Human Placental Trophoblasts Regulatory T Cells by + Activation

Activation of CD8+ Regulatory T Cells by Human Placental Trophoblasts Ling Shao, Adam R. Jacobs, Valrie V. Johnson and Lloyd Mayer This information is current as of September 23, 2021. J Immunol 2005; 174:7539-7547; ; doi: 10.4049/jimmunol.174.12.7539 http://www.jimmunol.org/content/174/12/7539 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Activation of CD8؉ Regulatory T Cells by Human Placental Trophoblasts1

Ling Shao,2* Adam R. Jacobs,† Valrie V. Johnson,† and Lloyd Mayer*

The immunological basis by which a mother tolerates her semi-allogeneic fetus remains poorly understood. Several mechanisms are likely to contribute to this phenomenon including active immune regulation by regulatory T cells. In this article, we report that human placental trophoblasts activate a clonal population of CD8؉ T cells with regulatory function. These cells are not MHC class I restricted, but require costimulation through a member of the carcinoembryonic Ag family present on early gestation trophoblasts. These regulatory T cells express the mucosal markers CD101 and CD103 and display selective usage of the TCR gene V␤9. ␤CD8؉ T cells isolated from the peripheral blood of pregnant mothers (16–28 wk) also demonstrate expansions in the same V family (V␤9), signaling a possible role for these cells in preventing fetal rejection in vivo. We have previously characterized a ؉ subset of CD8 regulatory T cells activated by the combination of the nonclassical class I molecule CD1d and a costimulatory Downloaded from molecule of the carcinoembryonic Ag family present on the intestinal epithelium. These data support the concept that distinct regulatory T cell populations exist at different sites and may be regulated locally by unique restriction elements, costimulatory signals, and Ags. The Journal of Immunology, 2005, 174: 7539–7547.

llogeneic tissue grafts are typically rejected rapidly and commensal ﬂora at the interface between the external environment

require exogenous immunosuppressive medications to and the mucosal immune system. Interestingly, patients with in- http://www.jimmunol.org/ A be tolerated. In contrast, the human fetus, which resem- flammatory bowel disease are deficient in the expression of the bles a semi-allogeneic graft, is accepted by the maternal immune CEA subfamily costimulatory molecule, which correlates with an system in the majority of cases without intervention. Potential inability of these patients to be tolerized to orally administered mechanisms underlying this phenomena are likely to be Ags (12). complex (1). Regulatory T cells (reviewed in Ref. 13) comprise a heteroge- Anatomically, placental trophoblasts occupy a unique position neous class of cells that suppress immune responses via cognate at the interface between mother and child. These epithelial cells interactions (CD4ϩCD25ϩ), soluble factors such as TGF-␤ or possess several intriguing features that suggest a role for these IL-10 (Th3 and Tr1 cells, respectively), or some combination of cells in preventing fetal rejection. Trophoblasts express little or no the two. These cells appear to play a role in controlling inflam- by guest on September 23, 2021 classical MHC class I (HLA-A, -B, and -C) (2–5), but do express matory immune responses in a number of settings including auto- a number of nonclassical MHC class I molecules. Examples in- immune and chronic inflammatory diseases. Recently, a role for clude HLA-E (6) and HLA-G (7, 8), which can modulate the in- regulatory T cells has been demonstrated in maintaining fetal tol- nate immune response and prevent NK (6) and NK T cell (9)- erance as well. CD4ϩCD25ϩ regulatory T cells play a critical role mediated cytotoxicity. Additionally, trophoblasts have been in mice during pregnancy after allogeneic matings (14). In hu- recently shown to express the nonclassical MHC class I molecule, mans, CD4ϩCD25ϩ cells expressing the regulatory transcription CD1d, on the surface of first-trimester human trophoblasts (10). factor FoxP3 (15), OX-40, and glucocorticoid-induced TNFR fam- We have previously described a subset of CD8ϩ regulatory T ily-related gene are expanded in the peripheral blood as well as at cells in humans that is restricted by CD1d and a costimulatory the maternal-fetal interface (16), implying an important role for molecule of the carcinoembryonic Ag (CEA)3 family expressed on these cells in fetal tolerance. intestinal epithelial cells (IEC). These cells use a restricted TCR In this report, we show that trophoblasts can activate a subset of repertoire (V␤5.1/V␣18), are CD28Ϫ, and can suppress immune CD8ϩ T cells that are independent of classical MHC class I but are responses in vitro (11). In the intestine, these cells may play an dependent upon a CEA subfamily member. These T cells have a important role in controlling inflammatory responses to normal restricted TCR repertoire, coexpress the mucosal markers CD101 and CD103, and demonstrate in vitro regulatory properties. Fi- nally, we provide evidence that these CD8ϩ T cell subsets are *Immunobiology Center and †Department of Obstetrics and Gynecology, Mount Si- expanded in the peripheral blood of pregnant mothers, suggesting nai School of Medicine, New York, NY 10029 a potential role for these cells in vivo. Received for publication October 18, 2004. Accepted for publication April 8, 2005. 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. Materials and Methods Trophoblast isolation 1 This work was supported by Public Health Service Grants AI23504, AI24671, AI44236, and AI07605 (to L.M.). Placentae from 7- to 18-wk elective abortions performed at the Mount 2 Address correspondence and reprint requests to Dr. Ling Shao, Mount Sinai School Sinai Medical Center or full-term (38–40 wk) pregnancies were collected of Medicine, 1 Gustave L. Levy Place, Department of Immunobiology, Box 1089, in accordance with the Mount Sinai Institutional Review Board guidelines. New York, NY 10029. E-mail address: [email protected] Trophoblasts were isolated as described previously (17, 18), with modifi- 3 Abbreviations used in this paper: CEA, carcinoembryonic Ag; IEC, intestinal epi- cations. Briefly, placental specimens were washed twice in PBS, and mem- thelial cell; FasL, Fas ligand; BGP, biliary glycoprotein; NCA, nonspecific cross- branes were manually removed. The placenta was then digested in PBS reacting Ag. containing 1.5 mg/ml Dispase (Roche Applied Science) and 300 ␮g/ml

