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PROBLEM: We wanted to further study the mechanisms of immune suppression and suppression inducing capacities by choriocarcinoma products, e.g. both crude human choriocarcinoma supernatant (HCS) and especially an active fraction obtained by high performance liquid chromatography (HPLC) from the culture supernatant of the Jeg-3 human choriocarcinoma cell line, since it appeared by weight and charge criteria to be a different molecular species than the low molecular weight fraction previously isolated from mouse and human term placenta. It was important to know whether the purified material was active in vivo as it was in vitro. Therefore, we tested the effects of HCS in vivo in three systems: prevention of fetal demise in the CBA/J x DBA/2 abortion prone murine mating combination, where the effects of the HPLC purified fraction were also monitored as well as by a cell transfer system, where the suppression is revealed by a local GVH/HVG assay, and finally enhancement the survival of a mildly immunogenic tumor allograft. METHODS: An active fraction was isolated from HCS by ion exchange HPLC. Female CBA J were mated with DBA/2 and the influence of 3 intraperitoneal injections of both crude HCS and the active fraction was evaluated by monitoring the percentage of fetal resorptions. Simultaneously, on the day when resorptions were counted, maternal splenocytes from these females were harvested and were injected by the subcutaneous way in C3H/HEJ hind feet. The lymph node reactivity (HVG + GVH) was assessed by [ 3 H]thymidine intake by cells harvested from the draining popliteal lymph nodes. For assessment of influence of HCS on allograft rejection, BALB/b (H-2 b ) mice received a subcutaneous injection of allogeneic P815 tumor cells (H-2 d ). The influence of HCS injections on tumor survival was analyzed by regular measurements of the mean tumor diameter. RESULTS: Intraperitoneal injection of HCS reduced fetal resorptions from 24.7 to 13%. Injection of the in vitro active fraction induced the same rate of reduction. The mean intensity of HvG GvH reaction was 13400 cpm per lymph node when splenocytes from the control group were injected compared to 2900 cpm when splenocytes from treated mice were used. P815 tumor allografts were completely rejected in all cases after 21 days. Weekly subcutaneous injections of HCS prolonged tumor survival in all cases up to at least 30 days. CONCLUSION: The fraction isolated from HCS increased very efficiently the survival of allografts as well as those of allogeneic fetuses in a resorption prone murine mating. The choriocarcinoma cell line might prove to be a useful source of immunosuppressive materials, which could otherwise be important for the fetal-maternal tolerance and a successful pregnancy.

CBA x DBA/2 placentae are quantitatively or qualitatively deficient in their production of the anti-inflammatory Th2-type cytokines IL-4 and IL-10 compared with the nonresorption-prone CBA x BALB/c mating combination. Wastage in this mating combination is accompanied by increased levels of local inflammatory cytokines. In addition, alloimmunization enhances the placental production of IL-4 and IL-10 in CBA x DBA/2 matings. Furthermore, rIL-10 by itself completely reverses the high incidence of fetal resorption after i.p. injection. Conversely, anti-IL-10 increases the resorption rate, but only in CBA x DBA/2 matings. On the other hand, injecting either anti-IFN-gamma or pentoxifillin (an anti-TNF agent) partially reduces the resorption. When given together, they produce a synergistic remission of fetal loss. Finally, we report that recombinant ovine trophoblast protein, an IFN-tau which is known to influence reproductive outcome in ruminants, can also counteract increased CBA x DBA/2 fetal resorption. It simultaneously induces increased placental IL-4 and IL-10 production in this mating combination. These results indicate that the placentally produced anti-inflammatory cytokines can play a vital role in the survival to term of the fetal allograft, by counteracting deleterious inflammatory cytokines.

Pregnancy is controlled primarily, though not exclusively, by a delicate equilibrium between locally acting growth factors and cytokines, some under steroid control. The hypothesis considered here is that stress is able to influence the equilibrium between cytokines and thus lead to abortions or implantation failure. We thus detailed the studies on that topic in order to explore the psycho-neuro-immunological mechanisms concerned. The duration of stress, the patient's strategy for coping with this and the social context might be able to produce some opposite immunological effects. Thus, the link between stress and the immunological events induced is complex, and much care is needed for such patients.

