Immunoregulatory activity of cells from lymph nodes draining the uterus of allopregnant mice

Journal of Reproductive Immunology, 17 (1990) 239-252 Elsevier Scientific Publishers Ireland Ltd.

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JR1 00651

Immunoregulatory activity of cells from lymph nodes draining the uterus of allopregnant mice Sahar Kamel and Gary W. Wood Department of Pathology and Oncology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, KS66103 (U.S.A.) (Accepted for publication 20 February 1990)

Summary The presence of a local immunosuppressive environment in the vicinity of the developing fetus has been suggested to explain survival of the semi-allogeneic fetus in a potentially hostile maternal immunologic environment. The presence of nonspecific suppressor cells in the uterus of pregnant mice has been well-documented. It has been suggested that the local immunosuppressive environment extends to the lymph nodes draining the uterus of pregnant mice. Studies undertaken to investigate this hypothesis have provided conflicting data. The current study was performed as an attempt to resolve some of the controversial results obtained from previous studies and to characterize more extensively the nature of lymph node suppressor cells. Our results clearly demonstrated that neither specific nor non-specific immunosuppression was expressed within lymph nodes draining the uterus of allopregnant mice. Cells obtained from draining lymph nodes consistently exhibited a normal capacity to respond to alloantigens whether by proliferation or through cytotoxic T lymphocyte (CTL) generation. We conclude that immunosuppression fails to develop in draining lymph nodes during pregnancy and therefore plays little or no role in controlling the development of antifetal immune responses. Key words: suppressor cells; T lymphocytes;

mixed lymphocyte reaction;

cytotoxic Tlymphocytes; immunosuppression.

Correspondence to: Gary W _Wood. 0165-0378/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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Introduction Allogeneic pregnancy is a natural example of successful transplantation across major histocompatibility barriers. Despite early expression of histocompatibility and embryonic antigens by the developing fetus (Gill, 1979; Johnson and Calarco, 1980), the maternal immune system fails to reject the fetus. One possible explanation for this enigma is that local immunosuppressive mechanisms are operative in the environment surrounding the fetus. Several lines of evidence have lent support to this hypothesis. Early studies by Beer and Billingham (1974) demonstrated that skin allografts transplanted to a decidual tissue bed enjoyed prolonged survival. Several studies have provided strong evidence for the presence of immunoregulatory cells in the uterus of pregnant mice (Slapsys and Clark, 1982; Hunt et al., 1984; Tawfik et al., 1986a) which non-specifically suppress maternal-anti-paternal immune responses (Slapsys and Clark, 1982; Tawfik et al., 1986a,b) as well as mitogen-induced lymphocyte proliferation (Hunt et al., 1984). Suppression was shown to be mediated through the production of soluble suppressor factors (Kirkwood and Bell, 1981; Clark et al., 1983; Badet et al., 1983; Tawfik et al., 1986b). Prostaglandins (PGs), specifically PGE,, have been shown to be largely responsible for the soluble suppressor activity both in murine (Tawfik et al., 1986b; Matthews and Searle, 1987) and human pregnancy (Lala et al., 1988). Transforming growth factor-beta has also been implicated in soluble factor-mediated immunosuppression (Clark et al., 1988). Lymph nodes draining the uterus increase in size and weight during allogeneic pregnancy (Maroni and de Sousa, 1973; Ansell et al., 1978) and those changes are eliminated if the mother has been tolerized to paternal transplantation antigens prior to mating (Beer et al., 1975). Those observations provoked Clark and McDermott (1978) to suggest that the cellular reaction in the draining lymph nodes during pregnancy may represent activation of suppressor mechanisms which prevent immune responses harmful to the fetus. Clark and McDermott (1978) demonstrated suppressor cells in draining lymph node cells of pregnant mice undergoing an allogeneic pregnancy. Draining lymph node cells showed impaired reactivity in the graft-vs.-host mortality assay and in the generation of CTL after immunization both in vitro and in vivo. Suppression was reported to be non-specific and was associated with soluble suppressor activity (Clark et al., 1980). Suppressor cells appeared to be identical to those previously detected in the uterus (Slapsys and Clark, 1982). O’Hearn and Hilgrad (1981) also demonstrated a decreased-responsiveness of draining lymph node cells in a graft-vs.-host assay. However, their results differed from those of Clark et al. (1980) in that the former showed that hyporesponsiveness was specific for alloanti-

