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.     

Kinetics of lymphoproliferative responses of lymphocytes harvested from the uterine draining lymph nodes during pregnancy in rats

Paraaortic lymph node (PALN) 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 was analyzed in mixed lymphocyte reactivity (MLR) against mitomycin-C treated-paternal strain or unrelated cells. Mitogen stimulation of the cell 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 strongly suppressed in the pre-implantation and implantation stages of pregnancy. Specific and non-specific alloreactivities were enhanced at mid-pregnancy and normalized by the end of pregnancy. Reactivity to polyclonal mitogens is enhanced at mid-pregnancy, and the CD4+/CD8+ ratio is very low during all phases of pregnancy.     

Immunological changes in normal pregnancy.

During pregnancy the mother must tolerate intra-uterine allogenic fetal tissue. Failure of this tolerance may cause spontaneous abortion. The immunological changes occurring in normal pregnancy are poorly understood. The aim of this study was to investigate the immunological changes occurring in pregnancy. Thirty women in the first trimester; 10 in the second and 10 in the third trimester of pregnancy were studied and compared to age matched non-pregnant controls. In normal pregnancy there was an increase in the total white cell count with no change in the lymphocyte count. There was a fall in total T cell numbers and activated T cell numbers, with no change in helper/inducer or suppressor/cytotoxic T cell numbers. [3H]Thymidine uptake in response to three different mitogens was increased. This implies an increase in potential for the cells to respond to mitogens. There was no change in interleukin-2 receptor levels, suggesting that despite this increased potential there was no general activation of the immune system. A rise in IgM and IgG was found after mitogen stimulation of peripheral blood lymphocytes, suggesting an increase in potential antibody production. These results demonstrate that lymphocytes from pregnant women have an increased potential rather than an increased activity.     

Cellular events associated with autoimmune oophoritis and ovarian tumorigenesis in neonatally thymectomized mice

Thymectomy at 3 days of age (Tx-3) in (C3H/HeMs x 129/J)F1 (C31) female mice results in post-pubertal ovarian dysgenesis associated with high levels of circulating auto-oocyte antibodies (AOA) prior to ovarian tumor formation. Evidence suggests that the etiology for the ovarian dysgenesis resulting from Tx-3 is autoimmune and involves helper T cell abnormalities. The present study characterized circulating leukocytes and mitogenic activity using concanavalin A (ConA) with serologically selected spleen T cells. We observed no sustained abnormalities in either number of circulating leukocytes or percentages of granulocytes or lymphocytes. Circulating mononuclear cells with positive immunofluorescence for Thy 1.2 and Lyt 1.1+Lyt 1.2 cell markers were similar in all mice. However, the spleen cells from Tx-3 mice with ovarian dysgenesis remaining after adsorption with antisera to the Lyt 2.1+Lyt 2.2 antigens (helper T cells remaining) showed increased incorporation of [3H]thymidine compared to the intact mice. This stimulated activity occurred during the periods of early ovarian dysgenesis and active tumor growth. Apparently, the autoimmune oophoritis results from an imbalance within the Lyt 1 cells which may represent a primary insult to the ovary that results in later ovarian tumor development.     

Altered mitogen- and alloantigen-induced lymphoproliferative responses in primary lymphoid organs of pregnant mice

The effect of syngeneic murine pregnancy on the response of lymphocytes from the primary lymphoid organs to mitogenic and alloantigenic stimulation was investigated. Thymocytes and bone marrow cells from gravid animals were found to exhibit elevated reactivity to T cell mitogens and allogeneic stimulator cells. In contrast, the LPS response of bone marrow cells from pregnant mice did not differ significantly from that of virgin animals. These findings indicate that the immune reactivity of T cells derived from the primary lymphoid organs is altered during syngeneic murine pregnancy. Pregnancy-induced modifications in T lymphocyte reactivity may contribute to the ability of the maternal immune system to recognize and react against fetal antigens.     

The immunology of HIV disease and pregnancy and possible interactions

HIV infection may impair a large portion of the human immune response. Infection of CD4+ T cells results in depletion of this population, leading to dysfunction of T-cell-dependent activities. Numerous other immune functions are directly or indirectly impaired, including the function of CD8+ T cells and decreases in total lymphocytes, IL-2 secretion, IL-2R expression, proliferative response to mitogens, NK activity, ADCC, and several other measures. Immune alterations seen in normal pregnancy include decreases in CD4 and T cells, the proliferative response to mitogen, IL-2 secretion, and NK activity. These changes are largely cellular and usually are not apparent clinically. Normal pregnant women are immunocompetent. When pregnancy is complicated by asymptomatic HIV infection, the obstetric outcome does not seem to be adversely affected. Preliminary studies show that the course of HIV disease may not be adversely affected in these women, but this matter is the subject of intense investigation.     

