EUROP.
J. OBSTET. GYNEC.
REPROD.
BIOL., 1975, 5/i-2,
147-154.
EXCERPTA
MEDICA
lmmunobgical aspects of normal and abnormal cw-nancv W. D. Billington
Exactly 50 years ago, the geneticist1C.C. Little drew attention to the fact that the mammalian conceptus occupies a most privileged position, developing in intimate contact with maternal tissues in the foreign environment of the uterus and yet tolerated throughout the entire period of pregnancy. Now that the basic principles of transplantation immunology have been defined, it is clear that the paternally-inherited histocompatibility antigens of the conceptus should present to the mother an antigenic challenge capable of initiating immune responses leading to embryonic tissue rejection. Nevertheless, pregnancy extends well beyond the rejection time for surgically constructed allografts and there appears to be little evidence for any maternal-fetal incompatibility reactions. The reason for the apparently unqualified success of the conceptus as an intrauterine allograft has been sought for many years, and a number of hypotheses proposed. Three conditions must obtain in order for graft rejection to take place: firstly, the graft must possess foreign transplantation antigens in sufficient quantity and strength, and presented in a suitable form for recognition; secondly, for sensitization to occur the host must have an established vascular connection with the graft, and be capable of recognizing and reacting against the antigens - in other words must be immunologically competent; and, thirdly, the products of the immune response must be able to gain access to the graft. It would seem reasonable to consider that failure at some level of the afferent and/or efferent arm of this im-
munological reflex could be primarily responsible for the survival of the allografted conceptus. Whatever mechanisms are involved in holding the immunological forces of the mother in abeyance during normal pregnancy it is likely that these, as with other biological control systems, are potentially capable of breaking down and leading to pathological changes. The following review will therefore give brief consideration to the possible factors protecting normal pregnancy and also to a number of clinical abnormalities in which immunological components are implicated or suspected.
Possible factors involved in the maintenance of the conceptus Mode of vascularization
Unlike surgically constructed grafts, the conceptus shows no ingrowth of host vessels. Although the circulatory systems of the mother and embryo come into exceedingly close contact within the complex architecture of the hemochorial placenta they remain entirely independent. This is generally considered to be an important factor in reducing the level of sensitization of the mother to fetal antigenic stimulus. Experimental connection of the vascular systems of an F, hybrid and a parental strain animal leads to the condition of parabiosis intoxication, where severe immunological reactions follow the free transfer of competent lymphocytes to the hy-
148
W. D. Billington, Abnormal immunology
brid parabiont. Similar reactions appear to occur when the placenta is bypassed and the rat fetus is connected directly to the maternal tissues (A. E. Beer, personal communication). Although lack of vascular continuity with the mother may reduce primary sensitization, it may not be capable of protecting the conceptus against a preexisting maternal immunity, since a nonvascularized graft can undergo rejection in an immune host (Merwin and Hill, 1954).
Antigenic immaturity of the conceptus
If the conceptus is low or lacking in its expression of transplantation antigens then there can be no specific antagonistic response by the mother. However, many studies have reported that organized fetal tissues possess antigens recognizable in various experimental systems, and this hypothesis is now usually dismissed. In actual fact, the antigenic status of the conceptus, and especially the placenta (which is the only fetal element in direct contact with uterine tissues), is rather ill defined. Much more information is required on the time of appearance, precise nature, distribution, relative strength and immunogenicity of fetal antigens. It is becoming apparent that these play a major role in fetal-maternal immunological relationships.
Immunological unresponsiveness of the mother
It is possible that the pregnant state may in some way influence the ability of the female to mount an effective immune response. There are two main ways in which this could operate: I. nonspec@c depression of immunity. This may result from high circulating levels of hormones or serum factors such as glycoproteins or seromucoids. Corticosteroids can depress immune reactivity by producing a transient lymphocytopenia and also by having a weak immunosuppressive action. The protein hormones HCG and HPL have been shown to impair lymphocyte transformation in vitro, as have various pregnancy serum factors. 2. speciJic unresponsiveness.
Exposure
to fetal
antigen could result in a state of classical immunological tolerance in the mother. More recently, evidence has been marshalled to implicate ‘blocking’ antibody or immune complexes, operating in a manner similar to that described for tumor survival in immune hosts (HellstrGm and Hellstr(im, 1974). Since the antigenic stimulus from the fetus is likely to be rather prolonged, and at a low dose level, the second of these specific states is a more attractive possibility since this would favor the induction of humoral antibody or other blocking factors capable of impairing normally effective cell-mediated responses. Although it is well known that pregnant females generally show little or no alteration of reactivity to allogeneic skin grafts and are capable of normal humoral responses to injected antigen, it is now evident that more subtle regulations of the immune response occur during pregnancy. It may well be that both nonspecific and specific factors play their part, perhaps at different times and at different levels of suppression.
Immunological privilege of the uterine environment
It has been suggested that the uterus may share with such sites as the anterior chamber of the eye and the brain the property of immunologicalprivilege. The reason for this usually lies in the absence of a normal vascular supply and/or lack of lymphatic drainage. However, the uterus is not overtly deficient in this respect, and it has recently been shown to be an effective. route for the induction of imniunity. Rats and mice can be immunized by intrauterine instillation of allogeneic cells, and allografts of skin and other tissues undergo rejection under certain circumstances (Beer and Billingham, 1971). Nevertheless, this hypothesis cannot be completely discarded, since most of these experiments involved the nonpregnant uterus, and the absence of the decidual tissue which is a sine qua non of pregnancy may possibly have allowed immune recognition or reaction not found under normal pregnancy conditions. It might be thought that the occasional occurrence of ectopic implantation in our own species would argue against the uterus having any special immunological properties. ,But here again tht: existence of a
149
W. D. Billington, Abnormal immunology
decidual component, albeit rather restricted in some cases, might be an important factor. There is in fact experimental evidence for an immunologically protective role of decidual tissue (Kirby, Billington and James, 1966; Beer, Billingham and Hoerr, 1971).
Local hormone action
In addition to the suggested general suppressive effect of steroid and protein hormones, it is also possible that extremely high concentrations of HCG may reside at the site of production on the surface of the syncytiotrophoblast and play a significant part in the ability of the placenta to resist maternal immune attack. The hormone could prevent the reccognition of any antigens present on the placenta either by physical masking or by inhibiting lymphocyte approach. Since techniques are not yet available for the quantitative assay of cell surface hormone, and it is not even known whether antigens are present on the trophoblast membrane (see below: Presence of an anatomical barrier), this hypothesis is at present only a theoretical possibility. It should also be noted that HCG is a human placental hormone that may not have a counterpart in all mammalian species, nor even all those with a hemochorial placentation.
Invasive activity of trophoblast
The known ability of trophoblast to invade and phagocytose uterine tissues has led to the suggestion that any immunologically involved maternal leucocytes might be destroyed by this means before being able to initiate an immune response. There is no direct evidence for this suggestion, and it is unlikely to be a significant mechanism in the later stages of pregnancy when the invasive activity has ceased and phagocytosis considerably reduced.
Presence of an anatomical barrier
In theory, a barrier could inhibit, totally or partially, either the afferent or efferent arm of the immune response. Tn the pregnancy situation such a barrier must clearly interpose between fetal and
maternal tissues, and the only material in a suitable anatomical position is the trophoblast, since this provides a continuous front to the embryonic elements throughout the entire period of pregnancy. The fetal membranes should also be considered as a barrier during the later stages of pregnancy, but these present a rather special problem, fully discussed elsewhere (Jenkinson and Billington, 1974b).