DNase I (Calbiochem) for 15 min at 37°C with gentle agitation. Superna- perature. After extensive washing, blots were developed with SuperSignal tants were harvested and layered on FCS (Omega Scientific) and centri- (Pierce). fuged for 15 min at 1200 ϫ g. Pellets were resuspended in PBS and layered on a discontinuous Percoll density gradient (25/45/60%) and centrifuged Immunohistochemistry for 20 min at 1200 ϫ g. Trophoblasts were isolated from the 25/45% Percoll interface and washed twice in AIM-V serum-free medium (Invitro- Placentae were washed twice in sterile PBS. Specimens were then snap gen Life Technologies). In some cases, cells were preincubated with Abs frozen in OCT compound (Miles) in liquid nitrogen. Eight-micrometer against CD1d (mAb D5, MsIgG2b; a kind gift from Drs. R. Blumberg frozen sections were cut and immediately fixed in 4% PBS-buffered for- (Brigham and Women’s Hospital, Boston, MA) and S. Balk (Beth Israel malin overnight. Sections were rinsed briefly in PBS, and the tissue was Deaconess Medical Center, Boston, MA)), gp180 (mAb B9, MsIgG1; see outlined by an ImmEdge hydrophobic pen (Vector Laboratories). Nonspe- Ref. 19), pan-MHC-I (W6/32, MsIgG2a), or isotype controls (BD Pharm- cific-Ab activity was blocked with normal horse serum for 30 min at room ingen) for 60 min at 4°C. Excess Ab was removed by extensive washing in temperature. Slides were incubated with 10 ␮g of either the B9 mAb or PBS before coculture. isotype control (IgG1; BD Pharmingen) for2hatroom temperature. Bi- otinylated secondary Abs and streptavidin-HRP from the Vectastain ABC T cell isolation and T trophoblast coculture (avidin-biotin complex) kit was added (Vector Laboratories) for 45 min at Blood from pregnant and nonpregnant donors was obtained with informed room temperature. Slides were developed with diaminobenzidine (Dako- consent. Three to 5 ml of heparinized whole blood was subjected to Ro- Cytomation), counterstained with hematoxylin (Fisher Scientific), and setteSep (StemCell Technologies) separation to isolate T cells (purity mounted with Vectashield (Vector Laboratories). Ͼ95%) according to the manufacturer’s instructions. A total of 2 ϫ 106 T cells was labeled with CFSE (Molecular Probes) (20) and cultured with Suppression assays 1 ϫ 106 freshly isolated trophoblasts (described above) in AIM-V serum- free medium (Invitrogen Life Technologies) for up to 7 days at 37°C, with Trophoblast-activated T cells were sorted, as described in Flow cytometry ϩ ϩ low and sorting, into proliferating and nonproliferating (based on CFSE stain- Downloaded from 5% CO2. CD4 and CD8 proliferating (CFSE ) and nonproliferating ϩ ϩ (CFSEhigh) T cells could be determined by flow cytometry. The data are ing) CD8 and CD4 fractions. These cells were added to fresh PBMC ϫ 5 presented as the percentage of CD8ϩ T cells proliferating in the cultures (1 10 cells/well), isolated from healthy volunteers stimulated with 1% (CD8ϩCFSElow/total CD8ϩ T cells). PWM (Sigma-Aldrich). Cell ratios (E:T ratio) varied between 1:2 and 1:8. All conditions were performed in triplicate. After 7 days, supernatants were Flow cytometry and sorting collected, and secreted Ig was determined by ELISA. In some experiments, sorted cells were separated from PWM-stimulated PBMC by a Transwell Cells were washed twice in PBS/1% BSA (Sigma-Aldrich). For phenotypic filter (Falcon). http://www.jimmunol.org/ studies, CFSE-labeled T cells were stained with mAbs against CD3(- In other experiments, these fractions were irradiated 2500 rad (cesium PerCP), CD8(-allophycocyanin), and one of the following Abs: IgG con- source) and cocultured in a nonrelated allogeneic MLR culture at a 1:1 and 1:2 trol(-PE), CD101(-PE), CD103(-PE), CD28(-PE), CTLA-4(-PE), or (effector CD8ϩ or CD4ϩ to responder T cell) ratio. [3H]Thymidine (MP Bio- CD178(-PE) (all from BD Pharmingen). For TCR repertoire studies, cells medical) incorporation was measured after 5 days of culture. Inhibition, ex- were stained with Abs against various TCR-V␤ family members conju- pressed as a percentage of the control MLR, was calculated by the following gated to PE (Immunotech), CD3(-PerCP), and CD8(-allophycocyanin) (BD ϭ ϩ ϫ formula: % of control (MLR effector T cell)cpm/MLRcpm 100. Pharmingen). Cells were analyzed on a FACSCalibur (BD Immunocytom- etry Systems) or sorted on a FACSVantage machine at the Mount Sinai ELISA Flow Cytometry Core. ␥ mRNA isolation and CDR3 display IFN- and IL-10-cytokine ELISA was performed using the OptEIA kit (BD Pharmingen) according to the manufacturer’s instructions. All sam- by guest on September 23, 2021 Proliferating and nonproliferating CD8ϩ T cells, as