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BOR - Papers in Press, published online ahead of print September 29, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.034108
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BIOLOGY OF REPRODUCTION 72, 338–345 (2005)
DOI: 10.1095/biolreprod.104.034108
© 2005 by the Society for the Study of Reproduction, Inc.

Blockade of CD86 Signaling Facilitates a Th2 Bias at the Maternal-Fetal Interface and Expands Peripheral CD4+CD25+ Regulatory T Cells to Rescue Abortion-Prone Fetuses1

Xiao-Yong Zhu, Yue-Hua Zhou, Ming-Yan Wang, Li-Ping Jin, Min-Min Yuan, and Da-Jin Li2

Laboratory of Reproductive Immunology, Institute of Obstetrics and Gynecology, Fudan University, Shanghai 200011, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intervention in B7 (CD80/CD86)/B7-ligand (CD28/CTLA-4) pathways is an effective way of preventing unwanted immune responses, such as allograft rejection. Pregnancy maintenance represents maternal tolerance to the fetal allograft, which is accompanied by a type 2 helper cell (Th2) bias at the maternal-fetal interface. Here, the costimulatory signal of CD86 was selectively blocked, and that of CD80 was kept unimpaired by administration of anti-murine CD86 monoclonal antibody at the early gestational stage in abortion-prone CBA/JxDBA/2 matings and normal pregnant CBA/JxBALB/c matings. It was demonstrated that in vivo blockade of CD86 costimulation could suppress maternal immune attack to the fetus by shifting cytokines from Th1 predominance to Th2 bias at the maternal-fetal interface, and expanding peripheral CD4+CD25+ regulatory T cells, which play an important role in the development and maintenance of maternal-fetal tolerance. Furthermore, the expression of CD28 and its ligands CD80/CD86 on peripheral lymphocytes was down-regulated, whereas that of CTLA-4 was up-regulated, which might facilitate the suppressive effect of CD4+CD25+ regulatory T cells on the alloreactive T cells. The maternal-fetal immunotolerance induced by CD86 blockade decreased fetal resorption in CBA/JxDBA/2 matings, but did not affect normal pregnant CBA/JxBALB/c matings. These results suggest that selective blockade of CD86 costimulation leads to maternal immune tolerance to embryo antigen, and might contribute to a rational immunoregulatory regimen for recurrent spontaneous abortion.

cytokines, embryo, immunology, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful outcome in pregnancy and allografts depends on alloimmune regulation [1–3]. In a number of models, fetus acceptance has been associated with a bias toward (local) type 2 help cell (Th2) cytokine production (e.g., interleukin-4 [IL-4], IL-10, transforming growth factor-ß [TGF-ß]) and away from Th1-type proinflammatory cytokines (e.g., interferon-{gamma} [IFN-{gamma}], tumor necrosis factor-{alpha} [TNF-{alpha}]) at the maternal-fetal interface. It is evident that an ongoing modulation occurs between prorejection (abortion) and antirejection (successful pregnancy) both in pregnancy and transplantation [3, 4–7].

Antigen-specific T cell activation is a critical step in the rejection of transplanted allografts. It has been known that productive activation of T cells occurs after concomitant engagement of T cell receptor with antigen presented on antigen-presenting cells (APCs) in association with major histocompatibility class molecules and the delivery of costimulatory signals resulting from the interaction of several ligand:coreceptor complexes [8, 9]. The important costimulatory interactions leading to rejection responses include but are not limited to the following: CD28 with CD80 (B7-1)/CD86 (B7-2), CD40L with CD40, 4-1BB and 4-1BB ligand [10], and ICOS with B7h [11]. A few molecular interactions, in contrast, lead to decreased rejection. Intervention in the B7/B7-ligand pathways is a selective way of preventing unwanted immune responses, such as in organ or tissue transplantation and autoimmune diseases. Binding avidity and dissociation rates of CD80/CD86 with CD28/ CTLA-4 (cytotoxic T lymphocyte-associated antigen 4, CD152) suggest specific roles for CD86-CD28 on the one hand and CD80-CTLA-4 on the other hand [12]. The CD80-CTLA-4 interaction is required to induce in vitro unresponsiveness, and CD80-deficient donor hearts were rejected under immunosuppressive treatment of CTLA-4 immunoglobulin (Ig), whereas hearts from CD80+ donors showed prolonged graft survival [13, 14]. Blockade of CD80 or CTLA-4 accelerated allogeneic heterotopic heart rejection in CD28-deficient mice, whereas CD86 blockade significantly prolonged graft survival. Moreover, CD28 is constitutively expressed on T cells, whereas CTLA-4 expression is up-regulated late after T cell activation, suggesting that CD86 is a preferable ligand for CD28, whereas CD80 is a preferable ligand for CTLA-4 [12, 15]. Thus, the relative levels of CD80 and CD86 expressed on APCs could affect the balance between CD28- and CTLA-4-dependent immune outcomes. As a result, treatment with anti-CD86 monoclonal antibody (mAb) alone, leaving CD80-CTLA-4 signaling unimpaired, could further enhance the induction of alloantigen-specific tolerance [16–18].