To determine if interleukin-16 (IL-16), IL-17, and IL-18 are present at the murine fetomaternal interface during pregnancy as a first step towards investigating their roles in fetomaternal relationship.Expression of IL-16, IL-17, and IL-18, was assessed by immunohistochemistry (IHC) in the BALB/c x BALB/k (H2d x H2k), and the CBA/J x BALB/c non-abortion prone, and CBA/J x DBA/2 abortion prone matings. Enzyme-linked immunosorbent assay (ELISA) were performed for the two latter cytokines to compare local production in the abortion prone CBA/J x DBA/2 versus the non-abortion prone CBA/J x BALB/c matings.Expression of IL-17 was borderline. The anti-IL-16 staining specifically localized in the uterine stroma and glandular epithelium and was rather low in the placenta. IL-18 staining started in the peri-implantation uterus in the basal proliferative stroma, and was also traced, although weaker, in the glandular epithelium. In the immediate post-implantation period, a weak stromal staining persisted but there was a strong labeling of the ectoplacental cone. Interestingly, when the ectoplacental cone differentiates into placenta having a major histocompatibility complex (MHC) class I + spongiotrophoblast and a (MHC class I-) labyrinth, a very strong transient labeling of uterine natural killer (u-NK) cells was found. Later in gestation, IL-18 was also produced by giant cell and spongiotrophoblast. Finally, we compared by ELISA the production of IL-17/-18 in CBA/J x DBA/2 and CBA/J x BALB/c matings. We detected significantly more IL-18 in the non-abortion prone combination decidua or placenta.The three cytokines IL-16, IL-17, and IL-18 were detected at the fetomaternal interface with a tissue specific, stage-dependent distribution. The predominance of IL-18 secretion in the non-resorption prone matings lead us to question the general validity of the classical T-helper (Th)1/2 paradigm.

Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.

In this study, coreceptor usage of HIV-1 other than subtype B in relation to HIV-1 transmission from mother to child was investigated. Repeated sampling of 42 HIV-1-seropositive, asymptomatic women in Cameroon during the second and third trimesters of pregnancy, at delivery, and 6 months postpartum were performed. Env subtyping was carried out from uncultured peripheral blood mononuclear cells (PBMCs) by heteroduplex mobility assay and, whenever necessary, by DNA sequencing. Virus isolates were tested for coreceptor usage on human cell lines-U87.CD4 and GHOST(3)-engineered to express stably CD4 and the chemokine receptors CCR1, CCR2b, CCR3, CCR5, or CXCR4, or the orphan receptors BOB/gpr15 or Bonzo/STRL33/TYMSTR. Transmission rate was 11.9%. Viruses were predominantly envelope subtype A and used CCR5 as coreceptor and, surprisingly, 4 of 28 (14.2%) isolates from mothers and 1 of 3 isolates from children used the orphan receptor Bonzo as well. In 2 transmitting mothers from whom sequential HIV-1 isolates were available, viral coreceptor usage evolved from CCR5 monotropic to CCR5/Bonzo dual tropic during pregnancy, and in 1 case transmission of this virus could be documented. Our data suggest that evolution of HIV-1 coreceptor usage to dual (or multi-) tropism may occur during pregnancy.

An atypical HIV-1 strain (CAM001) was identified in a pregnant Cameroonian woman in 1995. HMA subtyping of the env region was unsuccessful, and sequence analyses were performed. Unique sequence motifs were found at the V3 tip (GAGRALHA and GAGRAWIHA), and phylogenetic studies showed that the env C2-V5 sequence branched within group M but remained distinct from all known HIV-1 subtypes, while p17 gag branched with the subtype F sequences. Four other HIV group M viruses, undetermined by HMA, of African origin were found to cluster with CAM001 in the C2-V5 sequences. With the BLAST method, we found in databases three strains whose V3 sequences also clustered with CAM001. These unusual env sequences from eight HIV-1 strains derived from Cameroon formed a separate cluster in HIV-1 group M, which we designated k.