determined by CFSE ples were run in triplicate. For Ig detection, 96-well plates were coated with staining, were sorted and lysed in TRIzol (Invitrogen Life Technologies). goat anti-human polyspecific Ab (BioSource International) at a concentra- Total RNA was isolated according to the manufacturer’s instructions (In- tion of 6.25 ␮g/ml in 15 mM sodium carbonate/35 mM sodium bicarbonate vitrogen Life Technologies). RT-PCR was performed using primers spe- buffer overnight at 4°C. Nonspecific binding was blocked with PBS/0.5% cific for human V␤ genes described elsewhere (21) and a fluorescently Tween 20/1% BSA for1hatroom temperature. Samples were diluted 1/10 labeled constant TCR ␤-chain primer. Products were run on an ABI 377 and 1/40 in PBS/0.5% Tween 20 and incubated at room temperature for sequencer and analyzed with Genotyper software (Applied Biosystems). 2 h. After incubation, goat anti-human ␬ and goat anti-human ␭ Abs con- jugated to alkaline phosphatase (BioSource International) were added at a Real-time PCR concentration of 0.7 ␮g/ml each. Plates were developed with p-nitrophenyl phosphate (Sigma-Aldrich) in substrate buffer (9.7% diethanolamine, 0.5 Isolated trophoblasts were lysed in TRIzol reagent (Invitrogen Life Tech- ␮ M MgCl-6H2O (pH 9.8)). Plates were read on a uQuant (Bio-Tek Instru- nologies), and total RNA was collected according to the manufacturer’s ments) microplate reader (650 nm for cytokines and 405 nm for Ig) and instructions (Invitrogen Life Technologies). One microgram of total RNA analyzed with KC4 software (Bio-Tek Instruments). was reverse transcribed into cDNA. Real-time PCR was performed on the For TGF-␤ ELISA (Quantikine; R&D Systems), 200 ␮l of culture su- Roche LightCycler using the FastStart SYBR Green kit (Roche) and the pernatants or culture supernatants treated with 1 N HCl to activate latent following conditions: CD1d primers, 5Ј-GCAACGACTCGGACAC-3Ј ␤ Ј Ј TGF- were added to precoated 96-well plates and incubated for3hat and 5 -CTGTTTCCCTCGTCCA-3 . PCR conditions were as follows: room temperature. Plates were washed three times, and the detection Ab touchdown PCR was performed according to the following protocol: 1) was added and incubated for an additional 1.5 h. Plates were washed three 95°C for 10 min (enzyme activation); 2) 95°C for 5 s (denaturation); 3) more times, and 200 ␮l of substrate solution was added. After 20 min, 50 63°C for 5 s for the first 10 cycles, a 0.5°C temperature decrease over the next ␮l of stop solution was added, and the absorbance of the samples at a 16 cycles to a final annealing temperature of 55°C for 26 additional cycles (a wavelength of 450 nm was determined. total of 55 cycles); and 4) 72°C for 14 s (extension). Samples were run in duplicate. Quantitation was performed by second derivative maximum analysis. PCR product size was confirmed by gel electrophoresis. Cytotoxicity/redirected lysis assay Western blot Cells from trophoblast cocultures or cells from a conventional MLR were pretreated with OKT3 (1 ␮g/ml) or IgG2a isotype control Ab (1 ␮g/ml) for Whole placentae or isolated trophoblasts (see above) were lysed in 1% 1 h at room temperature and used as effector cells. FcR-expressing P815 digitonin lysis buffer containing 300 mM NaCl, 50 mM HEPES, 2.5 mM cells labeled with 51Cr were used as target cells in a redirected lysis assay. EDTA, and protease inhibitor mixture (Complete Mini; Roche) on ice for Triplicate wells of effector cells were incubated with 5 ϫ 103 labeled P815 4–6 h. Lysates were centrifuged at 12,000 ϫ g for 10 min. Supernatants targets in different ratios in V-bottom plates (Nunc) for4hat37°C, with ϫ ␮ were mixed with 4 SDS-loading buffer (100 mM Tris, 4% SDS, 0.2% 5% CO2. After incubation, 100 l of culture supernatant was harvested, bromphenol blue, and 20% glycerol) and resolved on 8% SDS-polyacryl- and the amount of released 51Cr in each triplicate sample was assayed by amide gels. After transfer to nitrocellulose membranes, these were blocked gamma counter. Spontaneous release of 51Cr was measured by incubating with 5% dry milk/PBS overnight at 4°C. Primary Abs were incubated for P815 cells alone, whereas maximal release was determined by solubilizing 2 h at room temperature, and then washed with PBS containing 0.1% labeled P815 cells in 1% Triton X-100. Specific lysis was calculated by the Tween 20 (Fisher Scientific). Secondary Ab (goat anti-mouse-HRP conju- following formula: Percent specific lysis ϭ (51Cr released Ϫ spontaneous gate; Invitrogen Life Technologies) was incubated for2hatroom tem- 51Cr)/(maximal 51Cr release Ϫ spontaneous 51Cr) ϫ 100. The Journal of Immunology 7541