Trophoblast as an immunological barrier
To remain effective, any form of barrier must possess at ieast some degree of immunological inertia, otherwise it would itself be susceptible to destruction. The primary question is therefore whether or not the trophoblast possesses transplantation antigens. And even if it does, whether they are expressed in a form recognizable by the mother and capable of stimulating an immune response. This question is not the simple one it might seem, since trophoblast exists in a variety of biological forms not only in different species but during particular developmental phases and in the mature placenta. In most cases it is also impossible to obtain in pure form for immunological analysis. Evidence relating to trophoblast antigenicity is confusing and controversial. It was originally proposed that trophoblast may be unique, having either an absence or severe intrinsic deficit of transplantation antigens (Simmons and Russell, 1962). An alternative hypothesis was that antigens may be present but prevented from being recognized to any detectable degree (Kirby, Billington, Bradbury and Goldstein, 1964; Currie and Bagshawe, 1967). The second possibility arose initially from electron-microscopical and histochemical studies on the mouse placenta demonstrating a fibrinoid-like material coating the surface of the trophoblast. Later investigation of trophoblast of the human placenta, and of hydatidiform mole trophoblast, indicated that the surface coating was actually a membranebound mucoprotein, and quite distinct from the gross fibrinoid deposits in other regions of the placenta (Bradbury, Billington, Kirby and Williams, 1970). The immunological implications of the ultrastructural and histochemical characteristics of the trophoblast cell surface have recently been considered in detail (Billington, 1975). Apart from
150
masking by mucoprotein, other possible mechanisms for lack of antigen recognition include masking by hormone (as described above, under Local hormone action), low antigen density, cell surface topography (e.g. antigenic determinants hidden in membrane convolutions), and antigen loss by shedding or internalization. It may well be that there is no common mechanism for all types of trophoblast. Experimental evidence for antigen masking by mucoprotein came from studies by Currie, Van Doorninck and Bagshawe (1968) on ectoplacental cone, a form of trophoblast that can be dissected from the conceptus early in mouse pregnancy. These workers demonstrated that following incubation with the enzyme neuraminidase the trophoblast was able to induce sensitization to subsequent skin grafts by injection into allogeneic mouse recipients. By analogy with similar experiments on tumor cells, the results were interpreted as indicating the unmasking of antigens by removal of neuraminit acid from cell surface sialomucoprotein. However, a later study by Simmons, Lipschultz, Rios and Ray (1971) failed to confirm these findings. Using a different approach, we have also investigated antigens of mouse ectoplacental cone trophoblast (Jenkinson and Billington, 1974a). In a cell-mediated microcytotoxicity assay, similar to that used byvandeputte and Sobis (1972) for trophoblastgiant cells, invitro cultures of trophoblast were shown to be insusceptible to lysis by immune lymphoid cells, even after preincubation in neuraminidase. Parallel cultures of embryonic fibroblasts and monolayers of embryonic cells from the same conceptus from which the trophoblast was dissected were extensively destroyed. Thus ectoplacental cone trophoblast appears not to possess, or present in an appropriate form, those antigenic determinants recognized by the effector cells on the allogeneic fibroblasts and embryonic tissue. Although the findings of Currie and his colleagues remain to be explained, it would seem that the early proliferating trophoblast of the mouse possesses at least a functional antigenic deficiency. This would be a most valuable property for a tissue having a fundamental role as a barrier protecting the embryo from potentially deleterious maternal immunity. As far as later stages of trophoblast are concerned, particularly those constituting the fetomaternal interface in the definitive p!acenta, the evidence is much less convincing. Numerous tech-
W. D. Billington, Abnormal immunology
niques have been used to investigate trophoblast in the placenta of mouse and man, but none have as yet fulfilled all the criteria for the unequivocal demonstration of histocompatibility antigens on the cell surface (Billington, Elson, Jenkinson 8z Searle, 1974).However, such studies, taken together with the not inconsiderable amount of indirect or circumstantial evidence, do indicate the likelihood that placental trophoblast possesses antigenic determinants (Billington, 1971; Edidin, 1972). Even if these exist at low surface density, and masked to some degree by mucoprotein, they may still have a significant role in the immunological relationship between mother and fetus. In addition to being insusceptible to maternal attack, they could be at least partially responsible for the induction of protective serum blocking factors in the mother, and might possibly also provide the placenta with an effective filtering system against the ingress of potentially harmful maternal antibody to the fetal tissues. Although the trophoblast provides a crucial barrier at the placental level it should be noted that this is not absolute. Certain maternal immunoglobulins are transmitted to the fetus, and there is some evidence for the passage of small numbers of fetal blood cells to the maternal circulation near to the time of parturition. The trophoblast is fundamentally, however, a highly selective macromolecular filter that also prevents the free passage of maternal lymphocytes that would lead to fetal damage or destruction. In summary, it seems possible to conclude that no single explanation can suffice for the survival of the conceptus as an intrauterine allograft. There is clearly a complex and dynamic immunological equilibrium between the maternal and fetal organisms that has arisen during the evolution of the viviparous state. This equilibrium probably involves the constant interplay of many factors, including at least some of those described above.
Immunological factors in pathological pregnancy
Although an immunological aetiology proposed for several clinical complications nancy, the evidence is in most cases rather tary or even circumstantial. The following
has been of pregfragmenis a brief
W. D. Billington, Abnormal immunology
summary of these phenomena, details of which can be found in the recent review by Jenkins (1975).
Spontaneous abortion
There are a number of lines of evidence that fetalmaternal incompatibility may be a causative factor in abortion. There is an increased frequency of abortions in ABO blood group incompatible matings (Matsunaga and Itoh, 1958; Wren and Vos, 1961) and it has been suggested that 2 % of all zygotes may die as a result of such incompatibility (Chung and Morton, 1961). A state of hypersensitivity to paternally derived antigens of the fetus is indicated in habitual aborters, who reject skin grafts from their husbands faster than those from unrelated males (Bardawil, Mitchell, McKeogh and Marchant, 1962) and also show an increased response to paternal antigens in a mixed lymphocyte reaction (Halbrecht and Komlos, 1968). There are a few cases of habitual aborters who have carried successfully to term following change of husband (Javert, 1962). AIthough it is claimed that aborters show a high level of antitrophoblast antibody in their serum (Montemagno and Di Stefano, 1965), their response to fetal transplantation antigen surprisingly appears to be reduced, since there are higher titers of HL-A antibody in secundigravida women who have had a normal first pregnancy than in those having had a previous spontaneous abortion (Burke and Johansen, 1974).
151
Rhesus disease
Isoimmunization of the mother to the Rh (and ABO) antigens of the fetal red cell is a well-described situation. The sensitizing bleed usually takes place at or around the time of parturition, when the placental barrier is breaking down, although it is now known that earlier bleeds may occur, particularly following therapeutic abortion (Freda, Gorman, Galen and Treaty, 1970). Choriocarcinoma
Evidence has been presented that this trophoblast tumor occurs more frequently in women who show a tendency towards compatibility with their husbands on the ABO system (Scott, 1962), although a much smaller study indicated no great difference from the expected distribution (Schmidt and Hertz, 1961). Azar (1962) reported a higher proportion of tumors amongst women in inbreeding communities, and concluded that consanguinity was an important factor. Robinson, Shulman, Ben-Hut-, Zuckerman and Neuman (1963) found that choriocarcinoma patients usually accepted a graft of their husband’s skin for prolonged periods in comparison with a similar graft from an unrelated donor, but reported later (Robinson, Ben-Hur, Zuckerman and Neuman, 1967) that there was no marked compatibility between the individuals on the basis of leucocyte antigen typing. This indicates the induction of a maternal tolerance to the paternal antigens of the tumor, rather than any predisposing genetic factors.
Preeclampsia Graft-versus-host disease
An immunological basis for preeclampsia has been sought for many years but the evidence is still inconclusive. Placental and renal pathology indicates an immune battleground, and abnormal serum protein levels are a common finding. Data are also beginning to accumulate suggestive of a shift in HL-A gene frequency towards an incompatibility between mother and father. One of the more persuasive arguments is that injection of placental extracts produces preeclampsia-like syndromes in experimental animals. This phenomenon is discussed in detail by Beer (p. 135 this volume).
