated with immobilised anti-CD3 and soluble anti-CD28 antibodies. After gating for CD4þ T cells, the fluorescence intensity of live CFSE-labelled cells after 72 h culture is shown as a histogram. The percentages in the figure represent undivided CFSEþ cells. These results are representative of 10 independent experiments. FOXP3, forkhead box P3.
compared with the normal pregnancy control group (P , 0.05; Table 3). Furthermore, compared with untreated mice, the proportion of CD4þCD25þFOXP3þ T cells in the spleen and decidua was significantly higher in mice treated
Effects of adoptive transfer of CD41CD251FOXP31 and TGF-b-induced Treg cells on abortion rate In the spontaneous abortion model, the embryo resorption rate was significantly higher in untreated mice (median 19.1%) compared with the normal pregnancy model (median 0%; P , 0.001; Table 4). The embryo resorption rates of mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (10.8% and 11.8%, respectively) were significantly lower than in untreated mice and did not differ significantly from that observed in the normal pregnancy model. The embryo resorption rate for mice treated with CD4þCD25 T cells (20.2%) did not differ significantly from that of untreated mice and was significantly higher the rates in mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (Table 4). There were no significant differences in fetal weights, placental
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Table 3. Phenotypic characterisation of splenic CD41 cells following T cell transfer and forkhead box P3 (Foxp3) protein and mRNA expression in deciduas of mice Data are the mean s.d. (n ¼ 10 per group). Significant differences were found by post hoc analysis with Bonferroni correction. zP , 0.05 compared with Group 1; yP , 0.05 compared with Group 2; *P , 0.05 compared with Groups 3, †P , 0.05 compared with Groups 4. Group 1, normal pregnancy model (CBA/ J BALB/c); Group 2, untreated abortion model; Group 3, abortion model treated with CD4þCD25þFOXP3þ Treg cells; Group 4, abortion model treated with transforming growth factor-b1-induced Treg cells; Group 5, abortion model treated with CD4þCD25 T cells Abortion models (CBA/J DBA/2)
Group 1 CD4þCD25þFoxp3þ T cells/CD4þ T cells (%) Spleen 7.25 1.11 Decidua 29.71 5.44 Splenic CD4þ cells CD25/CD4 (%) 7.64 1.98 Foxp3/CD4 (%) 7.25 1.11 Foxp3 expression Protein 0.805 0.110 mRNA 0.555 0.084 CD4þCD69þ T cells/CD4þ T cells (%) Spleen 4.77 1.25 Decidua 3.48 1.26 CD4þCD25þFoxp3þT cell/CD4þCD69þT cells (%) Spleen 1.64 0.57 Decidua 10.39 6.08 A
Group 5
P-valueA
7.47 0.58y* 21.67 3.62zy
3.46 0.48z† 13.58 3.02z†
,0.001 ,0.001
12.57 3.78zy 9.50 0.69zy
10.58 3.08y 7.47 0.58y*
4.88 1.73† 3.46 0.48z†
,0.001 ,0.001
0.638 0.058zy 0.481 0.052zy
0.556 0.045zy 0.384 0.032zy*
0.285 0.039z† 0.064 0.020z†
,0.001 ,0.001
Group 2
Group 3
3.3 0.48z 15.74 2.96z
9.5 0.69zy 23.86 4.06zy
5.01 1.88 3.30 0.48z 0.244 0.056z 0.075 0.020z
Group 4
4.45 1.30 3.52 1.05
5.38 1.95 3.66 1.10
5.14 1.44 3.34 1.01
4.92 1.25 3.84 0.96
0.82 0.35 4.83 1.49
1.99 0.75y 7.16 2.72
1.59 0.58 7.18 2.95
0.76 0.28† 3.74 1.15z
0.674 0.866 ,0.001 0.001
Determined by ANOVA.
