An in vitro and in vivo analysis of murine immunocompetence during pregnancy and lactation

Spleen cells from pregnant and lactating BALB/c mice were depressed in their cytolytic capabilities after in vivo immunization with the allogeneic EL4 lymphoma. However, in vitro spleen cells from both syngeneic (BALB/c X BALB/c) and allogeneic (BALB/c X C57BL/6J) matings responded with proliferative and cytolytic responses which were comparable to virgin controls. Upon secondary in vitro stimulation, in vivo primed maternal cells had responses which were similar to virgin controls. In addition, the in vivo sensitized maternal spleen cells adoptively immunized in irradiated allogeneic recipients responded like the virgin controls. In these studies, suppressor cells could not be detected in either nonimmune or immune maternal spleen cell populations.     

Deviation of humoral and cellular alloimmune reactions by placental extracts

Modifications of the alloimmune response at both the humoral and the cellular levels by placental extracts (PE) syngeneic to the recipient were studied in the mouse using two different H-2 strain combinations. CBA (H-2k) or C57BL/Ks (H-2d), immunized with A/J (H-2a) spleen cells. The tests included in vivo tumor allograft evolution (accelerated rejection or enhancement reactions), and in vitro analysis of the involved immune agents, both cellular and humoral, using mixed lymphocyte reactions (MLR) and biological activity studies of serum samples. Animals from the recipient strains exhibited a delayed rejection of A/J tumor Sa 1 allografts if preimmunization was carried out with 10(6) A/J spleen cells combined with PE syngeneic to the recipients, as compared to controls immunized with A/J cells only or supplemented with isogeneic liver extracts (LE). The serological analysis revealed that PE treatment did not modify the overall hemagglutinating antibody production but resulted simultaneously in both a decreased production of cytotoxic complement fixing antibodies and an increase of specific anaphylactic mast cell degranulating antibodies, as compared to controls. The sera from PE-treated donors also demonstrated enhancing activity following passive transfer to isogeneic recipients. MLR regulatory activity was exhibited by spleen cells from PE- and immunogen-treated mice although the same or stronger activity was obtained from mice immunized without the addition of PE. However, in vivo transfer of these cells to syngeneic recipients showed that PE treatment erased the accelerated rejection caused by allogeneic immunization in the absence of PE and could even cause some degree of allografted tumor enhancement. The cells responsible for this inhibitory effect were mainly IJ+ lymphocytes, since their elimination with a relevant anti-IJ serum and complement restored a secondary type rejection pattern. These results show that PE present during the onset of immunization can promote the activation of regulatory agents such as enhancing antibodies and suppressor cells favoring allograft survival.     

Existence of suppressor cells in the spleen of allogeneic and syngeneic primiparous pregnant mice

Mixed lymphocyte reaction (MLR) and in vitro induction of cytolytic cells against alloantigens were investigated using spleen cells from primiparous mice mated allogeneically or syngeneically. One-way MLR was reduced significantly in degree not only in allogeneic but also in syngeneic pregnant mice. MLR of virgin spleen cells was suppressed when mitomycin C-treated spleen cells taken from syngeneic or allogeneic pregnant mice were added as regulator cells. These suppressive effects disappeared when regulator cells were treated with anti-Thy 1 or anti-Lyt 2 serum plus complement. Generation of cytotoxic lymphocytes from syngeneic or allogeneic pregnancy spleen cells in MLR was depressed compared with that from virgin spleen cells. The addition of pregnancy spleen cells to MLR suppressed in vitro generation of cytotoxic lymphocytes from virgin spleen cells. These results indicated that reduction of in vitro cellular responses of pregnancy spleen cells was due to suppressor cells in the spleens. These cells suppressed non-specifically the reactions to alloantigens and could be detected both in allogeneic and syngeneic primiparous pregnancies.     

