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The Journal of Immunology, 2007, 179, 5211 -5219
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Link between PDL1 and T Regulatory Cells in Fetomaternal Tolerance1

Antje Habicht*, Shirine Dada*, Mollie Jurewicz*, Brian T. Fife{dagger}, Hideo Yagita{ddagger}, Miyuki Azuma§, Mohamed H. Sayegh* and Indira Guleria2,*

* Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115; {dagger} University of California San Francisco Diabetes Center, University of California, San Francisco, San Francisco, CA 94143-0540; {ddagger} Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan; and § Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Acceptance of the fetus expressing allogeneic paternal Ags by the mother is a physiologic model of transplantation tolerance. Various mechanisms contribute to fetal evasion from immune attack by maternal leukocytes. We have recently demonstrated that the inhibitory costimulatory molecule PDL1 plays a critical role in fetomaternal tolerance in that PDL1 blockade or deficiency resulted in decreased allogeneic fetal survival rates. CD4+CD25+ T regulatory cells (Tregs) have also been demonstrated to play an important role in fetomaternal tolerance. Since PDL1 is expressed on Tregs, we explored the interactions between PDL1 and Tregs in vivo in a mouse model of fetomaternal tolerance. Depletion of CD25+ T cells abrogated the effect of anti-PDL1 Ab indicating that the effect of PDL1 is possibly mediated by CD25+ Tregs. Adoptive transfer of Tregs from wild-type but not PDL1-deficient mice into PDL1-deficient recipients significantly improved fetal survival. The frequency, phenotype and placental trafficking of Tregs from PDL1-deficient mice were similar to those of wild-type controls, but were defective in inhibiting alloreactive Th1 cells in vitro. This is the first report providing evidence for a link between PDL1 and T regulatory cells in mediating fetomaternal tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The development of fetus in the mother can be considered as an in vivo model of allograft tolerance. Study of tolerance mechanisms enabling a state of pregnancy could therefore provide additional insight to our understanding of immunologic tolerance as a whole. Many factors promoting immune evasion have been explored in recent years (1). We have recently proposed and tested the hypothesis that cells present at the uteroplacental interface expressing PDL1, a negative costimulatory molecule, protect murine allogeneic concepti from maternal T cell mediated immunity. We observed that pregnant CBA mice treated with a blocking anti-PDL1 mAb lost allogeneic but not syngeneic concepti. Similarly, a deficiency of PDL1 resulted in substantial increase in the rate of spontaneous fetal resorption and decrease in fetal survival (2). This increase in the rate of resorption was coupled with increase in T cell infiltration in the placenta, thereby suggesting that PDL1-expressing decidual cells inhibit PD-1-expressing T cells that infiltrate the placenta in response to paternal Ags.

Recent studies have highlighted the significance of regulatory T cells during immune interactions (3, 4, 5). The CD4+CD25+ regulatory T cell (Treg)3 subset has been found to suppress autoimmune responses and to play a role in tolerating allogeneic organ grafts (3, 4, 5, 6). The acceptance of paternally derived tumor cells during pregnancy (7) suggests the involvement of systemic regulatory processes in pregnancy. Aluvihare et al. (8) have recently shown that Tregs expand in murine pregnancies and are essential for a successful allogeneic pregnancy. In another study involving CBAxDBA abortion prone model, accumulation of paternal alloantigen specific Th1 cells is suggested to be due to insufficient generation of pregnancy induced Tregs (9). The data in mouse are consistent with a similar function for Tregs in human pregnancy. It has been reported that decidual and/or peripheral blood CD4+CD25+high T cells increased during early pregnancy (10, 11) and returned to lower levels postpartum in the subjects studied (12).

Naturally occurring Tregs are characterized by the surface expression of CD4 and CD25 (13). In addition to sustained high surface expression of CD25, CTLA4, and glucocorticoid-induced TNFR-related protein (GITR) expression are features of suppressive Tregs (14, 15). Expression of the transcription factor Foxp3 has been shown to be confined to Tregs in mice and is critical to their development and regulatory function (15). Unlike other Treg cell markers, Foxp3 expression has been reported as not being up-regulated in conventional mouse T cells upon their activation (16, 17). Recently mice that express normal Foxp3 linked to a fluorescent reporter (18, 19) have been generated. These mice would be a very useful in tracking and studying Tregs, since these cells can be isolated live by flow sorting and then used in tracking or functional studies.

