A Placenta-Derived Suppressor Factor with a T-Cell Bias

Copyright 0 Munksgaard, 1999

American Journal of Reproductive Immunology ISSN 8755-8920

A Placenta-Derived Suppressor Factor with a T-cell Bias RAJ RAGHUPATHY, S.F.B. KHAN,P.V. SYAMASUNDAR, P. BANSAL,AND F. AZIZIEH

R~igliipatli~~ R, K l i ~ nSFB, S~~riiiirisiiiidwP V, Briiisal P, Azizieli F. A plriceiitcr-derioed siippressorfrrctor with N T-cell bicis. AJRI 1999; 42205-21 8 0Miiriksgurird, Copeiihngeii PROBLEM: Functional and mechanistic aspects of immunosuppression by murine placental supernatants (MPS) were investigated. METHOD OF STUDY: MPS and a low molecular weight fraction of the supernatant (MPSf) were tested for suppressive action on T-cell reactivity iii uitro and iii oiuo, on B-cell responses and on T-cell activation events. RESULTS: MPS and MPSf suppress mitogen-induced proliferation and mixed lymphocyte reactions of human and murine lymphocytes, antigen-induced proliferation of T cells in oitro and in oiuo, proliferation of CD8+ lymphocytes, proliferation induced by cross-linking of surface CD3 and the in uiuo response of mice to allogeneic stimuli. MPSf affects cell cycling of activated T cells and blocks interleukin (1L)-2 production. MPSf does not affect antibody production or the induction of MHC class I1 expression on B cells. CONCLUSIONS: MPSf is a potent inhibitor of T-cell responses iiz uitro and in uioo, with no demonstrable effect on B-cell function.

Key words: Immunosuppression, placenta, placental immunosuppressive factor, pregnancy RAJ RAGHUPATHY F. AZIZIEH Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait S.F.B. KHAN Department of Natural Studies, Jamia Millia Islamia, New Delhi, India P.V. SYAMASUNDAR P. BANSAL National Institute of Immunology, New Delhi, India Current address: Washington University School of Medicine, St. Louis, MO Address reprint requests to Raj Raghupathy, Department of Microbiology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat 13110 Kuwait. E-mail: [email protected] Submitted June 24, 1998; revised September 9, 1998; accepted September 10, 1998.

INTRODUCTION Maternal recognition of the fetoplacental unit has been suggested as being beneficial t o the fetus as this leads t o the production of cytokines that increase uterine vascularization and blood flow appropriate for placental function.' However, excessive stimulation of the maternal immune system may lead to AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 42, 1999

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adverse effects on the placenta, resulting in fetal damage or loss’.2; immunosuppression at the maternal-fetal interface may play a useful and critical role in countering these e f f e c t ~ . l . ~ - ~ The need for some form of immunomodulation is also predicated by the demonstration of the susceptibility of pregnancy to immunological effectors such as natural killer (NK) cells and ThI-type cytokines. N K cells’*9 and activated macrophagesI0 in the decidua can bring about fetal resorption. Abortions can be induced in mice by the administration of tumor necrosis factor (TNF)-a and interferon (1FN)y in vivo”; these cytokines affect the survival and proliferation of trophoblast cells and the development of embryos in vitro.”*13 In a murine model of immunologically-mediated pregnancy failure, we have shown previously that maternal strain lymphocytes respond to placental antigenic stimuli iiz vitro with the proliferation of CD8+ T cells and the production of high levels of interleukin (1L)-2, TNFa, and IFN-y.I4 The placentas of fetal resorptionprone mice also have substantially higher levels of mRNA corresponding to these cytokines as compared to placentas from normal pregnant mice.Is Hill and colleagues have demonstrated increased Th 1-type cytokine production by peripheral blood mononuclear cells from women with recurrent pregnancy loss after these cells have been activated by antigens from a trophoblast cell line.16 To keep such deleterious responses under check, it is argued that immunosuppression may be conducive, perhaps even vital, to pregnancy.1.7 Immunosuppressive molecules produced at the fetomaternal interface by the placental7 and the decidua,’’ including hormones and proteins,l”-” have been described in the literature and a variety of immunosuppressive effects have been attributed to these factors. Water soluble extracts of murine placentas have been shown to enhance the survival of allogeneic tumors” and to inhibit mixed lymphocyte reaction^,'^ cytotoxic T lymphocyte (CTL) activityz4 and antibody-dependent cellular cytotoxicity of spleen cells.’s Homogenized cell lysates of placentas have also been shown to have immunomodulatory effects on cellular and humoral responses iiz vitroz6 and on graft-versus-host reactions (GvHR).’~ Although some of the functional characteristics of murine placental supernatants (MPS) have been described previously, there is little definitive information on possible selective effects on cell-mediated immunity versus humoral immunity and the actual mode(s) of action of the suppressive activity. Furthermore, the apparent size of the active moiety (or moieties) is still in question, as different reports attribute the functional effects to different molecular 0 MUNKSGAARD, COPENHAGEN

weight fractions, and not all functions have been localized to the same fraction. It is in this context that we have investigated some functional and mechanistic aspects of MPS and report here that the observed immunosuppressive capabilities are exhibited by a 1-3 kDa fraction, which we have termed murine placental suppressor factor (MPSf). We show here that MPSf inhibits several T cell responses: (i) mitogen-induced proliferation of murine and human T cells, (ii) antigen-induced murine T-cell proliferation, (iii) proliferation of CD8+ T cells in a mixed lymphocyte reaction (MLR), (iv) proliferation of T cells in response to allogeneic challenge in vivo, and (v) stimulation of T cells via surface CD3 molecules. We find that MPSf is not toxic to cells nor is it a generalized anti-proliferative factor. MPSf blocks cell cycling of activated lymphocytes and the production of IL-2 by activated T cells suggesting effects on early events in T-cell activation. In contrast to its effects on T-cell activity, MPS/MPSf does not seem to affect B-cell function measured by (i) antibody production and (ii) non-proliferative activation of B cells by IFN-y and IL-4, leading us to speculate that this factor is T-cell-biased in its activity, which correlates with observations that the predominant maternal anti-fetal response is humoral and not ~ell-mediated.~~~~~