Maternal alloimmune reactions towards the murine conceptus and graft-versus-host reaction (GVHR). I. Priming for anti-paternal GVHR by gestation

In the H-2 compatible (but minor loci-incompatible) BALB/c-DBA/2 strain combination (both H-2d), intravenous injection of 1.3 X 10(7) BALB/c spleen cells from virgin females into DBA/2 newborn mice less than 18 h old does not result in a significant lethal graft-versus-host reaction (GVHR). A strong GVHR (79% lethal) is induced if the BALB/c donors have been preimmunized to DBA/2. Spleen cells from BALB/c mice pregnant by DBA/2 males are also able to induce a significant, but weaker, GVHR (16% lethal) indicating a cellular priming to paternal antigens by gestation. A significant difference exists between anti-DBA/2 GVH reactivity of spleen cells from primiparous (22% lethal) and multiparous (9% lethal) allopregnant BALB/c mice, indicating that the allogeneic boosters of successive allogestations act more on the target-protective side of immunity than on the target-aggressive one. Sera from allopregnant mice (BALB/c X DBA/2) inhibit the GVHR induced by their own cells, while sera from isopregnant ones (BALB/c X BALB/c) have no effect. Thymectomy performed at 6-wk of age, six weeks before gestation did not significantly modify the maternal reactivity. A similar priming by allogestation in the same strain combination was found for local GVHR (induced in adult F1 hybrids) resulting in higher (+132%, P less than 0.005) stimulation indices and seen to be specific for the paternal strain, the indices induced by the same cells being lower (-35%, P less than 0.05) compared to that induced by cells from virgin BALB/c, when injected into irrelevant F1 hybrids (BALB/c X CBA).     

Maternal immunoregulation: interrelationship between alloreactive and anti-self plus conventional antigen T sets of cells

In a previous paper we reported early immunoregulatory mechanisms involving not only the appearance of progressive suppression but also significant increases in alloreactive T levels in paraaortic lymph nodes (PALN) and spleen, not only in allogeneic but also in syngeneic pregnancies. Taking into account the hypothesis of the superposition of the alloreactive and the anti-self plus conventional antigens T sets of cells, we investigated whether immunization with conventional antigens was able to alter alloreactive T levels. Weekly i.p. doses of rabbit red bloods cells (RRBC) in BALB/c mice resulted in a dose-dependent increase in spleen alloreactivity as determined by graft-versus-host (GvH) assays in strain combinations differing at H-2 level but not in those sharing the same H-2 with BALB/c. The increases could be significantly decreased by an anti-idiotype anti-RRBC serum. Pretreatment with i.p. weekly doses of sheep red blood cells (SRBC) before mating was able to induce dose-dependent fetal damage when the parents differed at the H-2 level. SRBC immunization in one of the uterine horns induced increases in PALN weight which were much higher in progesterone-pseudopregnant than in virgin mice; T alloreactivity was significantly increased in the draining PALN only in pseudopregnant females. These results favour the postulation of the superposition between the alloreactive and the anti-self plus conventional antigens T sets of cells and suggest a possible role for conventional fetal antigens (non H-2) in triggering immunoregulatory mechanisms operating in pregnancy.     

Maternal cell traffic in allogeneic embryos

Blastocysts from CBA and/or C57BL/6 mice were transferred into the uterus of allogeneic, syngeneic or semi-syngeneic pseudopregnant female mice. Observations of embryo status, relative embryo position in the uterus and immunofluorescent staining of embryonic cells for determination of H-2 antigen type shows that cell movement occurs between the maternal host and the fetus and apparently among embryos. Both in-utero maternal cell traffic in the fetus and the rate of fetal resorptions depend on the proximity of allogeneic embryos in utero and the genetic inter-relationship of the surrogate mother to the fetus.     

Effect of the dam strain on the spontaneous incidence of cleft lip and palate and intrauterine growth of CL/Fr mouse fetuses

Purpose The effects of dam strain on the spontaneous incidence of the cleft lip and palate (CLP) and the intrauterine growth of transferred CLP-susceptible CL/Fr embryos were examined with an embryo transfer technique. The CL/Fr strain of embryos at the early blastocyst stage was transferred to the same dam strain and also to the CLP-resistant C57BL dam strain. A laparotomy was done on the 18th gestational day at which time the number of fetuses, the resorption sites and the fetus weight were recorded. Each fetus was checked for the presence of CLP. Five criteria to assess the reproduction and fertility as well as the fetus weight were then compared between both dam strains.Results The dam pregnancy rate and the fetus survival rate in the CL/Fr dam strain were both significantly lower than those in the C57BL dam strain. The resorption rate in the CL/Fr dam strain was significantly higher than that in the C57BL dam strain. The spontaneous incidence rate of CLP in the CL/Fr dam strain was also significantly higher than that in the C57BL dam strain. The fetus weight of the CL/Fr fetuses developed in the CL/Fr dam strain was significantly lighter than that of the CL/Fr fetuses developed in the C57BL dam strain.Conclusion The results indicated that the CLP-susceptible CL/Fr dam strain provided a less favorable uterine environment for the implantation, survival and intrauterine growth of the transferred CL/Fr embryos and also caused a higher spontaneous incidence rate of CLP. Thus, it can be concluded that the effect of the dam strain appears to play an important role on the spontaneous incidence of CLP and the intrauterine growth of the CL/ Fr strain embryos transferred to both CL/Fr and C57BL dam strains.     

