Fetus As Allograft

Peter M Johnson, Department of Immunology, University of Liverpool, Liverpool, UK

Copyright © 1998 Elsevier Ltd. All Rights Reserved.

Modern considerations of the haplo-nonidentical fetus as an allograft in outbred pregnancy have developed from initial postulates, put forward by Medawar, Brent and Billingham in 1953, that were derived from their work on transplantation tolcr-ance. In these, they proposed that I) the conceptus may not be immunogenic, 2) pregnancy may alter the immune response, 3) the uterus may be an immunologically privileged site, and 4} the placenta may be an effective immunological barrier between mother and fetus. They were also influenced by the earlier work in 1945 of RD Owen, who demonstrated transplantation tolerance between dizygotic twin cattle (who share a common placenta and blood supply).

In 1958, Woodruff showed that pregnant female rats could react immunologically against fetal tissue removed from one embryo and grafted to the thigh muscle, whereas the remaining intrauterine embryos were unaffected. This demonstrated that the 'transplantation privilege' normally offered to the fetoplacental unit in pregnancy depends on intrauterine mechanisms. However, several later studies in experimental animals have shown that tissue transplants (e.g. skin grafts) placed at intrauterine sites in hormonally primed or pregnant recipients are not exempt from immune rejection, although a degree of prolongation of graft survival times can occasionally occur. Thus, the pregnant uterus is not an immunologically privileged site. In addition, it is clear that pregnancies will normally survive in maternal hosts that have high levels of pre-existing alloimmunity from either natural immunization (previous pregnancy or blood transfusion) or experimental immunization with fetal or paternal cells.

The modern focus of attention on the fetus as an allograft has now been directed more toward the specialized features of fetal trophoblastic cells that appear to confer transplantation protection allowing viviparous reproduction. Trophoblast is the first cell type to differentiate following fertilization and forms the lining cell of the placenta and extraembryonic membranes encasing the conceptus; it represents the entire fetal tissue interface with maternal blood and decidualized endometrial tissue. In humans, the outermost trophoblastic layer of the chorionic villous placenta is syncytiotrophoblast, a continuous cell layer without intercellular junctions, that has a mononuclear cytotrophoblast layer immediately below within the villous structure. It is through syncytiotrophoblast that the fetus 'eats, breathes and survives', and molecular expression by this cell reflects its wide variety of essential biological functions. There is a separate population of invasive cxtravill-ous mononuclear cytotrophoblast that migrate into the decidualized endometrium and also proliferate laterally to form the chorionic membrane; in early pregnancy, these extravillous cytotrophoblast cells display many of the cell biological characteristics of tumor cclls in their proliferative and invasive properties, which largely cease after the first trimester of pregnancy. This entry will concentrate largely on human pregnancy, since there arc numerous anatomic differences in placentation between spccies, but will also refer to observations in other species when appropriate.

Passive resistance of trophoblast to cytotoxicity

It is now clear that fetal trophoblast utilizes several mechanisms which may critically protect it from maternal cytotoxic attack. First, human placental villous cytotrophoblast and syncytiotrophoblast do not express any classical polymorphic class I or II major histocompatibility complex (MHC) antigens (e.g. HLA-A, -B, -DQ or -DR), and it appears that constitutive HLA expression may not be induced by any of the known exogenous upregulators (e.g.

interferon y, IFN-y). Classical MHC antigens are thus not expressed throughout gestation, from the tro-phoectoderm of the preimplantation blastocyst to the syncytiotrophoblast of the term placenta. The genetic mechanisms of gene expression and control in syncytiotrophoblast, a true cellular syncytium, are difficult to envisage. However, there would appear to be little doubt that the lack of expression of classical class I MHC molecules at this site in humans is of central importance in protecting the fetal tissue interface from cytotoxic attack by maternal T cells or allo-antibody. There may also be other, more subtle implications, since syncytiotrophoblast can express endogenous retroviral particles; these are likely to be involved with syncytial cell formation, but could also strongly influence expression of other genes. In rodent pregnancies, in contrast, there is evidence of placental expression of some classical class I MHC antigens; the immunobiological implications of this are still far from clear.