The PD-1 receptor and its ligands, PDL1 and PDL2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses (20, 21, 22, 23). In addition to the markers for Tregs discussed above, PD-1 mRNA is highly expressed in CD4+CD25+ Tregs and anergic T cells. This could suggest a mechanism by which PD-1 could be regulating T cell responses (24, 25). The ligand for PD-1, PDL1 is also expressed on regulatory T cells (26) and very recently, PDL1 expression on vascular endothelium was shown to be critical for the immunoregulatory effect of CD4+CD25+Foxp3+ T cells (27). In the present study, we investigated the interactions between PDL1 and Tregs in regulating the maternal alloimmune response against paternal Ags on the fetus. We provide definitive evidence for a novel link between PDL1 and Tregs in mediating fetomaternal tolerance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

CBA/CaJ (CBA) (H-2Kk, H-2Dk, H-2Lk) and C57BL/6 (B6) (H-2Kb, H-2Db, H-2Lb), were obtained from The Jackson Laboratory. PDL1-deficient mice (PDL1–/–) were generated as previously described (2) and were maintained as a breeding colony in our animal facility. All mice were used at 8–12 wk of age and were housed in accordance with institutional and National Institutes of Health guidelines.

Timed matings and resorption rates

Naive female C57BL/6 or PDL1–/– mice were mated with male allogeneic CBA mice. Females were inspected daily for vaginal plugs and sighting a vaginal plug was designated as day 0.5 of pregnancy. Plugged females were either sacrificed at gestational day 10.5 –13.5 days post coitum (dpc) or were monitored until parturition and the number of pups born was recorded.

Treatment protocol

Pregnant female C57BL/6 mice were injected on days 6.5, 8.5,10.5,12.5 dpc with the blocking anti-mouse PDL1 mAb with the dosage of 500, 500, 250, and 250 µg, respectively (2). This anti-PDL1 (MIH6) mAb was generously provided by Dr. Miyuki Azuma (28). To deplete CD4+CD25+ T cells, naive female B6 mice were injected with the depleting anti-CD25 mAb (PC61) (250 µg/day) 6 days (day –6) and 1 day before (day –1) they were mated with male CBA. The degree of CD4+CD25+ depletion in the spleens was assessed by flow cytometry and was consistently >90%. All mAbs were given i.p.

ELISPOT assay

Splenocytes from pregnant mice (female CBA mated with C57BL/6 male), which have been treated with anti-CD25 or control IgG, were obtained as single cell suspensions and used as responder cells. Splenocytes from male C57BL/6 (or CBA as syngeneic control) mice were irradiated and used as stimulator cells. The ELISPOT assay was adapted to measure IFN-{gamma} secreting cells as described before (2).

T cell purification

Spleens and lymph nodes were harvested from female C57BL/6 or PDL1–/– mice and CD4+ T cells were purified by magnetic bead negative selection (Miltenyi Biotec). To purify CD4+CD25+ T cells, the enriched CD4+ T cells were incubated with PE-conjugated anti-CD25 for 10 min at 4°C, washed, and then incubated with anti-PE microbeads (Miltenyi Biotec) for 15 min at 4°C. Magnetic separation was performed using a MS positive selection column according to the suggested protocol (Miltenyi Biotec). The purity was consistently >95% for CD4+CD25– and CD4+CD25+ T cell preparations.

Adoptive cell transfer

Female PDL1–/– mice were i.v. injected with total RBCs depleted splenocytes (50 x 106 cells/mouse) from wild-type (WT) controls or PDL1–/– mice, and mated with male CBA mice on the same day. The mice were followed to determine the number of pups delivered. In a second experiment female PDL1–/– mice received sorted CD4+CD25+ T cells (4 x 105) from either WT or PDL1–/– mice on day of mating with CBA males and were monitored until parturition. As an autologous/syngeneic control, PDL1–/– females were mated with PDL1–/– males. Tregs from WT or PDL1–/–-deficient mice were then transferred to these autologously mated females and litter size was determined.

Histology

At predetermined intervals, placentae were removed for histological analysis and immunostaining. Placentae were embedded in Tissue-Tek O.C.T. compound and frozen in liquid nitrogen. Immunohistochemistry was performed on 5 µm frozen tissue sections with Ab to Foxp3 (FJK-16s, Rat IgG2a) to detect Tregs. Sections were fixed in cold acetone, washed in PBS and quenched in 0.3% hydrogen peroxide in PBS. The sections were then washed in PBS, blocked with normal serum and then incubated with primary Ab overnight at 4°C. After washing with PBS, slides were incubated with biotinylated Ab for 40 min at room temperature. Slides were incubated with avidin-conjugated peroxidase (Vectastain ABC vector Elite kit) and developed with DAB (3,3'-diaminobenzidine). The tissue sections were then lightly counterstained with H&E and positive staining was indicated by a red-brown coloration as described before (2). Isotype-matched Ab (Rat IgG2a) was used as negative control.

Suppression Assay

Isolated 1 x 105 CD4+CD25– T cells from female C57BL/6 were cultured with 6 x 105 irradiated (3000 rad) allogeneic splenocytes from male CBA. Decreasing numbers of sorted CD4+CD25+ T cells starting at 1 x 105 from WT or PDL1–/– mice were added to the culture. T cell proliferation was measured by [3H]TdR incorporation in the last 12 h after a 3-day culture. In some suppression assays anti-PDL1 mAb or control IgG was added in vitro at 2, 20, or 50 µg/ml and IL-17 was measured by Luminex (see below) in the culture supernatants after 48 h of culture.