To understand the mechanisms preventing and/or facilitating maternofetal transmission of human immunodeficiency virus (HIV)-1 across the placenta during pregnancy.Current experimental data were reviewed.The data about the production of cytokines by placental cells and explants, taken together with information indicating selective passage of certain HIV-1 variants across the placental trophoblast, suggest an intricate regulatory network operating at the fetomaternal interface. The data show a differential differentiation of early and late trophoblasts, as far as HIV entry routes are concerned. We believe this explains the relative predominance of the early infection window, as far as in utero infection is concerned. Whether such a differentiation state can be transiently induced on term placental trophoblasts by several differentiation agents, including cytokines, is being investigated. Whatever the results may be, it is obvious that infection of placental cells is an excellent model of passage infection by HIV of/through a mucosal barrier.

PROBLEM: The mechanisms of the immunosuppressive and immunosuppression-inducing capacities of Jeg-3 human choriocarcinoma cell line supernatants (HCSs) are not yet completely understood. The influence on interleukin (IL)-2. IL-4 and interferon (IFN)-γ production: IL-2 receptor (IL-2R) α-, β-, and γ-chain; and the signaling pathway molecules Janus kinase (Jak) 1, Jak3, signal transducers and activators of transcription (Stat) 1, Stat3, and Stat5 should be investigated. METHOD OF STUDY: For assessment of IL production, whole peripheral venous blood from healthy donors was stimulated with phorbol-myristate-acetatz and ionomycine. Secretion of ILs was blocked with monensine. Intracellular ILs were analyzed by flow cytometry. For IL-2R and signaling pathway molecule analysis, peripheral blood lymphocytes were stimulated with phytohemagglutinin (PHA). IL-2R chains were measured by flow cytometry, and Jaks. Stats by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. RESULTS: Phorbol-myristate-acetate and ionomycine strongly increase the percentage of IL-2 + cells: an additional 50% HCSs significantly suppresses the percentage to. or below the level of unstimulated cells. IFN-γ production is strongly decreased by HCSs in some cases, but not in others. PHA stimulates IL-2R α-, β-, and γ-chain expression and their signaling pathway molecules Jakl. Jak3, Stat1, Stat3, and Stat5. 50% HCS downregulates the α-chain and slightly upregulates the β-chain. Jakl. Jak3. Stat1, Stat3, and StatS expression is suppressed approximately to, or below the level of unstimulated cells. CONCLUSIONS: HCS forcefully blocks the production of IL-2; the IL-2R α-chain; and Jakl. Jak3. Stat1, Stat3, and Stat5 expression, The observed phenomena might be caused by downregulation of an IL-2R regulation gene, and might play a key role in the expansion of choriocarcinoma, and possibly in the survival of the fetal allograft.

To determine the mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses the placenta into the fetal blood, 12 matched samples of serial maternal blood, term placentas, and infant blood obtained from a cohort of pregnant women in Cameroon identified as predominantly infected by subtype A viruses were studied. HIV-1 env sequences were detected by polymerase chain reaction (PCR) in both chorionic villi and enriched trophoblastic cells of all 12 placentas but at variable rates of detection. Heteroduplex mobility assay analysis showed the presence of multiple HIV-1 env quasispecies in sequential maternal peripheral blood mononuclear cell samples, but only a small number of env variants were found in chorionic villi and enriched trophoblastic cells. These data indicate that HIV-1 env sequences are always present in term placentas of seropositive women, contrasting with the low frequency at which infection is diagnosed by PCR in neonates with tat, gag, and env primers. Maternal HIV-1 variants appear to undergo a strong negative selection by different cell populations within the placental villi.