Recent studies have shown comprehensively that the extravillous cytotrophoblast population in humans selectively expresses the nonclassical class 1 MHC antigen, HLA-G, which has limited genetic polymorphism; the HLA-G gene lacks an intact inter-feron-stimulated response element (ISRE) for upreg-ulation. To date, HLA-G expression has not been noted in adult tissues and this class I MHC locus may have evolved for a specialized immunogenetic function in pregnancy. HLA-G protein expression in fetal tissues other than trophoblast remains controversial, notably in fetal thymus, where positive selection of HLA-G-restricted T cells could possibly occur. There has been speculation that HLA-G might serve in cytotrophoblast invasion and proliferation in endometrial tissue, or as a passive cell surface class I MHC molecule protecting this cytotrophoblast population from MHC-nonrestricted natural killer (NK) cell attack (see below) without presenting a classical class I MHC antigen that could act as a target for MHC-restricted T cell cytolysis. An opposing argument is that trophoblastic HLA-G expression may be a redundant evolutionary relic. This could be supported by observations that j32-microglobulin-defkient mice, obtained by homologous recombination in germ-line genes and which do not express constitutive class I MHC molecules, are capable of reproducing readily and with normal litter size; however, these mice lack functional CD8" cytolytic T cells and would not have the ability to distinguish self from nonself.

The final mechanism that trophoblast employs throughout gestation to protect itself from maternal cytotoxicity is to express a high level of complement regulatory proteins on its surface, notably membrane cofactor protein (MCP: CD46), decay accelerating factor (DAF: CD55) and membrane attack complex inhibitory factor (CD59). The former is of particular interest since, for many years, a polymorphic cell surface antigen system termed trophoblast-leukocyte common (or cross-reactive) antigen (TLX antigen) attracted attention from reproductive immunologists as a potentially immunoregulatory cell surface polymorphic antigen expressed by both fetal trophoblast and maternal immunocompetent cells. It is now recognized that this TLX antigen is identical to CD46; its strong expression by all trophoblast sub-populations serves to protect this cell type from complement-mediated damage consequent to either maternal antibody attack or local tissue restructuring and hemostatic alterations that may promote persistent low-level local complement activation.

Active maternal immunoregulatory responses

A central creed in reproductive immunology is that there is active maternal immune recognition of pregnancy (i.e. of fetal trophoblast) that leads to cellular, antibody or cytokine responses which protect the fetal allograft. Taking antibody responses first, there has been much evidence that so-called blocking antibodies (which impair in vitro lymphocyte response assays) can be found in normal pregnancy sera and placental eluates. However, the data often are not convincing that these systemic effects can always be ascribable to specific antibody, rather than other serum factors, and that they occur in all successful pregnancies. For example, agammaglobulinemic women do not suffer undue problems of pregnancy failure other than indirectly following their susceptibility to infection. It is probable that the blocking antibody phenomenon is an effect rather than a cause of the immunologic success of outbred pregnancy.

Of more interest may be local cellular adaptations within the maternal endometrium and placental bed site. It is now recognized that mature CD3-positive T cells and, particularly B cells are comparatively rare at these sites. The predominant cell types are: I ) activated class II MHC antigen-positive macrophages (which serve as an immediate defense to infection at these vital tissue sites in pregnancy, but may also be providing nonspecific immunosuppressive mediators such as PGE2); and 2) CD3 CD 16 CD56bnsht granulated lymphocytes (endometrial granulated lymphocytes, eGL). These latter cells have attracted much attention regarding their apparent novel phenotype and function. They may be hor-monally regulated in that they occur in endometrial tissue in significant numbers during the secretory rather than proliferative phase of the cycle, and are normally lost by menstruation. In pregnancy, their numbers increase still further in first-trimester endometrium (or, in ectopic pregnancies, in any decidualized tissue), but most CD3~ eGL have been lost by term. What is their function? Their phenotype most closely resembles that of NK cells and pre-NK cells, although most CD3" eGL may not proliferate rapidly in response to interleukin 2 (IL-2) and do not express other NK cell molecules (e.g. CD16 or CD57). One postulate is that they are a form of NK cell in arrested maturation, perhaps because of persistent expression of HLA-G on the target invasive cytotrophoblast?