Natural history of fetal cell microchimerism during and following murine pregnancy

In humans, fetal cells enter the maternal circulation during all pregnancies and can persist for decades. Human studies, however, are often limited by the number of subjects and the availability of healthy and diseased tissues for analysis. We sought to develop a murine model to establish the natural history of fetal cell microchimerism in various maternal tissues during and after healthy pregnancies resulting from congenic and allogenic matings. We bred C57BL/6J and DBA/2J virgin female mice to C57BL/6J males transgenic for the enhanced green fluorescent protein (GFP), which shows autosomal dominant inheritance with complete penetrance and is under the control of a ubiquitous chicken beta-actin promoter and a cytomegalovirus enhancer. During pregnancy and at different times after delivery, female mice were sacrificed. Tissues were collected and the presence of the gfp transgene and GFP+ cells was assessed by real-time quantitative PCR and by immunofluorescence. During pregnancy, microchimerism was detected in all tissues from mice carrying GFP+ fetuses. Fetal cells were often mononuclear. The frequency of fetal cells in the lungs was significantly higher compared to other tissues. The level of microchimerism was also significantly higher in congenic compared to allogenic matings. After delivery, the frequency of fetal cells decreased and fetal cells were undetectable at 2 and 3 weeks after the first delivery. However, some mice that had three gestations had detectable fetal cells 3 weeks after their last delivery. Using sensitive methods of detection, we demonstrate that fetal cell microchimerism occurs during all murine pregnancies. We describe a useful model for the study of the consequences of this phenomenon.     

Prevention of graft rejection by donor type II CD8(+) T cells (Tc2 cells) is not sufficient to improve engraftment in fetal transplantation

OBJECTIVES: Tc2 cells, a subset of CD8(+) T cells, are able to facilitate engraftment in a murine model of postnatal allogeneic bone marrow transplantation. The purpose of this study was to evaluate whether Tc2 cells could improve engraftment in fetal transplantation. METHODS: Gestational day 13 C57BL/6 (H-2(b)) fetal mice were used as recipients, adult B6D2F(1) mice (C57BL/6 x DBA/2, H-2(b/d)) as donors, and splenocytes from B6C3F(1) (C57BL/6 x C3H/He, H-2(b/k)) mice were used as stimulators in cultures used to generate the Tc2 cells from B6D2F(1) mice. Peripheral blood chimerism was examined monthly for 3 months. Thereafter, recipients were sacrificed to evaluate the levels of peritoneal, splenic and bone marrow chimerism. The T-cell responses of recipient splenocytes to cells of host origin were measured as a proliferative response in mixed lymphocyte cultures. RESULTS: Low levels of peripheral blood cell chimerism (<0.3%) were observed at 1 month of age, which declined further by 3 months of age. The levels of donor cells in the spleen, bone marrow and peritoneal cavity were usually not more than 0.05%. The peritoneal cavity tended to have higher levels of donor cells with 1 recipient sustaining as high as 25.03% at the age of 3 months. Higher peritoneal chimerism correlated with a lower donor-specific T-cell response. CONCLUSIONS: Transplantation of Tc2 cells was insufficient to improve bone marrow engraftment in utero, suggesting that graft rejection is not the major barrier to successful in utero transplantation. Donor cells can persist in the peritoneal cavity and might play an important role in inducing immune tolerance in fetuses.     