Table 4. Comparisons of embryo resorption rates in pregnant CBA/J mice Data are presented as the mean s.d. (with P-values determined by ANOVA) or as median values with the interquartile range in parentheses (and P-values determined by the Kruskal–Wallis test). The number of embryos from each mouse ranged between 6 and 11. *P , 0.05 compared with Group 1; †P , 0.05 compared with Group 2, zP , 0.05 compared with Groups 3 and 4. Group 1, normal pregnancy model (CBA/J BALB/c); Group 2, untreated abortion model; Group 3, abortion model treated with CD4þCD25þFOXP3þ Treg cells; Group 4, abortion model treated with transforming growth factor-b1-induced Treg cells; Group 5, abortion model treated with CD4þCD25 T cells; FOXP3, forkhead box P3 Group 1
No. mice No. embryos No. resorptions Resorption rateA (%) A
10 8.7 1.5 0.0 (0.0, 1.0) 0.0 (0.0, 9.1)
Abortion model
P-value
Group 2
Group 3
Group 4
Group 5
10 9.3 1.1 2.0 (1.0, 2.0)* 19.1 (11.1, 22.2)*
10 8.6 1.7 1.0 (0.0, 1.0)† 10.8 (0.0, 14.3)†
10 8.3 1.6 1.0 (0.0, 1.0)† 11.8 (0.0, 14.3)†
10 8.8 1.5 2.0 (1.0, 3.0)*z 20.2 (11.1, 33.3)*z
0.658 ,0.001 ,0.001
The resorption rate was calculated as (no. resorptions/no. embryos) 100.
weights or their ratio (P . 0.05 for all; see Table S1 available as Supplementary Material to this paper). Effects of adoptive transfer of CD41CD251FOXP31 and TGF-b-induced Treg cells on TH1 and TH2 responses Analysis of serum cytokines indicated that concentrations of serum IFN-g were significantly higher in untreated mice or mice treated with CD4þCD25 T cells (P , 0.001 for all; Fig. 3a). However, there was no significant difference in serum IFN-g levels among mice in the normal pregnancy group and those treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (P . 0.05; Fig. 3a). In addition, serum concentrations of TGF-b1 and IL-10 in mice treated with CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells were significantly higher than in the other three groups (P , 0.001 for all; Fig. 3b, c).
Analysis of IL-10 and TGF-b1 levels produced by CD4þCD25 T cells and CD4þCD25þFOXP3þ or TGF-b1induced Treg cells revealed that IL-10 and TGF-b1 secretion by CD4þCD25 T cells was significantly lower than that of either CD4þCD25þFOXP3þ or TGF-b1-induced Treg cells (P , 0.001 for all; see Table S2). Discussion Although adoptive transfer of CD4þCD25þ Treg cells from pregnant mice prevents spontaneous abortion (Zenclussen et al. 2005), their relatively low yield may limit their therapeutic potential. Because Treg cells isolated from non-pregnant mice can be induced by anti-CD3 and anti-CD28 antibodies and IL-2 into therapeutic Treg cells (Yin et al. 2012), the aim of the present study was to investigate whether T cells induced with anti-CD3
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(a) 80
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5000
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Abortion models Fig. 3. Adoptive transfer of CD4þCD25þFOXP3þ and transforming growth factor (TGF)-b1-induced regulatory T (Treg) cells modulated T helper (TH) 1 and TH2 responses towards TH2. FOXP3, forkhead box P3. Peripheral blood samples were obtained from individual mice on Day 14 of pregnancy (n ¼ 10 per group) and serum concentrations of (a) interferon (IFN)-g, (b) interleukin (IL)-10, and (c) TGF-b1 were determined using ELISA. Group 1, normal pregnancy model; Group 2, untreated abortion model; Group 3, abortion model treated with CD4þCD25þFOXP3þ Treg cells; Group 4, abortion model treated with TGF-b1-induced Treg cells; Group 5, abortion model treated with CD4þCD25 T cells. Data are the mean s.d. Significant differences were determined by post hoc analysis with Bonferroni correction. *P , 0.05 compared with Group 1; †P , 0.05 compared with Group 2; zP , 0.05 compared with Group 3; yP , 0.05 compared with Group 4.
