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Immunologic sensitization prior to birth
CURRENT DEVELOPMENTS
Immunologic DONALD HEINZ
V. W.
sensitization
prior to birth
CRAMER KUNZ
THOMAS
J.
GILL
Pittsburgh,
Pennsylvania
III
A series of clinical and experimental reports have been presented in which there is euidence to indicate that prenatal sensitization of the fetus to specific antigens can occur during apparently normal pregnancies. It appears that the mechanism involved in the alteration of the immune response may be the prenatal passage of antigen and/or maternal celts to the developing fetus and the production of specific immunologic reactivity. If future clinical and experimental investigations define more clearly the mechanism by which this event occurs, methods of specifically altering the immune response of the offspring could be developeii. ‘This type of “immunologic engineering” could afford the ofispring an enhanced immune response against infectious agents and vertically transmitted oncogenic agents or could induce specific tolerance to selected tissue antigens.
A N U M B E R 0 F careful investigations in the past several years have made it clear that the mammalian fetus acquires the capacity for immunologic responsiveness early in its development. These studies have been conducted primarily in the larger animals with a long gestation period such as the sheep, cow, and? to a more limited degree. the human neonate.l-” The functional and morphologic development of the fetal imFrom the Department University of Pittsburgh Medicine.
mune system in these species shares a number of characteristics in common. Both the structure of the lymphatic system and the ability to mount an immunologic response develop in an orderly, sequential manner. These processes begin in the first trimester of gestation, and by the third trimester the fetus has acquired the ability to respond to some antigenic stimuli in a fashion equal to that of the adult. During gestation the developing immune system shares with other organ systems the of the maternal environment. protection The placenta provides a physical barrier protecting the fetus and preventing the passage of potentially damaging agents which could affect critical phases of organ system development.’ Since the developing immune system shares this environment, studies of the immune response in the fetus had to be conducted under conditions of direct rx-
of Pathology, School of
The work in the authors’ supported by grants from Znstitutes of Health (AI GM 00135), the Beaver Society, the Samuel and Foundation, and the Tim Memorial Cancer Fund.
laboratory was the National 10611 and County Cancer Emma Winters Coracio
Reprint requests: Dr. Donald V. Cramer, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. 431
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Cramer,
Kunz, and Gill
posure to the antigen.’ Under 1110~ normal conditions, the placenta selectively prevents the passage of most antigens to the fetus. However, there is a growing body of clinical evidence indicating that the mother and her offspring frequently share sensitivities to a variety of specific antigens. If it is assumed that the placenta does not provide an absolute protective barrier against certain antigens, this shared sensitization may reflect the prenatal passage of antigen, cells, 01 immunologic mediators such as transfer factor to the fetus prior to birth with a subsequent active response by the developing infant. The purpose of this review is to summarize these reports of prenatal sensitization and to discuss the possible mechanisms responsible for the observations in the context of current experimental work. Shared
prenatal
sensitization
A number of recent reports indicate that at birth the infant frequently shares with its mother immunologic reactivity to specific antigens. This immunologic reactivity is usually measured by humoral antibody production (especially fetal immunoglobulin M [ IgM] ) , by skin testing, or by demonstrating increased in vitro lymphocyte responsiveness to the specific antigens. Those antigens which have been included in this group are some infectious agents, a few common allergens, and a group of ubi
i~lllntlrloglobulirl III utllbilic;~l c.o~tl blood )s an important diagnostic l13t. Of nwre it Iterest for the. jxq)oses of tlii5 wview. hog ever, arc those maternal infections in \vhich there is little c,vidence that fetal infection has occurred. and y-t the wonate is sensitizrd to certain antigenic components of the agent. Included within this group are such agents as tuberculosis, mumps, lkherichia c-oli, and Salmonella
typlwrcr.
In tuberculosis there can be shared sensitization to tuberculin proteins betwwn the mother and the apparently normal, uninfected offspring. In a series of 1 102 neonates born to mothers infected with tuberculosis, jentgens!’ described 5 apparently normal children with positive skin tests at birth. ‘These tests were only transiently positive, and most were ne,yative 2 to -I weeks later. In none of thr cases was there evidence of active infection in the offspring. Similarly, Mohr’” has reported that of 11 children born to 5 purified protein derivative mothers 5 had positive ( P. P.D. ) --positive intradermal reactions to P.P.D. In the same series, none of 78 children from tuberculinnegati\rc mothers had a positive reaction. ‘This type of response by the newborn infant implies direct exposure to antigenic components of the agent or to the passage of specific immunologic information from the mother, perhaps in the form of intact cells. In addition to concordant positive tuberculin reactions, there have been reports that in vitro cellular responses to P.P.D. are also shared by mother and child. Field and Caspary” conducted a survey designed to examine the dr,gree and extent of shared sensitivities between mother-child pairs to a number of antigens. Included within the survey are pairs with normal mothers and mothers having a variety of inflammatory and neoplastic diseases. Five pairs of healthy mothers and their newborn babies showed lymphocyte sensitivity to P.P.D. The test system employed involved the inhibition of guinea pig macrophage migration by incubation with lymphocytes which were exposed to the test antigen. The significance of these results is not clear, however. since the
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specificity of the test system and the significance of a positive response remain unexplained. Astor and Frickr2 reported 6 similar maternal-fetal pairs with in vitro inhibition of leukocyte migration and concordant positive skin tests to P.P.D., but the significance of this report also is not clear since documentation of these observations was not complete. Maternal exposure to mumps virus can also produce an altered immune response in the offspring of infected mothersl” Ten years after a mumps epidemic, 12 Eskimo children born to mothers pregnant during the epidemic were evaluated with regard to their immune status. All of the mothers had serologic evidence of exposure to the virus, which was the first and apparently only epidemic of mumps in this group of people. The offspring that potentially had been exposed to the virus in utero had a positive mumps skin test but had no evidence of neutralizing antibodies to the virus. This failure to develop a humoral response despite the presence of an active cellular response was not observed in the other children investigated. The children born prior to the epidemic had both a positive antibody response and a positive skin test, while those born after the epidemic had only occasional positive skin tests and antibody responses. The authors concluded that mumps infection during pregnancy had led to the exposure of the embryo or the fetus to viral antigens with subsequent immunologic sensitization. These clinical observations were confirmed in pregnant rhesus monkeys by infecting the animals during the first third of gestation and demonstrating that the infant monkeys had an immune response to mumpsI Four term infant monkeys were challenged with mumps virus and demonstrated delayed hypersensitivity to the agent, but they were unable to produce antibody. The authors were able to demonstrate that the fetal monkeys were infected for only short periods of time (approximately one week), and they concluded that under these conditions “split” immunologic responses may have occurred because the fetuses had received a small dose