MATERIALS AND METHODS Preparation of MPS Syngeneically pregnant BALB/c mice were sacrificed on day 15 of gestation and the placentas dissected out. The placentas were each minced in 1 mL of RPMI 1640 medium (GIBCOBRL, USA) supplemented with 10% fetal calf serum (FCS) (Boehringer Mannheim Gmbh, Germany), 100 mg/L of streptomycin and 100 units/L of penicillin and then incubated at 37°C in a humidified 5% CO’ incubator for 24-48 hr. The supernatants were pooled, centrifuged twice at 4000 rpm for 30 min to remove cellular contamination, filter-sterilized and stored at - 70°C until use. MPS was fractionated by sequential ultrafiltration on an Amicon Diaflo YMlO membrane (molecular weight cut-off 10 kDa) (Amicon, USA) followed by ultrafiltration on a YM3 membrane (molecular weight cut-off 3 kDa). The material under 3 kDa was then dialyzed against PBS (50 mM, pH 7.4) in a dialysis membrane with a molecular weight cut-off of 1 kD (Spectra/Por, Spectrum, USA). The 1-3 kDa fraction contains the suppressive activity (see Results) and is referred to as murine placental suppressor factor (MPSf).

PLACENTAL SUPPRESSOR FACTOR WITH A T-CELL BIAS

Preparation of Lyiiiplzocytes Murine splenic lymphocytes were obtained from spleens of adult BALB/c, C57BL/6, and C3H/He mice after selective lysis of red blood cells (RBCs) by Tris-buffered ammonium chloride (90% 0.16M NH,Cl in 0.17M Tris, pH 7.6), washed twice in RPMI medium and then suspended in medium supplemented with 2% newborn calf serum (GIBCOBRL, USA). Human peripheral blood lymphocytes were obtained by Ficoll-Paque (LKB-Pharmacia, Sweden) density gradient centrifugation of peripheral blood obtained by venipuncture of healthy donors, washed three times in RPMI medium, and resuspended in medium supplemented with 10% heat-inactivated human AB serum.

Mitogeiz -induced Proliferation Murine lymphocytes (5 x lo5 cells/well) were challenged in triplicate with 1 pg/mL of Con A (Sigma Chemical Co., USA) or 10 pg/mL of lipopolysaccharide (LPS) (Sigma) in the presence or absence of MPS or MPSf and incubated for 48 hr. Human lymphocytes (2 x lo5 cells/well) were stimulated with 4 pg/mL of phytohemagglutinin (PHA) (Sigma) or 1 pg/mL of pokeweed mitogen (PWM) (Sigma) in triplicate in the presence or absence of MPS or MPSf and incubated for 72 hr. Cells were pulsed with 1 pCi/well of [3H]thymidine (Amersham, UK) for 18 hr after which the cells were harvested onto glass fiber filters (Wallac, Finland) and the radioactivity measured in a Betaplate 1205 scintillation counter (LKB-Pharmacia, Sweden).

MLR Murine splenocytes (5 x lo5 cells/well) or human peripheral blood lymphocytes (2 x lo5 cells/well) were challenged in triplicate with allogeneic cells at different stimu1ator:target ratios in the presence or absence of MPS in 96 well tissue culture plates (Costar, USA). After incubation at 37°C in 5% C 0 2 for 72 hr (for murine cells) or 120 hr (for human cells), the wells were pulsed with [3H]thymidine, harvested, and the radioactivity estimated.

Coiistitutive Proliferatiori of Cell Lines The human myelogenous leukemia cell line K562, mouse T-cell lymphoma EL4 and rat B cell hybridoma 187.1 were maintained in RPMI 1640 medium supplemented with 10% FCS. Varying numbers of these cells (2,500-20,000 cells/well) were seeded in triplicate into 96-well tissue culture plates with or without MPS or MPSf. The wells were immediately pulsed with 1 pCi [3H]thymidine, incubated for 18 hr after which the plates were harvested and the radioactivity counted.

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Toxicity Testirzg of MPS and MPSf In order to ascertain whether MPS is cytotoxic, mouse splenocytes were incubated with MPS or MPSf for 24 or 48 hr at 37"C, after which the viability of the cells was estimated by trypan blue exclusion and by propidium iodide staining; for the latter, cells were exposed to a 1% solution of propidium iodide (Sigma) in phosphate-buffered saline (PBS) (50 mM, pH 7.4) and followed by analysis on an EPICS 751 Flow Cytometer (Courter Electronics Ltd., UK). CD8+-MHC Class I-restricted M L R Splenocytes obtained from BALB/c (H-2d) spleens were depleted of CD4+ .T cells and B cells by sequential incubation with anti-CD4 antibodies (GK1.5 hybridoma, ATCC) and affinity-purified goat anti-mouse Ig followed by incubation with rabbit complement (Lowtox-M, CedarLane, Canada). The purity of the remaining CD8 T cells was determined by staining with a monoclonal anti-CD8 antibody (TIB105 hybridoma, ATCC). 5 x lo5 CD8 cells were challenged in triplicate with semi-log titration of irradiated EL4 cells (H-2b) in the presence or absence of MPS or MPSf. After 48 hr, cells were pulsed with 0.5 pCi [3H]thymidine per well for 18 hr. Plates were harvested and the radioactivity counted as described earlier. +