The response of the pregnant ewe to challenge with foetal and paternal lymphocytes

The efferent lymph from the popliteal lymph nodes of pregnant ewes challenged between 93 and 127 days with specific paternal or foetal lymphocytes was examined. There was an increased efflux of lymphocytes and blast cells from the challenged node, similar to that observed during the response of normal, non-pregnant ewes to allogeneic cells. Additionally, there was a decrease in the mixed lymphocyte and mitogen responsiveness of the efferent lymphatic cells that was comparable with that evoked by challenge of non-pregnant sheep. While cytotoxic cells could not be detected in the lymph after challenge of normal or pregnant animals, specific cytotoxic antibody was invariably produced in both instances. The present observations that the immune responsiveness of the pregnant ewe to foetal lymphocytes remains normal are consistent with an earlier report on the antifoetal reactivity of maternal cells in vitro. It is inferred that the in vitro reactivity of maternal lymphocytes was a valid reflection of the capacity of these cells in the intact ewe.     

Subpopulations of t lymphocytes and lymphocyte proliferative activity in normal pregnancy]

T lymphocyte subpopulations (CD3+, CD4+, CD8+) and the lymphocyte proliferative responses to mitogens (PHA, Con A, PWM), in the environment of fetal calf serum (FCS) were examined in 16 normal primigravidas in the third trimester of pregnancy and in 15 healthy nonpregnant women. In normal pregnant women significantly lower absolute and percentage numbers of CD3+ and CD4+ T cells and almost twice reduced CD4+/CD8+ ratio were found, in comparison with nonpregnant subjects. Despite the shifts among particular T lymphocyte subsets in the peripheral blood, non disorders were found in the lymphocyte proliferative responses to mitogens in normal pregnancy.     

Cord blood lymphocyte subpopulations and mitogenic activity in whole blood microculture

Comparative studies on cord and adult blood showed that cord blood contained at least twice as many lymphocytes as adult blood. Relatively, the percentage of T cells (E-RFC) was significantly lower in cord blood lymphocytes. The percentage of B cells (EAC-RFC and SmIg bearing cells), as well as the total number of T and B cells (mm(-3)), was significantly higher in cord blood. In vitro mitogen transformation of cord and adult lymphocytes in while blood, cultured for different times and diluted to contain equivalent numbers of lymphocytes per culture, showed significant qualitative and quantitative differences. Responses to the T cell mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A) were examined from 3 to 6 days in culture. Cord blood lymphocytes were significantly more responsive when cultured for 3 to 4 days, similar to adult cells after 5 days, but significantly less responsive after 6 days in culture. The optimal levels of T cell mitogen responsiveness in cord cells (Day 4) were similar to adut cells (Day 6). Spontaneous transformation of unstimulated lymphocytes and B cell mitogen transformation with pokeweed mitogen (PWM) and staphylococcal protein A (SpA) were all significantly higher in cord blood than in adult whole blood cultured for 5 days.     

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.     

Characterization of hormone-dependent suppressor cells in the uterus of mated and pseudopregnant mice

The survival of the implanted "fetal allograft" has been attributed to the action of local decidua-associated suppressor cells. These suppressor cells are Fc-receptor positive small lymphocytic cells lacking T-cell markers which arise following implantation, are localized at the implantation site, and block the action of IL-2 that stimulates NK and T effector cells. Kinetic studies have demonstrated the occurrence of an earlier peak of suppressive activity occurring 2-3 days after mating prior to implantation. The cells associated with pre-implantation suppression differs significantly from post-implantation suppressor cells. Velocity sedimentation studies show that early suppression is associated with large cells sedimenting at a modal velocity of 6-7 mm/hr. Suppressive activity from cells of similar size is also present in the uterine lining of hormonally-treated, pseudopregnant mice. In addition, suppressor cells can be demonstrated in the non-pregnant uterus at the time of estrus. These observations suggest suppressor cell activity may be hormonally regulated. The suppressor cell(s) in pseudopregnant mice bears Lyt 2.1 and Thy 1.2 surface antigens and suppressor(s) present in the pregnant animals bears Lyt 2.1 and Lyt 1.1. Although the suppressor cell was large, it did not appear to be a macrophage because it was resistant to antibodies to Mac-1 and FcR cell surface markers but susceptible to anti-T cell reagents. Furthermore, suppression was not mediated by a soluble factor that has been associated with the small lymphocytic suppressor. Thus, the suppressor activity present in the pre-implantation uterine lining appears to differ significantly from the suppressor cell activity found after implantation. The possible role of a hormone-activated suppressor T cell in the success of the pregnancy is discussed.     