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sensitization
prior
to birth
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of antigen which led to the development of cellular immunity without humoral immunity. Two other infectious agents have been shown to be capable of sensitizing the embryo or fetus in utero. When lymphocytes from randomly chosen neonates were cultured in vitro with E. coli antigens, a certain proportion reacted positive1y.l” In order to determine whether this reactivity was compatible with in utero antigen contact, a series of newborn children from women with significant E. coli bacteriuria or pyelonephritis during pregnancy was examined.lG The lymphocytes from children born to infected women all showed positive reactions when incubated with E. coli antigen. In the control groups, only 2 of 11 children had positive reactions, and, in one of those, the mother had a history of pyelonephritis caused by E. coli prior to and during pregnancy. Similarly, an infant born to a mother immunized with one dose of typhoid-paratyphoid vaccine prior to delivery had circulating IgM hemagglutinating antibody to the somatic antigen of S. typhosa. I7 Since IgM antibody does not cross the placenta in any significant amounts, the authors felt that the antibody was fetal in origin and had been produced in response to in utero exposure to Salmonella antigens. These observations on fetal sensitivity to infectious agents may represent instances of in utero exposure to antigens with immunologic sensitization being the only manifestation of the process. If this hypothesis were based only on clinical observations in infected mothers, it would have to be made with care. The placenta could have been damaged and the fetus could have been exposed to the intact, pathogenic microorganisms, leading to brief subclinical infections. In the case of tuberculosis, however, there is experimental evidence in rats and mice that tuberculin antigens alone can produce prenatal sensitization. When pregnant female mice were inoculated intravenously with tuberculin protein (P.P.D.) , postnatal challenge of the offspring with the protein caused accelerated lymph node
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Kunz,
and
Gill
development and positive skin tests.l Similarly, pregnant rats given heat-killed M. tuberculosis bacilli gave birth to offspring, of which 26 per cent had positive skin tests to tubercu1in.l:’ These two reports lend support to the concept that the sensitivity to tuberculin protein in the neonate resulted from prenatal transfer under physiologic rather than pathologic conditions. A variety of other antigens which are nonpathogenic are associated with shared sensitization in human subjects. Leikin and Oppenheim2”-22 demonstrated that cord blood lymphocytes from newborn children are commonly reactive to such antigens as diphtheria toxoid, tetanus toxoid, soluble dental plaque, bovine serum albumin, streptolysin 0, and pneumococcal polysaccharide. The response of the lymphocytes from a child to a particular antigen was not necessarily associated with a response to the other antigens tested. This finding was interpreted as indicating that the neonatal response was specific. Since in vitro responses to these antigens require prior sensitization of the lymphocyte, the response of the neonate’s lymphocytes was interpreted as being the result of passage of sensitizing antigen across the placenta and active immunization of the developing fetus.2Z Some common allergens also have been reported to produce shared sensitivity. Studies with allergic patients showed that their newborn infants can be sensitized at birth, perhaps as the result of prenatal exposure to the antigensZ3 Kaufman’* reported a case in which both the mother and newborn child shared sensitization to grass pollen. At birth the child had a positive skin test to the antigen and had specific immunoglobulin E (IgE) antibody in the cord blood that could be detected by means of the Prausnitz-Kiistner (P-K) reaction. In a similar report 22 children were examined at birth, and 4 were found to be sensitive to extracts of common allergens such as mixed ragweed, pollen, molds, grass pollen, and dustZ5 The sensitization seen in these children and the responses of cord blood lymphocytes to a number of ubiquitous
antiCgem indicate that prerlatal cuposure ttj antigen, or to maternal culls, may havr occurred. If antigen or cells passed to the fetus and produced immunologic stimulation. it most likely occ,urred in the absence of significant placental damage. Possible
mechanisms
of prenatal
sensitization
The clinical reports suggest that immunologic information can be passed from mother to fetus under normal physiologic conditions. Experimental work lends support to this concept and provides important clues to the possible mechanisms of transfer. Although the means by which the maternal environment can influence the immunologic development of the offspring is unknown, the experimental studies suggest several mechanisms, none of which need be mutually exclusive: (1) passage of maternal antibody with alteration of the fetal immune response by specific antibody; (2) passage of immunologic informational products, such as transfer factor, from the mother; (3) active migration of sensitized maternal lymphocytes to the fetus with proliferation and participation in the immune response or recruitment of fetal lymphocytes for a specific response; (4) the transplacental passage of antigen to the fetus and direct sensitization of its lymphocytes. The possibility that maternal antibody is responsible for neonatal immunologic sensitization is unlikely. Maternal antibodies, mainly immunoglobulin G (IgG) , are readily passed from the mother to her offspring during pregnancy. This antibody can alter the immune response of the newborn infant by inhibiting the response to its specific antigen, and this effect is one of the major deterrents to early vaccination.“‘-?” Maternal antibody may also be able to enhance antibody production when present in quite small amounts.3”W”,’ However, these effects are essentially passive functions associated with control of the immune response. In order for maternal antibody to produce sensitization in the neonate, it would be necessary for the antibody to confer specificity. Since cellular immunity cannot be transferred \vith serum, it would
