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.     

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.     

Pregnant and pseudopregnant mice produce a low cytotoxic alloantibody response when challenged by fetal but not adult F1 alloantigens

The tertiary cytotoxic alloantibody response of syngeneically pregnant, pseudopregnant and non-pregnant BALB/c mice immunised with semi-allogeneic lymphocytes from fetal and adult (CBA X BALB/c)F1 donors was investigated by the microcytotoxicity method. The pregnant and pseudopregnant animals showed a significantly lower response to fetal alloantigens compared to that towards adult alloantigens (P less than 0.001). In contrast, control non-pregnant recipients showed no diminution of response to fetal alloantigens. These observations suggest that the hormonal status of pregnancy, even in the absence of a conceptus, permits a low cytotoxicity antibody response when challenged specifically by fetal, but not when challenged by adult, F1 alloantigens.     

Effects of pseudopregnancy on immunoglobulin-secreting cells in mice

Pregnant mice characteristically show elevated numbers of immunoglobulin-secreting cells in their enlarged spleen and para-aortic lymph nodes. A comparative study of pseudopregnancy and syngeneic pregnancy in CBA/Ca mice was made to evaluate the role of maternal hormones in the induction of these changes. To induce pseudopregnancy, CBA/Ca female mice were mated with vasectomized males, the duration of pseudopregnancy induced in this way is 8-10 days. Serum progesterone levels were monitored periodically throughout pseudopregnancy using RIA technique. Changes were recorded in weight of the thymus, spleen and para-aortic lymph nodes, levels of serum IgG and IgM (ELISA-technique), and content of splenic IgG- and IgM-secreting cells (protein A plaque assay). Thymus involution was observed in pregnant and pseudopregnant mice. Patterns of change in the weight of the spleen and the para-aortic lymph nodes (PALN) were similar in pregnant and pseudopregnant mice until day 8. Ig-secretion in the spleen increased slightly day 5-8 in both pregnant and pseudopregnant mice, but to a lesser extent in the latter. No differences were observed in serum Ig levels between the two groups until day 8. A marked decrease in serum IgG levels and, to some extent, IgM levels between days 8 and 12 was observed in pregnant animals.     

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).     

Transfer of susceptibility to experimental autoimmune orchitis from responder to non-responder substrains of BALB/c mice

Substrains of BALB/c mice differ in their susceptibility to experimental autoimmune orchitis (EAO), with BALB/cJ representative of the non-responders and BALB/cBy representative of the responders. We examined whether the susceptibility of these two substrains could be altered by reciprocal adoptive transfer of lymphoid cells. The cells transferred were of three types, normal spleen cells, T cell-enriched spleen and lymph node cells from mice immunized with testis homogenate (TH) in complete Freund's adjuvant (CFA) and given an extract of Bordetella pertussis (BP) and the latter cells activated by in vitro culture with TH antigen for 48 h. Controls were given buffer alone. Cell or buffer recipients were immunized with TH + CFA + BP three weeks later and examined for testicular histopathology 25-28 days after immunization. The cultured, immune T-enriched cells were consistently effective in transferring susceptibility from BALB/cBy to BALB/cJ. In the reverse experiments, non-responsiveness could be transferred from BALB/cJs to BALB/cBys most effectively with immune, non-cultured T-enriched cells. Transfer of cultured, immune T-enriched cells from BALB/cJs to other BALB/cJs had no significant effect on susceptibility to EAO. The results suggest that susceptibility to EAO in BALB/c mice depends on the T cell responses in the mice and not on differences at the level of the testis.     

Allogeneic reactivity of maternal lymphoid cells during the course of gestation. Modifications and sex differences in a local GVH assay

The immune reactivity of lymphoid cells from pregnant mice was studied during the course of pregnancy in primiparous and multiparous animals either " isopregnant " (male and female of same strain) or " allopregnant " (male and female differing at H-2), using a local GVH assay (CBA lymphoid cells injected into (CBA X A/J)F1 recipients). The findings were as follows: The lymphoid cell number in the para-aortic lymph nodes ( PALN ) was increased at all stages of gestation. The peak occurred in the 2nd week in primiparity and as early as 60 h after fertilization in multiparity. PALN cell alloreactivity was weak at the beginning and higher than normal in the third week of pregnancy. Spleen cell alloreactivity was increased in the second week and decreased in the third week in primiparous compared with multiparous animals. Anti-paternal alloreactivity exhibited by spleen cells of allogestation was decreased (as compared to cells of isogestation ) especially in primiparous mice, particularly in the third week. At this time, the anti-paternal alloreactivity of PALN cells was increased. The influence of the recipient's sex on GVHR intensity was reversed when the cells were obtained from a pregnant donor, becoming stronger in male compared with female hybrids.     