CD4+CD25+ regulatory T cells (Treg) have emerged as a unique population of suppressor T cells that maintain peripheral immune tolerance [19]. These cells constitutively express CTLA-4 and can induce dendritic cells to express indoleamine 2,3-dioxygenase by an interaction with its ligand, CD80/CD86. In turn, expression of indoleamine 2,3-dioxygenase promotes maternal-fetal tolerance [20–22]. However, the intensity of CD28 costimulation (CD80/ CD86-CD28) correlates negatively with the suppressive capacity of the CD4+CD25+ regulatory T cells [23, 24].

In the present study, using CBA/JxDBA/2 matings as an abortion-prone model and CBA/JxBALB/c matings as a normal pregnant model [25, 26], we treated pregnant CBA mice with anti-CD86 mAb but not with anti-CD80 mAb at the early stage of gestation, and then evaluated the effects of CD86 blockade on pregnancy outcome and the balance between Th1 and Th2 cytokines at the maternal-fetal interface in the two experimental models. In addition, we analyzed changes in the proportion of CD4+CD25+ regulatory T cells as well as the expression pattern of costimulatory molecules on peripheral lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Inbred strains of 8-wk-old female CBA/J, male DBA/2, and male BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and subsequently maintained in the Laboratory Animal Facility of the Chinese Academy of Sciences (Shanghai, China). They were housed five to six per cage with a 12L:12D cycle and were given standard laboratory chow. Investigations were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals.

Antibodies and Treatment Regimen

A PO3 cell line producing anti-murine CD86 mAb, rat IgG2b, was generously provided by Professor K. Okumura (Tokyo, Japan). The mAb was purified by standard procedures with affinity chromatography (protein G column, Pharmacia) from ascites of nude mice following an i.p. injection of the PO3 cell line. The purity of mAb was verified by SDS-PAGE analysis [27].

After overnight cohabitation of CBA/J/J females with BALB/c or DBA/2 males, females with vaginal plugs (Day 0.5 of gestation) were segregated and randomized to different treatment groups.

The first group of female animals received an i.p. injection of 100 µg of anti-CD86 mAb in 200 µl of PBS on Days 4.5, 6.5, 8.5, and 10.5 of gestation, respectively. The second group received only one i.p. injection of 100 µg of anti-CD86 mAb on Day 4.5 of gestation. As a control, irrelevant-isotype-matched rat IgG2b (Sigma, St. Louis, MO) was administrated at the same dosage and same time.

Preparation of Decidual and Placental Explant Culture Supernatants

Mice were killed on Day 13.5 of gestation. The uterine horns were opened longitudinally, and the fetoplacental unit was separated from the uterine implantation sites. The whole placental and decidual unit was separated individually from the respective embryo and its implantation site, and was then cultured for 24 h in 2 ml of RPMI 1640 medium containing 10% fetal calf serum, 10 mM Hepes, 1 mM L-glutamine, 0.05 mM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin (all obtained from Life Technologies, Grand Island, NY) in 24-well tissue culture plates (Costar, Cambridge, MA) [28]. The supernatants were then collected individually and immediately frozen until further analysis.

Fetal Resorption

On Day 13.5 of gestation, 10 to 11 females per group were killed, the uteri were removed, and the implantation sites were evaluated. The resorbed embryos were identified by their small size, and necrotic hemorrhagic appearance was compared with that of normal embryos. The percentage of resorption was calculated as 100% x the ratio of resorption number to total implantation number (resorption plus normal implantation number).

Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis for Cytokine mRNA Expression

Total RNA was isolated from an ectoplacental cell cone (EPC) at Day 9.5 of gestation (n = 5) using TRIzol reagent (Life Technologies) and converted to cDNA. The entire implantation site (EPC) was used as the source of total RNA in the present study due to difficulties in separating the placenta from the decidua at this stage of gestation. The amount of polymerase chain reaction (PCR) amplifiable product in each reverse-transcribed sample was standardized by a preliminary amplification for the hypoxanthine phosphoribosyl-transferase (HPRT) sequence, and by readjusting the sample concentration according to the densitometry reading of the HPRT band.

The mRNAs for IFN-{gamma}, IL-4, IL-10, and IL-12p40 were then quantitated in the standardized cDNA samples by quantitative reverse transcription (RT)-PCR as described elsewhere [29]. Briefly, the initial mRNA level was normalized by amplification of HPRT following the synthesis of cDNA. An equivalent amount of standardized cDNA from each analytic sample was coamplified with an added constant concentration of competitor. The competitor and target DNA shared the same sense and antisense primers used for target amplification, and had an identical sequence to that of the target mRNA except for deletion of a fragment from the competitor sequence. Both the target cDNA and competitor DNA were coamplified with equal amplification efficiency in the same PCR reaction. The PCR products were distinguishable on an electrophoresis gel. The cycling conditions were 1 min at 95°C for denaturation, 1 min at 60°C for annealing, and 1 min at 72°C for extension, then 7 min at 72°C for further extension after the last cycle. The cDNA samples were amplified in 27 cycles for HPRT, 30 cycles for IFN-{gamma}, 37 cycles for IL-4, and 35 cycles for IL-10 and IL-12p40. The primer pairs used to generate HPRT, target IL-4, IL-10, IL-12p40, and IFN-{gamma} and the primers used to construct the competitor were described previously [29]. The expected size of HPRT PCR product is 165 base pairs (bp). The sizes of the target and competitor products, respectively, for the tested cytokines are 385 and 333 bp (IL-4), 186 and 140 bp (IL-10), 237 and 187 bp (IFN-{gamma}), and 302 and 256 bp (IL-12p40). The PCR products were visualized after electrophoresis through 2% agarose gels. The corresponding bands were scanned and quantitated by a densitometer (Alpha Innotech Corporation).

ELISA for Cytokines

Concentrations of IL-2, IL-4, IL-10, TGF-ß, IFN-{gamma}, and TNF-{alpha} in the culture supernatants (n = 5) were assayed using the commercially available Quantikine M kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions. The lowest limit of the cytokine ELISA kits was as follows: IL-2, 2.0 pg/ml; IL-4, 2.0 pg/ml; IL-10, 4.0 pg/ml; TGF-ß, 2.0 pg/ml; IFN-{gamma}, 2.0 pg/ml; and TNF-{alpha}, 5.1 pg/ml.

Flow Cytometry

Spleens were harvested from pregnant CBA/J mice at Day 9.5 of gestation (n = 5), and single-cell suspensions were prepared. The splenocytes were stained by a standard immunofluorescence assay with various combinations of fluorescence-conjugated antibodies. Fluorochrome-conjugated antibodies were all purchased from Caltag (Burlingame, CA). Antibodies were PE-CY5-conjugated rat anti-mouse CD4 (GK1.5), APC-conjugated rat anti-mouse CD25 (PC61 5.3), PE-conjugated rat anti-mouse CD80 (RMMP-1), PE-conjugated rat anti-mouse CD86 (RMMP-2), PE-conjugated hamster anti-mouse CD28 (37.51.1), PE-conjugated hamster anti-mouse CTLA-4 (UC10-4B9), and PE-conjugated rat anti-mouse IL-10 mAb (JES5-2A5). Samples were analyzed on a FACS Caliber flow cytometer using CellQuest software (BD Biosciences, San Jose, CA).