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gens of the fetus, while the latter group reported that localized suppression acted without specificity for fetal alloantigens. In contrast, a study by Gottesman and Stutman (1980) demonstrated that draining lymph nodes from pregnant mice bearing syngeneic or allogeneic fetuses have a low proliferative capacity in mixed lymphocyte reactions (MLR) while retaining the ability to generate normal levels of alloreactive CTL in vitro. In their study, the decreased proliferative capacity of draining lymph node cells was shown not to be due to the presence of a suppressor cell population. The current study was undertaken in an attempt to resolve some of the conflicting data obtained previously by other investigators. In addition, our previous studies have identified macrophages as potent immunoregulatory cells located in the vicinity of the developing fetus. Thus, we were also interested in determining whether or not pregnancy results in the activation of the immunosuppressive capacity of lymph node macrophages. The in vitro immune reactivity of draining lymph node cells from primiparous allopregnant mice was compared with that from age-matched virgin controls. The results demonstrated that draining lymph node cells of allopregnant mice have a normal capacity both to proliferate and to generate CTL in MLR. Moreover, irradiated draining lymph node cells exhibit no suppressive activity on either the proliferative response or the generation of CTL in maternalanti-paternal MLR. These results suggest that local immunosuppression in draining lymph nodes should not be used as an explanation for protection of the fetoplacental unit. Materials and methods Animals Young adult (6-8 weeks old) female BALB/c and male C57BL/6 mice were purchased from Harlan-Sprague/Dawley, Inc., and were maintained in the animal care facility of the University of Kansas Medical Center. For breeding purposes, female mice were housed in cages with males at a ratio of 2:l and were checked each morning for the presence of a vaginal plug. The day of sighting a vaginal plug was considered day 0 of pregnancy. Mice were killed by cervical dislocation following light ether anesthesia. Preparation of cell suspensions Spleen cells were prepared by sieving through stainless steel mesh. Cells were filtered through sterile gauze then washed twice with medium (RPM1 1640 containing penicillin and streptomycin) and resuspended in medium supplemented with 2.5% heat-inactivated group AB pooled human serum, 2 mM r_-glutamine and 5 x lo- M 2-mercaptoethanol (2-ME) (complete medium). Lymph node cells were prepared by washing tissue with medium, mincing

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with iris scissors, followed by sieving through a stainless steel mesh and resuspending in complete medium, as described above. Lymph nodes draining the uterus included para-aortic and renal lymph nodes, whereas peripheral lymph nodes included a pool of inguinal, brachial and axillary lymph nodes. Draining lymph nodes of pregnant mice were harvested on days 1419 of pregnancy. In some experiments, lymph node cells were isolated by enzymatic digestion. Tissue was minced with iris scissors, washed, suspended in collagenase (Type I, 1 mg/ml, Cooper Biomedical) plus protease (0.5 mg/ml, Sigma) in medium and agitated in a tissue digestion flask (Wheaton) for 60 min at room temperature. Following digestion, lymph node cells were filtered through gauze to remove undigested tissue, washed twice with medium and resuspended in complete medium. To prepare single cell suspensions of uterine cells, uteri were aseptically removed from 14-19-day pregnant BALB/c females mated with C57BL/6 males. The uteri were separated from visceral yolk sacs, fetuses and placentae and cell suspensions were prepared as described previously (Hunt et al., 1984). Briefly, tissue was minced with iris scissors, washed, suspended in collagenase (Type I, 1 mg/ml, Cooper Biomedical) plus protease (0.5 mg/ ml, Sigma) in medium and agitated in a tissue digestion flask (Wheaton) for 90 min at room temperature. Following digestion, the uterine cells were filtered through sterile gauze to remove undigested tissue, washed with medium and resuspended in complete medium. The yield of cells from uterine tissue ranged between 10’ and lo8 cells/g (wet weight) of tissue and viability was consistently above 90% as determined by trypan blue exclusion. Mixed lymphocyte reaction A standard mixed lymphocyte culture system was employed. Responder BALB/c spleen or lymph node cells were added to 96-well plates at a concentration of 5 x lo6 cells/ml in complete medium. Stimulator C57BL/6 spleen cells were also suspended at 5 x lo6 cells/ml and received 2500 rad of 13’Cs prior to addition to plates. Responder and stimulator cells were added to wells in volumes of 100 ~1 each. In some experiments, lymph node or uterine cells were added as regulators to BALB/c x C57BL/6 spleen cell MLR. Regulator cells were suspended in complete medium at a concentration of lo6 cells/ml and received 2500 rad of 13’Csprior to culture. They were added on day 0 of culture in a volume of 100 )Uwell. Cells were cultured at 37 OCin 5% CO,, 95% air for 4 days. [3H]thymidine (1 @i/well) was added 8 h prior to termination of culture. Cells were harvested with a Brandel Cell Harvester and counted in a liquid scintillation counter. Target cells for lympholysis assay P815, a mastocytoma cell line derived from DBA/2 mice (H-2d) and EL-4,