Immunosuppressive effect of pregnant mouse serum on allostimulatory activity of dendritic cells

In normal pregnancy, the maternal immune system is directed towards tolerance or suppression in order to prevent rejection of the semi-allogenic fetus. Antigen-presenting cells, especially dendritic cells (DCs), are key cells in initiation and regulation of immune responses. The presence of potent immunostimulatory DCs in the decidual tissue of pregnancy has been demonstrated. The aim of this study was to determine how allostimulatory activity of DCs could be affected during pregnancy. DCs were isolated from spleen of pregnant or non-pregnant Balb/c mice and co-cultured with allogenic T lymphocytes prepared from brachial lymph nodes of C57BL/6 mice. Some cultures of non-pregnant female DCs were treated by 2.5% serum obtained from pregnant mice at early, middle or late gestational periods, and were used in the same mixed lymphocyte reaction (MLR) settings. Cell proliferation was measured by 3H-thymidine incorporation, and cytokine production measured in supernatants of MLR cultures using ELISA. The effect of pregnant mouse serum on expression of DC surface markers was evaluated by flow cytometry. No significant difference was found between stimulatory potential of splenic DCs from pregnant and non-pregnant mice in induction of allogenic T cell proliferative response. Moreover, serum of early or late pregnancy did not have any effect on DC function in comparison with non-pregnant mouse serum, while mid-pregnancy serum significantly inhibited allostimulatory activity of DCs. IFNgamma production in co-culture of DCs treated with pregnant mouse serum was significantly lower than that of the control group; however, no significant difference in IL-10 production was observed. Treatment of DCs with pregnant mouse serum did not influence the percentage of cells expressing MHC-II, CD86, CD8alpha or CD11b. However, a marked reduction of the mean fluorescence intensity of MHC-II was observed. Collectively, our results concerning the diminished capacity of DCs to induce production of Th1 cytokines and allogenic T cell proliferation after treatment with pregnant mouse serum reveal a new way of immunologic tolerance against the semi-allogenic fetus.     

The immune response of offspring mice from mothers immunized during pregnancy with protein antigens

The immune system of offspring mice from mothers immunized with photo-oxidized timothy grass pollen antigen B (OX-AgB) or Trinitrophenyl-bovine gamma globulin (TNP-BGG) during their pregnancy was examined. Offspring mice immunized 6 or 8 weeks after delivery with the same antigen administered to their mothers have completely suppressed primary responses and greater than 80% suppressed secondary responses. The observed immunosuppression in offspring mice appears to persist until about 16 weeks after delivery, and is antigen-specific. Adoptive transfer studies show that spleen cells from adult OX-AgB primed mice injected i.v. into lethally X-irradiated (800 rads) syngeneic recipients and challenged with antigen produce significant levels of AgB-specific IgG antibody. Spleen cells (5 x 10(6] from offspring mice of mothers immunized with OX-AgB during their pregnancy were added to spleen cells from adult OX-AgB primed mice and injected i.v. into lethally X-irradiated syngeneic recipients and challenged with antigen. These recipients showed a significantly (greater than 85%) immunosuppressed secondary response. The observed immunosuppression appears to be mediated by CD4+ and CD8+ T cells suggesting a requirement for both T suppressor inducer and effector cell populations. The reported findings are discussed in relation to possible mechanisms to explain the immunosuppression obtained in the offspring of mothers immunized during pregnancy.     

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.     