and anti-CD28 antibodies , IL-2 and TGF-b1 could also confer protective effects. After abortion-prone mice had been injected with TGF-b1-induced Treg cells, the rate of embryo loss was reduced from 19.1% to 11.8%, and was similar to that of abortionprone mice treated with CD4þCD25þFOXP3þ Treg cells. Thus, adoptive transfer of either CD4þCD25þFOXP3þ or TGF-b1induced Treg cells could prevent fetal loss. However, there was no significant difference in the rate of embryo loss between abortion-prone mice treated with CD4þCD25 T cells and the untreated controls. After the adoptive transfer of CD4þCD25þFOXP3þ or TGF-b1-induced CD4þCD25þ Treg cells, triple staining analysis revealed that the proportion of CD4þCD25þFOXP3þ cells in the spleen and levels of FOXP3 protein and mRNA in deciduas of abortion-prone mice increased markedly, but there was no such change in FOXP3 expression in
abortion-prone mice that were treated with CD4þCD25 T cells. The adoptive transfer of CD4þCD25þFOXP3þ or TGF-b1induced CD4þCD25þFOXP3þ T cells could protect fetuses from spontaneous abortion, possibly by increasing the secretion of TH2 cytokines and decreasing the secretion of TH1 cytokines. Thus, the autologous transfer of in vitro TGF-b1-induced Treg cells in the early stage of pregnancy may represent a novel therapeutic strategy for women with unexplained recurrent spontaneous abortions. In cases of human miscarriage, the proportion of FOXP3þCD4þ T cells is reduced compared with normally progressing pregnancies (Inada et al. 2013). In the present study, the decidual expression of FOXP3 was significantly higher in mice receiving the adoptive transfer of CD4þCD25þFOXP3þ and TGF-b1-induced cells. A transcription factor, FOXP3 is
Induced Treg cells prevent spontaneous abortion
expressed in Treg cells but not in other types of T cells (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003). Mice deficient in FOXP3 exhibit serious defects in their Treg cells and develop severe autoimmune diseases, and CD4þCD25 T cells ectopically expressing FOXP3 acquire a regulatory phenotype (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003). Furthermore, there was a significant association between polymorphisms in the Foxp3 gene and unexplained recurrent spontaneous abortion in Chinese Han patients (Wu et al. 2012). Thus, FOXP3 appears to be a master gene governing the immunosuppressive activity of CD4þCD25þ Treg cells. TGF-b1 converts naı¨ve CD4þCD25 cells into a population that is indistinguishable from CD4þCD25þFOXP3þ Treg cells in phenotype and suppressive activity (Sakaguchi et al. 2001; Zheng et al. 2002; Chen et al. 2003; Park et al. 2004; Fantini et al. 2004; Piccirillo and Shevach 2004; Wahl and Chen 2005; Selvaraj and Geiger 2007; So and Croft 2007). When naı¨ve CD4þCD25 T cells were activated with TGF-b1, the expression of Treg cell-associated markers, such as CD25, CTLA-4 and FOXP3, was upregulated. These TGF-b1-induced changes occur in CD25þ Treg cell-depleted cultures, confirming that TGF-b1 actually transforms CD4þCD25 cells, as opposed to merely expanding CD4þCD25þ cells migrating from the thymus (Park et al. 2004). TGF-b1 induced CD4þCD25þ Treg cells had a contact-dependent mechanism of action that was not affected by anti-TGF-b1 or anti-IL-10 antibodies and they were shown to be potent inhibitors of CD8þ T cell activation (Piccirillo and Shevach 2001; Yamagiwa et al. 2001). This mechanism of action distinguishes TGF-b1-converted Treg cells from TH3 and Type 1 regulatory T (Tr1) cells, the activities of which are solely dependent on soluble TGF-b1 and IL-10, respectively, and are contact independent. After stimulation with anti-CD3 and anti-CD28 antibodies, TGF-b1 and IL-2 for 5 days in the present study, the proportion of cells expressing CD25 and FOXP3 increased significantly. In addition, the TGF-b1-induced CD4þCD25þ T cells significantly inhibited the proliferation of responder T cells, confirming their immunosuppressive function that was indistinguishable from CD4þCD25þFOXP3þ Treg cells. TGF-b1-induced Treg cells are potentially vital for clinical immunosuppression. In addition to reducing fetus resorption rates in abortion-prone mice (Zenclussen et al. 2005; Yin et al. 2012), Zheng et al. (2006) showed that ex vivo induction of Treg cells with TGF-b1 can act like a vaccine that generates host suppressor cells with the potential to protect major histocompatibility complex (MHC)-mismatched organ grafts from rejection. Weber et al. (2006) showed that adaptive TGF-b1-induced Treg cells may control diabetes in part by impairing the survival of islet-specific TH1 cells, and thereby inhibiting the localisation and response of autoaggressive T cells in the pancreatic islets. In addition, data from Fantini et al. (2006) suggest that Treg cells expanded by TGF-b1 emerge as a class of regulatory T cells with therapeutic potential in T cell-mediated chronic intestinal inflammation. These studies led us to postulate that adoptive transfer of TGF-b1-induced CD4þCD25þ Treg cells may represent a therapeutic solution for maternal–fetal immune tolerance and prevent fetal loss in abortion-prone mice with the added advantage of the possibility of one-to-one adaptive transfer given the high yield of cells.