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a T cell lymphoma of C57BL/6 (H-2b) origin were purchased from American Type Culture Collection (ATCC). Both lines were maintained in RPM1 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics. To prepare Con A-induced lymphoblasts, C57BL/6 spleen cells were isolated as described above and suspended at a concentration of 5 x lo6 cells/ml in RPM1 1640 supplemented with 10% FBS, L-glutamine (2 mM), antibiotics, 2-ME (5 x 10m5 M) and 5 pg/ml Con A (Sigma Chemical Co., St Louis, MO). The cells were incubated in 100 mm tissue culture Petri dishes for 48 h at 37OC in 5% CO,, 95% air. At the end of that period, viable cells were separated on a ficoll-hypaque gradient before use as targets in “Cr release assay. Target cells were labeled for 1 h at 37OC with 0.3 mCi Na;CrO, (ICN Radiochemicals, 5 mCi/ml)/ 10’ cells/ml of RPM1 1640 supplemented with 10% FBS, L-glutamine (2 mM), non-essential amino acids (lx), sodium pyruvate (1 mM), 2-ME (5 x lo+ M) and antibiotics. Cells were washed three times with medium and adjusted .to a concentration of lO’/ml. CTL assay

To test for CTL activity in sensitized lymphocyte populations, a 4-h “Cr release assay was employed. Effector cells were harvested from MLR induction cultures and sedimented over ficoll-hypaque to remove dead cells. Viable cells were washed twice and suspended in complete medium. Variable numbers of effector cells were added to a constant number of target cells in 96-well round bottom plates, to obtain the desired effectorkarget (E:T) ratios. Target cell concentration was lo5 cells/ml, in 100 $/well. The cultures were incubated at 37OC in 5% CO,, 95% air for 4 h. At the end of the incubation period, plates were centrifuged for 5 min at 400 x g and 100 ~1 aliquots of supernatants were removed and assessed for released isotope in a beta-scintillation-counter. Three replicate cultures were used at each point and percentage specific Wr release was calculated using the following formula: % specific Yr release = 100 X (sample cpm - spontaneous cpm)/ (maximum cpm - spontaneous cpm). Spontaneous release was obtained by incubating the target cells in culture medium alone, whereas maximum release was obtained by culturing target cells with a detergent (4% cetrimide). Results of representative experiments are presented. Each experiment has been repeated at least three times with reproducible results. Statistical analysis was performed using Student’s t-test, or one way analysis of variance (ANOVA). Results Proliferation by draining lymph node cells in MLR

Draining lymph node cells (para-aortic and renal) were used as responder

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cells and were cultured with irradiated stimulator C57BL/6 spleen cells in a 4-day MLR. No statistically significant differences were detected when the levels of proliferation of cells obtained from pregnant draining lymph node cells were compared with either those of draining lymph nodes from agematched virgin BALB/c mice, or peripheral lymph nodes from pregnant animals (Fig. 1). Immunomodulatory effect of draining lymph node cells Irradiated (2500 rad) draining lymph node cells were used as regulator cells in a 4-day maternal-anti-paternal MLR. The ability of draining lymph node cells to modulate maternal-anti-paternal MLR was compared with that of lymph node cells from virgin mice or cells from peripheral lymph nodes of pregnant animals, as demonstrated in Fig. 2. In order to test the possibility that the method of preparation of lymph node cell suspensions might have an effect on the immunoregulatory capacity of those cells by selecting for certain cell populations and discriminating against others, the immunoregulatory capacity of lymph node cells prepared by enzymatic digestion of minced tissue was compared with that of mechanically-prepared lymph node cells (Fig. 2). Enzymatic digestion of lymph nodes resulted in 1.5 -2.0-fold increase in cell yield (data not shown) compared with the conventional mechanical method of cell dissociation. The results depicted in Fig. 2 200 -