Lectin-binding characteristics of mouse oviduct and uterus associated with pregnancy block by autologous antiprogesterone monoclonal antibody

Oviducts and uteri were removed from BALB/cJ and F1 (CBA/Ca X BALB/cJ) mice at known stages post coitum and following treatment with an antiprogesterone monoclonal antibody (DB3) or a non-specific immunoglobulin (DNP). Thin sections of tissue were prepared and reacted with fluorescent conjugates of a wide range of lectins to determine if saccharide alterations were associated with the pregnancy-inhibiting effect of the DB3 antibody in BALB/c, but not F1, individuals. The ampullary region of the DB3-treated BALB/c mice showed the most marked changes, with an almost total inhibition of lectin binding, particularly for N-acetylglucosamine residues. There was also a reduced affinity for a lectin reactive with N-acetylgalactosamine in the uteri of DB3-treated BALB/c mice, associated with an extended expression during gestation of this saccharide in the proximal region of the oviduct in such mice. These are the first biochemical alterations in reproductive tract epithelia to be associated with the efficacy of the DB3 antibody in preventing pregnancy.     

Allograft enhancement during normal murine pregnancy

The immunizing effect of pregnancy was assessed using the cardiac allograft technique to estimate maternal reactivity against paternal antigens. In this study the following immunological activities were observed in pregnant or post partum mice. Cardiac allografts expressing paternal antigens were accepted in pregnant or post partum mice longer than in virgin recipients. The cardiac allograft enhancing effect was strain specific, was directly transferable by maternal lymphoid cells but not serum and could be shown to remain in effect in post partum female mice for at least five weeks after delivery.     

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.     

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.     

Antibody isotype produced by pregnant mice immunised with fetal allogeneic lymphocytes is mainly IgG1

Virgin and Pregnant BALB/c mice were immunized three times with (BALB/c X CBA)F1 fetal or adult lymphocytes. An enzyme linked immunosorbent assay (ELISA) was developed for analysing the isotypes and specificities of the alloantibody produced. Pregnant mice immunized with fetal lymphocytes showed an immunodeviation towards IgG1 production compared with other groups.     

Levels of serum amyloid P-component associated with pregnancy and collagen-induced arthritis in DBA/1 (H-2q) mice

The levels of the acute-phase reactant serum amyloid P-component (SAP) were measured by quantitative rocket immunoelectrophoresis in pregnant and non-pregnant DBA/1 female mice with or without collagen-induced rheumatoid arthritis (CIA). Non-pregnant animals with CIA showed elevated SAP titres related to the severity of the disease. Pregnancy alone also caused increased SAP levels equivalent to those found in animals with established CIA but which were virgin. The clinical remission seen in arthritic animals during pregnancy was not associated with reductions in circulating SAP levels. Increasing parity, however, caused a lowering of SAP levels in animals with or without CIA compared to the primiparous individuals. Pregnancy causes a strain-dependent elevation of serum SAP which is not further elevated by CIA, thus limiting the usefulness of SAP measurements in assessment of disease progression or remission during gestation.     

Maternal alloimmune reactions towards the murine conceptus and graft-versus-host reaction (GVHR). II. Inhibition of priming by placental extracts

Gestation can induce a priming for a GVHR towards paternal strain antigens, although this priming is significantly lower than the one induced by experimental immunization. A role has been sought for placental substances in decreasing this priming through immunomodulation. BALB/c (H-2d) spleen cells do not usually induce a systemic, lethal GVHR in DBA/2 (H-2d) newborn mice except when the donors are preimmunized with DBA/2 cells. Placental extracts (as well as RPMI medium or liver extracts used as controls) were added to DBA/2 cells injected into BALB/c mice used as cell donors for GVH induction. The latter's spleen cells, harvested on day 6 after immunization, were used for systemic and local GVHR. In the systemic assay (lethal effect on DBA/2 newborn mice injected i.v. with BALB/c spleen cells) a significant protection was observed. In the local assay (popliteal lymph node assay in F1 hybrids injected with BALB/c spleen cells into the foot-pad) a highly significant inhibition of priming was detected in recipients injected with spleen cells from placental extract-treated donors. The stimulation index was even lower than that obtained with unprimed BALB/c spleen cells. The same type of local GVHR in (CBA/Ca X A/J) F1 hybrids injected with CBA cells led to similar results. In both situations (systemic and local GVHR) the observed inhibition was found to be specific to the priming cell strain. These results support the working hypothesis that placental substances are able to modify the systemic response of an organism towards both H-2 and non-H-2 alloantigens.     