Luminex cytokine assay

IFN-{gamma}, IL-17, IL-4, and IL-5 were measured in the cell supernatants from the suppressor assay. The multibead array Luminex (Linco Research) was used to quantify the cytokines, according to the manufacturer’s instruction, with RPMI 1640 medium as blank. A Luminex 100 IS instrument (Biosource) with the Star Station acquisition program (v2 Applied Cytometry Systems) was used to process the data. All samples were run in single wells, except the standard curve points, which were run in duplicate according to the manufacturer’s recommendations.

Flow cytometry

All Abs and reagents for flow cytometry analysis were purchased from BD Pharmingen or eBioscience. Foxp3 staining was performed using a commercially available kit (eBioscience). Cells were analyzed on a FACSCalibur (BD Biosciences) using CELLQuest software (BD Biosciences).

RNA extraction and real-time PCR (rt-PCR)

RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RNA was treated with DNase (Invitrogen), and 5 µg of RNA was then reverse transcribed to synthesize 60 µl of cDNA. Each quantitative PCR reaction consisted of 20 µl containing 250 ng of cDNA, 10 µl of SYBR Green master mix (Applied Biosystems), and 250 nmol of sense and antisense primer. Primers were designed using Primer Express software. The reaction conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min; fluorescence was measured during the annealing/extension phase. Expression was measured as copies of any given gene divided by copies of the housekeeping gene GAPDH.

Primer sequences

IFN-{gamma}. Sense: 5'-AACGCTACACACTGCATCTTGG-3'; anti-sense: 5'-GCCGTGGCAGTAACAGCC-3'.

IL-17. Sense: 5'-TTCAGGGTCGAGAAGATGCT-3'; anti-sense: 5'-AAACGTGGGGGTTTCTTAGG-3'.

IL-4. Sense: 5'-TCATCGGCATTTTGAACGAG-3'; anti-sense: 5'-CGTT TGGCACATCCATCTCC-3'.

IL-5. Sense: 5'-AAAGAGAAGTGTGGCGAGGAGA-3'; anti-sense: 5'-CACCAAGGAACTCTTGCAGGTAA-3'.

Foxp3. Sense: 5'-GGCCCTTCTCCAGGACAGAC-3'; anti-sense: 5'-TCCACAGTGGAGAGCTGATGC-3'.

GAPDH. Sense: 5'-GGCAAATTCAACGGCACAGT-3'; anti-sense: 5'-AGATGGTGATGGGCTTCCC-3'.

Statistics

Student’s t test was used for comparison of the means. A p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Regulatory T cells express PDL1

We have recently demonstrated that the inhibitory costimulatory molecule PDL1 plays a critical role in fetomaternal tolerance in that PDL1 blockade or deficiency resulted in decreased allogeneic fetal survival rates (2). Tregs have also been recently shown to expand in murine pregnancies and are required for a successful allogeneic pregnancy (8). To examine the link between PDL1 and Tregs in fetomaternal tolerance, we first studied the expression pattern of PDL1 on both effector and regulatory CD4+ T cells derived from naive female C57BL/6 mice. As shown in Fig. 1, PDL1 was highly expressed on CD4+CD25+Foxp3+ (35.3 ± 4.8%) but to a significantly lesser degree on CD4+CD25–Foxp3– (10.6 ± 1.7%) T cells. These data indicate that the PDL1 expression is increased on CD4+ T cells expressing a regulatory phenotype.


Figure 1
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  		FIGURE 1. CD4+CD25+Foxp3+ T cells express PDL1. Surface expression of PDL1 on lymphocytes prepared from spleens of naive female C57BL/6 mice was evaluated. A, Representative histograms of the CD4+CD25+Foxp3+ (Treg) and CD4+CD25–Foxp3– (Teff) population prepared from spleens. B, The percentage of PDL1+ was significantly increased within the CD4+CD25+Foxp3+ population as compared with CD4+CD25–Foxp3– population (n = 7 per group, p < 0.0001). The data represent the mean ± SD of the results obtained from three separate experiments.

 
CD25 depletion abrogates the effect of PDL1-blockade

To further explore the interactions between PDL1 and Tregs we used an established fully allogeneic mating setup of female C57BL/6 (H-2b) x male CBA (H-2k). Pregnant female mice were treated with a blocking anti-PDL1 mAb or control IgG as previously described (2). Furthermore, we used an anti-CD25 mAb to deplete Tregs before the mating. The depletion of Tregs can be achieved by using this anti-CD25 mAb (29), and our group has published a protocol with this mAb to deplete the CD25+ Tregs in vivo (26, 30, 31). Two doses of 250 µg of CD25-depleting Ab (day –6 and day –1) resulted in >99% depletion of CD4+CD25+ Tregs on day 0 and this depletion remained up to day 13.5 (Fig. 2A).