There are a few exceedingly rare cases of human male neonates showing the symptoms of a wasting disease believed to result from an immunological attack on the foetus by transplacentally transmitted maternal lymphocytes. This serves to emphasize the importance of a placental barrier in normal pregnancy. There is also a recent report that GVH disease can be detected in neonatal animals following immunization of the mother to paternal antigens either before or during pregnancy (Beer and Billingham, 1973), but independent studies have so far been unable to confirm this (unpublished results).
152
W. D. Billington,
Dysmaturity
Retardation of intrauterine growth is frequently associated with placental insufficiency, which can have histological features suggestive of an immune conflict. Certain types of dysmature babies show clinical symptoms of graft-versus-host disease, and have abnormal immunoglobulin levels. Fetal malformation It has been claimed that theincidence of congenital anomalies is higher in women with raised HL-A antibody levels during pregnancy (Terasaki, Mickey, Yamazaki and Vredevoe, 1970), but this has been questioned by a later study that failed to detect any such association. It is quite clear that much more information is needed on all these abnormalities of pregnancy. Many of the newer techniques of immunology, particularly for the assay of cell-mediated immunity, have yet to be employed for this purpose.
References Azar, H. A. (1962): Cancer in Lebanon and the Near East. Cancer, 15, 66.
Bardawil, W. A., Mitchell, G. W., McKeogh, R. P. and Marchant, D. J. (1962): Behaviour of skin homografts in human pregnancy. I. Habitual abortion. Amer. J. Obstet. Gynec.,
Physiol.,
5, 27.
Billington, W. D. (1975): Organization, ultrastructure and histochemistry of the placenta: immunological considerations. In: The Immunobiology of Trophoblast. Editors: R. G. Edwards, C. W. S. Howe and M. H. Johnson. Cambridge University Press, Cambridge. In press. Billington, W. D., Elson, J., Jenkinson, E. J. and Searle, R. F. (1974): Antigenicity of the trophoblast. In: Proc., Ist International Gynecology,
Congress
on Immunology
in Obstetrics
and
p. 111. Excerpta Medica, Amsterdam. Bradbury, S., Billington, W. D., Kirby, D. R. S. and Williams, E. A. (1970): Histochemical characterization of the surface mucoprotein of normal and abnormal human trophoblast. Histochem.
immunology
Burke, J. and Johansen, K. (1974): The formation of HL-A antibodies in pregnancy. The antigenicity of aborted and term foetuses. J. Obstet. Gynaec. Brit. Cwlth, 81, 222. Chung, C. S. and Morton, N. E. (1961): Selection at the ABO locus. Amer. J. hum. Genet., 13, 9. Currie, G. A. and Bagshawe, K. D. (1967): The masking of antigens on trophoblast and cancer cells. Lancet, I, 708. Currie, G. A., Van Doorninck, W. and Bagshawe, K. D. (1968): Effect of neuraminidase on the immunogenicity of early mouse trophoblast. Nature (Lond.), 219, 191. Edidin, M. (1972): Histocompatibility genes, transplantation antigens and pregnancy. In: Transplantation Antigens: Markers of Biological Individuality, p. 75. Editors: B. D. Kahan and R. A. Reisfeld. Academic Press, New York London. Freda, V. J., Gorman, J. G., Galen, R. S. and Treaty, N. (1970): The threat of Rh immunization from abortion. Lancet,
2, 147.
Halbrecht, I. and Komlos, L. (1968): Lymphocyte transfer in mixed wife-husband leucocyte cultures in abortions and hydatidiform moles. Obstet. and Gynec., 31, 173. Hellstrom, K. E. and Hellstrbm, I. (1974): Lymphocyte-mediated cytotoxicity and blocking serum activity to tumour antigens. Advanc. Immunol., 18, 209. Javert, C. T. (1962): Further follow-up on habitual abortion patients. Amer. J. Obstet. Gynec., 84, 1149. Jenkins, D. M. (1975): Pregnancy phenomena with a possible immunological basis. In: Immunology of Human Reproduction. Editors: J. S. Scott and W. R. Jones. Academic Press, London. In press. Jenkinson, E. J. and Billington, W. D. (1974a): Differential susceptibility of mouse trophoblast and embryonic tissue to immune cell lysis. Transplantation, 18, 286. Jenkinson, E. J. and Billington, W. D. (1974b): Studies on the immunobiology of mouse foetal membranes: the effect of cell-mediated immunity on yolk sac cells in vitro. J. Reprod. Fertil., 41, 403.
84, 1283.
Beer, A. E. and Billingham, R. E. (1971): Immunobiology of mammalian reproduction. Advanc. Immunol., 14, 1. Beer, A. E. and Billingham, R. E. (1973): Maternally acquired runt disease. Science, 179, 240. Beer, A. E., Billingham, R. E. and Hoerr, R. A. (1971): Elicitation and expression of transplantation immunity in the uterus. Transplant. Proc., 3, 609. Billington, W. D. (1971): Biology of the trophoblast. Advunc. Reprod.
Abnormal
.I., 2, 263.
Kirby, D. R. S., Billington, W. D., Bradbury, S. and Goldstein, D. J. (1964): Antigen barrier of the mouse placenta. Nature
(Lond.),
204, 548.
Kirby, D. R. S., Billington, W. D. and James, D. A. (1966): Transplantations, of eggs to the kidney and uterus of immunized mice. Transplantation, 4, 713. Little, C. C. (1924): Genetics of tissue transplantation in mammals. J. Cancer Res., 8, 75. Matsunaga, E. and Itoh, S. (1958): Blood groups and fertility in a Japanese population, with special reference to intrauterine selection due to maternal-foetal incompatibility. Ann. hum. Genet., 22, 111. Merwin, R. M. and Hill,E. L. (1954): Fate of vascularized and non-vascularized sub-cutaneous homografts in mice. J. nat. Cancer Inst., 14, 819.
Montemagno, U. and Di Stefano, M. (1965): Gli anticorpi antiplacenta nell’aborto ricercati con tecnica immunofluorescente. Momt. ostet-gmec., 36, 563. E., Ben-Hur, N., Zuckerman, H. and Neuman, Z. (1967): Further immunologic studies in patients with chorionepithe]iomaand hydatidiform mole. Cancer Res., 27,
Robinson,
1202.
W. D. Billing&m,
Abnormal
Robinson, E., Shulman, J., Ben-Hur, N., Zuckerman, H. and Neuman, Z. (1963): Immunological studies and behaviour of husband and foreign homografts in patients with chorionepithelioma. Lancet, I, 300. Schmidt, P. J. and Hertz, R. (1961): Blood group factors in women with choriocarcinoma as compared with those of their husbands. Amer. J. Obstet. Gynec., 82, 651. Scott, J. S. (1962): Choriocarcinoma: observations on the aetiology. Amer. J. Obstet. Gynec., 83, 185. Simmons, R. L., Lipschultz, M. L., Rios, A. and Ray, P. K. Failure of neuraminidase to unmask histocompatibility antigens on trophoblast. Nature New Biol., 231, 111. Simmons, R. L. and Russell, P. S. (1962): The antigenicity of mouse trophoblast. Ann. N.Y. Acad. Sci., 99, 111. Terasaki, P. I., Mickey, M. R., Yamazaki, J. N. and Vredevoe, D. (1970): Maternal-foetal incompatibility I. Incidence of HL-A antibodies and possible association with congenital anomalies, Transplantation, 9, 538. Vandeputte, M. and Sobis, H. (1972): Histocompatibility antigens on mouse blastocysts and ectoplacental cones. Transplantation,
153
immunology
14, 331.