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Antigen -induced T-cell Proliferation Foot pads of BALB/c mice were injected with ovalbumin (100 pg/animal) emulsified in complete Freund's adjuvant (Difco, USA). Seven days later, 5 x lo5 cells/ well of inguinal and popliteal lymph node cells suspended in Click's medium (GIBCOBRL, NY, USA) containing 10% FCS were challenged, in triplicate, with varying concentrations of ovalbumin in the presence or absence of MPS at a concentration of 25% in 96-well tissue culture plates. The cultures were pulsed with [3H]thymidine after 4 days, harvested and the radioactivity estimated.

Proliferation Induced by Cross Linking of Cell Suifcice CD3 Splenocytes obtained from BALB/c animals were cultured in tissue-culture plates coated with anti-CD3c antibodies in the presence or absence of MPSf for 48 hr. Plates were pulsed with 0.5 pCi [3H]thymidine, the cells harvested and the radioactivity counted as described.

Effect of MPS on the Allogeneic Response IIZ Vivo To induce a primary allogeneic response, BALB/c cells (5 x lo6 cells in 50 pL PBS) were injected into the

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footpads of adult C57BL/6 mice with or without MPS or MPSf. The contralateral footpad of each animal was injected with normal saline. Popliteal lymph nodes were removed 5 days later and allogeneic responses evaluated by measuring (i) lymph node weights and (ii) incorporation of thymidine by lymph node cells. The allogeneic response in terms of increase in lymph node weights was calculated as ratios of the weights of experimental versus contralateral lymph nodes. For thymidine incorporation, lymph node cells were seeded in triplicate into 96-well plates and immediately pulsed with 1 pCi [3H]thymidine. Lymphocytes were harvested 18 hr later and the radioactivity estimated.

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BALB/c mice were injected intra-peritoneally with 300 pg of ovalbumin (Sigma, USA) adsorbed on alum (Alhydrogel, Superfos, Denmark) in the presence or absence of an equal volume of MPSf. Mice were bled on day 14 and the sera assayed for ovalbumin-specific antibodies by enzyme linked immunosorbent assay (ELISA).

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The expression of MHC Class I1 molecules was induced with IFN-)I and with IL-4, and the effect of MPS on this induction was assessed by flow cytometric measurement of MHC Class I1 density on the B cell surface. The B cell line A20 (I-A") (kind gift of Dr. Satyajit Rath) was cultured with 100 U/mL of r-IFN-y (Genzyme, USA) for 48 hr in the presence or absence of MPSf. BALB/c splenocytes were similarly stimulated with 100 U/mL r-IL4 for 48 hr in the presence or absence of MPSf. Cells were harvested at the end of the incubation period and stained with anti-I-A" antibody (hybridoma HB 26, ATCC) followed by fluorescent isothiocyanate (F1TC)-conjugated goat anti-mouse immunoglobulin (1g)G (USB, USA) as the second antibody. The cells were analyzed on a BRYTE HS flow cytometer (BIORAD, Italy).

IL -2 AssCiy 5 x lo5 BALB/c splenocytes were stimulated with varying doses of Con A, i n the presence or absence of MPS or MPSf. Supernatants from these cultures were collected at different time points and IL-2 levels estimated in a bioassay. CTLL-2, an IL-2-dependent cell line was maintained in medium containing 10 U/mL of human r-IL-2. Just prior to the assay, the cells were washed and starved of IL-2 by incubation in medium without IL-2 for 6-8 hr. These cells were then 0 MUNKSGAARD, COPENHAGEN

seeded into 96-well plates at a density of 1 x lo4 per well. An equal volume (50 pL) of standard r-IL-2 or experimental culture supernatants were added and the cells incubated for 24 hr at 37°C. The plates were then pulsed with 0.5 pCi [3H]thymidine for 16 hr, the cells harvested and the radioactivity counted.

Espressioiz of IL-2 Receptor. BALB/c splenocytes were stimulated in the presence or absence of MPSf with 5 pg/mL of Con A for 36 hr. Cells were collected, stained with anti-IL-2R antibody (TIB-222, ATCC) and then were analyzed on a BRYTE HS flow cytometer (BIORAD) and the data plotted as bivariate displays of cell number versus fluorescence intensity.

DNA Cell Cycle Aiialysis BALB/c splenocytes were stimulated with Con A in the presence or absence of MPSf as described above and the cells fixed at several time points by adding 70% ice cold ethanol with vigorous vortexing. Cells were pelleted at 3000 rpm, resuspended in the residual ethanol and then incubated on ice for 30 min. To the pellet were added 200 pL propidium iodide (50 mg/ mL) and 10 pL of 1% (v/v) Triton X-100. Analysis was done on an EPICS 751 flow cytometer (Courter Electronics, USA). The data was acquired as bivariate histograms of cell number versus fluorescence intensity.