Figure 2
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  		FIGURE 2. Effect of PDL1 blockade is dependent on the presence of CD4+CD25+ regulatory T cells. CD25 depleted and non-depleted C57BL/6 female mice were mated with male CBA mice and treated with anti-PDL1 mAb or IgG. A, Onset and duration of antibody-mediated CD25 depletion was determined. Surface expression of CD4 and CD25 on lymphocytes prepared from spleens of naive C57BL/6 females treated with anti-CD25 mAb –6 d, 0 d, 5.5 d and 13.5 d before/after potential mating. B, The percentages of resorbing fetuses was assessed at day 13.5 dpc after detection of vaginal plugs in the pregnant mice. PDL1 blockade (n = 4) significantly increased the fetal resorption rates compared with control IgG-treated animals (n = 6) (p < 0.0001), prior depletion of CD25+ T cells (n = 7) significantly enhanced the fetal rejection rates (p < 0.002) and abrogated the effect of PDL1 blockade (n = 7) (NS). C, Pregnant mice were followed to term and fetal survival rate was evaluated by counting the litter size. Each dot represents the result from an individual mouse. D, ELISPOT analysis for IFN-{gamma} in the anti-CD25 depleted and control IgG-treated mice.

 
CD25 depleted and non-depleted C57BL/6 pregnant mice were either sacrificed on day 13.5 dpc to assess the percentage of resorbing embryos or followed to term to evaluate fetal survival by counting the litter size. As previously reported, in vivo PDL1 blockade resulted in a significant increase in fetal resorption as compared with control IgG-treated mice (62.0 ± 3.0% vs 18.8 ± 2.3%; n = 6; p < 0.0001). In keeping with a recent report by Aluvihare et al. in vivo antibody-mediated depletion of Tregs significantly increased the percentage of fetal resorption on day 13.5 dpc from 18.8 ± 2.3% in control IgG-treated mice up to 31.9 ± 2.1% in anti-CD25-treated animals (n = 6; p < 0.002). The anti-CD25-treated mice also had decreased fetal survival rates as compared with the control IgG-treated group (4.5 ± 0.22 vs 6 ± 0.26; n = 6; p = 0.01). Interestingly, the effect of PDL1 blockade seems to be dependent on the presence of Tregs, as their prior depletion abrogated the effect of PDL1 blockade (31.9 ± 2.1% vs 29.6 ± 3.2% respectively; n = 6; ns) (Fig. 2B). Consistent with its effect on day 13.5, anti-PDL1 treatment also significantly reduced fetal survival as measured by litter size (3.5 ± 0.2 pups/plugged female vs 6 ± 0.3; n = 6; p < 0.0001). Again, this effect was abrogated by prior depletion with anti-CD25 mAb (4.5 ± 0.2 pups/plugged female vs 4.3 ± 0.21; n = 6; ns) (Fig. 2C).

Although Tregs are characterized by the surface expression of CD25, activated CD4+ T cells also express this marker. In vivo antibody-mediated CD25 depletion could therefore also lead to the depletion of recently activated effector T cells which may be required for the rejection of the allogeneic fetus. However, the fact that treatment with anti-CD25 mAb resulted in a significant increase in fetal resorption and reduction in the number of fetuses delivered at term clearly indicate that alloreactive effector T cells are still present and are capable of mounting an effective alloimmune response. To further rule out this possibility we measured the frequency of IFN-{gamma} producing alloreactive T cells in CD25-depleted and non-depleted pregnant C57BL/6 females (mated with CBA males) at d13.5 dpc using an ELISPOT assay. No statistically significant difference in the frequency of IFN-{gamma} producing cells (after 24 h of culture) was observed between the CD25-treated group and the untreated control group (156 ± 30 spots/5 x 105 splenocytes in anti-CD25-treated group vs 116 ± 37 spots/5 x 105 in control IgG-treated group, n = 4; Fig. 2D). Taken together, these results also indicate that alloreactive effector T cells are still present and are capable of mounting an effective alloimmune response in vitro and in vivo.