Immunization with a syngeneic regressor tumor causes resorption in allo-pregnant mice

The purpose of our studies is to establish experimental systems in which one can deliberately disrupt the apparent maternal tolerance toward the semiallogeneic fetuses. Bases on the hypothesis that immunization against tumor-associated antigens may lead to a subsequent immune response directed against cross-reacting fetal antigens, we have immunized C57BL/6J female mice with a syngeneic regressor tumor. Mice were subsequently mated to B6D2F1, DBA/2, CBA/J or C57BL/6J males. We show that a high proportion of embryos sired by either B6D2F1 or DBA/2 males undergo resorption whereas those engendered by CBA/J or C57BL/6J males remain fully protected.     

Successful pregnancy in ovariectomized mice using a combination of heterotopic autotransplantation of ovarian tissues and embryo transfer

Aim:  The present report is the first to show that, after ovariectomy, female mice with autotransplanted ovarian sections can maintain pregnancy after embryo transfer (ET) independent of the transplantation site.
Methods:  Three-month-old ICR females were ovariectomized, and sections from their own ovaries were transplanted either under their kidney capsule (KC group) or into a subcutaneous space (SC group) just after ovariectomy. In vitro fertilized blastocysts were transferred into uterine horns of the pseudopregnant mice that had received the transplanted ovarian tissues. Cesarean sections were carried out 17 days after ET to deliver any live fetuses that were present, and the numbers of implantation sites and fetuses were noted. Transplanted ovarian sections were removed and fixed for histological analysis.
Results:  Of the 10 mice in the KC group that received 107 blastocysts, two females (20%) became pregnant; they showed 12 implantation sites (11.2%) and produced four pups (3.7%). In the SC group, 101 blastocysts were transferred to eight females, and three females (37.5%) became pregnant; there were seven implantation sites (6.9%), and three pups (3.0%) were born. There were no statistically significant differences between the two groups in any of the parameters evaluated. On histological examination, luteinization and vascularization of the ovarian sections that were transplanted in the pregnant SC and KC females were noted.
Conclusion:  The pregnancy and full-term fetal development were obtained in ovariectomized mice using a combination of heterotopic ovarian tissue autotransplantation and transfer of embryos produced by in vitro fertilization.     

Characterisation of the humoral immune response during murine pregnancy

Blood samples from female C57BL/10 mice mated with CBA/Ca males were obtained before, during and after both first and second pregnancies. A cellular enzyme-linked immunospecific assay (CELISA) was used to detect maternal antibodies against antigens on paternal splenocytes. Alloantibodies were detected in 48% of mice during or 9 days after a first pregnancy and in 82% of mice by the ninth day after the second pregnancy; these antibodies were first observed on day 10 of the first pregnancy. In two of four active multigravid sera tested, an increase in IgG1 concentration was detected; the level of all other isotypes remained within normal limits. Weak binding of alloantibody to an antigen of approximate molecular weight 44,000 was detected on CBA/Ca splenocytes by immunoblotting sera from multiparous animals. These sera also recognised an antigen of similar molecular weight on H-2b identical 129J splenocytes but not on splenocytes from the maternal strain. These results provide further information on the maternal humoral immune response during murine pregnancy.     

An investigation of allogeneic pregnancy in multiparous mice subjected to in vivo depletion of CD8 (Ly2)-positive lymphocytes by monoclonal antibody treatment

Adult thymectomized C57/Bl (H-2b) and DBA/1 (H-2q) female mice were subjected to treatment with rat anti-mouse CD8 and mouse anti-rat Ig (kappa) prior to entering their third pregnancy with CBA/Ca (H-2k) males. The treatment protocol drastically reduced the number of CD8 (Ly2)-carrying lymphocytes (T-cytotoxic/suppressor phenotype) in the spleen and para-aortic lymph nodes, as assessed by immuno-staining. All mice were investigated on day 18 of their third gestation. The following data were collected from experimental and control groups: (1) resorption frequency, (2) weight of the placenta, fetuses, spleen and para-aortic lymph nodes, (3) immunohistochemical analysis of maternal lymphoid tissues, (4) level of anti-paternal IgG serum antibodies, (5) content of "background" IgM and IgG-secreting cells in spleen and para-aortic lymph nodes. Neither the resorption frequency nor placental/fetal weight was affected by anti-CD8 treatment. However, the formation of anti-paternal antibodies was enhanced in anti-CD8 treated C57/Bl mice.     