FIGURE 1. Trophoblasts express CD1d and a CEA subfamily member. A, Real-time PCR shows the presence of CD1d mRNA as early as 7 wk of gestation. A decrease in the cycle number at which the PCR reaches the log-linear phase signifies an increased mRNA abundance at 10 and 12 wk. Below, Agarose gel electrophoresis confirms the appropriate product size. Positive control is a plasmid containing CD1d. B, Immunohistochemistry showing specific B9 mAb reactivity in preterm trophoblasts (12 wk) and not interstitial tissue. The left panel

represents the isotype control. C, Im- Downloaded from munohistochemistry showing no B9 mAb reactivity in term placenta. Con- trol staining shows speciﬁc reactivity using an Ab against placental alkaline phosphatase (mAb 8B6), but not an isotype control. D, Western blot dem-

onstrating B9 mAb immunoreactivity http://www.jimmunol.org/ in 7-, 10-, 11-, and 12-wk trophoblasts and an IEC cell line control (lanes 1–5, respectively). by guest on September 23, 2021 Results testinal epithelium, gp180, which is 180 kDa (Fig. 1D, lane 5). Trophoblasts express CD1d and a CEA subfamily member Because term placentae do not express CD1d (10) and did not stain with mAb B9 (Fig. 1C), these molecules appear to be induced Trophoblasts express a number of nonclassical class I molecules, early in gestation and may be temporally regulated. including HLA-E (6), HLA-G (7, 8), MICA/B, and the neonatal FcR (22). We have previously characterized a subset of CD8ϩ regulatory T cells in the intestine that require a complex of the nonclassical class I molecule, CD1d, and a costimulatory molecule of the CEA subfamily for activation (11, 23). In agreement with previous data showing the expression of CD1d on human trophoblasts (10), quantitative PCR of mRNA isolated from human trophoblasts showed increasing CD1d transcripts present from ges- tational age wk 7–12 (Fig. 1A). To determine whether the costimulatory molecule of the CEA family was also present, placentae were subjected to immunohis- tochemical analysis using the B9 mAb. The B9 mAb was identified originally by its ability to block the generation of CD8ϩ regulatory T cells in T cell:IEC cocultures (11). Expression of the epitope recognized by mAb B9 was restricted to intestinal epithelium (19, 23) (not lung or skin) and cortical thymic epithelium. On normal IEC, it recognizes a dominant 180-kDa band by Western blot. Further characterization has shown that the mAb is specific for a shared epitope in the N-domain of number of CEA subfamily members (e.g., CEACAM1, CEACAM3, and CEACAM6). As seen in Fig. 1B, mAb B9 stained trophoblasts from 8- to 12-wk FIGURE 2. Trophoblasts preferentially expand CD8ϩ T cells. A, CFSE- placentae (12 wk shown) but not interstitial tissues. In contrast, no ϩ B9 staining was seen in term placenta (Fig. 1C). Western blot labeled CD3 T cells cocultured with trophoblasts show multiple divisions (decreased CFSE intensity, x-axis) after 7 days. CD8 staining intensity is analysis of resolved placental lysate revealed a single band with an shown on the y-axis. Both CD8ϩ and CD8Ϫ cells proliferate. B, Tropho- apparent molecular mass between 80 and 100 kDa (Fig. 1D). In- blasts (n ϭ 40) preferentially and significantly expand CD3ϩCD8ϩ T cells terestingly, this is in contrast to the size of the predominant mo- compared with the percentage of CD3ϩCD8ϩ T cells that proliferate in a .p Ͻ 0.01 ,ءء .(lecular species of mAb B9-reactive material expressed by the in- conventional MLR (n ϭ 11 7542 TROPHOBLASTS ACTIVATE CD8ϩ REGULATORY T CELLS

FIGURE 4. Trophoblast-activated CD8ϩ T cells demonstrate no cytotoxicity in the redirected lysis assay. Unsorted T cells activated in a MLR or unsorted T cells from trophoblasts cocultures were preincubated with OKT3 mAb or an IgG2a isotype control. Cells were then incubated with 51Cr-labeled P815 target cells for4hattheindicated E:T ratios in the redirected lysis assay. Culture supernatants were collected and analyzed by

gamma counting. Legend: E:T ratio, 25:1 (Ⅺ); 12.5:1 (f); 6.25:1 (z); Downloaded from 3.125:1 (o); 1.56:1 ( ); 0.78:1 ( ); 0.39:1 ( ); 0.19:1 ( ). This ﬁgure is representative of three experiments with similar results.

Trophoblasts preferentially expand CD8ϩ T cells in vitro

The presence of both CD1d and a form of the costimulatory mol- http://www.jimmunol.org/ ecule of the CEA family recognized by mAb B9, suggested the possibility that CD8ϩ regulatory T cells might be activated at the maternal/fetal interface (analogous to the intestine). Autologous (from pregnant patients) or allogeneic (from normal volunteers) peripheral blood T cells were labeled with CFSE and cocultured with freshly isolated trophoblasts from 9- to 16-wk placentae. No significant differences in T cell proliferation were observed between cultures using autologous vs allogeneic blood (autologous (n ϭ 6), 24.3 Ϯ 16.6%; allogeneic (n ϭ 34), 35.7 Ϯ 24.9% ( p ϭ by guest on September 23, 2021 0.09)). As seen in Fig. 2A, after 7–9 days of coculture, both proliferating CD4ϩ T cells and CD8ϩ T cells can be identified by a decrease in CFSE fluorescence intensity. Similar to T cells cultured with freshly isolated IEC, trophoblasts preferentially activate CD8ϩ T cells (Fig. 2B) when compared with a conventional MLR. Interestingly, CD4ϩ T cell expansion appears to occur in the absence of class II expression on trophoblasts.

Regulatory activity of trophoblast-activated CD8ϩ T cells To test the regulatory capacity of these CD8ϩ cells, proliferating and nonproliferating CD8ϩ T cells were sorted and cocultured with PBMC stimulated with 1% PWM. Suppression of PWM-induced Ig secretion is a well-established assay for Ag-nonspeciﬁc regulatory T cell activity (24) and is particularly useful in detecting