Murine pregnancy-associated modulations in lymphocyte reactivity to mitogens: identification of the cell populations affected

Lymphocytes from the thymus, spleen and inguinal lymph nodes of syngeneically pregnant and non-pregnant mice were compared in their responsiveness to polyclonal stimulation by mitogen. Pregnancy-associated changes in mitogen reactivity were detected, on a cell-per-cell basis, in thymocytes (increased) and spleen cells (decreased) but not in lymph node cells. The hyperreactivity of thymocytes during pregnancy correlated with physiological involution of the thymus occurring through the selective loss of relatively immature, non-mitogen-reactive, Lyt 1+2+ cells. The remaining cells were found largely to be mature Lyt 1+2- T cells with the capacity to respond to mitogenic stimulation. It is most likely the relative increase in the proportion of these Lyt 1+2- cells that causes the hyper-responsiveness of thymocytes to mitogens observed during pregnancy. On the other hand, while spleen cells from pregnant animals gave lower responses to mitogens than those from control virgin females, isolated splenic T cells from the two groups proved equally reactive to T cell mitogens. This supports the contention that at least some aspects of immunity during pregnancy are down-regulated by inhibitory cells within the non-T cell compartment. The results demonstrate the importance of identifying the reactive cell population in studies on changes in lymphocyte responsiveness in pregnancy.     

Anticollagen antibody responses in DBA/1 (H-2q) mice associated with type II collagen-induced arthritis and the effect of pregnancy

We have previously shown that DBA/1 mice immunized with heterologous type II collagen showed remission of the subsequent collagen-induced arthritis (CIA) when pregnant, but experienced exacerbation postpartum. Measurement of anticollagen antibody (aCa) responses by ELISA in primiparous mice immunized at day 1 of pregnancy revealed no significant difference compared to aCa titres in virgin animals, apart from slightly increased titres following the primary immunisation. When mice received collagen challenge during early pregnancy, however, the date at which maximal antibody titres was reached was delayed by 5 days. Pregnancy initiated following the intraperitoneal boost caused a ten-fold suppression in aCa titres with a rise post-partum. Measurements of aCa levels in individuals which showed fetal resorption indicated that suppression of humoral responses was dependent on the presence of a viable conceptus. Antibody titres declined in all animals after a period of time, which was more prolonged in multigravidae where aCa titres were higher than in nulliparous and primiparous mice. The results show that although pregnancy alters aCa responses during the course of gestation, no long-term modification of humoral immunity occurs, an observation in agreement with the clinical findings in these mice and in humans.     

过继转移FasL基因修饰的树突细胞对小鼠自然流产模型胚胎丢失的影响

目的:观察过继转移FasL基因修饰的树突细胞(DC)对小鼠自然流产模型胚胎丢失的影响,探讨它在诱导妊娠免疫耐受中的作用。方法:构建鼠源FasL(mFasL)真核表达载体pcDNA3.1-mFasL,用电转染法将它转染给DBA/2雄鼠骨髓来源的DC,将转染成功的mFasL-DC于交配前经腹腔注射给CBA/J母鼠。实验动物分为6组:(1)正常妊娠模型组(CBA/J×BALB/c);(2)未添加干预的流产模型组(CBA/J×DBA/2);(3)转输DC培养基(DCCM)的流产模型组;(4)转输单纯DC(DC)的流产模型组;(5)转输转染空质粒DC的流产模型组;(6)转输转染mFasL质粒DC的流产模型组,于妊娠第12~14天观察孕鼠胚胎丢失率。结果:转输mFasL-DC后孕鼠胚胎丢失率明显低于未添加干预或转输DC培养基的流产模型组(P<0.01),与转输单纯DC或带空质粒的DC组相比,其胚胎丢失率也明显下降(P<0.05),它与正常妊娠组相比胚胎丢失率无显著差异(P>0.05);转输单纯DC或空质粒的DC组与未添加干预流产模型组相比胚胎丢失率有所下降,但没有统计学差异;转输DC培养基组与未加干预组之间胚胎丢失率无统计学差异(P>0.05)。结论:过继转移mFasL-DC能诱导妊娠免疫耐受,降低小鼠自然流产模型孕鼠胚胎丢失率。