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.     

Active suppression of host-vs-graft reaction in pregnant mice. IV. Local suppressor cells in decidua and uterine blood

The mammalian fetus bears a wide variety of antigens against which the maternal immune system can respond. Although some of these antigens are transplantation antigens, the type of immune response mounted by the mother seems incapable of mediating graft rejection. We have previously demonstrated suppressor cells in the lymph nodes draining the uterus (DLN) that regulate the immune response in allogeneically pregnant C3H/HeJ and CBA/J mice. The suppressor cells were shown to be small lymphocytes (sedimenting at 3 mm/h at unit gravity) resistant to anti-T cell serum + complement that elaborated a soluble suppressor activity and selectively inhibited the generation of cytotoxic T lymphocytes (CTL) reactive with paternal alloantigens. Suppression could be induced in the DLN by syngeneic pregnancy or pseudopregnancy, and behaved as an anatomically localized activity during pregnancy. We now report that during first allogeneic pregnancy, the most potent suppressor cell activity is found in lymphocytes in uterine venous blood and in decidual lymphocytes. This suppressor cell population also sediments at 3 mm/h and is associated with production of a soluble suppressor factor. Substantial suppressor cell activity can also be obtained from the deciduomata of pseudopregnant mice. Local suppressor cell activity within the uterus may play an important role in ensuring the immunological success of the fetal allograft.     

Autologous mixed lymphocyte reaction (AMLR) in man. XVI. The AMLR and monoclonal antibody-defined T cell subsets and HNK 1+ natural killer cells in normal human pregnancy

Peripheral blood mononuclear cells from 27 pregnant women and 10 age-matched non-pregnant women were examined for monoclonal antibody-defined T cells, immunoregulatory T-cell subsets, natural killer cells, activated T cells and surface Ig+B lymphocytes using a fluorescence-activated cell sorter (FACS analyzer). The autologous mixed lymphocyte reaction (AMLR) and in vitro influence of interleukin 1 (IL-1) and interleukin 2 (IL-2) on the AMLR were also studied. No significant difference was observed in the proportions of Leu 3+ (helper/inducer phenotype) and Leu 2+ (suppressor/cytotoxic) T cells during all three trimesters of pregnancy and in post-partum period when compared to non-pregnant healthy control women. T cells expressing DR antigen (evidence of T-cell activation) were significantly increased during second trimester (P less than 0.02) and in post-partum period (P less than 0.05). However, Tac+ T cells (IL-2 receptor positive T cells, another but distinct marker for T cell activation) were normal throughout pregnancy and in the post-partum period. Leu 7+ (HNK 1+) lymphoid cells (containing a population of natural killer cells) were normal during all 3 trimesters of pregnancy but were increased during post-partum period. Surface Ig+B cells were comparable to control group throughout pregnancy and during post-partum period. The AMLR was significantly deficient (P less than 0.01) during first and third trimester of pregnancy. In vitro addition of purified IL-2 restored the AMLR to the baseline levels of the controls but the AMLR was still lower than the levels in controls with IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered humoral immunity during pregnancy in the guinea pig

A guinea pig model was developed to determine whether humoral immune responsiveness is altered during pregnancy. Pregnant animals were immunised at mid-gestation with haptenated protein. The humoral response to antigen was measured as numbers of antibody-producing cells in the spleen, the affinity of the antibody produced by spleen cells and the levels of IgG in the serum. The values obtained were compared with those from an age matched non-pregnant control group.

Early in the primary response, there was a significant decrease in the number of IgM antibody-producing cells with an associated decrease in serum IgM levels in pregnant animals. Late in the primary response, pregnant and control animals had similar levels of IgM antibody-producing cells. During the later stages of the response, many of the pregnant animals did not respond with IgG antibody-producing cells or IgG in the serum. When IgG-producing cells were detected, the antibody was of lower affinity than that observed with the control group.

A selective lack of responsiveness was detected in the primary response of pregnant guinea pigs. The reduced number of IgG antibody-producing cells in gravid animals suggests that an immune switch from IgM to IgG is impaired in pregnancy. The low affinity of antibody produced indicates the immunoglobulin produced in pregnancy may also be functionally limited.     