Statistical Analysis

Data were expressed as the mean ± SD, and analyzed with the aid of an SPSS database (SPSS Inc., Chicago, IL; www.spss.com). The significance of difference in the resorption rates was tested by a {chi}2 test. A Student t-test was performed to test the significance of treatment effects on cytokine expression, peripheral CD4+CD25+ T regulatory cell population, and peripheral costimulatory molecule expression. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Anti-CD86 mAb Administration Improved Fetal Viability in Abortion-Prone CBA/JxDBA/2 Matings

We wondered first whether anti-CD86 mAb alone could prolong fetus survival and prevent fetal wastage of CBA/ JxDBA/2 matings. To test this conjecture, the CBA/J females mated with DBA/2 or BALB/c males were administered anti-CD86 mAb at Day 4.5 of gestation (the putative time of murine implantation) or at Day 4.5, 6.5, 8.5, and 10.5 of gestation. The results shown in Table 1 indicate that the fetal resorption rate of CBA/JxBALB/c matings was neither altered significantly after CD86 blockade at Day 4.5 of gestation, nor was it altered after CD86 blockade at Days 4.5, 6.5, 8.5, and 10.5 of gestation, compared with that of controls. The 5% to 10% embryo resorption rate of CBA/ J females mated with BALB/c males may be explained by chromosomally abnormal embryos [30]. No effect was found with the isotype injections compared with that of control animals (data not shown).


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  		TABLE 1. Effect of anti-CD86 mAb on embryo resorption rate in CBA/JxBALB/c matings

In contrast, CBA/J females that were mated with DBA/ 2 males experienced embryo resorption rates of up to 20% to 30%. The results shown in Table 2 clearly demonstrate that blockade of CD86 both at Day 4.5 or at Days 4.5, 6.5, 8.5, and 10.5 of gestation significantly reduced the resorption rate of CBA/JxDBA/2 matings compared with that of controls, bringing the resorption rate to the normal level noted in CBA/JxBALB/c matings. It is interesting that administration of anti-CD86 mAb on Day 4.5 alone, the putative time of murine implantation, had similar effects to those of administration every other day starting at Day 4.5 of gestation. These results suggested that in the absence of an effective CD86 costimulatory signal (treatment with anti-CD86 mAb), the embryo resorption was abrogated. We therefore proposed that CD86 costimulation might be essential for the early events that lead to embryo resorption.


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  		TABLE 2. Anti-CD86 mAb improved fetal viability in abortion-prone CBA/JxDBA/2 matings

Anti-CD86 mAb Administration Altered Cytokine Transcription at the Maternal-Fetal Interface

Given that selective blockade of CD86 costimulation rescued the potentially doomed implantations from abortion, we wondered whether the fetus-rescuing effects of anti-CD86 mAb could be attributed to its influence on shifting the Th1 and Th2 balance at the maternal-fetal interface, because previous evidence showed that a Th2 bias at the maternal-fetal interface was advantageous for fetal viability, whereas abnormally elevated concentrations of Th1 cytokines were associated with spontaneous abortions [31, 32]. To confirm this hypothesis, the EPCs (at Day 9.5 of gestation) of pregnant CBA/J females mated with DBA/2 males were collected for quantitative RT-PCR analysis of cytokine mRNA expression. A pair of mutually counteracting cytokines, IL-4 and IFN-{gamma}, which promote helper cell type differentiation and stability in opposite directions, was analyzed for transcription. It was shown that selective blockade of CD86 costimulation resulted in up-regulation of IL-4 and IL-10 mRNA expression, whereas expression of IFN-{gamma} mRNA was concomitantly down-regulated (Fig. 1).



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  		FIG. 1. Quantitative analysis of cytokine mRNA expression in ectoplacental cone by RT-PCR. Quantitative PCR analysis was carried out for IL-4, IL-10, IL-12, IFN-{gamma}, and HPRT after anti-CD86 mAb administration, or treatment of isotype-matched rat immunoglobulin as control. A) RT-PCR products were separated on agarose gel. Relative expression was calculated as the ratio of PCR products of target to competitor. Results were reproducible in five different animals, and the photograph is a representative one. B) CD86 blockade resulted in a Th2 bias in transcription at the maternal-fetal interface. Student t-test: *P < 0.05 vs. control

Then we tested whether interference in CD86 costimulation could abrogate the effect of IL-12, a cytokine capable of eliciting Th1-associated cytokine production and involved in embryo resorption of CBA/JxDBA/2 matings [33]. It was found that IL-12 mRNA decreased significantly after the pregnant mice of CBA/JxDBA/2 matings had received anti-CD86 mAb administration (Fig. 1).