Wren, B. G. and Vos, G. H. (1961): Blood group incompatibility as a cause of spontaneous abortion. J. Obstet. Gynaec.
pregnant uterus are not? Wouldn’t you feel that these findings are an argument in favor of some protective role of the decidua? I know very well that Beer has done some very beautiful experiments which tend to disprove this point, at least with other tissues implanted. What is your feeling about it? I would agree entirely because they were also my own experiments so I happen to believe them. The reason why 1 didn’t go into that in great detail is because I know that Beer is going to talk about it later. But what I did perhaps not bring out is that a lot of the early experiments, claiming to show that one got tissue rejection in the uterus, to my mind were related to the fact that themodeldidnot allow thedecidual tissue to develop, and this was subsequently confirmed by Beer’s experiments. I think he will probably agree with me that there is a protective influence by the decidua in respect to the immunological interaction and that this is the interpretation of the experiments that you mention. In other words that those immune reactions which are responsible for the destruction of the embryo transplanted to the kidney, in the same animal cannot bring about rejection of the embryo within the uterus.
BILLINGTON:
Brit. Cwlth, 68, 637.
Discussion 1 must say that I am glad to hear that you find exactly the same results as the ones we have found with ectoplacental cones treated in vitro with sensitized lymphocytes. I think there is a further argument to indicate that the trophoblast cells do not display histocompatibihty antigens and this is based on our observations on ectoplacental cones transplanted in vivo under the kidney capsule of presensitized allogeneic mice. As in this case also no rejection is observed, the phenomenon applies not only to in vitro studies but also to in vivo experiments. Moreover, Dr. H. Sobis, who is working in my laboratory, has done some electron microscopy on the ectoplacental cones implanted under the kidney capsule. Not until four to five days after implantation did she observe the presence of an outer cell layer of mucopolysaccharides. Therefore, this layer of mucopolysaccharides seems to be a secondary phenomenon and not the cause of the absence of rejection. In presensitized animals one would indeed expect morphological evidence of rejection earlier than 4 to 5 days after implantation. Now the second remark is that you reject the hypothesis that the uterus should be, or could be, an immunologically privilegedorgan. How wouldyouexplainin that case, the experiments of Kirby et al. in which, in preimmunized mice, allogeneic blastocysts implanted under the kidney capsule are rejected but allogeneic blastocysts in the pseudoVANDEPUTTE:
BEER: We know that transplantation antigens can be identified on the zygote from the two cell stage onwards. Park and Robertson have both shown us that there is an intense amount of mononuclear cell infiltration at the time of implantation of the mammalian zygote which is very reminiscent of the classic delayed type hypersensitivity reaction. This suggests that something has been recognized, and although perhaps I in the past have soft-pedalled the uterus as an immunologically privileged site, rethinking and redoing experiments, I believe that there is a brief period during which the uterus does afford an immunologically privileged site for the implanting embryo. I would hope to give you some data later to support that thesis.
I am amazed that under rare conditions, malignant trophoblastic cells, for instance choriocarcinoma, may express transplantation antigens resulting in an antigen-antibody reaction. We all know that some patients, even with vaginal and lung metastases, will have spontaneous regression and healing of the disease. This means to me that the maternal immune mechanisms can defeat the malignant choriocarcinoma. How do you feel about that? VORHERR:
BILLINGTON: I have spoken on this point to Bagshawe, and he says that if one gets a cellular infiltration then the prognosis tends to be better than if one doesn’t get a cellular infiltration and, obviously, this would appear to indicate that histocompatibility antigens, HL-A antigens, are present on choriocarcinema. But there is no direct evidence of this and the possibility of their spontaneous regression being due to an immune response is really conjectural. I don’t know whether Park would challenge that.
154
W. D. Billington, Abnormal immunology
PARK: I agree with that. I don’t think matters have been taken any further during the last several years. The possible role of transplantation antigens in relation to spontaneous regression is still quite obscure. It was Bagshawe who made the first attempts to understand this by doing skin grafting of the patients with skin from husbands and the like. But not only did that fail to influence the course of the disease, it failed to produce convincing evidence that transplantation antigens were playing any significant part at all.
But how can an organism defeat full blown lung metastases if there is no immune response?
VORHERR:
BILLINGTON: Lung metstases of what? Do you mean pulmonary embolism?
No, I am talking about lung metastases of choriocarcinoma clearly proven on X-ray; a few months later everything is gone and the patient survives. Such cases have been reported. VORHERR:
One may very abnormal genetic noma and the patient; case. I don’t think BILLINGTON:
possibly implicate, in that situation, a relationship between the choriocarcibut that certainly is not normally the I can say anything more on that.
PARK: Just one further comment; the regression of choriocarcinema is the regression of a tissue which, as we were discussing earlier, has a finite life span. For some reason that, as far as I know, is still quite obscure, normal trophoblast is controlled by a strictly run biological clock which sees to it, in the appropriate species, that after a time the tissue begins to become seriously senescent and impaired. Now this a queer carcinoma; in it, we see the malignant expression of tissue of this type, tissue which has a much more limited span than any other tissue, even including such things as deciduous teeth, which have a life span much less than that of the organism as a whole. I can’t help feeling that in some way the curious behaviour of choriocarcinoma on these occasions is related in a degree to that limited life-span aspect of trophoblast behavior just as it may be to the transplantation aspect of choriocarcinoma.
Yes, I think I would support that, but if I might add one point which is slightly to the contrary. A number of people have tried to develop continuous cell lines of trophoblast for various reasons and have failed abjectly. However, there is a report - I don’t know if it has been substantiated that it is possible to develop a continuous cell line from choriocarcinoma. So, although the biological clock of the two may be very closely related, there must be certain methods by which the latter can escape from this control. BILLINGTON:
ROBERTSON: Could I ask a question which is out of total ignorance? One thing which has always intrigued me is what happens to the breakdown products of spermatozoa before
conception occurs? It is rather like asking the question, “where do all the flies go in winter?“. What happens to all those millions of spermatozoain theejaculate? Their numbers, as we understand it, reduce very, very considerably until a few panting survivors reach the ampulla of the tube. What happens to all those that don’t make it? BEER: Now
you are tempting me because I think this is something which we as immunobiologists have totally neglected, the chronic repetitive inoculation which the female has to undergo, whether for pleasure or procreation. 1 hope to make a good case that this accomplishesan immunologic conditioning of the uterus. It makes it more conducive, not only to deciduagenesis but also to implantation of the embryo against which the sensitivity is directed; a kind of backward look. I’m not sure whether I understand it but I hope at least to play a game with you to try to help you to help me understand. ROBERTSON: I always thought it was rather different. Obviously the gastrointestinal tract is in something of the same position. When one looks at the normal gastrointestinal tract one sees mucosa that looks as though it would be pretty well protected by a cellular reaction against potential foreign antigens with plenty of lymphocytes and plasma cells in the mucosa. But the round cell component of the endocervix, of the endometrium and of the fallopian tube is minimal compared with the gastrointestinal tract. BILLINGTON: I think perhaps that I should add that the presence of spermatozoa, and possible induction of immunity as a result, is not essential for normal pregnancy, because it is perfectly possible to transplant fertilized embryos into the uterus of a previously virgin female and they develop quite normally.
Yes, but it’s a question of degree. If one takes two groups of tubal ligated animals, one group mated with only a vibrator on the day of estrus to induce pseudopregnancy, the other group mated with males of an unrelated genetic strain, and four days later challenges each group to implant genetically foreign blastocysts the females whoseuteri had been exposed to seminal products will demonstrate higher reproductive efficiency than those not exposed. I think we have been a little bit naive or too easily satisfied that we could transplant allogeneic embryos to females that have never met an allogeneic male. We have never studied the degree of hospitality. BEER:
Is there that degree of relevance in our own species, where normally there is only one embryo and either it implants or it fails’? Most often this question of degree relates to polytocous species where there are a number of eggs.
BILLINGTON:
BEER: Human
non.
sex ratio data would suggest a similar phenome-
J. OBSTET. GYNEC.
REPROD.