Adoptive transfer of WT splenocytes rescues pregnancies in PDL1-deficient mice

Given these data suggesting a role for Tregs in PDL1-mediated fetal survival we performed further adoptive transfer studies. 50 million splenocytes from either female C57BL/6 or PDL1-deficient mice were i.v. injected into PDL1-deficient mice on the day of mating with CBA males (1x), on day 0.5 and day 6.5 dpc (2x), or three times (day 0.5, day 6.5 and day 10.5) after the vaginal plug was documented. We then assessed fetal survival rates by allowing pregnant animals to go to term. As shown in Fig. 3A, the fetal survival rate in PDL1-deficient mice in this allogeneic mating set up is dramatically reduced as compared with that in C57BL/6 controls (2.3 ± 0.4 pups/litter; n = 6; vs 8.5 ± 0.3 pups/litter; n = 6; p < 0.0001). While the adoptive transfer of PDL1-deficient splenocytes had no effect on fetal survival rates even when injected three times (2.4 ± 0.2; n = 9; ns), the transfer of WT splenocytes significantly increased the number of animals born in a dose dependent manner from an average of 3.5 ± 0.4 pups/litter with one injection (n = 6; p < 0.05), to 4.5 ± 0.2 pups/litter with two injections (n = 6; p < 0.0001) and up to 5.2 ± 0.1 pups/litter with three injections (n = 9; p < 0.0001). These results suggest that PDL1 expressing Tregs in the splenocyte pool from C57BL/6 mice could be, at least to some degree, responsible for tolerance to fetal alloantigens.


Figure 3
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  		FIGURE 3. Adoptive transfer of WT splenocytes and Tregs increases fetal survival rate in PDL1–/– mice. Female PDL1–/– mice were mated with allogeneic male CBA. A, Female mice were injected with 50 x 106 total splenocytes from either female PDL1–/– or C57BL/6 mice on day of mating (1x), day 0.5 and day 6.5 after detection of vaginal plugs (2x) or on day 0.5, day 6.5 and day 10.5 after detection of vaginal plugs (3x). Fetal survival rate at term was evaluated by counting the litter size. The fetal survival rate in PDL1–/– mice is significantly reduced as compared with that in C57BL/6 controls (p < 0.0001). While the adoptive transfer of PDL1–/– splenocytes had no effect on fetal survival rate, even when injected 3 times, the transfer of WT splenocytes significantly increased the litter size in a dose-dependent manner (p < 0.05, p < 0.0001, and p < 0.0001 respectively). Values represent the mean ± SD of each group (n = 6). B, Representative FACS profile of isolated CD4+CD25+ T cells from PDL1–/– and WT mice showing similar percentage of Foxp3 expressing cells. C, Female PDL1–/– mice were injected with 4 x 105 CD4+CD25+ T cells from either female PDL1–/– mice or WT C57BL/6 mice. On the day of the adoptive transfer, the mice were mated with CBA males. The number of pups born at term were determined for each mouse successfully mated within 4 d of adoptive transfer. While adoptively transferred PDL1–/– Tregs did not alter fetal survival rate (NS), the transfer of WT CD4+CD25+ T cells significantly increased the fetal survival rate (p < 0.0001). Values represent the mean ± SD of each group (n = 6).

 
PDL1-deficient Tregs exhibit defective regulatory functions against paternal alloantigens in vivo

To compare the regulatory function of CD4+CD25+ T cells derived from C57BL/6 or PDL1-deficient female mice in vivo, we isolated CD4+CD25+ T cells with magnetic bead separation from both groups and adoptively transferred the same cell number (4 x 105 cells) into PDL1-deficient females on the day of mating with CBA males. Both C57BL/6 and PDL1 deficient freshly isolated CD4+CD25+ T cells expressed high levels of Foxp3 (>95%) and expression levels were comparable between the two groups (Fig. 3B). Again, the pregnancy outcome was evaluated by counting the number of pups delivered at term. Interestingly, the transfer of WT (4.3 ± 0.21 pups/litter; n = 6, p < 0.0001) but not PDL1 deficient (2.2 ± 0.3 pups/litter; n = 6, ns) CD4+CD25+ T cells significantly increased the fetal survival rate as compared with controls (2.3 ± 0.4 pups/litter; n = 6), suggesting an impaired regulatory function of PDL1-deficient regulatory T cells in vivo (Fig. 3C). These results indicate an important role for PDL1 expression on Tregs in maintaining tolerance at the fetomaternal interface. The data demonstrating no change in fetal outcomes following Treg transfer in autologous/syngeneic matings is shown in Table I.


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  		Table I. Effect of Tregs on litter size in autologous mating set up

 
Frequency, phenotype and placental trafficking of Tregs in PDL1-deficient and WT mice

We then studied the potential mechanisms of defective regulation by PDL1-deficient Tregs. First, we compared the frequency and regulatory phenotype of Tregs in spleens of PDL1-deficient and WT female mice by flow cytometric analysis. The frequency of CD4+ T cells expressing CD25 in PDL1-deficient mice was similar to that observed in C57BL/6 controls (6.7 ± 0.6%; n = 10; vs 8.5 ± 1.1%; n = 10; respectively; ns). Furthermore, the expression of markers such as Foxp3 (79.6 ± 4.1% vs 80.2 ± 3.5% respectively; ns), CTLA4 (59 ± 6.3 vs 50.1 ± 4.9 respectively; ns), GITR (80.1 ± 2.1% vs 78 ± 3.8%; ns) and CD103 (46.5 ± 6.8% vs 47.7 ± 7%; ns) on CD4+CD25+ T cells did not differ between the two groups (Fig. 4A). Finally, histochemical analysis with Foxp3 immunostaining (Fig. 4B) also showed similar number of Foxp3+ cells in the placenta of PDL1 deficient and WT mice (9.6 ± 1.67 in WT vs 10 ± 2 in PDL1-deficient placenta; n = 5). All together, these results indicate that CD4+CD25+Foxp3+ Tregs are present in PDL1-deficient mice in a similar frequency as in WT mice, that these cells exhibit a similar phenotype to conventional Tregs, and that PDL1-deficient Tregs can traffic to the placenta.