Anti-CD86 mAb Administration Influenced Cytokine Expression at the Maternal-Fetal Interface

We had previously shown that a decreased expression of IFN-{gamma} and TNF-{alpha} in CBA/J (mated with DBA/2) splenic CD4+ T cells was accompanied by a parallel increased expression of IL-4 and IL-10 after costimulation blockade [34]. To further demonstrate the role of CD86 blockade in shifting the cytokine phenotype at the maternal-fetal interface, we cultured placental and decidual units from CBA/ JxDBA/2 or CBA/JxBALB/c matings, and analyzed the expression of IL-2, IFN-{gamma}, TNF-{alpha}, IL-4, IL-10, and TGF-ß in the supernatant. The tissues of pregnant CBA/J females mated with DBA/2 males showed significantly lower production of TNF-{alpha} and IFN-{gamma} in the supernatants of the anti-CD86 mAb-treated groups than that of controls (Fig. 2A), whereas the culture supernatants of tissues from CBA/ JxBALB/c mice treated with anti-CD86 mAb showed no significant differences in cytokine production compared with that of controls (Fig. 2A). Moreover, the excessive expression of IFN-{gamma} and TNF-{alpha} during the gestation of abortion-prone CBA/JxDBA/2 matings had been ameliorated by CD86 blockade to the level resembling its counterpart, the normal pregnant CBA/JxBALB/c matings. There was no detectable IL-2 production at the maternal-fetal interface in either CBA/JxDBA/2 or CBA/JxBALB/ c matings (data not shown).



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  		FIG. 2. Blockade of CD86 costimulation shifted the cytokine protein profiles from Th1 predominance to Th2 bias at the maternal-fetal interface. Expression of IL-2, IFN-{gamma}, TNF-{alpha}, IL-4, IL-10, and TGF-ß at the maternal-fetal interface was analyzed by ELISA in the culture supernatants of placental and decidual units from CBA/JxDBA/2 or CBA/JxDBA/2 matings treated with anti-CD86 mAb on Day 13.5 of gestation (n = 5), treatment of isotype-matched rat immunoglobulin as control. A) Th1 cytokines were decreased by CD86 blockade. There was no detectable IL-2 (data not shown). B) Blockade of CD86 resulted in a Th2 bias in translation at the maternal-fetal interface in CBA/JxDBA/2 matings. No further protective effects were observed in CBA/JxBALB/c matings (A and B). Student t-test: *P < 0.05 vs. control

The secretion of IL-10, TGF-ß, and IL-4 from placental and decidual tissues of CBA/JxDBA/2 or CBA/JxBALB/ c matings are shown in Figure 2B. There was a significant elevation of IL-10, IL-4, and TGF-ß in CBA/JxDBA/2 tissues following anti-CD86 mAb treatments compared with that of controls (Fig. 2B). The protecting effect of anti-CD86 mAb administration was confined to the CBA/ JxDBA/2 mating combination. Anti-CD86 treatment did not further decrease the resorption rate of normal pregnant CBA/JxBALB/c matings.

Anti-CD86 mAb Administration Expanded Peripheral CD4+CD25+ T Regulatory Cell Population in CBA/ JxDBA/2 Matings

Aluvihare et al., [35] recently demonstrated that the expanded population of splenic CD4+CD25+ T cells from normal pregnant female mice contained suppressor activity, which could abolish fetal rejection. However, CD80/CTLA-4 mediated inhibition of resting T cells required the presence of CD4+CD25+ regulatory T cells [23]. To test these possibilities and to further elucidate the mechanisms involved in the fetus-rescuing effects of CD86 blockade, the splenic CD4+CD25+ T cell subset of pregnant CBA/J mice was analyzed at Day 9.5 of gestation after administration with anti-CD86 mAb at Day 4.5 of gestation. Blockade of CD86 costimulation resulted in a peripheral expansion of the CD4+CD25+ T cell subset (Fig. 3). It should be pointed out that although regulatory T cells are characterized by a surface expression of CD25, the activated CD4+ T cells also express this marker. Expansion of CD4+CD25+ T cells could also be due to an expansion of the alloreactive effector T cell population responding to paternal antigens expressed by the fetus, or to an expansion of the regulatory T cell population suppressing antifetal rejection (or both).