In this context, it could be relevant that several rodent models of recurring fetal loss in pregnancy appear to operate via a final mechanism involving attack by NK cells of macrophage-derived products against the fetoplacental unit. Unfortunately, clear immunopathological evidence for cellular immune damage at the fetal tissue interface in cases of human recurrent spontaneous abortion has been difficult to obtain. In susceptible rodent immunogenetic breeding combinations, several other potent immunomod-ulators can cause fetal loss (e.g. lipopolysaccharide treatment). Hence, examples of pregnancy failure in rodents may reflect endogenous genetic factors together with exogenous pathogens, endotoxins, or environmental stimuli. Nevertheless, scid/beige double mutant mice (that lack T cell, B cell and NK cell function) reproduce normally, although these mice do still bear a granulated lymphoid cell type in endometrial tissue. In the absence of environmental factors, an intact immune system may not be necessary for reproductive success.

More recent attention has been directed toward cytokine cross-talk that may occur at the fetomater-nal tissue interface in pregnancy. One hypothesis has been that fetal trophoblast antigens (e.g. HLA-G) or other products might stimulate maternal lymphocytes in endometrial tissue to produce cytokines and growth factors that act in a paracrine manner beneficial to trophoblast growth and differentiation (the so-called immunotrophism hypothesis). Although the relatively low numbers of mature T cells in endometrial tissues could limit this effect, it is also well established that decidualized endometrial tissue extracts are particularly potent in demonstrating immunosuppressive effects. It is now clear that other cell types in this tissue are highly active in cytokine secretion (e.g. eGL and glandular epithelial cells). For example, there is evidence for the significant production of the colony stimulating factors (CSFs), tumor necrosis factor a (TNFa), IL-6 and trans forming growth factor /3 (TGFjS) within decidual tissue. Similarly, fetal syncytiotrophoblast would appear to be excessively endowed with growth factor receptors, including those for the CSFs, IFNy, TNFc*. TGF/3, epidermal growth factor and transferrin. Thus, a complex interactive cytokine network may be operative within uteroplacental tissue that provides both immunosuppressive and growth-promoting signals.

Based on the above considerations, it had been proposed that certain cases of unexplained recurrent spontaneous abortion in humans could result from a failure of active maternal immune adaptation to the fetal allograft in pregnancy and might be assisted by deliberate prior immunization with paternal or third-party cells to promote these missing protective responses. However, the most recent information now suggests that there is no strong conceptual framework for this hypothesis acting at a systemic level, and that alloimmunization in such cases may not always significantly improve subsequent successful pregnancy outcome rates in fully controlled clinical efficacy studies.

Fetal protection from prenatally transferred maternal IgG antibody

In man and certain other species, maternal im munoglobulin G (IgG) is selectively transferred prenatally into the fetal circulation following initial recognition by specific transporting Fey receptors on placental syncytiotrophoblast. This provides transient passive immunity to assist postnatal immune protection following parturition and release from the sterile intrauterine environment into pathogen-rich environments at a time prior to development of neonatal immunocompetence. Significant maternal IgG transfer occurs in humans from around the 20th-22nd week of gestation. However, together with beneficial antimicrobial immunity, deleterious maternal autoantibodies and fetal-specific alloantibodies could also potentially be transferred into fetal circulation. Hence, elegant mechanisms have evolved to protect the fetus from adverse maternal IgG antibody, notably maternal anti-HLA alloantibody (which may-have developed from a previous pregnancy) and that would be expected to have catastrophic effects on the developing fetal immune system if allowed to penetrate further than extraembryonic tissue.