RESULTS MPS Inhibits Pr.ol$er.ritive Respoiises of Muriiie aiid Huiiiaiz T Lyriiphocytes Murine responses to Con A, PWM, and LPS are inhibited dramatically by MPS as are the proliferative responses of human lymphocytes to PHA. MPS at a concentration of 12.5Y0 inhibits Con A- and LPS-induced proliferation of murine lymphocytes by 95% (Fig. 1A,B, respectively) and inhibits the proliferation of human peripheral blood monocular cells (PBMC) in response to PHA (85Y0 inhibition at 25Y0 MPS) (Fig. 1C) and PWM (63% inhibition at 25%) MPS) (Fig. 1D). Using the inhibition of response to Con A as the standard assay, the optimal time of collection of MPS from explant cultures was found to be 24 hr (data not shown). MPS (at a concentration of 12.50/) was shown to strongly suppress bi-directional MLR of both human (80% inhibition) and murine lymphocytes (85% inhibition) (Fig. 1E).

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Clzaracterizcitioi? of Murille Placeiztnl Suppressor. Factor.

membrane with a cutoff of 10 kDa followed by ultrafiltration on a membrane with a cutoff of 3 kDa. This was followed by extensive dialysis against PBS. Immunosuppressive activity was assayed at each stage by

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Effect of MPS on niitogen-induced proliferative responses and MLR. The proliferation of murine splenocytes in response to Con PBL stimulated with PHA and PWM, respectively. Data are expressed as mean of triplicate values with standard errors always less than 20%. Murine and human MLR (E) are a h inhibited dramatically by MPS; data are expressed as mean of triplicate values. Panel F demonstrates that MPSf does not inhibit the constitutive proliferation of a B-cell hybridonia and that, on the other hand, 25% MPS brings about substantial suppression; data are expressed as mean of six values with standard error less than 20%.

A (A) and LPS (B) is suppressed by MPS. C and D depict the inhibition of responses of human

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2. Characteflristics of the suppressive factor in MPS. MPS (crude supernatant) and MPSf (the 1-3 kDa fraction) show identical suppressive capabilities whereas the > 10 kDa fraction shows no suppression. Further characterization reveals that the active moiety has a molecular weight between 1 and 3 kDa and that heat treatment has no affect on its activity. Fig.

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MPSf Does Not Inhibit the Constitutive Prolgercition of Cell Lines To ascertain whether the suppressive activity of MPS and MPSf can be attributed to a general block in proliferation, we examined the effect of MPS and MPSf on the constitutive proliferation of a few selected cell lines. A leukemia cell line (K562), a lymphoma cell line (EL4), and a B cell hybridoma (187. I ) were tested; while MPS blocked the proliferation of all cell lines tested in a non-cell-type-specific manner, purified MPSf did not affect the constitutive proliferation of the cell lines to any discernible extent. Fig. IF depicts the effects of MPS and MPSf on the proliferation of the B cell hybridoma as a representative of similar effects on K562 and EL4. The observation that MPS suppresses constitutive proliferation while MPSf does not, suggests that a factor or factors other than MPSf may have a general anti-proliferative effect. 0 MUNKSGAARD, COPENHAGEN

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M P S and MPSf Are Not Cytotosic MPS and MPSf were found to be non-toxic to splenocytes after extended exposure of murine spleen cells to these preparations. Viability of cells estimated after 24 and 48 hr of exposure to MPS or MPSf was not significantly different from the viability of control cells exposed only to medium as measured by trypan blue exclusion; 75 & 5% versus 82 f 4% viability in the absence and presence of MPS, respectively. Propidium iodide staining of cells exposed to 50% MPS shows that such cells are no less viable than are cells in medium alone (Fig. 3). MPSf was similarly found to be non-toxic to cells (data not shown).

MPSf Inhibits Antigen -induced Proliferation MPSf mediates a profound suppression of the proliferation of murine T cells responding to antigen-specific stimuli. Lymphoid cells of mice immunized with ovalbumin respond vigorously to in Gitro challenge with ovalbumin as shown in Fig. 4A, but this response is strongly inhibited by the presence of MPSf at a concentration of 25%.

MPSf Inhibits the Proliferation of CD8+ T Cells MPSf was examined for its ability to inhibit the proliferative response of murine CD8+ T cells to an allogeneic stimulus. CD8+ T enriched cells from BALB/c (H-2") mice were incubated with EL4 (H-2b) cells in either 25% MPSf or in medium alone. Proliferation of CD8+ T cells was inhibited by MPSf as shown in Fig. 4B.

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MPSf Inhibits Prolifercitioiz Induced by CD3 Cross-linking When T cells are stimulated by cross-linking their CD36 molecules with antibody, a strong proliferative response ensues as expected (Fig. 4C); in the presence of MPSf, however, a profound suppression in the proliferation is observed.

MPSf Suppresses Allogeneic Responses IIZ Vivo MPSf was examined for its effect on allogeneic responses induced in uiuo in local lymph nodes upon injection of allogeneic lymphocytes. BALB/c cells were injected into the footpads of C57BL/6 along with MPSf (final concentration = 50%). As can be seen in Fig. 5, MPSf causes a profound inhibition of the in uiuo allogeneic response measured on day 5 as a function of the increase in the weights of popliteal lymph nodes (88% inhibition) and thymidine incorporation by lymph node cells in culture (72% inhibition).

MPSf Does Not Suppress Antibody Production In a bid to ascertain the effect of MPSf on antibody production, we immunized mice with an antigen, ovalbumin, along with or without MPSf and then com-

pared the levels of antibodies produced in the two cases. Three-hundred micrograms of ovalbumin adsorbed on alum were injected along with an equal volume of MPSf or normal saline; antibody levels were estimated by ELISA 14 days later. As can be seen in Fig. 6 , the inclusion of MPSf along with the immunogen does not affect subsequent antibody titers.