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seem unlikely that the in vitro expressions of of cell-mediated immunity are the result of maternal antibody. This is particularly true in a number of clinical and experimental reports in which the effect persists beyond the time maternal antibody is present in detectable amounts. The possibility that informational molecules, such as transfer factor or nucleic acid complexes, may influence the immune response of the offspring has received little or no attention. A small molecule such as transfer factor (molecular weight 10,000 Dalton Units) could be involved with prenatal sensitization, provided that it is capable of transmitting specific immunologic information.35 If transfer factor were to act as a nonspecific adjuvant, the fetal cells would have to be primed by prior antigen exposure in order to exhibit a specific response.36 The passage of intact maternal cells to the fetus must be considered as an impo’rtant possibility in view of reports that significant numbers of maternal cells may traverse the placenta in normal animals. In mice, for example, Tuffrey and co-worker+’ 38 have reported that large numbers of maternal cells can be detected in the offspring. However, this report of placental porosity was soon disputed39 and has been the center of some controversy.40 Similarly, Beer and colleagues 41~ 42 have conducted a number of experiments in rats that suggest the transfer of significant numbers of maternal cells to the fetus with the production of graft-versushost reactions. When syngeneic or allogenic lymphocytes sensitized to the paternal component of the fetal cells were injected during pregnancy, a high percentage of the offspring died with the signs of a graft-versus-host reaction. These studies provide strong circumstantial evidence that viable cells have traversed the placenta, but direct evidence of foreign cells in the tissues of the affected animals will be necessary to confirm this concept. In man, there is cell movement in both directions between the fetus and the mother. The passage of cells from the fetus to the mother causes, for example, maternal isoim-
Immunologic
sensitization
prior
to birth
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munization and Kh hemolytic disease of the newborn infant. The extent of the passage of maternal cells to the fetus varies. Maternal erythrocytes regularly pass to the fetal circulation in normal pregnancies, and studies with the use of various types of labeled erythrocytes infused into the maternal circulation showed that maternal-to-fetal transfer occurred to 12 per cent to more than 50 per cent of pregnancies.‘” In addition to red cells, small numbers of leukocytes and platelets pass from mother to child.“4-4F These cells apparently do not persist for any period of time under normal conditions.47-4g If the newborn infant has an immunologic deficiency, however, maternal lymphocytes can be responsible for graft-versus-host reactions.50, 51 Although the number of lymphocytes that traverse the placenta appears to be small and they persist for only short periods of time, they might play a significant role in some of the observations discussed above. In those cases in which in vitro lymphocyte transformation to specific antigens is thought to represent a secondary response by fetal cells, it is possible that small numbers of maternal cells may be responding instead. The numbers would have to be small in order to escape detection by chromosomal studies,ll which have failed to detect female cells in cell cultures from male infants. Even with small numbers of cells, it is possible that they are capable of passing information to the fetal cells or recruiting fetal cells during a response. In any event, trauma during delivery may be associated with the passage of maternal cells to the newborn infant at birth, and this possibility will have to be carefully considered in evaluating the in vitro studies of lymphocytes in cord blood. The passage of antigen across the placenta with subsequent sensitization of the fetus has received some experimental attention. Much of this attention has been devoted to the possibility that prenatal exposure of the fetus to foreign histocompatibility antigens might lead to tolerance in the offspring. Experimental studies showed that neonatal exposure to tissue antigens led to tolerance in rats and mice
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Kunz,
and
Gill
and that a number of factors were involved in determining the degree of tolerance. including age, sex, genetic background. and dose.“, X: In those animals given small doses of antigen, sensitization rather than tolerance was observed.‘“, 53, 5* Billingham and his coworkers”’ showed that young rats exposed to foreign tissue antigens during development rejected skin grafts in an accelerated fashion. Similarly, Stastnyl” showed that when homografts were placed on previously sensitized pregnant rats 4 to 10 days prior to delivery the offspring showed accelerated graft rejection, Skin grafts placed on the mothers before or after this period were ineffective in producing sensitization of the offspring. Even when tolerance was induced in the mothers prior to homografting, the offspring still showed accelerated skin graft rejection. It would not be expected that tolerant maternal cells would cross the placenta and participate in the reaction. Stastny concluded that antigen, rather than maternal cells, had passed to the fetus during the mid-gestational period and had actively sensitized the fetus. Other experimental work with similar results may be explained, at least in part, by the same assumption. Hypersensitized female rabbits have offspring which rejected skin transplants in an accelerated fashion.“” While it was felt that the transplacental agent responsible for the accelerated rejection was maternal antibody, the prenatal passage of maternal cells or antigen is also a distinct possibility. When neonatal rabbits from normal mothers were skin-grafted, they rejected adult skin in a significantly shorter period of time compared to the adult.“‘; Billingham and co-worker?* suggested that the latter observation may be the result of the heterogeneous nature of the rabbit population, leading to weak, transitory sensitization of the neonates to parental tissue antigens. Other studies suggested that prenatal sensitization may be caused by the transplacental passage of antigen. In guinea pigs, horse serum proteins given intravenously to the mother immediately prior to birth induced hypersensitivity in the offspring.3’ Since there was insufficient time for the pregnant female ani-
n~;~ls to develop antibodies to the horse set’um proteins ;Ind since they did not appear ill colostrum, the proteins probably passed to the fetus transplacentally and produced active immunization. These observations are manifestations of the relationship between immunologic stimulation and tolerance that exists in the adult animal? When an animal is exposed to an antigen, there is a balance between stimulation and paralysis that depends upon the chemistry of the antigen and the genetic background of the host. The chemical properties of the antigen set the level and range of dosage that can be used in stimulating an antibody response. The factors determining whether the antigen will induce stimulation or paralysis are the dose, the time over which it is given, the rate of antigen degradation, and the genetic background of the host. The same type of relationships probably also exist in the developing immune system of the fetus. In our laboratory the genetic control of the immune response to synthetic polypeptides, which are models for native proteins, has been studied extensively in inbred rat.““-” The response to these antigens is controlled by at least two genes and is linked to the major histocompatibility locus. When females of a poorly responding or highly responding strain are immunized with the aggregated antigen and then bred, the immune response of the offspring can be altered, depending upon the genotype of the strain.‘“-“C This effect requires aggregation of the antigen with methylated bovine serum albumin in order to slow its metabolic degradation and provide for its slow release from the injection site. For example, in the low-responding F344 strain, the immune responses of the 8- to lo-week-old offspring from sensitized mothers are significantly higher than those observed in the offspring from nonimmunized mothers. Kadiolabeled portions of the antigen can be detected in the fetal liver and blood prior to birth and in the bone marrow after birth.““2 (is7GOThese studies are compatible with the direct passage of antigen to the fetus prenatally, and they suggest that transplacental sensitization
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of the fetus may be an important spread phenomenon.