Spontaneous resorption in the CBA x DBA/2 model is attributed to NK cells, macrophages, and Th1-type cytokines. In vivo depletion of NK cells by anti-asialoGM1 Ab or macrophage depletion by silicon dioxide treatment reduced abortion rates, which could no longer be boosted by injecting TNF-alpha (which activates NK cells) or IFN-gamma (which activates macrophages). TNF-alpha + gamma-IFN coadministration aborted80% of the embryos whether or not NK cells or macrophages had been depleted or estradiol + progesterone was injected to correct potential reduction in ovarian function by cytokines. The cytokines also aborted IRF1+/+ C57BL/6 but not IRF1-/- females pregnant by IRF1+/+ DBA/2. Both spontaneous and cytokine-boosted abortions in CBA x DBA/2 were blocked by Ab to fgl2 prothrombinase [corrected] expressed by cytokine-stimulated vascular endothelial cells and monocytes; in vivo Ab depletion of granulocytes also prevented TNF-alpha + IFN-gamma-induced abortions. Cytokine-triggered thrombotic/inflammatory processes in maternal uteroplacental blood vessels causes abortion.

Maternal LIF is essential for embryo implantation in mice and it may also be the case in humans. We recently reported that endometrial LIF secretion from infertile women presenting repeated failures of embryonic implantation or unexplained sterility was significantly lower than the secretion of explants from fertile women. We now report on the modulation of the endometrial LIF secretion according to the obstetrical status. CSF-1 has little effect or increases LIF secretion from fertile women whereas it inhibits secretion from infertile women with repeated failures of embryonic implantation.

This review deals with maternal T cell reactivity in pregnancy. Since other chapters consider recurrent spontaneous abortion in human, we focus(very briefly) only on data in such situations when they cast light on maternal T cell status in normal human pregnancy. It is necessary, however, to deal with abortion models in mice since these are models for study of T cell reactivity during gestation.

Human endometrium from fertile women secrete HILDA/LIF in vitro. In endometrial explants from women suffering from an unexplained sterility or women whose fertilized eggs do not implant the in vitro secretion is significantly reduced. HILDA/LIF secretion is increased by IL-1 and decreased by IL-4. CSF-1 has little effects on secretion from fertile women but clearly inhibits secretion from some infertile women. Altogether, the results suggest that HILDA/LIF might be involved in human implantation.

In this brief review, we recall the antigenic status of the embryo and the possible threats that the maternal immune system poses to fetal survival even at implantation stage. We then quickly recall the existence of Early Pregnancy Factor (EPF), the available data for early embryo derived suppressor and suppressor inducer molecules, point to the existence of cloned progesterone dependent T cell derived suppressor molecule, disquisite on immunoregulatory properties of tau interferons, and finish by dealing with Interleukin 10 and the TH1/TH2 balance concept.

Stress is known to induce abortions, but underlying mechanisms are unknown. Both alloimmunization and injection of antibody to the asialoGM1 determinant of natural killer cells have been shown to prevent stress-triggered abortion in mice. DBA/2J-mated CBA/J female mice were used to investigate the influence of stress during early gestation on systemic hormone levels and on cytokines in the decidua that are thought to be relevant to abortion in nonstress-related murine abortion. Lowered levels of progesterone did not occur as a result of stress. In stressed mice, increased levels of the abortogenic cytokine tumor necrosis factor alpha (TNF alpha) were associated with decreased levels of pregnancy-protective transforming growth factor beta 2-related suppressive activity in uterine decidua. In the alloimmunized animals where stress failed to boost the abortion rate, these effects were abrogated. Production of TNF alpha may be stimulated by the neurotransmitter substance P (SP); after injection of an SP receptor antagonist or SP-antibody, stress failed to increase the abortion rate above the background level. The increased levels of TNF alpha we observed in the stressed animals were completely abrogated in the animals that had received the SP receptor antagonist; stress also failed to decrease the pregnancy-protective suppressive activity in the decidua of these animals. The data indicate that stress may inhibit protective suppressor mechanisms and promote secretion of abortogenic cytokines such as TNF alpha via neurotransmitter SP.