MPSf does not Affect Non-proliferative Activcition of B Cells The effect of MPSf on IFN-y-induced increase in MHC class I1 expression on B cells was tested using a B cell line; these cells were treated with r-IFN-y in the presence or absence of 25% MPSf for 48 hr after which they were stained with an anti-MHC class I1 antibody to measure the density of surface class I1 molecules by flow cytometry. r-IFN-y induces the expression of surface class I1 molecules (Fig. 7A) and this expression is not affected by the presence of MPSf in culture (Fig. 7B); thus, MPSf does not affect enhancement of MHC class I1 expression brought about during B-cell activation. Similarly, murine splenocytes were incubated for 48 hr with 100 U/mL of r-IL-4 in the presence or absence of 25% MPSf; an enhancement in class I1 expression is seen after induction with IL-4 (Fig. 7C) and this induction is not affected by the presence of MPSf (Fig. 7D). These results indicate that MPSf does not inhibit non-proliferative activation of B cells.

MPSIMPSf Inhibits IL-2 Secretion T lymphocytes respond to mitogen-induced activation by secreting IL-2, which acts in an autocrine/paracrine loop giving a critical stimulus that leads to T-cell proliferation. Fig. 8 depicts a dramatic suppression in the amount of IL-2 secreted by cells stimulated in the presence of MPS or MPSf. In the control wells devoid of MPS/MPSf, IL-2 was detected shortly after stimulation, whereas in the presence of either MPS or MPSf, IL-2 was not detected even after 36 hr of incubation, demonstrating that MPS and MPSf suppress IL-2 secretion by activated T lymphocytes.

MPSf Bloclcs Eizlianceinent of IL-2 Receptor Exp ressioii

Fig. 3. Lack of toxicity of MPS. Flow cytometric estimation of staining by propidium iodide of cells exposed to 5 0 % MPS for 24 hr indicates no difference between normal and MPS-treated cell populations (A). Panel B shows the unstained and methanol-treated (killed cells) controls.

Along with the secretion of IL-2 as one of the early events in T-cell activation, the expression of IL-2 receptor IL-2R is upregulated in stimulated cells to increase the quantum of the response to IL-2 being secreted. As shown in Fig. 9, Con A-stimulated T lymphocytes express significant levels of IL-2R, the expression of which is profoundly suppressed by MPSf.

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Effect of MPSlMPSf 012 Cell Cycling The S (synthetic) phase of the cell cycle indicates a preparative phase in the cell just prior to replication. This is characterized by a shift from the G,/G, state to the G2/M phase of the cell cycle. The top panel in Fig. 10 shows the progressive induction of the S phase in a time-dependent manner. The middle and lower panels of Fig. 10 depict the absence of induction of the S phase in the presence of MPS or MPSf illustrat0 MUNKSGAARD, COPENHAGEN

Fig. 4. Effect of MPS on proliferative responses. (A) Ovalbumin-primed lymph node cells from BALB/c animals are inhibited from proliferating in response to ovalbumin in the presence of MPSf. (B) MLR between purified CD8 + T cells (BALB/c, H-2d) and allogeneic cells (EL4 cell line, H-2') is suppressed by MPSf. (C) MPSf inhibits the proliferation of T cells in response to cross-linking of surface CD3 by anti-CD3 antibodies. Data are expressed as mean of triplicate values with standard error less than 20%.

ing the lack of cell cycling in the presence of MPS or MPSf; this indicates a block at an early event in the proliferative cascade.

DISCUSSION Placental supernatants have been shown to suppress proliferative responses of human and murine lymphocytes in uitr.o.''-" Human placental superna-

PLACENTAL SUPPRESSOR FACTOR WITH A T-CELL BIAS / 213

and supernatants of human trophoblast lines7.33-35have been shown to inhibit mitogen-induced proliferation, antigen-induced proliferation and allogeneic responses iii vitro and iii uivo. Here, we show that MPS and a suppressor factor-enriched fraction inhibit the proliferation of T cells in response to stimulation by (i) mitogen and antigen (ii) MLR (iii) allogeneic immunization iiz uivo and (iv) surface CD3 cross-linking. We have demonstrated that the suppressive capability of this substance is not attributable to a general anti-proliferative effect. MPSf does not inhibit the normal constitutive proliferation of cell lines. Interestingly, the crude supernatant contains an activity that inhibits the normal proliferation of cell lines; thus, MPS may contain another substance that non-specifically abrogates the proliferation of cell lines. The suppressive effect of MPSf is also not attributable to toxicity; MPS is not toxic to cells even after substantial exposure of cells to high concentrations of MPS and MPSf, demonstrating that the suppression that we have observed is not due to mere cell killing. We report for the first time that a substance under 3 kDa in size constitutes the active suppressive moiety(s) in MPS. The suppressive activity of watersoluble extracts of murine placentas was previously reported to be associated with substances of high molecular weight.” Chaouat and colleagues have demonstrated a functionally similar low molecular weight human placental factor that inhibits CTL activity, MLR and NK function in human and murine systems, and local and systemic allogeneic reactions and GvHR in mice”; this human placental suppressor factor is under 1 kDa in size.3’ The active moiety in the supernatant of human choriocarcinoma cell lines is also a low molecular weight substance of about 5-6 kDa.35 Heating does not affect the ability