Immunologic
and wide-
Implications
These experimental and clinical studies represent an immunologic phenomenon which has received little attention to date. In each case, there is reason to believe that the fetus is actively sensitized to foreign antigens during pregnancy. In the clinical studies this sensitivity is most commonly expressed as shared lymphocyte reactivity between the mother and the newborn infant. If the mother and fetus share similar antigen sensitivity, the implication is that in some manner the antigen and/or appropriate antigenie information has reached the fetus during development. Since the newborn infant has lymphocytes responsive to only a few antigens, the responses are not simply nonspecific reactions of the cells. The experimental work relevant to prenatal sensitization consist primarily of a collection of observations in which antigen exposure of the mother has in some manner altered the immune response of the offspring. Because there has been little concerted effort so far to approach this problem, the reports have been sporadic and frequently contradictory. This is not surprising, since previous work with the induction of tolerance to tissue antigens and synthetic polypeptides in rats and mice has clearly shown that timing, dosage, and genotypes are among a few of the critical variables that influence the immune response. In those cases in which sensitization of the offspring occurred, there are a number of indications that the initial priming occurred in utero. In human subjects there is need for more complete studies of the phenomenon of shared lymphocyte sensitivities: The types and numbers of antigens involved and the specificity of the reponse need to be more carefully documented. At the present time there are only scattered indications that the transfer of prenatal sensitization observed in experimental animals is related to the shared lymphocyte responses observed in human subjects. The mechanism by which sensitization occurs in experimental
sensitization
prior
to birth
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animals is unknown, but the direct transfer of antigen or transfer of maternal cells to the fetus seems to be the best possibility. We favor the former, based on several lines of indirect evidence : ( 1) previous experimental work demonstrating the capacity of the fetus to respond at the appropriate time to a number of antigens, (2) the inability of a number of investigations to demonstrate consistently the participation of maternal cells in either in vivo or in vitro responses, (3) the transfer of sensitivity to the offspring of mothers in which tolerance to the specific antigen was induced, and (4) the evidence that prenatal antigen transfer is associated with altered immune responses in the offspring of immunized rats. If a systematic study shows that immunization during pregnancy can specifically alter the immune response of the developing fetus, at least three major applications of this type of “immunologic engineering” are apparent. First, in many areas of the world, infectious diseases early in life are still important causes of infant death. Under these conditions the ability to stimulate a primary immune response in utero could be an important vaccination procedure. This would be particularly true if immunization could be conducted prior to the transfer of significant amounts of maternal antibody to the offspring, since suppression of the immune response by maternal antibody is one of the important reasons for delayng early tetanus and diptheria vaccination. The same problem exists in sheep with regard to vaccination with Brucella abortus, but it has been successfully circumvented by in utero immunization in the absence of maternal antibody.67g 6R In human subjects, maternal immunization just prior to the twentieth week of gestation may allow the development of a primary immune response without interference form maternal antibody. In the case of tetanus and diphtheria toxoids, there have been reports of shared lymphocyte sensitivity between mother and child, and they may represent good candidates for clinical investigation. Second, the possibility that antigen presented in the proper dose at an early stage during gestation
438
Cramer,
could it),
Kunz,
produce may
to
to
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provide
life.
This
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Ratner, B., Jackson, H. C., and Gruehl, H. L.: J. Immunol. 14: 303, 1927. Gill, T. J., III: The chemistry of antigens and its influence on immunogenicity, in Borek, F., editor: Immunogenicity, Frontiers in Biology, Amsterdam, 1972, vol. 25, NorthHolland. Gill, T. J., III, Kunz, H. W., Stechschulte, D. J., and Austen, K. F.: J. Immunol. 105: 14, 1970. Gill, T. J., III, and Kunz, H. W.: J. Immunol. 106: 980, 1971. Kunz, H. W., Gill, T. J., III, and Borland, B.: J. Immunogenet. In press. Gill, T. J., III, and Kunz, H. W.: J. Immunol. 106: 274, 1971. Gill, T. J., III, Kunz, H. W., and Bernard, C. F.: Science 172: 1346, 1971. Sloan, B. P., and Gill, T. J., III: Immunochemistry 9: 677, 1972. Davis, B., Kunz, H. W., and Gill, T. J., III: Unpublished observations. Carpenter, C. B., Gill, T. J., III, and Mann, L. T., Jr.: J. Immunol. 98: 236, 1967. Richardson, M., Beck, C. C., and Clark, D. T.: J. Immunol. 101: 1363, 1968. Richardson, M., and Conner, G. H.: Infect. Immun. 5: 454, 1972. Ioachim, H. L., Keller, S. E., Gimovsky, M. L., and Shepherd, S.: Cancer Res. 33: 547, 1973. Dressman, G. R., Pinkerton, H., Holtwick, E. S., and Suriano, J. R.: Proc. Sot. Exp. Biol. Med. 137: 1337, 1971.