Reduction in popliteal lymph node graft-versus-host reactivity by homologous and heterologous pregnancy serum

The popliteal lymph node assay was used to investigate the effect of pregnancy on graft-versus-host reactivity (GvHR) of mouse spleen cells. After local injection of splenocytes from primiparous syngeneically pregnant (by BALB/cJ males) or allogeneically pregnant (by CBA/Ca males) mice no differences in lymph node weight gain were observed in F1 recipients (CBA/Ca x BALB/cJ) when compared to injections of cells from age-matched non-pregnant BALB/cJ mice. However, lymphocytes of pregnant BALB/cJ females which had previously been pregnant between 4 and 6 times by CBA/Ca males induced a significantly lower GvHR compared to cells of matched non-pregnant multiparous mice. These results suggested an inhibitory effect of gestation on cells possibly primed towards paternal antigens by multiple pregnancies. To test this hypothesis, virgin BALB/cJ mice were actively immunized with lymphocytes of male CBA/Ca mice. Before injection into F1 recipients, spleen cells of immunized animals were incubated for 1 h at 37 degrees C in heat-inactivated serum of primiparous pregnant or virgin non-pregnant mice. Pre-incubation in pregnancy serum had no effect on unprimed cells, but GvHR of cells derived from immunized donors was significantly depressed in female recipients. In male animals this effect was only irregularly observed. Inhibition of GvHR was also observed with serum from pregnant but not non-pregnant pigs. Depression of cellular immune response was observed as early as days 4-9 post-coitum (p.c.) with mouse serum and days 16-19 p.c. with pig serum. These results indicate that pregnancy serum contains factor(s) which modulates the GvHR of primed lymphocytes in both a species- and an antigen-non-specific manner while reactivity of naive spleen cells is not changed.     

Reactivity to alloantigens and polyclonal mitogens and CD4+/CD8+ cell ratio shifts of cervical lymph node and spleen cells during pregnancy in rats

Cervical lymph node (CLN) cells and spleen cells were harvested from virgin and pregnant rats bearing syngeneic or allogeneic fetuses at all stages of pregnancy including the pre-implantation period. The specific and non-specific alloreactivity of these cells were analyzed in MLR against mitomycin-C treated paternal strain or unrelated cells. Mitogen stimulation of the cell cultures utilized PHA, Con-A and PWM. Cells bearing T cell markers were labeled in an indirect assay using the monoclonal antibodies W3/25 and MRC OX 8. Specific alloreactivity is enhanced at mid-pregnancy in both cell populations. Non-specific alloreactivity was suppressed in the cervical lymph node cells. Spleen cells demonstrated an increased non-specific alloreactivity and T polyclonal mitogen reactivity (PHA and Con-A) at mid-pregnancy. Reactivity to Con-A was depressed in the early phase and at the end of allogeneic pregnancy in the CLN. The CD4+/CD8+ ratio was very low during all phases of pregnancy.     

Maternal background strain influences fetal-maternal trafficking more than maternal immune competence in mice

The objective of this study was to determine if fetal-maternal cell trafficking is affected by maternal immune competence and/or parental background strain using fluorescence-activated cell sorting (FACS). In our experience the sensitivity of FACS allows for the detection of 5 fetal in 10(7) maternal cells and assessment of cell surface phenotype. Wild-type C57BL/6J (n=18), FVB/NJ (n=15), and immunodeficient B6129S7-Rag1(tm1Mom)/J (n=16) female mice were mated to C57BL/6J males homozygous for the green fluorescent protein (GFP) transgene. Single cell suspensions of maternal lung, liver, spleen, bone marrow, and blood were analyzed between late gestation (day e16-18) and 1 day post-partum for the number of GFP-positive fetal cells in relation to 10(7) maternal cells and the percentage of GFP-positive cells that expressed the surface markers CD11b, CD29, CD34, CD44, or CD105. The highest relative proportions of GFP-positive fetal cells were observed in maternal lungs and livers from immunocompetent allogenic females. Among congenic matings, fetal cell microchimerism was higher in immunodeficient compared with immunocompetent females. Maternal strain and strain differences between the mother and father statistically significantly affected both the numbers of fetal cells and the relative distribution of cell types in maternal organs. The highest relative proportion of fetal cells was observed in allogenic matings with immunocompetent females. Since allogenic matings are more similar to those that occur in humans, future studies using animal models of microchimerism should consider incorporating this type of experimental design.     