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Fig. 1. Comparison of the proliferative capacity of lymph node cells draining the uterus of allogenically pregnant (BALB/c x C57BL/6) female mice (DLNC), lymph node cells draining the uterus of virgin BALB/c females (VLNC) and peripheral lymph node cells (PLNC) isolated from allogeneic pregnant (BALB/c x C57BL/6) females in MLRs. Values represent the mean and standard deviation of five replicate cultures.

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Fig. 2. Comparison of the immunotegulatoty effect of lymph node cells draining the uterus of allogeni(BALB/c x C57BL/6) female mice (DLNC), lymph node cells draining the uterus of vitgin BALB/c females (VLNC) and peripheral lymph node cells (PLNC) isolated from allogeneic pregnant (BALB/c X C57BLI6) females on the BALB/c X C57BLI6 MLR. Values represent the mean and standatd deviation of five replicate cultures. tally pregnant

demonstrate that none of the tested lymph node cell populations exerted any suppressive effect on the proliferative response, whether they were enzymatitally- or mechanically-prepared, when compared with cultures to which no regulator cells were added. i.e., none of the lymph node populations tested had any immunomodulatory effect on MLR. Control cultures were performed in which regulator uterine cells from pregnant animals were added to MLR as regulator cells. This treatment resulted in > 90% suppression of the basic proliferative response (Fig. 2). Generation of alioreactive CTL from draining lymph nodes Responder draining lymph node cells from allogeneic pregnant (BALB/c x C57BL/6) females were mixed with irradiated C57BL/6 stimulator spleen cells in a 4-day MLR. At the end of the culture period, cells were harvested from wells and tested for cytotoxic activity in a 4-h “Cr release assay. As

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shown in Fig. 3, those cells specifically lysed Con A-induced C57BL/6 splenocytes and EL-4 tumor cell targets, both of which are histocompatible with stimulator cells (H-2b). However, the data in Fig. 3 also exhibit no differences in the percent cytotoxicity obtained whether draining lymph node cells or lymph node cells from virgin animals were used as a source of responder cells in the induction cultures. P815 tumor targets (H-2d) served as a negative control in this assay. Negligible levels of P8 15 target cell lysis were obtained regardless of whether effector cells were generated from lymph nodes from pregnant or virgin females (Fig. 3). Suppression of CTL generation Irradiated draining lymph node cells were added to maternal-anti-paternal MLR, as described above. The ability of cells from pregnant animals to modulate CTL generation was compared with cells from virgin animals. Cells were harvested from wells on day 4 of MLR and their specific cytotoxic activity was assayed in a 4-h 51Crrelease assay. Effector cells harvested from cultures including no regulator cells exhibited specific cytotoxic activity against both EL-4 (H-2b) and Con A-stimulated C57BL/6 splenocytes (H2b), as demonstrated in Fig. 4. However, neither cells from pregnant nor cells from virgin mice exhibited any suppressive activity on the basic CTL response (Fig. 4). 5’Cr-labeled P815 cells (H-2d) served as control target cells

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Fig. 3. Comparison of CTL activity generated following in vitro sensitization of uterine draining lymph nodes (DLNC) of allopregnant and virgin mice. Data are reported as the mean of triplicate cultures from a representative experiment.