Immunologic DONALD HEINZ
V. W.
sensitization
prior to birth
CRAMER KUNZ
THOMAS
J.
GILL
Pittsburgh,
Pennsylvania
III
A series of clinical and experimental reports have been presented in which there is euidence to indicate that prenatal sensitization of the fetus to specific antigens can occur during apparently normal pregnancies. It appears that the mechanism involved in the alteration of the immune response may be the prenatal passage of antigen and/or maternal celts to the developing fetus and the production of specific immunologic reactivity. If future clinical and experimental investigations define more clearly the mechanism by which this event occurs, methods of specifically altering the immune response of the offspring could be developeii. ‘This type of “immunologic engineering” could afford the ofispring an enhanced immune response against infectious agents and vertically transmitted oncogenic agents or could induce specific tolerance to selected tissue antigens.
A N U M B E R 0 F careful investigations in the past several years have made it clear that the mammalian fetus acquires the capacity for immunologic responsiveness early in its development. These studies have been conducted primarily in the larger animals with a long gestation period such as the sheep, cow, and? to a more limited degree. the human neonate.l-” The functional and morphologic development of the fetal imFrom the Department University of Pittsburgh Medicine.
mune system in these species shares a number of characteristics in common. Both the structure of the lymphatic system and the ability to mount an immunologic response develop in an orderly, sequential manner. These processes begin in the first trimester of gestation, and by the third trimester the fetus has acquired the ability to respond to some antigenic stimuli in a fashion equal to that of the adult. During gestation the developing immune system shares with other organ systems the of the maternal environment. protection The placenta provides a physical barrier protecting the fetus and preventing the passage of potentially damaging agents which could affect critical phases of organ system development.’ Since the developing immune system shares this environment, studies of the immune response in the fetus had to be conducted under conditions of direct rx-
of Pathology, School of
The work in the authors’ supported by grants from Znstitutes of Health (AI GM 00135), the Beaver Society, the Samuel and Foundation, and the Tim Memorial Cancer Fund.
laboratory was the National 10611 and County Cancer Emma Winters Coracio
Reprint requests: Dr. Donald V. Cramer, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. 431
432
Cramer,
Kunz, and Gill
posure to the antigen.’ Under 1110~ normal conditions, the placenta selectively prevents the passage of most antigens to the fetus. However, there is a growing body of clinical evidence indicating that the mother and her offspring frequently share sensitivities to a variety of specific antigens. If it is assumed that the placenta does not provide an absolute protective barrier against certain antigens, this shared sensitization may reflect the prenatal passage of antigen, cells, 01 immunologic mediators such as transfer factor to the fetus prior to birth with a subsequent active response by the developing infant. The purpose of this review is to summarize these reports of prenatal sensitization and to discuss the possible mechanisms responsible for the observations in the context of current experimental work. Shared
prenatal
sensitization
A number of recent reports indicate that at birth the infant frequently shares with its mother immunologic reactivity to specific antigens. This immunologic reactivity is usually measured by humoral antibody production (especially fetal immunoglobulin M [ IgM] ) , by skin testing, or by demonstrating increased in vitro lymphocyte responsiveness to the specific antigens. Those antigens which have been included in this group are some infectious agents, a few common allergens, and a group of ubi
i~lllntlrloglobulirl III utllbilic;~l c.o~tl blood )s an important diagnostic l13t. Of nwre it Iterest for the. jxq)oses of tlii5 wview. hog ever, arc those maternal infections in \vhich there is little c,vidence that fetal infection has occurred. and y-t the wonate is sensitizrd to certain antigenic components of the agent. Included within this group are such agents as tuberculosis, mumps, lkherichia c-oli, and Salmonella
typlwrcr.