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The study of Saini et al. (2011) revealed that an imbalance in the TH1/TH2 response is associated with the development of unexplained recurrent spontaneous abortion, and that modulation of TH1 and TH2 responses towards TH2 can suppress maternal rejection of the fetus. In the present study, we found that serum concentrations of IFN-g were significantly higher in mice with abortions than in mice with normal pregnancies, supporting the notion that strong TH1 responses contribute to the development of spontaneous abortion. Adoptive transfer of CD4þCD25þFOXP3þ or TGF-b-induced Treg cells markedly decreased the serum concentrations of IFN-g and increased IL-10 and TGF-b1 levels, which coincided with reduced rates of spontaneous abortion. These data indicate that modulation of pro- and anti-inflammatory cytokine responses in abortionprone mice inhibits spontaneous abortion, and that adoptive transfer of CD4þCD25þFOXP3þ or TGF-b-induced Treg cells tends to maintain immune tolerance during pregnancy. These findings are clinically relevant because Treg cells from pregnant women can be collected, induced and transferred back early in pregnancy. Given the possible role of IL-6 transduction signalling in blocking Treg cell expansion (Arruvito et al. 2009), further studies will assess the effects of adaptive Treg cell transfer on this signalling pathway. The present study is limited in that in vivo models do not always represent the pathophysiology observed in patients. In addition, the mechanism underlying the protective effects was not examined, but will be determined in future studies. Furthermore, the transfer of Treg cells to the pregnancy tissues was not determined and needs to be assessed using cell tracker experiments in future studies. Finally, although we added anti-CD3 and anti-CD28 antibodies, TGF-b1 and IL-2 into the culture medium to induce cell phenotype switching based on previous studies (Park et al. 2004), we did not examine Ki67 expression in the TGF-b1-induced cells to differentiate them from expanding cells; again, this requires investigation in future studies. In conclusion, we found that isolated Treg cells can be induced by anti-CD3 and anti-CD28 antibodies, IL-2 and TGF-b1 in vitro, and that the adoptive transfer of these cells could prevent spontaneous abortion in mice by increasing the secretion of TH2 cytokines and decreasing TH1 cytokines. These studies are clinically relevant in that Treg cell isolation from pregnant women, in vitro expansion and autologous transfer may represent a novel therapeutic strategy for women with unexplained recurrent spontaneous abortions. Acknowledgement This study was supported by a grant from the National Natural Science Foundation of China (No. 81100474).
References Arruvito, L., Billordo, A., Capucchio, M., Prada, M. E., and Fainboim, L. (2009). IL-6 trans-signaling and the frequency of CD4þFOXP3þ cells in women with reproductive failure. J. Reprod. Immunol. 82, 158–165. doi:10.1016/J.JRI.2009.04.010 Bao, S. H., Wang, X. P., De Lin, Q., Wang, W. J., Yin, G. J., and Qiu, L. H. (2011). Decidual CD4þCD25þCD127dim/– regulatory T cells in patients with unexplained recurrent spontaneous miscarriage. Eur. J. Obstet. Gynecol. Reprod. Biol. 155, 94–98. doi:10.1016/J.EJOGRB.2010.11.007
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