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  		FIG. 3. Expansion of peripheral CD4+CD25+ regulatory T cell population driven by anti-CD86 mAb administration in CBA/JxDBA/2 matings. At Day 9.5 of gestation, splenocytes were prepared from CBA/J mice (n = 5), mated with DBA/2 males and treated with anti-CD86 mAb at Day 4.5 of gestation, treatment of isotype-matched rat immunoglobulin as control. The cells were stained with anti-CD4 and anti-CD25mAb, permeabilized, and stained with anti-IL-10 or anti-CTLA-4 mAb, respectively. Frequency of CD4+CD25+ T cells increased in splenocytes after anti-CD86 mAb administration. The majority of these expanded CD4+CD25+ T cells were characterized by an intracellular expression of IL-10 and CTLA-4, the characteristics of CD4+CD25+ regulatory T cells. Mean values are shown as bars. Student t-test: *P < 0.05 vs. control

Because regulatory T cells can be distinguished from the recently activated T cells by the expression of intracellular CTLA-4 and IL-10, we performed flow cytometry to analyze the expression of intracellular CTLA-4 and IL-10 in the expanded CD4+CD25+ T cell population driven by CD86 blockade. We observed the majority of the expanded splenic CD4+CD25+ T cells expressing intracellular CTLA-4 or IL-10, respectively (Fig. 3), suggesting that an expansion of the CD4+CD25+ regulatory T cell subset had likely occurred. Based on this expression pattern, the results suggested that CD86 blockade is likely involved in promoting the suppressive effect of CD4+CD25+ regulatory T cells on maternal antiembryo rejection and inducing maternal-fetal immunotolerance in CBA/JxDBA/2 matings.

Anti-CD86 mAb Administration Altered Costimulatory Molecule Expression on Splenocytes of Pregnant CBA/J Females Mated with DBA/2 Males

We next investigated whether the fetus-rescuing effect of CD86 blockade could be due to enhancement of the CD80-CTLA-4 pathway or to impairment of CD86-CD28 interaction (or both), whereas the antifetal rejection might be attributed to the predominance of the CD86-CD28 interaction or impairment of CD80-CTLA-4 engagement (or both). After administration with anti-CD86 mAb at Day 4.5 of gestation, the expression patterns of CD28/CTLA-4 and their shared ligands CD80 and CD86 on splenocytes of pregnant CBA/J mice mated with DBA/2 males were analyzed at Day 9.5 of gestation. There existed a reciprocal relationship between expression of these molecules (Fig. 4). The expression of CD28 and its ligands CD80 and CD86 both decreased, but that of CTLA-4 increased. These results suggest that the level of CD28 costimulation (CD28-CD80/ CD86) correlates negatively with the capacity of the CD4+CD25+ regulatory T cells to suppress CD4+CD25– T cells [23, 24], while the up-regulated expression of CTLA-4 may be associated with the expansion of CD4+CD25+ regulatory T cell population, owing to the constitutive expression of CTLA-4 on CD4+CD25+ regulatory T cells [20].



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  		FIG. 4. Expression patterns of CD80, CD86, CD28, and CTLA-4 on splenocytes induced by anti-CD86 mAb administration in CBA/JxDBA/2 matings. After administration with anti-CD86 mAb or isotype-matched rat immunoglobulin at Day 4.5 of gestation, splenocytes were prepared from CBA/J mice mated with DBA/2 males at Day 9.5 of gestation (n = 5), and then surface-stained by PE-conjugated anti-CD80, anti-CD86, anti-CD28, and anti-CTLA-4 mAb, respectively. Frequencies of these cell surface markers on the splenocytes were analyzed by FACS. Mean values are shown as bars. Student t-test: *P < 0.05 vs. control, **P < 0.01, vs. control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There was a predominance of Th2 over Th1 cytokines at the maternal-fetal interface when pregnancy was successful, although the mechanism responsible for shifting the local cytokine secretion pattern is not yet clear. It has been suggested that CD80 and CD86 provide distinct signals for the Th0 differentiation into either a Th1 (IFN-{gamma}-producing, IL-2-producing) or a Th2 (IL-4-producing, IL-10-producing) cytokine phenotype [36]. It was demonstrated that selective CD80 blockade ameliorated disease by shifting the cytokine phenotype to a Th2 pattern, whereas selective CD86 blockade worsened experimental autoimmune encephalomyelitis (EAE), a model of Th1-mediated disease, which indicates that Th1 responses require CD80 costimulation, whereas Th2 responses require CD86 costimulation [37]. By contrast, in the nonobese diabetic (NOD) mouse, another model Th1-mediated disease, selective CD80 blockade worsened disease, whereas selective CD86 blockade had a beneficial effect, leading to the conclusion that CD86 was a major costimulatory ligand in a Th1 response [38]. Although results produced by selective CD80 or CD86 blockade in vivo have been contradictory, the roles of CD80 and CD86 may vary depending on the status of the immune response and the APC being used in the response.