In tuberculosis there can be shared sensitization to tuberculin proteins betwwn the mother and the apparently normal, uninfected offspring. In a series of 1 102 neonates born to mothers infected with tuberculosis, jentgens!’ described 5 apparently normal children with positive skin tests at birth. ‘These tests were only transiently positive, and most were ne,yative 2 to -I weeks later. In none of thr cases was there evidence of active infection in the offspring. Similarly, Mohr’” has reported that of 11 children born to 5 purified protein derivative mothers 5 had positive ( P. P.D. ) --positive intradermal reactions to P.P.D. In the same series, none of 78 children from tuberculinnegati\rc mothers had a positive reaction. ‘This type of response by the newborn infant implies direct exposure to antigenic components of the agent or to the passage of specific immunologic information from the mother, perhaps in the form of intact cells. In addition to concordant positive tuberculin reactions, there have been reports that in vitro cellular responses to P.P.D. are also shared by mother and child. Field and Caspary” conducted a survey designed to examine the dr,gree and extent of shared sensitivities between mother-child pairs to a number of antigens. Included within the survey are pairs with normal mothers and mothers having a variety of inflammatory and neoplastic diseases. Five pairs of healthy mothers and their newborn babies showed lymphocyte sensitivity to P.P.D. The test system employed involved the inhibition of guinea pig macrophage migration by incubation with lymphocytes which were exposed to the test antigen. The significance of these results is not clear, however. since the
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120 3
specificity of the test system and the significance of a positive response remain unexplained. Astor and Frickr2 reported 6 similar maternal-fetal pairs with in vitro inhibition of leukocyte migration and concordant positive skin tests to P.P.D., but the significance of this report also is not clear since documentation of these observations was not complete. Maternal exposure to mumps virus can also produce an altered immune response in the offspring of infected mothersl” Ten years after a mumps epidemic, 12 Eskimo children born to mothers pregnant during the epidemic were evaluated with regard to their immune status. All of the mothers had serologic evidence of exposure to the virus, which was the first and apparently only epidemic of mumps in this group of people. The offspring that potentially had been exposed to the virus in utero had a positive mumps skin test but had no evidence of neutralizing antibodies to the virus. This failure to develop a humoral response despite the presence of an active cellular response was not observed in the other children investigated. The children born prior to the epidemic had both a positive antibody response and a positive skin test, while those born after the epidemic had only occasional positive skin tests and antibody responses. The authors concluded that mumps infection during pregnancy had led to the exposure of the embryo or the fetus to viral antigens with subsequent immunologic sensitization. These clinical observations were confirmed in pregnant rhesus monkeys by infecting the animals during the first third of gestation and demonstrating that the infant monkeys had an immune response to mumpsI Four term infant monkeys were challenged with mumps virus and demonstrated delayed hypersensitivity to the agent, but they were unable to produce antibody. The authors were able to demonstrate that the fetal monkeys were infected for only short periods of time (approximately one week), and they concluded that under these conditions “split” immunologic responses may have occurred because the fetuses had received a small dose
Immunologic
sensitization
prior
to birth
433
of antigen which led to the development of cellular immunity without humoral immunity. Two other infectious agents have been shown to be capable of sensitizing the embryo or fetus in utero. When lymphocytes from randomly chosen neonates were cultured in vitro with E. coli antigens, a certain proportion reacted positive1y.l” In order to determine whether this reactivity was compatible with in utero antigen contact, a series of newborn children from women with significant E. coli bacteriuria or pyelonephritis during pregnancy was examined.lG The lymphocytes from children born to infected women all showed positive reactions when incubated with E. coli antigen. In the control groups, only 2 of 11 children had positive reactions, and, in one of those, the mother had a history of pyelonephritis caused by E. coli prior to and during pregnancy. Similarly, an infant born to a mother immunized with one dose of typhoid-paratyphoid vaccine prior to delivery had circulating IgM hemagglutinating antibody to the somatic antigen of S. typhosa. I7 Since IgM antibody does not cross the placenta in any significant amounts, the authors felt that the antibody was fetal in origin and had been produced in response to in utero exposure to Salmonella antigens. These observations on fetal sensitivity to infectious agents may represent instances of in utero exposure to antigens with immunologic sensitization being the only manifestation of the process. If this hypothesis were based only on clinical observations in infected mothers, it would have to be made with care. The placenta could have been damaged and the fetus could have been exposed to the intact, pathogenic microorganisms, leading to brief subclinical infections. In the case of tuberculosis, however, there is experimental evidence in rats and mice that tuberculin antigens alone can produce prenatal sensitization. When pregnant female mice were inoculated intravenously with tuberculin protein (P.P.D.) , postnatal challenge of the offspring with the protein caused accelerated lymph node
434
Cramer,
Kunz,
and
Gill
development and positive skin tests.l Similarly, pregnant rats given heat-killed M. tuberculosis bacilli gave birth to offspring, of which 26 per cent had positive skin tests to tubercu1in.l:’ These two reports lend support to the concept that the sensitivity to tuberculin protein in the neonate resulted from prenatal transfer under physiologic rather than pathologic conditions. A variety of other antigens which are nonpathogenic are associated with shared sensitization in human subjects. Leikin and Oppenheim2”-22 demonstrated that cord blood lymphocytes from newborn children are commonly reactive to such antigens as diphtheria toxoid, tetanus toxoid, soluble dental plaque, bovine serum albumin, streptolysin 0, and pneumococcal polysaccharide. The response of the lymphocytes from a child to a particular antigen was not necessarily associated with a response to the other antigens tested. This finding was interpreted as indicating that the neonatal response was specific. Since in vitro responses to these antigens require prior sensitization of the lymphocyte, the response of the neonate’s lymphocytes was interpreted as being the result of passage of sensitizing antigen across the placenta and active immunization of the developing fetus.2Z Some common allergens also have been reported to produce shared sensitivity. Studies with allergic patients showed that their newborn infants can be sensitized at birth, perhaps as the result of prenatal exposure to the antigensZ3 Kaufman’* reported a case in which both the mother and newborn child shared sensitization to grass pollen. At birth the child had a positive skin test to the antigen and had specific immunoglobulin E (IgE) antibody in the cord blood that could be detected by means of the Prausnitz-Kiistner (P-K) reaction. In a similar report 22 children were examined at birth, and 4 were found to be sensitive to extracts of common allergens such as mixed ragweed, pollen, molds, grass pollen, and dustZ5 The sensitization seen in these children and the responses of cord blood lymphocytes to a number of ubiquitous