stimulated PBMCs. This inhibition is contact dependent because the effect is completely abrogated by separating the effector and target cells by a FIGURE 3. Regulatory activity of trophoblast-activated CD8ϩ T cells. Transwell filter. Nonproliferating CD8ϩ T cells also can inhibit Ig secre- ,p Ͻ 0.05). C ,ء ;p Ͻ 0.01 ,ءء) (A, CD3ϩCD8ϩ proliferating T cells in trophoblast cocultures demonstrate tion (35% inhibition at E:T ratio of 1:8 in vitro regulatory activity. CD3ϩCD8ϩ proliferating T cells, but not non- Trophoblast-activated T cells do not act as third-party suppressor cells in proliferating CD8ϩ T cells, efficiently (Ͼ80% suppression at 1:2 and 1:4 an allogeneic MLR. Trophoblast-activated T cells were sorted into prolif- E:T ratios) suppress Ig secretion by PWM-stimulated PBMCs. In contrast, erating and nonproliferating CD4ϩ and CD8ϩ T cells as described in Ma- CD4ϩ T cells do not suppress Ig secretion at similar E:T ratios. This figure terials and Methods. A total of 2 ϫ 105 irradiated trophoblast-activated T p Ͻ 0.01). B, cells was added in the indicated ratios to unrelated allogeneic MLRs in ,ءء) is representative of five experiments with similar results Sorted CD3ϩCD8ϩ T cells were added to PBMCs stimulated with 1% 96-well plates. Plates were incubated for 5 days, and 1 ␮Ci of [3H]thymi- PWM at 1:4 and 1:8 E:T ratios. Effector cells were either added directly to dine was added during the last 18 h of culture. [3H]Thymidine incorpora- target PBMCs or separated from target cells by a Transwell filter. Super- tion was measured by scintillation counting. Inhibition, expressed as a natants were collected after 7 days and analyzed by ELISA for total Ig. percentage of the control MLR, was calculated by the following formula: ϩ ϩ ϭ ϩ ϫ ϭ CD3 CD8 proliferating T cells in trophoblast cocultures efficiently % of control (effector MLR)cpm/(MLR alone)cpm 100%. p no (Ͼ75% at 1:4 and 1:8 E:T ratios) blocked the secretion of Ig by PWM- significant differences. The Journal of Immunology 7543 the regulation of T cell-dependent B cell differentiation (25). Pro- a pleotropic cytokine with well-known immunosuppressive prop- liferating CD8ϩ cells efficiently suppressed the secretion of Ig in erties. The regulatory T cell subsets Tr1 and, to a lesser extent Th3 these cultures compared with nonproliferating CD8ϩ T cells and cells, use IL-10 to help mediate suppression. As shown in Fig. 5, proliferating and nonproliferating CD4ϩ T cells cells at E:T ratios supernatants from cocultures of trophoblasts with peripheral blood as low as 1:8 (Fig. 3, A and B). Separating effector CD8ϩ T cells T cells demonstrate a significant increase in IL-10 compared with from target PBMC, via Transwell inserts, completely abrogated T cells or trophoblasts alone (Fig. 5A). This increase was most suppression, suggesting that the mechanism of suppression is con- significant at 48 h but was evident at 24 h (data not shown). In tact dependent (Fig. 3B). In contrast to CD8ϩ regulatory T cells contrast, neither IFN-␥ nor TGF-␤ were detectable in the same that our laboratory has characterized previously in the intestine, supernatants (Fig. 5, B and C). ϩ trophoblast-activated CD8 T cells were unable to act as third- ϩ party suppressor cells in an unrelated allogeneic MLR (Fig. 3C). Phenotype of trophoblast-activated CD8 regulatory T cells ϩ Proliferating CD4 T cells exhibited no suppressive activity in any We next characterized surface phenotype of trophoblast-activated assay (Fig. 3, A and C). This may suggest a potential regulatory CD8ϩ T cells. CD8ϩ T cells activated by trophoblasts use the ␣␤ ϩ niche for trophoblast-activated CD8 regulatory T cells in regu- TCR and show an up-regulation of the mucosal markers CD101 lating Ab responses during pregnancy. and CD103 (Fig. 6). In addition, these cells do not express detect- To define the mechanism of suppression, we initiated a series of able CTLA-4, the NK marker CD56, or Fas ligand (FasL) (Fig. 6). additional studies. Given the dependence upon cell contact for tro- Cytotoxic T cells may also initiate apoptotic programs via Fas- ϩ phoblast-activated CD8 T cell-mediated regulatory activity, one FasL. These data further support the notion that trophoblast-acti- ϩ ϩ possibility is that the proliferating CD8 T cells mediate their vated CD8 regulatory T cells do not suppress immune responses Downloaded from suppression via cytotoxicity. To test this possibility directly, we by cytotoxicity. Interestingly, in contrast to the CD8ϩ regulatory T radiolabeled FcR-expressing P815 target cells with 51Cr and used cells our laboratory characterized in the intestine, these cells ex- these cells as targets for our trophoblast-activated T cells in a re- press high levels of CD28. Differences between trophoblast- and directed lysis assay. Total cytotoxic activity was measured by IEC-activated regulatory CD8ϩ T cells in terms of regulatory func- cross-linking CD3 using the OKT3 mAb or an IgG2a isotype con- tion (inability of trophoblast-activated regulatory CD8ϩ T cells to trol. As seen in Fig. 4, T cells activated in a conventional MLR suppress the MLR) and CD28 expression suggested that these two http://www.jimmunol.org/ exhibited dose-dependent lytic activity. In contrast, trophoblast- regulatory populations were, in fact, distinct. activated T cells did not show any appreciable cytolytic activity at ϩ the same E:T ratios. This suggests that the mechanism by which TCR bias in trophoblast-activated CD8 T cells trophoblast-activated T cells suppress inflammatory immune re- To investigate this further, we examined the activation require- sponses is not likely to be mediated through cytotoxic effects. ments of trophoblast-activated CD8ϩ regulatory T cells. The addition of mAb B9 to trophoblasts before coculture, compared with Cytokine profile of trophoblast-activated CD8ϩ regulatory T an isotype control mAb (msIgG1), consistently inhibited between cells ϩ 10 and 40% of the expansion of CD8 T cells (Fig. 7A). This is Other contact-dependent regulatory T cells have been shown to consistent with what was seen in T:IEC cocultures. Interestingly, by guest on September 23, 2021 enhance their activity via cytokine production. For example, nat- in contrast to IEC-activated regulatory T cells, the addition of an ϩ ϩ urally occurring CD4 CD25 regulatory T cells use a combina- anti-CD1d mAb (mAb D5) did not block the expansion of CD8ϩ tion of IL-10 and TGF-␤ to suppress immune responses. IL-10 is T cells (Fig. 7A) in trophoblast/T cell cocultures when compared with an isotype control. mAbs to classical class I MHC (mAb W6/32) did not inhibit CD8ϩ T cell proliferation in these cultures as well. Thus, trophoblast-activated T cells seemed to differ from IEC-activated regulatory T cells in their requirements for CD1d recognition, but remained dependent on costimulation through a member of the CEA family recognized by the B9 mAb, and are class I independent.

FIGURE 6. Phenotype of CD3ϩCD8ϩ T cells cocultured with trophoblasts. CFSE-labeled T cells were cocultured with trophoblasts for 7 days FIGURE 5. Cytokine secretion of trophoblast-activated CD8ϩ T cells. and analyzed by four-color flow cytometry. Top, Isotype control staining Trophoblast/T cell cocultures (n ϭ 5) secrete IL-10 but not IFN-␥ or (left) and ␣␤ TCR staining (right) gated on total CD3ϩCD8ϩ T cells. TGF-␤. T cells were cocultured with trophoblasts for 48 h. Supernatant Bottom, CD3ϩCD8ϩ T cells were divided into proliferating and nonpro- was collected and analyzed by ELISA. Trophoblast/T cell cocultures had liferating T cells based on CFSE staining. The left column shows histo- significantly increased levels of IL-10 (A), but had nondetectable levels of grams for nonproliferating CD3ϩCD8ϩCFSEhigh T cells. The right column -p Ͻ 0.05). C, Before activation with 1 N HCl, TGF-␤ was shows histograms for proliferating CD3ϩCD8ϩ CFSElow T cells. This fig ,ء)(IFN-␥ (B undetectable in culture supernatants. ure is representative of four experiments with similar results. 7544 TROPHOBLASTS ACTIVATE CD8ϩ REGULATORY T CELLS Downloaded from