In the abortion-prone CBA/JxDBA/2 mating combination, IFN-{gamma} and IL-12 are involved in eliciting Th1 cytokine production and subsequent embryo wastage [33]. In this scenario, IL-12 would most likely up-regulate IFN-{gamma}, which would activate macrophages to produce TNF-{alpha}, which in turn, would lead to activation of natural killer cells to produce more IFN-{gamma}. As a result, this positive feedback evokes a Th1 cytokine milieu at the maternal-fetal interface [33, 39]. In the present study, we demonstrated that blockade of CD86 costimulation could abolish this feedback at the transcriptional level. Moreover, it seems likely that the surviving implants in the anti-CD86 mAb-treated CBA/JxDBA/ 2 pregnant female mice were due to the effect of selective CD86 blockade on shifting the balance from Th1 to a protective Th2 cytokine bias. This local effect of the costimulatory blockade at the maternal-fetal interface was consistent with its systemic effect on the cytokine polarization from Th1 to Th2 [34]. These alterations in the cytokine profiles achieved by CD86 blockade resulted in a physiologic environment that could be advantageous for maintaining and developing a normal pregnancy. However, the overall ability to resist infections (and other problems) would likely not be impaired in this circumstance, because the excessive expression of Th1 cytokines (IFN-{gamma} and TNF-{alpha}) during the gestation of abortion-prone CBAxDBA2 matings had been ameliorated by CD86 blockade to the level resembling its counterpart of the normal pregnant CBAxBALB/c matings, which was sufficient to achieve the pregnant physiological functions. Although it has been known that successful pregnancy is a "Th2 phenomenon," IFN-{gamma}, a particular Th1 cytokine, contributes to initiation of pregnancy-induced uterine vascular modification, maintenance of decidual integrity, and regulation of maturation and senescence of the uterine natural killer cell population [40]. Once abnormally elevated, IFN-{gamma} is deleterious and can result in embryo resorption [33]. Thus, a moderate concentration of IFN-{gamma} achieved by CD86 blockade is essential for normalcy of implantation sites and maintenance of decidual cell viability in the abortion-prone matings.

Evidence suggests that CD80 but not CD86 is the functional ligand for CTLA-4. In the studies using vascularized cardiac allografts, CD80 was identified as the ligand for CTLA-4 being responsible for prolonged allograft survival [13, 14]. It is interesting that the role of CTLA-4 can be revealed by use of CD86 blocking antibodies in these experiments. In the present study, fetal survival could result from enhanced CD80-CTLA-4 interactions, which are free to proceed in the absence of CD86.

It has been suggested that CD80 can direct CTLA-4 mediated inhibition of resting T cells, which requires the presence of CD4+CD25+ regulatory T cells [20]. Moreover, expansion of CD4+CD25+ T cells or augmentation of their activity can suppress allograft rejection [41–43]. In the present study, selective CD86 blockade left CD80-CTLA-4 signaling intact on one hand, and the expanded population of peripheral CD4+CD25+ T cells may facilitate suppressing the aggressive allogeneic response directed against the fetus on the other hand [34]. It has been demonstrated that CD80-CTLA-4 interactions occur more effectively in the absence of CD86 expression [18]. In the model of nonabortion-prone CBA/JxBALB/c, Th2 cytokines are predominant at the maternal-fetal interface, and in such a noninflammatory context, CD80 may preferentially interact with CTLA-4, which restricts T cell activation, acting to increase the threshold for activation. It is important to note that resting T cells do not express CTLA-4 until after activation. However, CD4+CD25+ regulatory T cells are likely the source of CTLA-4 under such circumstances. Indeed, there is support for a role of CTLA-4 in CD4+CD25+ regulatory T cell function [20, 44, 45].