antiCgem indicate that prerlatal cuposure ttj antigen, or to maternal culls, may havr occurred. If antigen or cells passed to the fetus and produced immunologic stimulation. it most likely occ,urred in the absence of significant placental damage. Possible
mechanisms
of prenatal
sensitization
The clinical reports suggest that immunologic information can be passed from mother to fetus under normal physiologic conditions. Experimental work lends support to this concept and provides important clues to the possible mechanisms of transfer. Although the means by which the maternal environment can influence the immunologic development of the offspring is unknown, the experimental studies suggest several mechanisms, none of which need be mutually exclusive: (1) passage of maternal antibody with alteration of the fetal immune response by specific antibody; (2) passage of immunologic informational products, such as transfer factor, from the mother; (3) active migration of sensitized maternal lymphocytes to the fetus with proliferation and participation in the immune response or recruitment of fetal lymphocytes for a specific response; (4) the transplacental passage of antigen to the fetus and direct sensitization of its lymphocytes. The possibility that maternal antibody is responsible for neonatal immunologic sensitization is unlikely. Maternal antibodies, mainly immunoglobulin G (IgG) , are readily passed from the mother to her offspring during pregnancy. This antibody can alter the immune response of the newborn infant by inhibiting the response to its specific antigen, and this effect is one of the major deterrents to early vaccination.“‘-?” Maternal antibody may also be able to enhance antibody production when present in quite small amounts.3”W”,’ However, these effects are essentially passive functions associated with control of the immune response. In order for maternal antibody to produce sensitization in the neonate, it would be necessary for the antibody to confer specificity. Since cellular immunity cannot be transferred \vith serum, it would
Volume Number
120 3
seem unlikely that the in vitro expressions of of cell-mediated immunity are the result of maternal antibody. This is particularly true in a number of clinical and experimental reports in which the effect persists beyond the time maternal antibody is present in detectable amounts. The possibility that informational molecules, such as transfer factor or nucleic acid complexes, may influence the immune response of the offspring has received little or no attention. A small molecule such as transfer factor (molecular weight 10,000 Dalton Units) could be involved with prenatal sensitization, provided that it is capable of transmitting specific immunologic information.35 If transfer factor were to act as a nonspecific adjuvant, the fetal cells would have to be primed by prior antigen exposure in order to exhibit a specific response.36 The passage of intact maternal cells to the fetus must be considered as an impo’rtant possibility in view of reports that significant numbers of maternal cells may traverse the placenta in normal animals. In mice, for example, Tuffrey and co-worker+’ 38 have reported that large numbers of maternal cells can be detected in the offspring. However, this report of placental porosity was soon disputed39 and has been the center of some controversy.40 Similarly, Beer and colleagues 41~ 42 have conducted a number of experiments in rats that suggest the transfer of significant numbers of maternal cells to the fetus with the production of graft-versushost reactions. When syngeneic or allogenic lymphocytes sensitized to the paternal component of the fetal cells were injected during pregnancy, a high percentage of the offspring died with the signs of a graft-versus-host reaction. These studies provide strong circumstantial evidence that viable cells have traversed the placenta, but direct evidence of foreign cells in the tissues of the affected animals will be necessary to confirm this concept. In man, there is cell movement in both directions between the fetus and the mother. The passage of cells from the fetus to the mother causes, for example, maternal isoim-
Immunologic
sensitization
prior
to birth
435
munization and Kh hemolytic disease of the newborn infant. The extent of the passage of maternal cells to the fetus varies. Maternal erythrocytes regularly pass to the fetal circulation in normal pregnancies, and studies with the use of various types of labeled erythrocytes infused into the maternal circulation showed that maternal-to-fetal transfer occurred to 12 per cent to more than 50 per cent of pregnancies.‘” In addition to red cells, small numbers of leukocytes and platelets pass from mother to child.“4-4F These cells apparently do not persist for any period of time under normal conditions.47-4g If the newborn infant has an immunologic deficiency, however, maternal lymphocytes can be responsible for graft-versus-host reactions.50, 51 Although the number of lymphocytes that traverse the placenta appears to be small and they persist for only short periods of time, they might play a significant role in some of the observations discussed above. In those cases in which in vitro lymphocyte transformation to specific antigens is thought to represent a secondary response by fetal cells, it is possible that small numbers of maternal cells may be responding instead. The numbers would have to be small in order to escape detection by chromosomal studies,ll which have failed to detect female cells in cell cultures from male infants. Even with small numbers of cells, it is possible that they are capable of passing information to the fetal cells or recruiting fetal cells during a response. In any event, trauma during delivery may be associated with the passage of maternal cells to the newborn infant at birth, and this possibility will have to be carefully considered in evaluating the in vitro studies of lymphocytes in cord blood. The passage of antigen across the placenta with subsequent sensitization of the fetus has received some experimental attention. Much of this attention has been devoted to the possibility that prenatal exposure of the fetus to foreign histocompatibility antigens might lead to tolerance in the offspring. Experimental studies showed that neonatal exposure to tissue antigens led to tolerance in rats and mice
436
Cramer,
Kunz,
and
Gill