Human interleukin for DA cells/leukaemia inhibitory factor (HILDA/LIF) is a cytokine with pleiotropic effects involved in successful murine implantation. We evaluated human uterine HILDA/LIF production by monitoring its in-vitro secretion by endometrial explant cultures obtained from individuals in either normal or pathological conditions. The cytokine secretion was standardized using the day 5:day 1 ratio of HILDA/LIF concentration in supernatants of such cultures, hereby termed HILDA/LIF production index (HLPI). Our results confirmed that HILDA/LIF is secreted by the human endometrium as assessed by secretion at every phase of the cycle in either normal fertile women, or women bearing intrauterine devices. This was also the case for samples obtained from infertile women presenting repeated failures of embryonic implantation or unexplained primary sterility. However, the HLPI were significantly lower in those latter two groups when compared to fertile women. These results suggest an abnormal regulation of HILDA/LIF secretion in such circumstances, and the clinical implication of those data is discussed.

Current evidence support the hypothesis that trophoblast interferons play a key role in preventing maternal immunologal rejection of the embryonic semi-allograft. The information of this review is divided in two sections. In the first section we described molecular and biological characteristics of type I (alpha, beta, omega, tau and spl) and type II (gamma) interferons. In the second section we emphasize studies on immunoendocrine functions of IFN-tau (oTP-1 or trophoblastins) in the network of cytokines and hormonal environment at the uterine embryonic interface.

Suppression of rejection of the conceptus is a thesis arising from the paradigm that the fetus resulting from mating of genetically dissimilar individuals is an allograft that paradoxically is highly successful. Cells able to suppress the generation of cytotoxic T lymphocytes (CTL) against paternal Class I MHC were found in paraaortic lymph nodes draining the uterus in most strains of allogeneically mated mice and, where deficient (i.e., C57B110ScSn, CBA/J), were detectable in the decidua (Clark 1991). Deficient decidual suppressor cell activity was found in mice with a high rate of spontaneous resorption (Clark et al 1986, 1990a, 1991a). It is important to note that kinetic studies of suppressor cell activity in decidua during mouse pregnancy demonstrated two types of suppressor cells, a CD8+ T cell population present during the pre- and peri-implantation phase of pregnancy, and a non-T non-B cell population which developed 4–5 days after implantation (Clark et al 1991b). Both were antigen non-specific, and the latter non-T non-B suppressor cell was particularly interesting as it released a soluble suppressor factor closely related to TGF-β2, and developed just before the usual time of onset of abortion in mice (Clark et al 1990b, 1991b, Lea et al 1992).

Problem: The in vivo gene delivery offers an alternative for affecting the expression of genes without deriving transgenic animals. In order to investigate the biological functions of genes during implantation, the in vivo transfection of the murine uterus was assessed using a non-cationic agent. Material and Methods: Various routes of administration were tested using complexes of JetPEI (Polyplus-Transfection) and pEGFPluc plasmid injected into mice. The luciferase activity was quantified and the GFP cell localisation was investigated in the uterus. The consequences of the injections on the pregnancy were estimated. Results: The efficiency of the in vivo transfection in the uterus as well the type of transfected cells depends on the route of injection. No apparent effect was seen on the pregnancy outcome. Conclusion: Investigating the function of a gene during implantation in the mouse appears feasible using the jetPEI-based method ofgene delivery. Targeted analyses of specific genes are in progress.

Treatment of pregnant CBA/J females with CFA at day 0.5 and 7.5 of pregnancy significantly reduced the fetal resorption rates from 45% to 29% (P less than 0.05). Supernatants of progesterone-treated spleen cells from CFA treated CBA/J females pregnant of DBA/2 males significantly reduced natural cytotoxicity, while those of untreated identically pregnant mice had no effect. Supernatants of CFA-treated virgin mice blocked natural cytotoxicity to the same extent as those of CFA-treated pregnant mice. These data suggest that nonspecific immunostimulation induces progesterone receptors in spleen cells of CBA mice and that these receptors allow a progesterone dependent suppressive pathway to exert an antiresorptive effect.