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Fig. 6. Lack of inhibitory effect of MPSf on antibody production. BALB/c mice injected with 300 pg/animal of ovalbumin adsorbed

on aluminum hydroxide with or without MPSf show no difference in antibodies generated. Data are represented as means of six animals in each group. Error bars indicate standard deviation.

of MPS and MPSf to induce suppression, ruling out the possibility that a prostaglandin E2-like molecule is responsible for suppression in this particular system. Our data clearly show that MPSf suppresses functions associated with ap T cells including antigen presentation in the context of MHC Class I1 molecules and alloreactivity restricted by both MHC Class I and MHC Class I1 molecules. It is interesting to note that a high number of T cells bearing the y6 TCR has been reported in the close vicinity of the fetomaternal interface.36 It is possible that what we have reported here may actually represent a T cell subset-specific phenomenon in which only the ap’ T cells are affected, much the same as Chaouat’s demonstration that the human placental suppressor factor inhibits ap T cell-mediated cytotoxicity but not y6 T cellmediated cytotoxicity.30 It would be highly illuminating to examine the effect of this factor on T cells

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Fig. 5. Effect of MPSf on the primary allogeneic response in cico. BALB/c cells were injected into C57BL/6 mice along with 50‘% MPSf and the allogeneic response measured as a function of increase in lymph node weights (A) and in lymphoid cell proliferation (B). MPSf inhibits the allogeneic response as demonstrated by the suppression of increase in the weight of popliteal lymph nodes (A), and increase in lymphoid cell proliferation (B). Values are expressed as means & S D of ten mice.

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RAGHUPATHY ET AL. r-IL-4

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bearing the y6 phenotype. One may speculate that y6-bearing T cells are not affected by this factor and that it inhibits only those immune responses that are associated with c@ TCR-bearing T cells. This would enable the local maternal immune system to tolerate the fetal allograft while at the same time retaining the ability to mount a successful immune response if necessary using the non-classical MHC-y6 T cell pathway. MPSf suppresses the production of IL-2 and consequently, as could be predicted, the expression of IL-2 receptor by activated T cells. In the absence of MPSf, IL-2 secretion is evident when tested 3 hr post-activation, while the addition of MPSf results in significant inhibition of IL-2 production even at 36 hr. Since IL-2 secretion is one of the early events in T-cell activation, it is clear that MPSf acts early during the activation process. That MPSf acts early in the proliferative cascade is supported by the observation that it blocks cell cycling in mitogen-stimulated cells. The absence of a sub-Go peak in cells incubated with MPSf (Fig. 10) rules out the possibility of induction of apoptosis by MPS/MPSf, reiterating an immunosuppressive, as opposed to a cytotoxic, role for MPSf. In contrast to its potent effects on T-cell responses in vitro and in vivo, MPS/MPSf does not seem to affect B-cell function measured in terms of (i) antibody production and (ii) non-proliferative activation of B cells by IFN-y and IL-4. Admittedly, only two B-cell responses have been studied here, but the lack of effect on B-cell function is still striking. We have tested the effect of MPSf on irz vivo antibody production by injecting increasing concentrations of MPSf at 0 MUNKSGAARD, COPENHAGEN

Fig. 7. Lack of inhibition of non-proliferative activation of B cells by MPSf. Activation of a B cell line by r-IFN-y (A) and IL-4 (C) results in an increase of class I1 expression; this enhancement in expression is not affected by the presence of MPSf (B, D)indicating a lack of effect on cytokineinduced Class I1 expression on B cells. Thiu dotted line = control, unstained cells; thick clotted h e = control, unstimulated, stained cells; thick dotted line = stained cells stimulated with r-IFN or IL-4.

several time points and still found no suppressive effect on antibody production (data not shown). The increase in IFN-y and IL-4-stimulated Class I1 expression on B cells is similarly unaffected by MPSf. A previous report has described the lack of inhibitory effects of crude MPS on T-independent B-cell funct i ~ n Thus, . ~ ~ MPSf appears to have, if not an exclusive T-cell specificity, at least a T-cell bias. The suppressive material produced by human choriocarcinoma cell lines was also found to suppress several T-cell responses with no effect on the proliferation of B cell hybridomas and the production of antibodies iiz oitro and in uivo.4.33-35This preferential inhibitory effect on T cells reflects the situation in normal pregnancy in which humoral responses are unaffected or even enhanced while cell-mediated immunity at the same time is dampened.3.'9~38Clearly there is a need to protect the placenta and the fetus from activated maternal T cells, especially those of the IL-2-, TNF-aand IFN-y-secreting Thl -type,7 which could conceivably mediate direct attack on the trophoblast or could mediate indirect effects via lymphokine-activated killer cells'9 and activated macrophages.'O We have not demonstrated that the suppressive activity is secreted by placental trophoblast cells and it may therefore be argued that the factor is a product of placental macrophages or other cell types. However, we have examined the effects of supernatants of murine splenocytes and lymph nodes and have determined that these supernatants do not manifest similar suppression of T-cell responses (unpublished observations); this would suggest that placenta-mediated suppression is not likely to be due to macrophages or

PLACENTAL SUPPRESSOR FACTOR WITH A T-CELL BIAS

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Medium -+-ConA +MPS+Con

A

+MPSf+Con A

A

1

3

6

12

A -

24

Fig. 8. Suppression of IL-2 production by MPS and MPSf. Mitogen-induced production of IL-2 is suppressed by MPS and MPSf.