It is now recognized that maternal HLA-specific alloantibody with specificity for the fetal HLA type will be efficiently bound by nontrophoblastic cells bearing fetal HLA antigens (i.e. macrophages, fibroblasts, endothelium) within the villous mesenchyme of placental tissue, and will not reach the fetal circu lation. Similarly, any other maternal antibody to a fetal antigen expressed within placental tissues will be sequestered by binding to an antigen-expressing cell or by binding as a soluble immune complex to high avidity Fey receptor-bearing macrophages and fetal stem vessel endothelium that are abundant in these extraembryonic tissues; the latter have specificity solely for complexed rather than monomeric IgG. Hence, these potentially deleterious maternal antibodies are trapped by the placental 'sink' or 'sponge'. Indeed, many immunohistological studies have drawn attention to the extensive IgG and complement deposition within term placental villous tissues - essentially, the placenta needs only to survive this immunopathologic onslaught for up to 5 months, unlike other body organs.

The exceptions that escape placental trapping of adverse maternal IgG antibodies are also of interest. For example, maternal IgG rhesus (D)-specific or platelet-specific antibodies will meet their cognate antigen first on fetal erythrocytes or platelets, respectively, within placental stem vessels. Hence, erythrolysis or thrombocytopenia will occur prior to trapping of immune complexes on fetal Fey receptor-positive placental vessel endothelium. In addition, maternal organ-specific autoantibodies reactive with autoantigens not represented within the placenta will not be sequestered and retained at this site. Thus, for example, maternal IgG thyroid-specific autoantibodies can reach the fetal thyroid and can sometimes directly cause transient symptoms of autoimmune thyroid disease during the neonatal period of persistence of passively transferred maternal IgG autoantibody.

Conclusions

Our understanding of the immunobiology of the fetal allograft has progressed considerably over the past 40 years. Current attention in human pregnancy is focused on the probable specialized role of selective HLA-G expression at the fetal tissue interface and on elucidating the function of the numerous maternal

CD3-negative granulated lymphocytes that can be found in the surrounding decidualized endometrial tissue, together with the complex cytokine networks that may exist within these tissues. It is possible that all of the components and mechanisms contributing to the success of the fetal allograft in immunocompetent pregnant hosts have now been identified, even if not all of the details are yet known. However, it is impossible to test this hypothesis in humans, and difficult to test in experimental animals. Hence, further novel approaches to unraveling the immuno-biological strategies employed for successful viviparity can be expected.

See also: Alloantigens; Cytokines; Cytotoxic T lymphocytes; Graft rejection; Hemolytic disease of the newborn; Maternal antibodies; MHC, functions of; Natural killer (NK) cells.

Further reading

Bronson RA, Alexander NJ, Anderson DJ et al (eds) 11996) Reproductive Immunology. Cambridge, VIA: Blackwell Science. Bulmcr JN and Johnson PM (1995) Immunopathology of pregnancy. In: Fox H and Wells M (eds) Haines and Taylor: Obstetrical and Gynaecological Pathology, 4th edn, pp 1807-1835. Edinburgh: Churchill Livingstone. Hunt JS (ed) (1994) Immunobiology of Reproduction.

Serono Symposia USA. New York: Springer-Verlag. Johnson PM (1993) Reproductive and maternofetal relations. In: I.achmann PJ, Peters DK, Rosen FS and Walport MJ (eds) Clinical Aspects of Immunology. 5th edn, pp 755-767. Boston: Blackwell Scientific. Johnson PM (1993) Reproductive immunopathology. In: Lachmann PJ, Peters DK, Rosen FS and Walport M| (eds) Clinical Aspects of Immunology, 5th edn. pp 2137-2152. Boston: Blackwell Scientific. Scott JS and Bird HA (eds) (1990) Pregnancy. Autoimmunity and Connective Tissue Disease. Oxford: Oxford University Press, Vince GS and Johnson PM (1995) Maternofetal immunobiology in normal pregnancy and its possible failure in recurrent spontaneous abortion. Human Reproduction 10 (suppl 2): 107-113.

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