This review considers the potential reduction of embryo mortality in vitro and in vivo in ruminants. Data on cytokines provided by different fields of reproductive immunology and biology were collated. Because of the crucial importance of the local interactions between the embryo and its dam, the expression of growth-factor and cytokine genes was analysed in the embryo proper, trophoblast, oviduct and endometrium by reverse transcriptase polymerase chain reaction in sheep and in cattle during the pre- and periimplantation periods. Many deleterious cytokines, such as tumour necrosis factor-alpha, interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and beneficial cytokines, such as transforming growth factor-beta, leukaemia inhibiting factor, colony-stimulating factor-1 (CSF-1), granulocyte-macrophage CSF, IL-1, IL-3, IL-4, IL-6, IL-10 and IFN-tau appeared to be involved in embryo survival in ruminants and other species. Their administration is efficient in a murine experimental model (CBA/J x DBA/2) of embryonic and fetal mortality. For instance, recombinant ovine IFN-tau (roIFN-tau) injected at the moment of implantation drastically reduces embryonic mortality in this model. In ruminants, roIFN-tau and recombinant bovine IFN-tau are very efficient in maintaining progesterone luteal secretion in cyclic animals. The involvement of IFN-tau in the mechanisms of maternal pregnancy recognition are particularly detailed in relation to inhibition of 13,14 dihydro-15-keto-prostaglandin F2 alpha (PGFM) pulses and oxytocin uterine receptivity. A synthetic model of the anti-luteolytic effects of IFN-tau on the endometrial cell is proposed. Finally, the particular potential of serum pregnancy-specific proteins (PSPs: PSPB, PSP60, pregnancy-associated glycoprotein) for monitoring embryo survival, with examples given for cattle and sheep is underlined.

CBA/J female mice have a high rate of fetal resorption when mated with DBA/2J males. This fetal wastage can be dramatically reduced by immunizing the female with BALB/cJ but not DBA/2J spleen cells. We report here that immunization with BALB/cJ (but not DBA/2J) spleen cells leads to 1) anti-paternal MHC antibody that is predominantly of the IgG1 isotype, and which disappears from the serum during pregnancy; 2) increased active suppression in both the spleen and placenta; and 3) an ability to adoptively transfer the fetal protection and placental suppression with serum from the immunized mice. Congenic absorption studies before adoptive transfer indicate that the active component of the serum is also directed against the paternal MHC haplotype. These results indicate that maternal humoral immunity can lead to increased fetal protection in correlation with local active suppression in the placenta. They also suggest an expansion of the placental immunoabsorbent hypothesis to include the induction of active suppression against maternal cell-mediated immunity.

Summary Supernatants from short-term cultures of mid-term murine trophoblast cells were assayed for their in vitro regulatory potential. They markedly inhibited cell-mediated lympholysis and mixed lymphocyte reaction in a non-specific, non-restricted fashion. By contrast, the mitogenic response to optimal doses of ConA was unaffected, while the plaque-forming cell response to sheep red blood cells was either unmodified or slightly enhanced. These data suggest that placenta-derived cells secrete factors which selectively impair some cell-mediated immune responses. It is suggested that these factors play an important role in the lack of generation of cytolytic T lymphocytes towards paternal alloantigens expressed on the trophoblast during allopregnancy.

Placental and trophoblast-enriched cells from iso or allopregnant mice (14 to 17 days of gestation) strongly suppress natural killing by mouse splenocytes. The suppression is not strain restricted and acts at the level of the effector cell. It was not reduced by pretreatment of trophoblast cells with RNA and protein synthesis inhibitors, nor by prostaglandin synthetase inhibitors. However, treatment with trypsin abolished the suppressive activity. Because CTL and ADCC cytotoxicity were also inhibited by trophoblast cells at the effector stage, we postulate that trophoblast cells in vivo are endowed with the ability to inactivate various types of cytotoxic cells in their vicinity that are potentially harmful for the conceptus.