Figure 4
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  		FIGURE 4. Comparable number and phenotype of regulatory T cells in PDL1–/– and WT mice. A, Surface expression of CD4 and CD25 on lymphocytes prepared from spleens of naive female C57BL/6 mice (n = 6) compared with PDL1–/– mice (n = 6). The percentage of CD4+CD25+ T cells did not differ between the two groups as assessed by flow cytometric analysis. Furthermore, no significant differences were found in the expression of markers associated with regulatory T cell function such as Foxp3, CTLA4, GITR, and CD103. Representative dot plots and histograms of three separate experiments. B, Immunohistochemical analysis for Foxp3 in the placentae of PDL1–/– and WT mice (13.5 dpc). Positive brown staining showing Foxp3+ cells is marked by arrows.

 
PDL1-deficient regulatory T cells exhibit diminished suppressive properties in vitro

We then analyzed the regulatory function of CD4+CD25+ T cells derived from WT or PDL1-deficient mice in vitro. For this purpose we stimulated female WT CD4+CD25– T cells with irradiated male CBA splenocytes and added increasing numbers of either WT or PDL1 deficient CD4+CD25+ T cells to the culture. WT or PDL1-deficient Tregs were anergic in vitro after stimulation with male CBA splenocytes (data not shown). In keeping with the in vivo data, Tregs from PDL1-deficient mice displayed defective ability to suppress the proliferation of CD4+CD25– T cells (Fig. 5A). We also determined the levels of Th1 and Th2 type cytokines in the culture supernatants of the suppressor assay described above. Consistent with the proliferation assay results, Tregs from PDL1-deficient mice were defective in their ability to suppress IFN-{gamma} production as compared with Tregs from WT mice as determined by Luminex analysis (Fig. 5B).


Figure 5
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  		FIGURE 5. PDL1–/– CD4+CD25+ T cells exhibit diminished suppressive properties in vitro. A, The proliferation of 1 x 105 CD4+CD25– T cells from female C57BL/6 mice in response to 6 x 105 irradiated splenocytes from allogeneic male CBA is suppressed by increasing numbers of WT Tregs, but not PDL1–/– Tregs. Cell proliferation was estimated in all cases by [3H]thymidine incorporation. Data shown are mean percent inhibition of proliferation from three sets of experiments. B, Culture supernatants from these suppressor assays were analyzed by Luminex for IFN-{gamma}. The data shown is the amount of IFN-{gamma} (in pg/ml) and represents mean of triplicate experiments. C, Real-time PCR analysis of IFN-{gamma}, IL-17, and IL-4, IL-5, and Foxp3 in the placenta of PDL1–/– and WT mice. Data shown is from 5 to 6 mice per group.