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Time (in hours)

related cell types. The demonstration of similar suppressive activity by a factor secreted by human trophoblast cell lines33-35suggests that MPSf may also be a trophoblast-derived factor. Given the dichotomy between Thl- and Th2-type responses in normal pregnancy and pregnancy failure,7*29it would be of great interest to investigate the effects of such pregnancy-related immunoregulatory factors on these T-cell subsets and their responses. An important role has been ascribed to the 34 kDa progesterone-induced inhibitory factor (PIBF) which suppresses mitogen-induced T-cell proliferation, activation of NK cells and T N F production by NK cells4' and protects against resorption in mice.20 SzekeresBartho and colleagues have recently demonstrated the ability of PIBF to shift the cytokine production pattern of activated splenocytes towards a ThZtype p r ~ f i l e . ~We ' have not investigated the effect of MPSf on Thl versus Th2 reactivity, but it is likely that factors such as the one reported here play useful roles in downregulating Thl-type reactivity since robust maternal Thl-type activity is deleterious for pregnancy, while normal pregnancy appears to be Th2biased.7.16.29 In summary, we report in this paper a low molecular weight murine placental factor which appears to be T cell-biased and which inhibits the proliferation of T cells activated via different pathways.

Acknowledgeinents Supported by Kuwait University Research Administration Project No. MI 090.

3001

Log Fluorescsnce Intensity

Fig. 9. Inhibition of IL-2 receptor expression by activated T cells. Mitogen-induced activation results in the expression of IL-2 receptor seen as a shift in the curve of anti-IL-2 receptor stained cells by flow cytometry (A). The addition of MPSf results in the suppression of IL-2 receptor as evinced by a lack of a shift in the profile of stained cells (B). Dotted lines = Control, non-specific antibody; solid liries = anti-IL-2 receptor antibody-stained cells.

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 42, 1999

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Fi,q. 10. Effect of MPS and MPSf on cell cycling. Activated cells were analyzed by flow cytometry after staining with propidium iodide a t different time periods after activation. Thin dotted curves represent tinstimulated cells and thick solid curves represent mitogen-stimulated cells. Control cells show a progressive induction of the S phase in a time-dependent manner. MPS and MPSf-treated cells show a lack of induction of the S phase indicating suppression of cell cycling in the presence of MPS o r MPSf. Dotted lines = control untreated cells, solid lines = cells treated with Con A.

MPSf

MPS

12 hwn

PLACENTAL SUPPRESSOR FACTOR WITH A T-CELL BIAS

REFERENCES 1. Beaman KD: Nidation, tolerance and immunotrophism. Am J Reprod Immunol 1990; 23:54-56. 2. Meegdes BH, Ingenhoes R, Peeters LL, Exalto N: Early pregnancy wastage: Relationship between chorionic vascularization and embryonic development. Fertil Steril 1985; 49:216-220. 3. Voisin G, Raghupathy R: Immunodeviation of the immune response during pregnancy. In Immunology of Reproduction, M Kurpisz, N Fernandez (eds). Oxford, UK, BIOS Scientific Publishers, 1995, pp 335-353. 4. Raghupathy R, Krishnan L: Immunosuppressive mechanisms in normal pregnancy. Central Eur J Immunol 1996; 21: 133- 140. 5. Arck PC, Clark DA: Immunobiology of the decidua. Curr Top Microbiol Immunol 1997; 222:45-66. 6. Chaouat G, Menu E, Mognetti B, Moussa M, Cayol V, Mostefaoui Y, Dubanchet S, Khadel P, Volumenie JL, kongieres CB, Delage GL: Immunopathology of early pregnancy. Inf Dis Obstet Gynecol 1997; 5:73-92. 7. Raghupathy R: ThI-type immunity is incompatible with successful pregnancy. Immunol Today 1997; 18:478482. 8. de Fougerolles R, Baines M: Modulation of natural killer activity influences resorption rates in CBA x DBA/2 matings. J Reprod Immunol 1987; 11:147-153. 9. Kinsky RG, Delage G, Rosin M, Thang N, Hoffman M, Chaouat G: A murine model of N K mediated resorption. Am J Reprod Immunol 1990; 23:73-79. 10. Baines MG, Duclos AJ, Antecka E, Haddad EK: Decidual infiltration and activation of macrophages leads to early embryo loss. Am J Reprod Immunol 1997; 37:471-477. 11. Chaouat G, Menu E, Clark DA, Dy M, Minkowski M, Wegmann TG: Control of fetal survival in CBA x DBA/ 2 mice by lymphokine therapy. J Reprod Fertil 1990; 89:447-458. 12. Yui J, Garcia-Lloret M, Wegmann TG, Guilbert LJ: Cytotoxicity of tumor necrosis factor-alpha (TNF-a) and gamma-interferon (IFN-y) against primary human trophoblasts. Placenta 1994; 15:819-835. 13. Hill JA, Haimovici F, Anderson DJ: Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. J Immunol 1987; 13912250-2259. 14. Tangri S, Wegmann TG, Lin H, Raghupathy R: Maternal anti-paternal reactivity in natural, immunologicallymediated spontaneous fetal resorptions. J Immunol 1994; 152:4903-49 1 1. 15. Tangri S, Raghupathy R: Expression of cytokines in placentas of mice undergoing immunologically-mediated spontaneous fetal resorptions. Biol Reprod 1993; 49:850-856. 16. Hill JA, Polgar K, Anderson DJ: T-helper I-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA 1995; 273: 1933- 1937. 17. Menu E, Kaplan JH, Andreu G, Denver L, Chaouat G: Immunoactive products of human placenta I. An immunoregulatory factor obtained from explant cultures of human placenta inhibits CTL generation and cytotoxic effector activity. Cell Immunol 1989; 119:341-352. 18. Clark DA, Flanders KC, Banwatt D, Millar-Brook W, Manuel J, Stedronska-Clark J, Rowley B: Murine pregnancy decidua produces a unique immunosuppressive