In the model of abortion-prone CBA/JxDBA/2, Th1 cytokines are predominant at the maternal-fetal interface, resulting in increased expression of CD86 on APC cells leading to a higher maternal response to paternal alloantigen, which provides antagonistic signals and suppressive action to CD4+CD25+ regulatory T cells. It has been demonstrated that CD28 and T cell receptor stimulation can antagonize the suppressive function of CD4+CD25+ regulatory T cells [20, 21, 46]. Modulating CD86 expression is crucial to effective T cell activation that is detrimental to fetal survival, not simply by the delivery of costimulatory signals but by overriding CD80-CTLA-4 interactions and inhibitory effects of CD4+CD25+ regulatory T cells. The costimulatory molecules CD80 and CD86 could present opposing roles in regulating the suppressive activity of CD4+CD25+ regulatory T cells [47]. Therefore, in abortion-prone CBA/ JxDBA/2 matings, CD4+CD25+ regulatory T cells might be diminished, or their suppressive capacity might be abolished to some extent (or both), and this might be restored by selective CD86 blockade to alter the relative balance between CD80 and CD86 costimulation. In addition to the suppressive effects on CD4+CD25– T cells, CD4+CD25+ regulatory T cells have been shown to inhibit the function of APCs by down-regulating the expression of costimulatory molecules [48]. It is suggested by the present study that CD86 blockade in vivo could lower the expression of costimulatory molecules to a level at which direct suppression of CD4+CD25+ regulatory T cells can resultantly operate. Among these, the decreased expression of CD80 and CD86 may to some degree be attributed to the effect of IL-10 [49].

There are two broad categories of naturally occurring and induced CD4+CD25+ regulatory T cells. The naturally occurring CD4+CD25+ regulatory T cells are antigen nonspecific and interact directly with other T cells to inhibit their activation, while the induced CD4+CD25+ regulatory T cells produce immunosuppressive cytokines such as IL-10, act directly on other T cells or APC, and are antigen-specific in some but not in all systems [50]. The induced CD4+CD25+ regulatory T cells can suppress normally responding CD4+CD25– populations, rendering them unable to respond to the subsequent antigen restimulation. Moreover, these altered CD4+CD25– cells in turn suppress syngeneic CD4+ T cells via the production of inhibitory cytokines [51, 52]. Therefore, CD4+CD25+ regulatory T cells can mediate "infectious tolerance." This secondary suppressive activity of CD4+CD25+ regulatory T cells is transferred in a cell-cell contact-independent manner and is mediated by the well-known immunomodulatory factors IL-10 and TGF-ß [51, 52, 53]. We demonstrated here that selective CD86 blockade could augment expression of IL-10 and expand the CD4+CD25+ regulatory T cell population; IL-10 itself was involved in the generation of induced CD4+CD25+ regulatory T cells, and in turn was secreted by CD4+CD25+ regulatory T cells. As a result, a positive feedback between IL-10 and CD4+CD25+ regulatory T cells can facilitate fetal allograft survival and development.

In conclusion, our results provide new insight into the immunoregulatory events in maternal-fetal immunotolerance. Our observations indicate that the physiological function of the costimulatory pathway may be imbalanced in certain pathological pregnancies, such as allogeneic fetal wastage, and selective CD86 blockade was effective in preventing maternal rejection of the allogeneic fetus via restoring the physiological balance of Th1/Th2 at the maternal-fetal interface, and expanding CD4+CD25+ regulatory T cell population, which are crucial to the maintenance of normal pregnancy. Thus, treatment with anti-CD86 mAbs may provide a new more rational immunotherapy for the immune-associated recurrent spontaneous abortion.


   ACKNOWLEDGMENTS
 
We thank Prof. K. Okumura (Juntendo University School of Medicine, Tokyo, Japan) for the generous gift of the PO3 cell lines.


   FOOTNOTES
 
1 Supported by the National Natural Science Foundation of China (grant 30200299 to X.-Y.Z. and grant 39770773 to D.-J.L.) and by grant 985B36 from Fudan University to X.-Y.Z. Back

2 Correspondence: Da-Jin Li, Laboratory of Reproductive Immunology, Institute of Obstetrics and Gynecology, Fudan University, Shanghai 200011, China. FAX: 86 21 63457331; djli{at}shmu.edu.cn Back

Received: 7 July 2004.

First decision: 27 July 2004.

Accepted: 16 September 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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