 
Increased Th1 cytokines and IL-17 in the placentae of PDL1-deficient mice

Recently, a subset of effector CD4 IL-17-producing T (Th17) cells distinct from Th1 or Th2 cells has been described and shown to play a crucial role in the induction of autoimmune responses (32, 33). It is thought that Th17 effector cells and regulatory T cells differentiate from the same precursor T cell depending on the balance of cytokines in the environment, and the balance between Tregs and Th17 cells determines the outcome of the immune response (34). In our previous study, we had demonstrated an up-regulation of the proinflammatory Th1 type cytokine IFN-{gamma} in the placentae of anti-PDL1 treated as well as pregnant PDL1-deficient mice (2). To assess the role of Th17, Th1, Th2 and Tregs in our allogeneic pregnancy model we analyzed the expression of IFN-{gamma}, IL-17, the Th2 cytokines IL-4, IL-5, and Foxp3 by real-time PCR in placentae of pregnant PDL1-deficient and WT mice on day 13.5 dpc. Interestingly, we detected significantly higher message level of IL-17 and IFN-{gamma} and decreased message levels of IL-4 and IL-5 in placentae of PDL1-deficient mice, as compared with WT controls (Fig. 5C). No differences in the message of Foxp3 (Fig. 5C) were observed, confirming that the number of protective Tregs is the same in PDL1 deficient and WT mice, while the number of pathogenic Th17 cells is increased in the placentae of PDL1-deficient pregnant mice. Interestingly, IL-17 levels were not different in culture supernatants of suppression assay with Tregs from WT or PDL1-deficient mice, thereby suggesting a role for alloreactive IL-17-producing cells locally in the placenta at the site of fetal rejection. Indeed, we have performed the suppression assay using T effector cells and WT Tregs with and without anti-PDL1 Ab added in vitro and measured IL-17 levels in the culture supernatant by Luminex assay. We did not observe any significant change in IL-17 levels following anti-PDL1 treatment (anti-PDL1 378 ± 58 pg/ml vs control IgG 358 ± 60 pg/ml at 20 µg/ml of Ab; ns). These data suggest that alloreactive T cell pool is specifically increased locally in the placenta of PDL1-deficient mice (Fig. 5C, IL-17 real-time PCR data) and not systemically in the peripheral lymphoid organs such as spleen. The skewing of T cells toward Th17 cells occurs dominantly in the local tissue with the alloreactive Th1 cells and cells secreting IL-17 leading to fetal rejection.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We had earlier shown that PDL1 plays a critical role in fetomaternal tolerance as blockade or deficiency of PDL1 results in compromised pregnancy (2). A critical role for Tregs in both murine and human pregnancy has been recently shown (8, 9, 10). In the present study we examined the relationship between PDL1 and Tregs as PDL1 has been shown to be expressed on Tregs (26). Our data with anti-CD25 mAb treatment which depletes mice of Tregs in vivo (29, 31) suggests that the PDL1 mediated effect on fetomaternal tolerance is possibly mediated through Tregs as in mice depleted of these cells, anti-PDL1 treatment had no effect. In addition, adoptive transfer of Tregs from WT mice to PDL1–/– mice resulted in improvement in pregnancy outcomes of the PDL1–/– mice while transfer of Tregs from PDL1–/– to PDL1–/– recipients was ineffective in improving the litter size. This data suggests an impaired regulatory function of Tregs from PDL1-deficient mice in vivo and imply an important role for CD4+CD25+ Tregs in PDL1-mediated fetal survival. These data are in line with some of the recent results reported which indicate that Tregs are important for successful pregnancy outcomes (8, 9). Also, the finding that the proportion of Tregs in the decidua of spontaneous abortion subjects is much lower than that in elective abortion subjects (10) is consistent with a requirement for their presence at the maternal-fetal interface. However, specifically the expression of PDL1 on regulatory cells conferring fetomaternal tolerance has not been previously reported in murine or human studies. Our results suggest that PDL1 expressing Tregs at least to some degree are responsible for regulating alloimmune responses to fetal Ags at the materno-fetal interface. However, though the transfer of CD4+CD25+ T cells from WT mice to PDL1–/– females significantly increased the litter size, this did not reach the fetal survival rate observed in normal pregnancies (4.3 ± 0.2 vs 8.5 ± 0.3 pups/litter, p < 0.0001). Therefore, this partial effect suggests that either: 1) the marked increase in alloreactive T cell expansion observed in PDL1-deficient mice (2) requires a higher number of Tregs to control the alloimmune response; and/or 2) that lack of local expression of PDL1 in the placenta also contributes to the reduced fetal survival rate in PDL1-deficient mice independent of Tregs. The number of Tregs we transferred is comparable to that reported in literature (9, 35) and hence the expression of PDL1 in the decidua could more likely be the explanation for the results obtained by us.

Other studies have implicated a role for PDL1 in the induction of Tregs as well (36, 37). For example, Selenko-Gebauer et al. have shown that blocking of PDL1 on dendritic cells (DC) results in an increase in T cell activation thereby suggesting that PDL1 on DC plays an important role in the induction and maintenance of T cell anergy (36). However whether this is a direct effect involving DC to the Ag-specific T cell interaction or an indirect one via activation of Tregs through PDL1 requires further investigation. In another study, in a cardiac transplant model PD-1-PDL1 interaction was suggested to be essential for induction of Tregs as blockade of PD-1 and PDL1 resulted in an increase in allograft rejection in an adoptive transfer model. The cells transferred were whole splenocytes and not purified Tregs (37), this being a limitation of this study thereby implicating the involvement of Tregs in this system without confirming it.

Our results with adoptive transfer of Tregs clearly indicate that the in vivo function of Tregs from PDL1-deficient mice could still be impaired even though the number, phenotypic markers and trafficking were similar to those of Tregs from WT mice. Tregs from PDL1-deficient mice were also defective in suppressing the proliferation of CD4+CD25– effector T cells in a MLR utilizing allogeneic stimulators. In line with the proliferation assay results, Tregs from PDL1-deficient mice were also impaired in their ability to suppress IFN-{gamma} production as compared with Tregs from WT mice.