The potentiating effect of pregnancy on humoral immune responses of mice

Previous work has shown that in the mouse, pregnancy significantly increases the level of immunoglobulin secreting cells in the lymph nodes draining the uterus. In this report it is demonstrated that there is also a widely disseminated potentiating effect on levels of immune responsiveness. T-cell responses, manifest as bystander B-cell responses, T-dependent and T-independent B-cell responses to injected antigens and non-specific “responses” to injected mitogens, have all been shown to be potentiated. Ablation of the fetus with retention of a healthy placenta leads to a loss of the potentiating effect. Feeding pregnant mice on indomethacin results in a lowering of immunoglobulin-forming cell levels in the para-aortic lymph node but has no effect on the disseminated potentiation effect detected by the popliteal lymph node assay. It is concluded that the fetus or the placenta under direct control of the fetus, is the source of the “pregnancy factor” responsible for the disseminated potentiation effect, that this factor is unlikely to be a prostaglandin and that the B-cell is the likely target of the potentiating factor. The possible nature of the “factor” is discussed.     

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.     

Maintained allogeneic pregnancy in rats depleted of T cytotoxic/suppressor cells by OX8 monoclonal antibody treatment

It has been suggested that CD8 positive suppressor T-cells might be of importance in the non-rejection of the fetus. In the present investigation allogeneically pregnant (Lewix x DA) rats were subjected to in vivo treatment with monoclonal OX8 antibodies, reactive with the rat equivalent of CD8 receptor. This treatment protocol drastically reduced the numbers of OX8 positive cells in spleens and para-aortic lymph nodes. On the day of delivery these rats, together with normal IgG treated controls, were dissected and analysed for effects on: (1) fetal survival; (2) weight and immunohistology of spleens and para-aortic lymph nodes; (3) total numbers of IgM- and IgG-secreting cells within these organs. The OX8 treated rats passed through pregnancy as successfully as did the controls. Both groups showed the same type of pregnancy-induced changes in their lymphoid organs, including dramatic growth of the para-aortic lymph nodes and increase in Ig-secretors within both spleen and para-aortic lymph nodes. This pattern was the same in all pregnant rats investigated, including untreated syngeneically mated Lewis rats. It is concluded that OX8 positive T "suppressor" cells do not play an important role in the maintenance of the fetal-placental unit.     

The responses of isolated uterine arteries from pregnant sows to vasoconstrictive agents

Isometric contractions of isolated uterine arteries, mesenteric arteries and the thoracic aortae of nonpregnant and pregnant sows were measured in a modified Krebs-Henseleit solution in order to investigate the characteristics of the uterine artery responsiveness to vasopressor substances during pregnancy. Contractile response (delta T) of the uterine artery from pregnant sow to angiotensin II(A II) was significantly smaller than that from nonpregnant animal. On the other hand, delta T of uterine artery from pregnant sow to norepinephrine (NE) was greater than that from nonpregnant animal. NE-induced delta T of preparations from both pregnant and nonpregnant sow were suppressed nearly to the same level following the treatment of phentolamine, or verapamil in the incubation medium of 2.5mM Ca2+. In the Ca2+-free (EDTA 1mM) incubation medium, the responses decreased to the minimum degree. These results imply that conspicuous refractoriness of the uterine artery to A II during pregnancy is due to the changes in the characteristics of the uterine vascular wall, and the enhanced responsiveness to NE of the uterine artery may be due to the increased sensitivity in alpha-adrenergic receptor on the vasculature with the increase in Ca ion influx.     

Immunological relationships between murine surrogate mothers and transplanted embryos, as studied by local GVH reactivity

C57BL/Ks (H-2d) female mice were transplanted with early (stage 2) embryos of the A/J (H-2a) strain. Spleens from mice exhibiting successful pregnancies were tested at days 16 to 19 of gestation in a local graft versus host (LGVH) assay using (C57BL/Ks X A/J)F1 recipients and proved to be significantly more reactive than virgin controls or mice carrying transplanted syngeneic fetuses. This increased reactivity was specific for the transplanted embryo's strain. Other controls included donors with semi-allogeneic (F1) transplanted fetuses and females naturally pregnant by allogeneic males which did not give reactions significantly different from virgin control spleen cells. Para-aortic lymph node cells (PALN) obtained from the same A/J embryo-transplanted females showed a strong T suppressive activity both on their own spleen cell (SC) reaction as well as on the reaction obtained with virgin SC. This suppressive activity also appeared to be embryo-strain specific. Serological tests revealed the presence of mast cell-degranulating (anaphylactic) antibodies but not of hemagglutinating or complement-fixing cytotoxic activities. The A/J offspring obtained after embryo transfer to C57BL/Ks females presented at the age of two months significantly lower LGVH reactivity against the surrogate mother's strain. The differences in the responsiveness of the mice transplanted with allogeneic embryos compared with those with conventional pregnancies are discussed.