FIGURE 8. B9 mAb recognizes NCA in placental lysates. A, Tropho- blast lysates were subjected to Western blot analysis using the anti-CEA mAb (Col-1) and the anti-BGP mAb (5F4). No immunoreactive bands FIGURE 7. Trophoblasts activate a restricted subset of regulatory were detected in trophoblast lysates. The Col-1 mAb detects CEA in the http://www.jimmunol.org/ ϩ ϩ ϩ CD8 T cells. A, CD3 CD8 T cell expansion in trophoblast cocultures IEC cell line, T84, and the 5F4 mAb detects BGP in both resting and can be inhibited consistently by the B9 mAb specific for a CEA subfamily anti-CD3-stimulated T cells. Western blot analysis using the F106-88 mAb member (n ϭ 4), but not by the D5 mAb specific for CD1d (n ϭ 3) or the reveals a band of ϳ100 kDa similar to that seen with the B9 mAb (arrow- W6/32 mAb (n ϭ 3), a pan-MHC class I Ab. Percent inhibition indicates heads). B, Trophoblast lysates were immunoprecipitated with the B9 mAb, ϩ ϩ low the percentage of CD3 CD8 CFSE T cells with B9, D5, or W6/32 and then resolved by SDS-PAGE. Western blotting with the B9 mAb and ϩ ϩ low mAb divided by the percentage of CD3 CD8 CFSE T cells induced to the NCA mAb reveals bands of the same molecular mass at ϳ80–100 kDa proliferate in cocultures with trophoblasts in the presence of the respective (arrows). isotype control Abs. TCR repertoire of trophoblast-activated CD8ϩ T cells. B, Proliferating and nonproliferating CD3ϩCD8ϩ T cells from trophoblast T cell cocultures or conventional MLRs were sorted, and their TCR rep- several experiments, we sequenced the CDR3 region of by guest on September 23, 2021 ϩ ϩ ertoire was examined by CDR3 display. CD3 CD8 proliferating T cells CD8ϩV␤9ϩ-expressing T cells but were unable to demonstrate a ␤ ␤ ␤ showed oligoclonal expansion of T cells using V 9, V 11, and V 12 but common motif (Fig. 7E), suggesting that the activation of these not other V␤s(n ϭ 15). One representative oligoclonal expansion is ϩ ϩ cells is, in part, Ag driven by distinct Ags. shown. C, Oligoclonal expansion of V␤9 CD3 CD8 T cells can be ϩ Thus, the difference in the CD8 T cell population activated by blocked by mAb B9 in trophoblast/T cell cocultures, but similar inhibition was not seen in the conventional MLR. D, Conversely, oligoclonal expan- trophoblasts vs IECs could be explained in part by the difference sion of V␤9 CD3ϩCD8ϩ proliferating T cells in the MLR can be blocked in CD1d dependence. Alternatively, there were also differences in by the pan-MHC class I mAb W6/32, but not in trophoblast/T cell cocul- the size of the protein in trophoblasts recognized by mAb B9 by tures (n ϭ 3). E, Sequencing of the CDR3 region of three V␤9 TCR using Western blot (Fig. 1D). We therefore sought to examine the nature CD8ϩ T cell clones from trophoblast/T cell cocultures revealed no com- of the molecule in the placenta recognized by the mAb B9 more mon CDR3 length or motif. closely. Lysates from early gestation placentae were resolved by SDS-PAGE and probed with Abs to CEA (CEACAM5), biliary glycoprotein (BGP) (CEACAM1), nonspecific cross-reacting Ag We next initiated studies assessing the restriction of the TCR (NCA) (CEACAM6), and mAb B9. As shown in Fig. 8A, only the repertoire in cells activated by trophoblasts. CD8ϩ regulatory T F106-88 mAb (specific for NCA), and not the Co1–1 mAb (spe- cells in the intestine show a restricted TCR repertoire usage, cific for CEA) or the 5F4 mAb (specific for BGP), identified a V␤5.1/V␣18. T cells activated by freshly isolated human tropho- protein with an apparent molecular mass of ϳ100 kDa similar to blasts also show a limited but distinct V␤ repertoire. CD8ϩ T cells the band revealed by the B9 mAb (arrowheads). To determine were sorted into populations of proliferating and nonproliferating whether this was the band initially identified by mAb B9 (Fig. 1D), cells based on CFSE intensity. RT-PCR using specific V␤ gene we performed immunoprecipitation studies using mAb B9. The primers (CDR3 display) demonstrated oligoclonal expansions of immunoprecipitates were resolved by SDS-PAGE and transferred V␤9, V␤11, and V␤12 populations in proliferating CD8ϩ T cells to nitrocellulose, and the membranes were probed with either mAb (Fig. 7B). In total, V␤9ϩCD8ϩ T cells were found to be clonally B9 or mAb F106-88. In both cases, a band of ϳ100 kDa was expanded in 6 of 15, V␤11 in 5 of 15, and V␤12in5of15 detected (Fig. 8B, arrows). Additional higher molecular mass cocultures. Interestingly, V␤9 CD8ϩ T cell clones were dependent bands were identified as well. Because CEA subfamily members upon the CEA subfamily member for expansion because oligo- can exhibit variable glycosylation patterns, these bands may rep- clonal expansions could be blocked by the B9 Ab but not by either resent posttranslational modifications of NCA (CEACAM6). an Ab against MHC class I (Fig. 7, C and D) or the D5 mAb (data Taken together, these data suggest that the differences in CD8ϩ not shown). In contrast, expansion of V␤9 CD8ϩ T cell clones in T cells activated by trophoblasts from those present in the intestine MLR cultures were not inhibited by mAb B9, but could be inhib- reflect differences in the CEA subfamily member (NCA in the case ited by the anti-MHC class I mAb W6/32 (Fig. 7, C and D). In of trophoblasts, and CEA in the case of IECs) and the requirement The Journal of Immunology 7545

for CD1d. The differences are also seen in distinct TCR gene us- 1.51 1.13

23 age, cellular phenotype, and function. T cells Ϯ ␤ Ϯ 2.61 3.19 ϩ A possible regulatory role for CD8ϩ T cells in vivo during CD8