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Fig. 4. Comparison of the ability of uterine draining lymph node cells (DLNC) and virgin draining lymph node cells (VLNC) to modulate CTL generation.

in this assay. Figure 4 shows that no killing of P815 targets was detected under any of the experimental conditions tested. The same experiment was repeated using enzymatically prepared populations of draining lymph node cells or lymph node cells from virgin animals with essentially the same results (data not shown). Discussion The developing fetus has the ability to induce alloimmune responses and has been shown to do so when transplanted to extrauterine sites (Simmons and Russell, 1962). Nevertheless, immune rejection of the fetus has not been documented during its normal development in the uterus, and cellular alloimmunity has not been demonstrated regardless of the number of natural stimulations. In attempting to explain these facts, several hypotheses have been developed. One is that immune regulation functions in some way to protect the semiallogeneic fetus. Immune regulation could function at several levels. For example, it could be effected through specific systemic immune suppression. It has been hypothesized that, during pregnancy, suppressor cells specific for the relevant alloantigens circulate throughout the immune system (Taylor and Hancock, 1975; Carter, 1976; Chaouat et al., 1979; Chaouat and Voisin, 1980). Despite extensive investigation, consistent support for this possibility has not been generated. In fact, well-doc-

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umented examples of biologically relevant specific immunoregulation are rare. An alternative possibility which has been considered is that the maternal immune system is non-specifically compromised during pregnancy (Murgita and Tomasi, 1975; Fabris et al., 1977; Pasta and Pejtsik, 1977). Despite anecdotal reports of increased incidence of infection during pregnancy (Thong et al., 1973), most evidence indicates that the maternal immune system is completely intact. Since systemic immune responses do not seem to be compromised either specifically or non-specifically, the suggestion has been made that some form of local immune regulation develops in tissues surrounding the fetus (Beer and Billingham, 1980; Siiteri and Stites, 1982). There would seem to be two general ways, one specific and the other nonspecific, in which a local immunoregulatory environment could be generated. Alloantigens could reach the maternal immune system in a form which would specifically inhibit rather than stimulate immune responses. Since alloantigens emanate from fetal tissues, the effect should be greatest in the local environment. This presupposes that alloantigen-positive fetal cells do not cross the placental barrier and enter the maternal circulation. Soluble histocompatibility antigen(s) would either be picked up by uterine antigen presenting cells and transported to lymph nodes and spleen or would reach lymphatic tissues directly through blood and lymphatics. Either way, maximum effects should be exhibited in lymph nodes draining the uterus. A local non-specific immunosuppressive environment could be generated through release of any of a variety of immunosuppressive factors from the fetus or extra-fetal tissues. Such factors could, for example, activate cells which have the capacity for inhibiting immune responses within the uterus or local lymph nodes. The results of the current study clearly demonstrated that neither specific nor non-specific immunosuppression was expressed within lymph nodes draining the uterus during normal pregnancy. Previous studies (Clark et al., 1980) demonstrated that draining lymph nodes contain a normal complement of CTL precursors; data which these studies confirmed. Also, in our study, no cells were present which were capable of suppressing alloimmune responses. Moreover, we were unable to detect suppressor cells by using enzymatic digestion to increase the proportion of stromal cells such as macrophages and dendritic cells (Frangakis et al., 1982; Monfalcone et al., 1986), a population known to be enriched in non-specific suppressor cells. Another argument which might be made relative to these data is that the third trimester of murine pregnancy is not the optimal time to look for immunoregulation; that immune suppression may have been manifested in lymph nodes earlier during pregnancy. The logic which we used in choosing this time period was that; (1) if immunosuppression was generated earlier, it would have to be sustained throughout pregnancy in order to be effective,