BIOL., 1975, 5/i-2,
147-154.
EXCERPTA
MEDICA
lmmunobgical aspects of normal and abnormal cw-nancv W. D. Billington
Exactly 50 years ago, the geneticist1C.C. Little drew attention to the fact that the mammalian conceptus occupies a most privileged position, developing in intimate contact with maternal tissues in the foreign environment of the uterus and yet tolerated throughout the entire period of pregnancy. Now that the basic principles of transplantation immunology have been defined, it is clear that the paternally-inherited histocompatibility antigens of the conceptus should present to the mother an antigenic challenge capable of initiating immune responses leading to embryonic tissue rejection. Nevertheless, pregnancy extends well beyond the rejection time for surgically constructed allografts and there appears to be little evidence for any maternal-fetal incompatibility reactions. The reason for the apparently unqualified success of the conceptus as an intrauterine allograft has been sought for many years, and a number of hypotheses proposed. Three conditions must obtain in order for graft rejection to take place: firstly, the graft must possess foreign transplantation antigens in sufficient quantity and strength, and presented in a suitable form for recognition; secondly, for sensitization to occur the host must have an established vascular connection with the graft, and be capable of recognizing and reacting against the antigens - in other words must be immunologically competent; and, thirdly, the products of the immune response must be able to gain access to the graft. It would seem reasonable to consider that failure at some level of the afferent and/or efferent arm of this im-
munological reflex could be primarily responsible for the survival of the allografted conceptus. Whatever mechanisms are involved in holding the immunological forces of the mother in abeyance during normal pregnancy it is likely that these, as with other biological control systems, are potentially capable of breaking down and leading to pathological changes. The following review will therefore give brief consideration to the possible factors protecting normal pregnancy and also to a number of clinical abnormalities in which immunological components are implicated or suspected.
Possible factors involved in the maintenance of the conceptus Mode of vascularization
Unlike surgically constructed grafts, the conceptus shows no ingrowth of host vessels. Although the circulatory systems of the mother and embryo come into exceedingly close contact within the complex architecture of the hemochorial placenta they remain entirely independent. This is generally considered to be an important factor in reducing the level of sensitization of the mother to fetal antigenic stimulus. Experimental connection of the vascular systems of an F, hybrid and a parental strain animal leads to the condition of parabiosis intoxication, where severe immunological reactions follow the free transfer of competent lymphocytes to the hy-
148
W. D. Billington, Abnormal immunology
brid parabiont. Similar reactions appear to occur when the placenta is bypassed and the rat fetus is connected directly to the maternal tissues (A. E. Beer, personal communication). Although lack of vascular continuity with the mother may reduce primary sensitization, it may not be capable of protecting the conceptus against a preexisting maternal immunity, since a nonvascularized graft can undergo rejection in an immune host (Merwin and Hill, 1954).
Antigenic immaturity of the conceptus
If the conceptus is low or lacking in its expression of transplantation antigens then there can be no specific antagonistic response by the mother. However, many studies have reported that organized fetal tissues possess antigens recognizable in various experimental systems, and this hypothesis is now usually dismissed. In actual fact, the antigenic status of the conceptus, and especially the placenta (which is the only fetal element in direct contact with uterine tissues), is rather ill defined. Much more information is required on the time of appearance, precise nature, distribution, relative strength and immunogenicity of fetal antigens. It is becoming apparent that these play a major role in fetal-maternal immunological relationships.
Immunological unresponsiveness of the mother
It is possible that the pregnant state may in some way influence the ability of the female to mount an effective immune response. There are two main ways in which this could operate: I. nonspec@c depression of immunity. This may result from high circulating levels of hormones or serum factors such as glycoproteins or seromucoids. Corticosteroids can depress immune reactivity by producing a transient lymphocytopenia and also by having a weak immunosuppressive action. The protein hormones HCG and HPL have been shown to impair lymphocyte transformation in vitro, as have various pregnancy serum factors. 2. speciJic unresponsiveness.
Exposure
to fetal
antigen could result in a state of classical immunological tolerance in the mother. More recently, evidence has been marshalled to implicate ‘blocking’ antibody or immune complexes, operating in a manner similar to that described for tumor survival in immune hosts (HellstrGm and Hellstr(im, 1974). Since the antigenic stimulus from the fetus is likely to be rather prolonged, and at a low dose level, the second of these specific states is a more attractive possibility since this would favor the induction of humoral antibody or other blocking factors capable of impairing normally effective cell-mediated responses. Although it is well known that pregnant females generally show little or no alteration of reactivity to allogeneic skin grafts and are capable of normal humoral responses to injected antigen, it is now evident that more subtle regulations of the immune response occur during pregnancy. It may well be that both nonspecific and specific factors play their part, perhaps at different times and at different levels of suppression.
Immunological privilege of the uterine environment
It has been suggested that the uterus may share with such sites as the anterior chamber of the eye and the brain the property of immunologicalprivilege. The reason for this usually lies in the absence of a normal vascular supply and/or lack of lymphatic drainage. However, the uterus is not overtly deficient in this respect, and it has recently been shown to be an effective. route for the induction of imniunity. Rats and mice can be immunized by intrauterine instillation of allogeneic cells, and allografts of skin and other tissues undergo rejection under certain circumstances (Beer and Billingham, 1971). Nevertheless, this hypothesis cannot be completely discarded, since most of these experiments involved the nonpregnant uterus, and the absence of the decidual tissue which is a sine qua non of pregnancy may possibly have allowed immune recognition or reaction not found under normal pregnancy conditions. It might be thought that the occasional occurrence of ectopic implantation in our own species would argue against the uterus having any special immunological properties. ,But here again tht: existence of a
149
W. D. Billington, Abnormal immunology
decidual component, albeit rather restricted in some cases, might be an important factor. There is in fact experimental evidence for an immunologically protective role of decidual tissue (Kirby, Billington and James, 1966; Beer, Billingham and Hoerr, 1971).
Local hormone action
In addition to the suggested general suppressive effect of steroid and protein hormones, it is also possible that extremely high concentrations of HCG may reside at the site of production on the surface of the syncytiotrophoblast and play a significant part in the ability of the placenta to resist maternal immune attack. The hormone could prevent the reccognition of any antigens present on the placenta either by physical masking or by inhibiting lymphocyte approach. Since techniques are not yet available for the quantitative assay of cell surface hormone, and it is not even known whether antigens are present on the trophoblast membrane (see below: Presence of an anatomical barrier), this hypothesis is at present only a theoretical possibility. It should also be noted that HCG is a human placental hormone that may not have a counterpart in all mammalian species, nor even all those with a hemochorial placentation.
Invasive activity of trophoblast
The known ability of trophoblast to invade and phagocytose uterine tissues has led to the suggestion that any immunologically involved maternal leucocytes might be destroyed by this means before being able to initiate an immune response. There is no direct evidence for this suggestion, and it is unlikely to be a significant mechanism in the later stages of pregnancy when the invasive activity has ceased and phagocytosis considerably reduced.
Presence of an anatomical barrier
In theory, a barrier could inhibit, totally or partially, either the afferent or efferent arm of the immune response. Tn the pregnancy situation such a barrier must clearly interpose between fetal and
maternal tissues, and the only material in a suitable anatomical position is the trophoblast, since this provides a continuous front to the embryonic elements throughout the entire period of pregnancy. The fetal membranes should also be considered as a barrier during the later stages of pregnancy, but these present a rather special problem, fully discussed elsewhere (Jenkinson and Billington, 1974b).