ϩ pregnancy 0.91 1.97 0.77 2.08 22 V The observation that particular V␤-expressing T cells were con- Ϯ ␤ Ϯ 9 CD3 3.44.24 1.86 1.74 7.55 6.34 ␤ sistently expanded in in vitro cocultures provided a means to search for these cells in vivo. Peripheral blood CD8ϩ T cells from pregnant women in their 16th to 29th wk of gestation were isolated 0.71 3.17 0.84 2.86 20 V ␤ Ϯ ␤ Ϯ and analyzed by a panel of V -specific Abs. We defined expansion 2.18 3.4 1.95 6.19 5.61 of a specific V␤ family if the percentage of these cells was Ͼ3 SDs above the mean of the controls. Intriguingly, 3 of 12 patients man- ϩ ␤ ϩ 0.46 3.38 0.22 2.77 ifested significant expansions in CD8 V 9 T cells consistent 18 V Ϯ ␤ Ϯ 1.87 0.3 0.98 1.89 4.84 with our in vitro data (Table I). A similar analysis was undertaken in term placenta (Table I). Lymphocytes isolated from term placenta did not show a signifi- ϩ 0.89 0.45 0.90 0.57 cant difference in the percentage of CD8 V␤9 T cells as compared 17 V Ϯ ␤ Ϯ 6.04 0.78 2.08 2.11 1.83 6.62 8.09 with the percentages found in normal peripheral blood. As men-

tioned above, term placentae do not express the necessary costimu- Downloaded from latory molecule recognized by the B9 mAb (Fig. 1C), and thus ϩ ϩ 0.36 4.91 0.40 4.47 ␤ 16 V may not facilitate the expansion of V 9 CD8 T cells. Ϯ ␤ Ϯ 1.17 2.17 Discussion Increasing evidence implicates an immunological component to 1.01 0.78 1.11 0.90 14 V fetal tolerance. Several studies have demonstrated that the mater- http://www.jimmunol.org/ Ϯ ␤ Ϯ 6) were used as controls. Three of 12 women showed percentages of V

ϭ nal immune system recognizes the semi-allogeneic fetus, but that it remains in a quiescent state (26, 27). The potential mechanisms underlying this phenomenon are likely to be complex and may 0.66 6.10 1.10 6.67 13.1 V Ϯ Ϯ involve several complementary or overlapping pathways to favor ␤ 1.35 ND 0.76 reproductive success. A cellular barrier comprised of placental trophoblasts separates a mother and her child. This arrangement would seem to implicate 0.58 3.39 0.32 3.41 12 V Ϯ ␤ Ϯ 1.49 ND 4.62 1.25 5.16 0.95 1.47 2.36 1.94 1.12 ND 5.29 0.89 ND 4.37 0.69 5.35 0.46 2.63 trophoblasts as a potential regulator of the immune environment at the maternal-fetal interface. Indeed, trophoblasts can inﬂuence the by guest on September 23, 2021 immune system during pregnancy through their expression of sol- 1.94 1.56 0.85 1.04

9V uble and cell surface-associated immunomodulatory molecules.

␤ Ϯ Ϯ 5.64 6.28 1.33 1.45 3.77 ND 0.93 5.39 0.33 2.83 3.72 1.45

14.67 For example, trophoblasts secrete IDO, which limits the availabil- ity of the essential amino acid tryptophan, consequently limiting lymphocyte proliferation (28, 29). Normal human trophoblasts 2.58 2.34 0.66 2.33 8V also lack the expression of MHC class II and the classical class I ␤ Ϯ Ϯ restriction elements HLA-A and HLA-B (2), but similar to IEC, express a variety of nonclassical class I molecules. Our laboratory has focused on a subset of regulatory T cells, the 2.41 5.29 0.86 3.08 ϩ 7V

␤ CD8 regulatory T cell. These cells were described originally in Ϯ Ϯ 1.16 1.44 0.99 6.17 5.66 the intestine where they are activated by an interaction with IEC through a combination of the nonclassical class I molecule CD1d and a costimulatory molecule of the CEA family recognized by the 1.10 4.05 0.84 3.01 5.1 V Ϯ Ϯ B9 mAb (11, 23). CD1d has been reported to be expressed by ␤ 9.23 trophoblasts during the early stages of pregnancy (10). We have extended these observations by demonstrating that CD1d mRNA is temporally regulated during the ﬁrst trimester, with increasing ex- 1.39 3.25 1.81 2.81 6) were subjected to three-color ﬂow cytometry. T cells from nonpregnant normal volunteers ( n 3V

␤ Ϯ Ϯ ϭ pression from 7 to 12 wk of gestation. Trophoblasts also express a form of the costimulatory molecule recognized by the mAb B9. Furthermore, similar to IEC, isolated trophoblasts preferentially ϩ regulatory T cell subsets during pregnancy peripheral blood T cells from 12 pregnant women in their 16th to 29th wk of gestation, term (36th to 40th wk gestation) placenta, and normal controls. Peripheral blood T cells isolated from mothers in their 16th 1.65 2.63 1.09 6.89

2V activate clonal populations of CD8 T cells. However, in contrast ϩ ϩ ␤ ϩ Ϯ Ϯ to IEC, CD8 T cell expansion by trophoblasts appears to be in- CD8

ϩ dependent of CD1d, and the CEA subfamily member expressed by these cells appears to be distinct. 2.51 5.68 1.81 5.41 1V Western blot analysis suggests that the protein recognized by the ␤ Ϯ Ϯ V B9 mAb in trophoblasts is NCA (CEACAM6), whereas in the