Naive murine CD4+ Th cells can differentiate to become either Th1/Th2, Th17 or Tregs depending on the cytokine microenvironment (38). IL-17 has been shown to be important in the development and progression of autoimmune as well as some inflammatory diseases (32, 33, 39, 40). A role for IL-17 in renal allograft rejection has also been shown (41). More recently a reciprocal relation between Th17 and Tregs has been suggested (34). We detected higher expression of IFN-{gamma} (Th1), IL-17 (Th17) and decreased expression of IL-4 and IL-5 (Th2) in placentae of PDL1-deficient mice, as compared with WT controls. Expression of Foxp3 (Treg) was similar between placentae of PDL1–/– and WT females. The number of Th17 cells is increased in the placentae of PDL1-deficient mice while the number of Tregs was similar in PDL1-deficient and WT mice. These data suggest that the altered balance between Th1 and Th17 effector cells on one hand and regulatory T cells on the other caused by the expansion of alloreactive effector T cells expressing IFN-{gamma} and IL-17 locally in the placenta and diminished suppressive activity of Tregs in PDL1-deficient mice is responsible for the decreased fetal survival rates. These conclusions are supported by recent reports that PDL1 may contribute to tolerance by limiting the expansion of alloreactive T cells (26, 42) possibly by cell cycle arrest (43), increasing apoptosis of alloreactive T cells (26, 44), or by active regulation of the alloimmune response by a subpopulation of CD4+CD25+ T cells (26, 45). It has been recently shown that skewing of T cells toward Th17 cells can be mediated by the presence of Tregs (46) in a proinflammatory cytokine microenvironment either by secreting immunosuppressive cytokine TGFbeta1 and/or by inhibition of Th1 and Th2 cells (46). In the present study, Tregs from WT mice inhibited IFN-{gamma} to a greater extent than Tregs from PDL1-deficient mice in the culture supernatants of suppression assay (Fig. 5B). Interestingly, there was no difference in IL-17 levels between Tregs from WT vs Tregs from PDL1–/– mice in the same suppression assay (WT 171 ± 25 pg/ml vs PDL1 deficient 142 ± 30 pg/ml at 1:1 Teffector/Treg ratio; ns). In addition, alloreactive Th17 cells secreting IL-17 were present locally at the site of fetal rejection at significantly higher levels in placentae of PDL1-deficient mice vs WT mice despite similar expression of Treg marker Foxp3 in the placentae of these mice. Collectively, our data suggests that 1) there is probably not a direct effect of Tregs on the differentiation of IL-17 cells and 2) that detrimental effect of IL-17 is more likely occurring locally in the placental tissue. A local effect of IL-17 in graft tissue has been reported recently. It was shown that IL-17 mRNA as well as protein are up-regulated locally in the broncheo alveolar lavage of lung transplant patients with acute allograft rejection (47). In another in vitro model system using cultured human renal epithelial cells, IL-17 was demonstrated to produce inflammatory mediators with the potential to stimulate early alloimmune responses (41). It has been recently proposed that skewing of responses toward Th17 or Th1 and away from Treg may be responsible for the development and/or progression of acute transplant rejection in humans (48). Blocking critical cytokines in vivo may result in a shift from a Th17 toward a regulatory phenotype could lead to prolongation of transplant function (48).

In contrast to other regulatory mechanisms such as IDO (49, 50), the "quantitative" rather than "all-or-none" effect of PDL1 on protection of the fetus (2) is relevant to the specific mechanism of action in that PDL1 probably functions at two levels; first, by affecting the alloreactive effector T (Th1/Th17) cell pool, and second, by affecting Tregs function. Together, these mechanisms regulate the alloimmune response locally protecting the fetus from rejection. Blockade or deficiency of PDL1 results in quantitative expansion of alloreactive T cells and reduction in function of Tregs, thus resulting in decreased fetal survival rather than an "all-or-none" phenotype or effect.

In all, our results clearly point to a novel link between PDL1 and Tregs in mediating fetomaternal tolerance. Further studies are required to gain understanding of the exact mechanisms of the interplay between PDL1, effector T cells (Th1 and Th17 cells), and regulatory T cells in vivo. Our studies have important implications for understanding physiologic mechanisms that promote fetomaternal and transplantation tolerance.


   Acknowledgment
 
We thank Dr. Jeffrey Bluestone for critical review and advice on the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R01 AI051559 and P01 AI056299 (to M.H.S.), American Society of Transplantation Basic Scientist Faculty Grant (to I.G.), and National Kidney Foundation Research Fellowship Grant (to A.H.). Back

2 Address correspondence and reprint requests to Dr. Indira Guleria, Transplantation Research Center, 300 Longwood Avenue, Boston, MA 02115. E-mail address: indira.guleria{at}tch.harvard.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; GITR, glucocorticoid-induced TNFR-related protein; dpc, days post coitum; WT, wild type. Back

Received for publication April 3, 2007. Accepted for publication August 20, 2007.


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 Introduction
 Materials and Methods
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 Disclosures
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