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molecule related to transforming growth factor TGFP2. J Immunol 1990; 144:3008-3014. 19. Stimson WH: The interface of pregnancy-associated serum proteins and steroids on the maternal immune response. In Immunology of Reproduction, TG Wegmann, TJ Gill (eds). New York, Oxford University Press, 1983, pp 283-294. 20. Szekeres-Bartho J, Chaouat G: Lymphocyte-derived progesterone induced blocking factor corrects resorption in a murine abortion system. Am J Reprod Immunol 1990; 23:26-28. 21. Ribbing SL, Hoversland RC, Beaman KD: T cell s u p pressor-factors play an integral role in preventing fetal rejection. J Reprod Immunol 1988; 14:83-95. 22. Chaouat G, Chaffaux S, Voisin GA: Immunoactive products of the placenta I. Immunosuppressive properties of crude and water soluble extracts. J Reprod Immunol 1980; 2:121-129. 23. Chaouat G, Kolb JP: Immunoactive products of placenta 11. Afferent suppression of maternal cell mediated immunity by supernatants from short-term cultures of murine trophoblast-enriched cell suspensions. Ann Immunol Inst Pasteur 1984; 135C:205-218. 24. Chaouat G, Kolb JP: Immunoactive products of murine placenta IV. Impairment by placental products of CTL function at the effector stage. J Immunol 1985; 135:9199. 25. Chaouat G, Kolb JP, Riviere M, Lankar D: Immunoactive products of placenta V. Soluble factors from murine placenta can block effector stages of maternal anti-paternal cell mediated immunity. Am J Reprod Immunol 1986; 12:70-77. 26. Duc HT, Masse A, Bobe P, Kinsky R, Voisin GA: Deviation of humoral and cellular alloimmune reactions by placental extracts. J Reprod Immunol 1985; 7:27-39. 27. Voisin JE, Kinsky RG, Voisin GA: Maternal alloimmune reactions towards the murine conceptus and graftversus-host reaction (GVHR) 11. Inhibition of priming by placental extracts. J Reprod Immunol 1986; 9:85-94. 28. Holland D, Bretscher P, Russel AS: Immunologic and inflammatory responses during pregnancy. Clin Lab Immunol 1984; 14:177. 29. Wegmann TG, Lin H, Guilbert L, Mosmann TR: Bidirectional cytokine interactions in the maternal-fetal relationship: Is successful pregnancy a Th2 phenomenon? Immunol Today 1993; 14:353-356. 30. Menu E, David V, Bensussan A, Chaouat G: Immunoactive products of human placenta 11. Direct inhibition of non-MHC restricted cytolytic activity of human CD3 $3 but not y6 expressing T cell clones. J Reprod Immunol 1989; 16:137-150. 31. Menu E, Kinsky R, Hoffman M, Chaouat G: Immunoactive products of human placenta IV. Immunoregulatory factors obtained from cultures of human placenta inhibit in vivo local and systemic allogeneic and graft versus host reactions in mice. J Reprod Immunol 1991 : 20: 195-204. 32. Chaouat G, Menu E, David F, Djian V, Kinsky R: Reproductive immunology 1989-92: Some important recent advances about the fetomaternal relationship. In Progress in Immunology VIII, Gergely J (eds). Springer Hungarica, 1993, pp 225-231. 33. Krishnan L, Menu E, Chaouat G, Talwar GP, Raghupathy R: Iii oirro and in cico immunosuppressive effects of supernatants from human choriocarcinoma cell lines. Cell Immunol 1991; 138:313-325.

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34. Krishnan L, Kinsky R, Chaouat G, Talwar GP, Raghupathy R: Suppression of local and systemic GVHR by supernatants from human choriocarcinoma cell lines. Cell Immunol 1993; 150:376-381. 35. Krishnan L, Sad S, Raghupathy R: Characterization of an immunosuppressive factor secreted by a human trophoblast-derived choriocarcinoma cell line. Cell Immunol 1995; 162:295-308. 36. Heyborne KD, Cranfill RL, Carding SR, Born WK, O’Brien RL: Characterization of y6-T lymphocytes at the maternal-fetal interface. J Immunol 1992; 149:2872-2878. 37. Bernadotte F, Mattsson R: Enhancement of murine splenic immunoglobulin secretion in uitro by supernatants from fetal and placental cell cultures. J Reprod Immunol 1988; 14~225-232.

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38. Dresser DW: The potentiating effect of pregnancy on humoral immune response of mice. J Reprod Immunol 1991; 20:253. 39. Drake BL, Head JR: Murine trophoblast cells can be killed by lymphokine activated killer cells. J Immunol 1989; 143:9-14. 40. Szekeres-Bartho J, Autran B, Debre P, Andrexu G, Denver L, Chaouat G: Immunoregulatory effects of a suppressor factor from healthy pregnant women’s lymphocytes after progesterone induction. Cell Immunol 1989; 122:281-294. 41. Szekeres-Bartho J, Wegmann TG: A progesteronedependent immunomodulatory protein alters the Thl/Th2 balance. J Reprod Immunol 1996; 3123195.