Trophoblast as an immunological barrier
To remain effective, any form of barrier must possess at ieast some degree of immunological inertia, otherwise it would itself be susceptible to destruction. The primary question is therefore whether or not the trophoblast possesses transplantation antigens. And even if it does, whether they are expressed in a form recognizable by the mother and capable of stimulating an immune response. This question is not the simple one it might seem, since trophoblast exists in a variety of biological forms not only in different species but during particular developmental phases and in the mature placenta. In most cases it is also impossible to obtain in pure form for immunological analysis. Evidence relating to trophoblast antigenicity is confusing and controversial. It was originally proposed that trophoblast may be unique, having either an absence or severe intrinsic deficit of transplantation antigens (Simmons and Russell, 1962). An alternative hypothesis was that antigens may be present but prevented from being recognized to any detectable degree (Kirby, Billington, Bradbury and Goldstein, 1964; Currie and Bagshawe, 1967). The second possibility arose initially from electron-microscopical and histochemical studies on the mouse placenta demonstrating a fibrinoid-like material coating the surface of the trophoblast. Later investigation of trophoblast of the human placenta, and of hydatidiform mole trophoblast, indicated that the surface coating was actually a membranebound mucoprotein, and quite distinct from the gross fibrinoid deposits in other regions of the placenta (Bradbury, Billington, Kirby and Williams, 1970). The immunological implications of the ultrastructural and histochemical characteristics of the trophoblast cell surface have recently been considered in detail (Billington, 1975). Apart from
150
masking by mucoprotein, other possible mechanisms for lack of antigen recognition include masking by hormone (as described above, under Local hormone action), low antigen density, cell surface topography (e.g. antigenic determinants hidden in membrane convolutions), and antigen loss by shedding or internalization. It may well be that there is no common mechanism for all types of trophoblast. Experimental evidence for antigen masking by mucoprotein came from studies by Currie, Van Doorninck and Bagshawe (1968) on ectoplacental cone, a form of trophoblast that can be dissected from the conceptus early in mouse pregnancy. These workers demonstrated that following incubation with the enzyme neuraminidase the trophoblast was able to induce sensitization to subsequent skin grafts by injection into allogeneic mouse recipients. By analogy with similar experiments on tumor cells, the results were interpreted as indicating the unmasking of antigens by removal of neuraminit acid from cell surface sialomucoprotein. However, a later study by Simmons, Lipschultz, Rios and Ray (1971) failed to confirm these findings. Using a different approach, we have also investigated antigens of mouse ectoplacental cone trophoblast (Jenkinson and Billington, 1974a). In a cell-mediated microcytotoxicity assay, similar to that used byvandeputte and Sobis (1972) for trophoblastgiant cells, invitro cultures of trophoblast were shown to be insusceptible to lysis by immune lymphoid cells, even after preincubation in neuraminidase. Parallel cultures of embryonic fibroblasts and monolayers of embryonic cells from the same conceptus from which the trophoblast was dissected were extensively destroyed. Thus ectoplacental cone trophoblast appears not to possess, or present in an appropriate form, those antigenic determinants recognized by the effector cells on the allogeneic fibroblasts and embryonic tissue. Although the findings of Currie and his colleagues remain to be explained, it would seem that the early proliferating trophoblast of the mouse possesses at least a functional antigenic deficiency. This would be a most valuable property for a tissue having a fundamental role as a barrier protecting the embryo from potentially deleterious maternal immunity. As far as later stages of trophoblast are concerned, particularly those constituting the fetomaternal interface in the definitive p!acenta, the evidence is much less convincing. Numerous tech-
W. D. Billington, Abnormal immunology
niques have been used to investigate trophoblast in the placenta of mouse and man, but none have as yet fulfilled all the criteria for the unequivocal demonstration of histocompatibility antigens on the cell surface (Billington, Elson, Jenkinson 8z Searle, 1974).However, such studies, taken together with the not inconsiderable amount of indirect or circumstantial evidence, do indicate the likelihood that placental trophoblast possesses antigenic determinants (Billington, 1971; Edidin, 1972). Even if these exist at low surface density, and masked to some degree by mucoprotein, they may still have a significant role in the immunological relationship between mother and fetus. In addition to being insusceptible to maternal attack, they could be at least partially responsible for the induction of protective serum blocking factors in the mother, and might possibly also provide the placenta with an effective filtering system against the ingress of potentially harmful maternal antibody to the fetal tissues. Although the trophoblast provides a crucial barrier at the placental level it should be noted that this is not absolute. Certain maternal immunoglobulins are transmitted to the fetus, and there is some evidence for the passage of small numbers of fetal blood cells to the maternal circulation near to the time of parturition. The trophoblast is fundamentally, however, a highly selective macromolecular filter that also prevents the free passage of maternal lymphocytes that would lead to fetal damage or destruction. In summary, it seems possible to conclude that no single explanation can suffice for the survival of the conceptus as an intrauterine allograft. There is clearly a complex and dynamic immunological equilibrium between the maternal and fetal organisms that has arisen during the evolution of the viviparous state. This equilibrium probably involves the constant interplay of many factors, including at least some of those described above.
Immunological factors in pathological pregnancy
Although an immunological aetiology proposed for several clinical complications nancy, the evidence is in most cases rather tary or even circumstantial. The following
has been of pregfragmenis a brief
W. D. Billington, Abnormal immunology
summary of these phenomena, details of which can be found in the recent review by Jenkins (1975).
Spontaneous abortion
There are a number of lines of evidence that fetalmaternal incompatibility may be a causative factor in abortion. There is an increased frequency of abortions in ABO blood group incompatible matings (Matsunaga and Itoh, 1958; Wren and Vos, 1961) and it has been suggested that 2 % of all zygotes may die as a result of such incompatibility (Chung and Morton, 1961). A state of hypersensitivity to paternally derived antigens of the fetus is indicated in habitual aborters, who reject skin grafts from their husbands faster than those from unrelated males (Bardawil, Mitchell, McKeogh and Marchant, 1962) and also show an increased response to paternal antigens in a mixed lymphocyte reaction (Halbrecht and Komlos, 1968). There are a few cases of habitual aborters who have carried successfully to term following change of husband (Javert, 1962). AIthough it is claimed that aborters show a high level of antitrophoblast antibody in their serum (Montemagno and Di Stefano, 1965), their response to fetal transplantation antigen surprisingly appears to be reduced, since there are higher titers of HL-A antibody in secundigravida women who have had a normal first pregnancy than in those having had a previous spontaneous abortion (Burke and Johansen, 1974).
151
Rhesus disease
Isoimmunization of the mother to the Rh (and ABO) antigens of the fetal red cell is a well-described situation. The sensitizing bleed usually takes place at or around the time of parturition, when the placental barrier is breaking down, although it is now known that earlier bleeds may occur, particularly following therapeutic abortion (Freda, Gorman, Galen and Treaty, 1970). Choriocarcinoma
Evidence has been presented that this trophoblast tumor occurs more frequently in women who show a tendency towards compatibility with their husbands on the ABO system (Scott, 1962), although a much smaller study indicated no great difference from the expected distribution (Schmidt and Hertz, 1961). Azar (1962) reported a higher proportion of tumors amongst women in inbreeding communities, and concluded that consanguinity was an important factor. Robinson, Shulman, Ben-Hut-, Zuckerman and Neuman (1963) found that choriocarcinoma patients usually accepted a graft of their husband’s skin for prolonged periods in comparison with a similar graft from an unrelated donor, but reported later (Robinson, Ben-Hur, Zuckerman and Neuman, 1967) that there was no marked compatibility between the individuals on the basis of leucocyte antigen typing. This indicates the induction of a maternal tolerance to the paternal antigens of the tumor, rather than any predisposing genetic factors.
Preeclampsia Graft-versus-host disease
An immunological basis for preeclampsia has been sought for many years but the evidence is still inconclusive. Placental and renal pathology indicates an immune battleground, and abnormal serum protein levels are a common finding. Data are also beginning to accumulate suggestive of a shift in HL-A gene frequency towards an incompatibility between mother and father. One of the more persuasive arguments is that injection of placental extracts produces preeclampsia-like syndromes in experimental animals. This phenomenon is discussed in detail by Beer (p. 135 this volume).