Influence of immunization with allogeneic spleen cells on the number of viable neonates in mice

Female CBA/J (H-2k) mice mated with male DBA/2J (H-2d) mice show a high level of fetal resorption, which can be reduced by immunization with BALB/c (H-2d) spleen cells. The morphologically defined fetal resorption rate upon which evaluation of the outcome of pregnancy has previously been based in this strain combination is not equivalent to the rate of production of viable neonates.     

Differences in immunogenicity indicating polymorphism of sperm antigens from mice of different inbred strains

Polymorphism of mouse sperm was investigated by analysis of immune sera generated in BALB/c female mice against sperm from 6 inbred strains. The immune sera were analyzed by immunofluorescence and Western blot techniques against sperm antigens from the 6 immunizing strains. Immunofluorescence revealed no differences in reactivity patterns or titers. However, several different reaction patterns were detected by Western blot technique which indicated that both the sperm extracts and the antisperm immune sera contained different components. Syngeneic (anti-BALB/c sperm) antisera showed far fewer reactive antibody species than allogeneic immune sera. The anti-BALB/c sera recognized an antigen of 23 kDa in sperm extracts from DBA/2J and C57BL/6 mice, and failed to react with an antigen of the same molecular weight when applied to sperm from A/J and 129/J mice, indicating antigenic differences between sperm from these inbred strains. Anti-C57BL/6 sera contained a unique antibody which reacted with an antigen of 80 kDa in all of the 6 sperm extracts, whereas others antisera did not detect this antigen. These findings indicate antigenic and immunogenic polymorphism in sperm from different inbred strains of mice.     

Inducible and endothelial nitric oxide synthase: genetic background affects ovulation in mice.

OBJECTIVE: Inducible nitric oxide synthase (NOS) and endothelial NOS are involved in female reproductive physiology. We sought to investigate the influence of the inducible (Nos2) and endothelial (Nos3) NOS genes as a function of genetic background on ovulatory capacity and early embryonic development in a mouse model. DESIGN: Observational study of genetically altered mice and their response to a superovulation protocol. SETTING: Academic research institution. ANIMALS: Wild-type mice and mice deficient for Nos2 or Nos3 were bred to C57BL/6J and 129/Sv genetic backgrounds. INTERVENTION(S): Superovulation protocol, oocyte culture. MAIN OUTCOME MEASURE(S): Number of oocytes harvested, early embryonic development of zygotes, evaluation of ovarian histology. RESULT(S): The mean number of oocytes was significantly reduced in Nos3 deficient mice on a C57BL/6J background compared with controls. Oocytes deficient for Nos3 on a C57BL/6J background also showed reduced progression to two-cell stage embryos after 24 hours, two-cell stage embryos to blastocyst stage embryos, and survival to 48 hours. Those effects were distinctly absent in mice deficient for Nos3 on a 129/Sv background and in mice deficient for Nos2 on either genetic background. CONCLUSION(S): Our data show that disruption of Nos2 had no effect on ovulation in our mice. The negative effect of Nos3 deficiency on ovulatory capacity and early embryonic development is modulated by genetic background. This suggests a role for strain-specific modifier genes in these processes.     

Increased maternal T cell autoreactivity associated with syngeneic murine pregnancy

In this study T cells isolated from isopregnant and virgin CBA/J mice were examined for reactivity to self antigen(s) in vitro and in vivo. The autoproliferative capacity of maternal versus virgin T cells was tested in vitro using autologous mixed lymphocyte reactions (AMLR). The popliteal lymph node (PLN) assay was used to compare the ability of maternal versus virgin T lymphocytes to mediate syngeneic graft-versus-host (SGvH) reactions in vivo. Splenic T cells obtained from pregnant animals near term were found to be approximately 10-fold more reactive towards syngeneic virgin non-T stimulator cells in AMLR than splenic T lymphocytes from age-matched virgin animals. In addition, T cells isolated from the spleens of gravid CBA/J mice displayed a significantly enhanced capacity to mediate SGvH reactions in virgin CBA/J females as measured by regional lymph node enlargement. These findings indicate that syngeneic murine pregnancy is accompanied by an increase in autoreactive T cell activity.