Expansion of CD8 intestine, mAb B9 recognizes CEA. CEA and NCA share 85% repertoire of CD3 sequence homology at the amino acid level within the IgV-like N 0013 00111221 D00011131 ␤ S 5678 4.959 2.44 3.47 6.08 8.23 ND 8.33 7.59 4.4 ND ND ND ND ND 3.02 ND 4.2 3.44 2.7 3.62 1.99 4.64 3.66 4.64 3.37 3.66 3.86 3.59 2.09 2.62 4.6 2.3 1.73 2.92 1.67 1.8 ND 1.23 0.95 3.64 4.87 6.08 3.85 7.93 0.7 6.02 ND 1.36 0.99 4.57 5.43 4.51 0.8 0.79 0.49 1.48 2.4 2.64 2.81 1.58 0.9 4 6.64 4.05 ND 4.41 2.19 3.86 3.22 1.45 4.47 4.52 1.11 5.9 0.65 1.72 3NDNDND 2 ND ND 5.25 3.68 1 5.05 ND 0.7 3.27 5.18 1.97 2.81 1.58 4.35 8.12 1.44 6.05 0.6 2.75 V 12 3.21 6.4 ND 4.3 5.2 4.53 1011 3.04 1.87 6.33 5.26 4.85 ND 3.37 3.96 ND 3.06 3.11 1.93 1.26 0.66 3.67 6.24 0.93 5.01 0.78 1.17 3.68 ND no. a domain and IgC-like A1 and B1 domains (30). Some evidence has Ͼ 3 Patient Term 3.85 Control 4.08 that were statistical outliers (shown in bold) when compared with normal controls ( Ͼ 3 SD from control mean). Table I. to 29th wk of gestation, and term placenta ( n already suggested that CEA and NCA might have similar functions 7546 TROPHOBLASTS ACTIVATE CD8ϩ REGULATORY T CELLS

(31), and we provide further evidence for functional similarity be- tory T cells. Similarly, the uterus may face a persistent, albeit tween the two molecules. Furthermore, the inability of the anti- temporary, antigenic challenge in the form of a semi-allogeneic CD1d mAb and a pan-MHC class I Ab to block CD8ϩ expansion fetus. Trophoblasts are anatomically poised to serve as modulators in trophoblast cocultures, but not in the MLR (class I only), sug- of the maternal immune system during pregnancy. We postulate gests that NCA recognized by mAb B9 in trophoblasts may be that the regulatory CD8ϩ T cells described in this study may rep- acting in conjunction with another nonclassical class I molecule. resent one of several subsets of regulatory T cells that exist during In concert with these data, CD8ϩ T cells expanding in tropho- pregnancy. Indeed, in mouse models, both CD4ϩ and CD8ϩ T blast cocultures use a limited TCR repertoire including V␤9. The cells have regulatory effects during pregnancy. Mouse models in absence of an invariant TCR gene sequence in different cocultures which there is a deficiency in CD4ϩCD25ϩ regulatory T cells have speaks to the role of distinct Ags driving the response. Similar to significant fetal resorption during allogeneic but not syngeneic regulatory CD8ϩ T cells in the intestine, trophoblast-activated matings (14). Likewise, CD8ϩ T cell depletion studies demon- CD8ϩ T cells express mucosal markers and efficiently suppress in strate that CD8ϩ T cells are critical in regulating the CBA/J ϫ vitro Ab production in an Ag-nonspecific manner by cell contact. DBA/2 model of spontaneous abortion in mice (49, 50). Several groups have shown an association of aberrant Ab re- In humans, the role for the various regulatory subsets are un- sponses with recurrent spontaneous abortion. The lupus anticoag- known. Although CD4ϩCD25ϩ regulatory T cells are expanded in ulant and anticardiolipin Abs are present in 1–3% of normal preg- the peripheral blood and decidua of pregnant women (15), the nancies but are markedly elevated in patients with recurrent fetal physiologic role of these cells in pregnancy has not been charac- loss (32, 33). CD8ϩ T cells are present at the maternal/fetal inter- terized fully. In this study, we explored the possibility that tropho- face, albeit at low numbers (34, 35). Furthermore, Yeaman et al. blasts could modulate the immune system through an interaction Downloaded from (36, 37) recently described uterine lymphoid aggregates consisting with CD8ϩ T cells. The specificity of trophoblast-activated CD8ϩ of predominantly CD8ϩ T cells and B cells in close association T cell for regulating T cell-dependent B cell responses and the during the proliferative phase of the estrous cycle. Indeed, Kemeny efficiency at which these cells are able to suppress Ig secretion and colleagues (38–40) have demonstrated that CD8ϩ T cells can suggests that these cells may occupy a regulatory niche during inhibit IgE secretion and have a regulatory role in allergic airway pregnancy. Overall, CD8ϩ regulatory T cells may contribute to the ϩ disease models. Additionally, CD8 suppressor T cells have also complex immune regulation that must occur during pregnancy. http://www.jimmunol.org/ been implicated in controlling systemic lupus erythematosus (41), an Ab-mediated autoimmune disorder, as well as suppressing thy- Acknowledgments rotropin receptor-specific HLA-DQA*0501-restricted CD4ϩ T We thank I. George for technical assistance with flow cytometry and cell cells that may be linked to the development of Grave’s disease sorting, T. Moran for assistance with the redirected lysis assay, and (42). This suggests the possibility that, in vivo, CD8ϩ T cell sub- P. Cortes, G. Randolph, and members of the Mayer laboratory for their sets may have a role in regulating B cells, potentially protecting the helpful advice and discussions. fetus from deleterious Ab effects. However, trophoblast-activated CD8ϩ T cells do not act as Disclosures third-party suppressor cells in allogeneic MLRs. This is in contrast The authors have no financial conflict of interest. by guest on September 23, 2021 to other regulatory cell populations such as the naturally occurring ϩ ϩ ϩ ϩ References CD4 CD25 regulatory T cell. One caveat is that CD4 CD25 1. Koch, C. A., and J. L. Platt. 2003. Natural mechanisms for evading graft rejec- regulatory T cells that can suppress allogeneic mixed lymphocyte tion: the fetus as an allograft. Springer Semin. Immunopathol. 25: 95–117. only do so at E:T ratios of 1:2 or greater (43, 44). This calls into 2. Blaschitz, A., H. Hutter, and G. Dohr. 2001. 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