There are a few exceedingly rare cases of human male neonates showing the symptoms of a wasting disease believed to result from an immunological attack on the foetus by transplacentally transmitted maternal lymphocytes. This serves to emphasize the importance of a placental barrier in normal pregnancy. There is also a recent report that GVH disease can be detected in neonatal animals following immunization of the mother to paternal antigens either before or during pregnancy (Beer and Billingham, 1973), but independent studies have so far been unable to confirm this (unpublished results).
152
W. D. Billington,
Dysmaturity
Retardation of intrauterine growth is frequently associated with placental insufficiency, which can have histological features suggestive of an immune conflict. Certain types of dysmature babies show clinical symptoms of graft-versus-host disease, and have abnormal immunoglobulin levels. Fetal malformation It has been claimed that theincidence of congenital anomalies is higher in women with raised HL-A antibody levels during pregnancy (Terasaki, Mickey, Yamazaki and Vredevoe, 1970), but this has been questioned by a later study that failed to detect any such association. It is quite clear that much more information is needed on all these abnormalities of pregnancy. Many of the newer techniques of immunology, particularly for the assay of cell-mediated immunity, have yet to be employed for this purpose.
References Azar, H. A. (1962): Cancer in Lebanon and the Near East. Cancer, 15, 66.
Bardawil, W. A., Mitchell, G. W., McKeogh, R. P. and Marchant, D. J. (1962): Behaviour of skin homografts in human pregnancy. I. Habitual abortion. Amer. J. Obstet. Gynec.,
Physiol.,
5, 27.
Billington, W. D. (1975): Organization, ultrastructure and histochemistry of the placenta: immunological considerations. In: The Immunobiology of Trophoblast. Editors: R. G. Edwards, C. W. S. Howe and M. H. Johnson. Cambridge University Press, Cambridge. In press. Billington, W. D., Elson, J., Jenkinson, E. J. and Searle, R. F. (1974): Antigenicity of the trophoblast. In: Proc., Ist International Gynecology,
Congress
on Immunology
in Obstetrics
and
p. 111. Excerpta Medica, Amsterdam. Bradbury, S., Billington, W. D., Kirby, D. R. S. and Williams, E. A. (1970): Histochemical characterization of the surface mucoprotein of normal and abnormal human trophoblast. Histochem.
immunology
Burke, J. and Johansen, K. (1974): The formation of HL-A antibodies in pregnancy. The antigenicity of aborted and term foetuses. J. Obstet. Gynaec. Brit. Cwlth, 81, 222. Chung, C. S. and Morton, N. E. (1961): Selection at the ABO locus. Amer. J. hum. Genet., 13, 9. Currie, G. A. and Bagshawe, K. D. (1967): The masking of antigens on trophoblast and cancer cells. Lancet, I, 708. Currie, G. A., Van Doorninck, W. and Bagshawe, K. D. (1968): Effect of neuraminidase on the immunogenicity of early mouse trophoblast. Nature (Lond.), 219, 191. Edidin, M. (1972): Histocompatibility genes, transplantation antigens and pregnancy. In: Transplantation Antigens: Markers of Biological Individuality, p. 75. Editors: B. D. Kahan and R. A. Reisfeld. Academic Press, New York London. Freda, V. J., Gorman, J. G., Galen, R. S. and Treaty, N. (1970): The threat of Rh immunization from abortion. Lancet,
2, 147.
Halbrecht, I. and Komlos, L. (1968): Lymphocyte transfer in mixed wife-husband leucocyte cultures in abortions and hydatidiform moles. Obstet. and Gynec., 31, 173. Hellstrom, K. E. and Hellstrbm, I. (1974): Lymphocyte-mediated cytotoxicity and blocking serum activity to tumour antigens. Advanc. Immunol., 18, 209. Javert, C. T. (1962): Further follow-up on habitual abortion patients. Amer. J. Obstet. Gynec., 84, 1149. Jenkins, D. M. (1975): Pregnancy phenomena with a possible immunological basis. In: Immunology of Human Reproduction. Editors: J. S. Scott and W. R. Jones. Academic Press, London. In press. Jenkinson, E. J. and Billington, W. D. (1974a): Differential susceptibility of mouse trophoblast and embryonic tissue to immune cell lysis. Transplantation, 18, 286. Jenkinson, E. J. and Billington, W. D. (1974b): Studies on the immunobiology of mouse foetal membranes: the effect of cell-mediated immunity on yolk sac cells in vitro. J. Reprod. Fertil., 41, 403.
84, 1283.
Beer, A. E. and Billingham, R. E. (1971): Immunobiology of mammalian reproduction. Advanc. Immunol., 14, 1. Beer, A. E. and Billingham, R. E. (1973): Maternally acquired runt disease. Science, 179, 240. Beer, A. E., Billingham, R. E. and Hoerr, R. A. (1971): Elicitation and expression of transplantation immunity in the uterus. Transplant. Proc., 3, 609. Billington, W. D. (1971): Biology of the trophoblast. Advunc. Reprod.
Abnormal
.I., 2, 263.
Kirby, D. R. S., Billington, W. D., Bradbury, S. and Goldstein, D. J. (1964): Antigen barrier of the mouse placenta. Nature
(Lond.),
204, 548.
Kirby, D. R. S., Billington, W. D. and James, D. A. (1966): Transplantations, of eggs to the kidney and uterus of immunized mice. Transplantation, 4, 713. Little, C. C. (1924): Genetics of tissue transplantation in mammals. J. Cancer Res., 8, 75. Matsunaga, E. and Itoh, S. (1958): Blood groups and fertility in a Japanese population, with special reference to intrauterine selection due to maternal-foetal incompatibility. Ann. hum. Genet., 22, 111. Merwin, R. M. and Hill,E. L. (1954): Fate of vascularized and non-vascularized sub-cutaneous homografts in mice. J. nat. Cancer Inst., 14, 819.
Montemagno, U. and Di Stefano, M. (1965): Gli anticorpi antiplacenta nell’aborto ricercati con tecnica immunofluorescente. Momt. ostet-gmec., 36, 563. E., Ben-Hur, N., Zuckerman, H. and Neuman, Z. (1967): Further immunologic studies in patients with chorionepithe]iomaand hydatidiform mole. Cancer Res., 27,
Robinson,
1202.
W. D. Billing&m,
Abnormal
Robinson, E., Shulman, J., Ben-Hur, N., Zuckerman, H. and Neuman, Z. (1963): Immunological studies and behaviour of husband and foreign homografts in patients with chorionepithelioma. Lancet, I, 300. Schmidt, P. J. and Hertz, R. (1961): Blood group factors in women with choriocarcinoma as compared with those of their husbands. Amer. J. Obstet. Gynec., 82, 651. Scott, J. S. (1962): Choriocarcinoma: observations on the aetiology. Amer. J. Obstet. Gynec., 83, 185. Simmons, R. L., Lipschultz, M. L., Rios, A. and Ray, P. K. Failure of neuraminidase to unmask histocompatibility antigens on trophoblast. Nature New Biol., 231, 111. Simmons, R. L. and Russell, P. S. (1962): The antigenicity of mouse trophoblast. Ann. N.Y. Acad. Sci., 99, 111. Terasaki, P. I., Mickey, M. R., Yamazaki, J. N. and Vredevoe, D. (1970): Maternal-foetal incompatibility I. Incidence of HL-A antibodies and possible association with congenital anomalies, Transplantation, 9, 538. Vandeputte, M. and Sobis, H. (1972): Histocompatibility antigens on mouse blastocysts and ectoplacental cones. Transplantation,
153
immunology
14, 331.