The genetic characteristics, surface markers, and mechanisms of action of early-acting, mitomycin-resistant, MLR suppressor T cells from multiparous mouse spleens were studied in MLR of various strain combinations. The cell activity appears to be genetically restricted at the suppressor/reactive cell level, and the major restriction occurs via the IC subregion. However, C3H.OL recombinant pointed toward a possible minor requirement for S-G subregion syngenicity. The suppressor T cell was characterized as an Ia+, Ly1-, Ly2+ subset. The use of Karush double chambers as well as of supernatants of regulated MLR provided evidence that the suppressor cells act via soluble product(s).

The cellular requirements for suppression of a mixed lymphocyte reaction (MLR) by in vivo alloantigen-activated spleen cells were investigated. We found that the suppression was mitomycin-resistant at days 3 and 5 after antigen injection. By contrast, it was radiation-sensitive on day 3 but not on day 5. Cell fractionation studies using a plate separation technique monitored by flow microfluorometry were performed. We found that neither purified Lyt-2- cells nor purified Lyt-2+ cells independently could suppress an MLR. By contrast, the reconstituted T population was still suppressive. It also was found that culture supernatant factors from allogeneically in vitro restimulated Lyt-2- cells could suppress an MLR if Lyt-2+ cells were present in the responding population. These data obtained by positive selection and reconstitution show that at least two surface immunoglobulin-negative cells interact for the generation of effective MLR suppression. The possibility that we are dealing with a suppressor-inducer, suppressor-effector system is discussed.

Trophoblast-enriched cell suspensions prepared by collagenase digestion from midterm murine placentae were found resistant to CTL-mediated lysis. Treatment of such cells by trypsin or neuraminidase rendered these cells susceptible to such lytic effectors. Collagenase-prepared cell suspensions could impair CTL action, whereas neuraminidase- or trypsin-treated cells did not retain this property. This effect was also observed with extracts. These results indicate that soluble factors (which we will characterize in another paper) released by trophoblast cells (in fact, spongiotrophoblast) can interfere in a dose-dependent fashion with the action of lytic effectors. We suggest that such active mechanisms are physiologic components of the placental barrier and might be defective in some cases of immunologic abortions.

CBA females (H-2k) mated with DBA2 males (H-2d) exhibit a high rate of fetal resorption (30%) when compared with the CBA female BALB/c male, CBA female/CBA male, DBA2 female/CBA male, DBA2 female/DBA2 male combinations (6 to 8%). Preimmunization of CBA females with spleen cells from DBA2, BALB/c, or CBA males were performed in order to test their effects on CBA maternal tolerance of (CBA X DBA2)F1 fetuses. Only preimmunization with BALB/c male cells was effective in decreasing resorption; cells from BALB/c females had no effect. In order to further test 1) the role of non-MHC-encoded antigens present in the BALB/c male background, 2) the necessity of an additional H-2 difference, and 3) whether or not the phenomenon is H-2d restricted, preimmunizations were performed by using cells from congenic BALB/k (H-2k), BALB/b (H-2b), or BALB/c (H-2d). Only the latter treatment was efficient, which suggests that the paternal H-2d haplotype must be presented in synergy with some non-MHC-encoded antigens in the BALB/c male background. Immunogenetic studies with cells from nine recombinant inbred strains that reassorted DBA2 and BALB/c genomes showed that three of them behave like BALB/c and six like DBA2. This would suggest that the genetic determinism of this phenomenon is simple.

There is little detailed information on the distribution of granulocytes-macrophages and lymphocytes at the pre- and peri-implantation period of the embryo in the uterus. Using two monoclonal antibodies (MAS-099C and MRC-OX41) we found that cells labelled with antibodies specific to either T lymphocytes and thymocytes or granulocytes-macrophages were present in the endometrium and myometrium before ovum implantation. Both types of labelled cells appeared to migrate from the uterine lumen to the deep endometrium, where their number peaked on day 4. At the early implantation period (days 5 to 7), there was a total lack of labelled cells around the conceptus. Specific changes before ovum implantation in the distribution or activation state of T lymphocytes and granulocyte macrophages may favour early embryo acceptance.