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(2) alloantigens are expressed maximally in fetal tissues after day 11 and (3) non-specific immunosuppression associated with tumor progression is expressed only late in the tumor growth phase. It is likely, therefore, that, to be effective, immunoregulation would have to be expressed in the second half of pregnancy. The results of the present study are in disagreement with observations by Clark and McDermott (1978), O’Hearn and Hilgrad (1981) and Gottesman and Stutman (1980), to varying extents. In our experimental system, evidence for an immunoregulatory role of draining lymph node cells was nonexistent, whether assayed by measuring proliferation or CTL generation. The discrepancies could be explained on the basis of differences in assay systems used and/or mouse strains employed. However, the importance of this study lies in the fact that it clearly demonstrated that local immune suppression is not a reproducible phenomenon. If localized immune suppression in draining lymph nodes is to be used as an explanation for maintenance of the integrity of allogeneic murine pregnancy, it should hold true for all mouse strains. Moreover, it should be evident using routine immunologic procedures such as the ones used during this study. This contrasts greatly with the localized immunosuppressive activity of decidual cells, which has been documented by numerous investigators using a variety of experimental systems, both in the mouse (Beer and Billingham, 1974; Slapsys and Clark, 1982; Hunt et al., 1984; Tawfik et al., 1986a,b; Mathews and Searle, 1987) and in the human (Golander et al., 198 1; Nakayama et al., 1985; Parhar et al., 1988). It would appear, then, that immunosuppression in local lymph nodes may not play a major role during pregnancy. An additional note of caution should be introduced at this point, which includes the studies performed with uterine cells. Removing cells from their in vivo environment can introduce artifacts which may not be directly reflected in the living animal. Thus, it will be important to demonstrate that immunosuppressive factors which are produced during culture also are produced in vivo and are immunosuppressive in vivo. It follows from these results that the enlargement of draining lymph nodes which is associated with allogeneic pregnancy (Maroni and de Sousa, 1973; Ansell et al., 1978) does not necessarily represent an activation of suppressor mechanisms against harmful anti-fetal immune responses, as suggested by others (Clark and McDermott, 1978); rather, it may be explained along the lines of the long-recognized phenomenon of ‘hybrid vigor‘ (Clark and Kirby, 1966; Beer et al., 1975). It has been reported that the placenta is larger in allogeneic pregnancies, and that preimmunization of the mother against paternal alloantigens increases the size of placentas, litters and draining lymph nodes. It has been hypothesized (Wegmann, 1984, 1987) that activation of maternal T cells by alloantigens expressed on trophoblast may result

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in the release of T cell-derived cytokines, such as IL-3 and GM-CSF, which directly influence trophoblast growth and function. Similar mechanisms may be operative in draining lymph nodes, resulting in their enlargement during allogeneic pregnancy. If maternal cellular immune responses are neither generated nor suppressed during pregnancy, how is it possible to explain that humoral immune responses are generated to embryonic antigens? Semiallogeneic tissue grafts are routinely rejected when placed in a variety of tissue sites. However, the ability to generate responses and thus be rejected by the immune system is not an inherent characteristic of transplanted tissue; it is necessary that histocompatibility antigens expressed within the graft gain access to the immune system. Thus, grafts to so-called priveleged sites are not rejected or are rejected in a delayed manner (Niederkorn and Streilein, 1983; Head and Billingham, 1985). Similarly, it is well-documented that cells within a graft, called passenger leukocytes, are responsible for sensitization of the recipient and the ultimate rejection of engrafted tissue (Steinmuller, 1967; Elkins, 1968). It is generally believed that those cells leave the graft and migrate to draining lymphoid tissue where T cell stimulation occurs. It is reasonable, therefore, to hypothesize that cellular immunity to alloantigens is only stimulated when the antigen is presented intact on a cell surface. Soluble histocompatibility antigens may induce antibody formation, but we would suggest that they are unable to stimulate a cellular immune response, e.g., cytotoxic T cells are not generated unless cells comparable to passenger leukocytes are able to reach lymphoid tissue. We hypothesize that, during pregnancy, cellular immunity is not generated because cells which express alloantigens at a density or in a format which would lead to an immune response do not reach the maternal circulation. We further hypothesize that antibody responses to histocompatibility antigens which occur even in primagravida mothers are generated in response to shed histocompatibility antigens. References Ansell, J.D., McDougall, G., Speedy, G. and lnchley, C.J. (1978) Changes in lymphocyte accumulation and proliferation in lymph nodes draining the pregnant uterus. Clin. Exp. Immunol. 31,397-407. Badet, M.T., Bell, S.C. and Billington, W.D. (1983) Partial characterization of immunosuppressive factors from short term cultures of murine decidual tissue. Ann. Immunol. (Inst. Pasteur) 134C, 321329. Beer, A.E. and Billingham, R. (1974) Host responses to intra-uterine tissue; cellular and fetal allografts. J. Reprod. Fertil. (Suppl). 21,59-74. Beer, A.E. and Billingham, R.E. (1980) Mechanisms of non-rejection of the feto-placental allografts. Folia Biol. 26,225-243. Beer, A.E., Scott, J.R. and Billingham, R.E. (1975) Histocompatibility and maternal immunological status as determinants of fetoplacental weight and litter size in rodents. J. Exp. Med. 142, 180-196.

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