Wren, B. G. and Vos, G. H. (1961): Blood group incompatibility as a cause of spontaneous abortion. J. Obstet. Gynaec.
pregnant uterus are not? Wouldn’t you feel that these findings are an argument in favor of some protective role of the decidua? I know very well that Beer has done some very beautiful experiments which tend to disprove this point, at least with other tissues implanted. What is your feeling about it? I would agree entirely because they were also my own experiments so I happen to believe them. The reason why 1 didn’t go into that in great detail is because I know that Beer is going to talk about it later. But what I did perhaps not bring out is that a lot of the early experiments, claiming to show that one got tissue rejection in the uterus, to my mind were related to the fact that themodeldidnot allow thedecidual tissue to develop, and this was subsequently confirmed by Beer’s experiments. I think he will probably agree with me that there is a protective influence by the decidua in respect to the immunological interaction and that this is the interpretation of the experiments that you mention. In other words that those immune reactions which are responsible for the destruction of the embryo transplanted to the kidney, in the same animal cannot bring about rejection of the embryo within the uterus.
BILLINGTON:
Brit. Cwlth, 68, 637.
Discussion 1 must say that I am glad to hear that you find exactly the same results as the ones we have found with ectoplacental cones treated in vitro with sensitized lymphocytes. I think there is a further argument to indicate that the trophoblast cells do not display histocompatibihty antigens and this is based on our observations on ectoplacental cones transplanted in vivo under the kidney capsule of presensitized allogeneic mice. As in this case also no rejection is observed, the phenomenon applies not only to in vitro studies but also to in vivo experiments. Moreover, Dr. H. Sobis, who is working in my laboratory, has done some electron microscopy on the ectoplacental cones implanted under the kidney capsule. Not until four to five days after implantation did she observe the presence of an outer cell layer of mucopolysaccharides. Therefore, this layer of mucopolysaccharides seems to be a secondary phenomenon and not the cause of the absence of rejection. In presensitized animals one would indeed expect morphological evidence of rejection earlier than 4 to 5 days after implantation. Now the second remark is that you reject the hypothesis that the uterus should be, or could be, an immunologically privilegedorgan. How wouldyouexplainin that case, the experiments of Kirby et al. in which, in preimmunized mice, allogeneic blastocysts implanted under the kidney capsule are rejected but allogeneic blastocysts in the pseudoVANDEPUTTE:
BEER: We know that transplantation antigens can be identified on the zygote from the two cell stage onwards. Park and Robertson have both shown us that there is an intense amount of mononuclear cell infiltration at the time of implantation of the mammalian zygote which is very reminiscent of the classic delayed type hypersensitivity reaction. This suggests that something has been recognized, and although perhaps I in the past have soft-pedalled the uterus as an immunologically privileged site, rethinking and redoing experiments, I believe that there is a brief period during which the uterus does afford an immunologically privileged site for the implanting embryo. I would hope to give you some data later to support that thesis.
I am amazed that under rare conditions, malignant trophoblastic cells, for instance choriocarcinoma, may express transplantation antigens resulting in an antigen-antibody reaction. We all know that some patients, even with vaginal and lung metastases, will have spontaneous regression and healing of the disease. This means to me that the maternal immune mechanisms can defeat the malignant choriocarcinoma. How do you feel about that? VORHERR:
BILLINGTON: I have spoken on this point to Bagshawe, and he says that if one gets a cellular infiltration then the prognosis tends to be better than if one doesn’t get a cellular infiltration and, obviously, this would appear to indicate that histocompatibility antigens, HL-A antigens, are present on choriocarcinema. But there is no direct evidence of this and the possibility of their spontaneous regression being due to an immune response is really conjectural. I don’t know whether Park would challenge that.
154
W. D. Billington, Abnormal immunology
PARK: I agree with that. I don’t think matters have been taken any further during the last several years. The possible role of transplantation antigens in relation to spontaneous regression is still quite obscure. It was Bagshawe who made the first attempts to understand this by doing skin grafting of the patients with skin from husbands and the like. But not only did that fail to influence the course of the disease, it failed to produce convincing evidence that transplantation antigens were playing any significant part at all.
But how can an organism defeat full blown lung metastases if there is no immune response?
VORHERR:
BILLINGTON: Lung metstases of what? Do you mean pulmonary embolism?
No, I am talking about lung metastases of choriocarcinoma clearly proven on X-ray; a few months later everything is gone and the patient survives. Such cases have been reported. VORHERR:
One may very abnormal genetic noma and the patient; case. I don’t think BILLINGTON:
possibly implicate, in that situation, a relationship between the choriocarcibut that certainly is not normally the I can say anything more on that.
PARK: Just one further comment; the regression of choriocarcinema is the regression of a tissue which, as we were discussing earlier, has a finite life span. For some reason that, as far as I know, is still quite obscure, normal trophoblast is controlled by a strictly run biological clock which sees to it, in the appropriate species, that after a time the tissue begins to become seriously senescent and impaired. Now this a queer carcinoma; in it, we see the malignant expression of tissue of this type, tissue which has a much more limited span than any other tissue, even including such things as deciduous teeth, which have a life span much less than that of the organism as a whole. I can’t help feeling that in some way the curious behaviour of choriocarcinoma on these occasions is related in a degree to that limited life-span aspect of trophoblast behavior just as it may be to the transplantation aspect of choriocarcinoma.
Yes, I think I would support that, but if I might add one point which is slightly to the contrary. A number of people have tried to develop continuous cell lines of trophoblast for various reasons and have failed abjectly. However, there is a report - I don’t know if it has been substantiated that it is possible to develop a continuous cell line from choriocarcinoma. So, although the biological clock of the two may be very closely related, there must be certain methods by which the latter can escape from this control. BILLINGTON:
ROBERTSON: Could I ask a question which is out of total ignorance? One thing which has always intrigued me is what happens to the breakdown products of spermatozoa before
conception occurs? It is rather like asking the question, “where do all the flies go in winter?“. What happens to all those millions of spermatozoain theejaculate? Their numbers, as we understand it, reduce very, very considerably until a few panting survivors reach the ampulla of the tube. What happens to all those that don’t make it? BEER: Now
you are tempting me because I think this is something which we as immunobiologists have totally neglected, the chronic repetitive inoculation which the female has to undergo, whether for pleasure or procreation. 1 hope to make a good case that this accomplishesan immunologic conditioning of the uterus. It makes it more conducive, not only to deciduagenesis but also to implantation of the embryo against which the sensitivity is directed; a kind of backward look. I’m not sure whether I understand it but I hope at least to play a game with you to try to help you to help me understand. ROBERTSON: I always thought it was rather different. Obviously the gastrointestinal tract is in something of the same position. When one looks at the normal gastrointestinal tract one sees mucosa that looks as though it would be pretty well protected by a cellular reaction against potential foreign antigens with plenty of lymphocytes and plasma cells in the mucosa. But the round cell component of the endocervix, of the endometrium and of the fallopian tube is minimal compared with the gastrointestinal tract. BILLINGTON: I think perhaps that I should add that the presence of spermatozoa, and possible induction of immunity as a result, is not essential for normal pregnancy, because it is perfectly possible to transplant fertilized embryos into the uterus of a previously virgin female and they develop quite normally.
Yes, but it’s a question of degree. If one takes two groups of tubal ligated animals, one group mated with only a vibrator on the day of estrus to induce pseudopregnancy, the other group mated with males of an unrelated genetic strain, and four days later challenges each group to implant genetically foreign blastocysts the females whoseuteri had been exposed to seminal products will demonstrate higher reproductive efficiency than those not exposed. I think we have been a little bit naive or too easily satisfied that we could transplant allogeneic embryos to females that have never met an allogeneic male. We have never studied the degree of hospitality. BEER:
Is there that degree of relevance in our own species, where normally there is only one embryo and either it implants or it fails’? Most often this question of degree relates to polytocous species where there are a number of eggs.
BILLINGTON:
BEER: Human
non.
sex ratio data would suggest a similar phenome-