and that a number of factors were involved in determining the degree of tolerance. including age, sex, genetic background. and dose.“, X: In those animals given small doses of antigen, sensitization rather than tolerance was observed.‘“, 53, 5* Billingham and his coworkers”’ showed that young rats exposed to foreign tissue antigens during development rejected skin grafts in an accelerated fashion. Similarly, Stastnyl” showed that when homografts were placed on previously sensitized pregnant rats 4 to 10 days prior to delivery the offspring showed accelerated graft rejection, Skin grafts placed on the mothers before or after this period were ineffective in producing sensitization of the offspring. Even when tolerance was induced in the mothers prior to homografting, the offspring still showed accelerated skin graft rejection. It would not be expected that tolerant maternal cells would cross the placenta and participate in the reaction. Stastny concluded that antigen, rather than maternal cells, had passed to the fetus during the mid-gestational period and had actively sensitized the fetus. Other experimental work with similar results may be explained, at least in part, by the same assumption. Hypersensitized female rabbits have offspring which rejected skin transplants in an accelerated fashion.“” While it was felt that the transplacental agent responsible for the accelerated rejection was maternal antibody, the prenatal passage of maternal cells or antigen is also a distinct possibility. When neonatal rabbits from normal mothers were skin-grafted, they rejected adult skin in a significantly shorter period of time compared to the adult.“‘; Billingham and co-worker?* suggested that the latter observation may be the result of the heterogeneous nature of the rabbit population, leading to weak, transitory sensitization of the neonates to parental tissue antigens. Other studies suggested that prenatal sensitization may be caused by the transplacental passage of antigen. In guinea pigs, horse serum proteins given intravenously to the mother immediately prior to birth induced hypersensitivity in the offspring.3’ Since there was insufficient time for the pregnant female ani-
n~;~ls to develop antibodies to the horse set’um proteins ;Ind since they did not appear ill colostrum, the proteins probably passed to the fetus transplacentally and produced active immunization. These observations are manifestations of the relationship between immunologic stimulation and tolerance that exists in the adult animal? When an animal is exposed to an antigen, there is a balance between stimulation and paralysis that depends upon the chemistry of the antigen and the genetic background of the host. The chemical properties of the antigen set the level and range of dosage that can be used in stimulating an antibody response. The factors determining whether the antigen will induce stimulation or paralysis are the dose, the time over which it is given, the rate of antigen degradation, and the genetic background of the host. The same type of relationships probably also exist in the developing immune system of the fetus. In our laboratory the genetic control of the immune response to synthetic polypeptides, which are models for native proteins, has been studied extensively in inbred rat.““-” The response to these antigens is controlled by at least two genes and is linked to the major histocompatibility locus. When females of a poorly responding or highly responding strain are immunized with the aggregated antigen and then bred, the immune response of the offspring can be altered, depending upon the genotype of the strain.‘“-“C This effect requires aggregation of the antigen with methylated bovine serum albumin in order to slow its metabolic degradation and provide for its slow release from the injection site. For example, in the low-responding F344 strain, the immune responses of the 8- to lo-week-old offspring from sensitized mothers are significantly higher than those observed in the offspring from nonimmunized mothers. Kadiolabeled portions of the antigen can be detected in the fetal liver and blood prior to birth and in the bone marrow after birth.““2 (is7GOThese studies are compatible with the direct passage of antigen to the fetus prenatally, and they suggest that transplacental sensitization
Volume Number
120 3
of the fetus may be an important spread phenomenon.
Immunologic
and wide-
Implications
These experimental and clinical studies represent an immunologic phenomenon which has received little attention to date. In each case, there is reason to believe that the fetus is actively sensitized to foreign antigens during pregnancy. In the clinical studies this sensitivity is most commonly expressed as shared lymphocyte reactivity between the mother and the newborn infant. If the mother and fetus share similar antigen sensitivity, the implication is that in some manner the antigen and/or appropriate antigenie information has reached the fetus during development. Since the newborn infant has lymphocytes responsive to only a few antigens, the responses are not simply nonspecific reactions of the cells. The experimental work relevant to prenatal sensitization consist primarily of a collection of observations in which antigen exposure of the mother has in some manner altered the immune response of the offspring. Because there has been little concerted effort so far to approach this problem, the reports have been sporadic and frequently contradictory. This is not surprising, since previous work with the induction of tolerance to tissue antigens and synthetic polypeptides in rats and mice has clearly shown that timing, dosage, and genotypes are among a few of the critical variables that influence the immune response. In those cases in which sensitization of the offspring occurred, there are a number of indications that the initial priming occurred in utero. In human subjects there is need for more complete studies of the phenomenon of shared lymphocyte sensitivities: The types and numbers of antigens involved and the specificity of the reponse need to be more carefully documented. At the present time there are only scattered indications that the transfer of prenatal sensitization observed in experimental animals is related to the shared lymphocyte responses observed in human subjects. The mechanism by which sensitization occurs in experimental
sensitization
prior
to birth
437
animals is unknown, but the direct transfer of antigen or transfer of maternal cells to the fetus seems to be the best possibility. We favor the former, based on several lines of indirect evidence : ( 1) previous experimental work demonstrating the capacity of the fetus to respond at the appropriate time to a number of antigens, (2) the inability of a number of investigations to demonstrate consistently the participation of maternal cells in either in vivo or in vitro responses, (3) the transfer of sensitivity to the offspring of mothers in which tolerance to the specific antigen was induced, and (4) the evidence that prenatal antigen transfer is associated with altered immune responses in the offspring of immunized rats. If a systematic study shows that immunization during pregnancy can specifically alter the immune response of the developing fetus, at least three major applications of this type of “immunologic engineering” are apparent. First, in many areas of the world, infectious diseases early in life are still important causes of infant death. Under these conditions the ability to stimulate a primary immune response in utero could be an important vaccination procedure. This would be particularly true if immunization could be conducted prior to the transfer of significant amounts of maternal antibody to the offspring, since suppression of the immune response by maternal antibody is one of the important reasons for delayng early tetanus and diptheria vaccination. The same problem exists in sheep with regard to vaccination with Brucella abortus, but it has been successfully circumvented by in utero immunization in the absence of maternal antibody.67g 6R In human subjects, maternal immunization just prior to the twentieth week of gestation may allow the development of a primary immune response without interference form maternal antibody. In the case of tetanus and diphtheria toxoids, there have been reports of shared lymphocyte sensitivity between mother and child, and they may represent good candidates for clinical investigation. Second, the possibility that antigen presented in the proper dose at an early stage during gestation
438
Cramer,
could it),
Kunz,
produce may
to
to
specific
provide
life.
This
the
immune
tive
in
a means
of purify
responsible Selected
for antigens
induction the major
acceptance
current
membranes
and
used
specifically
nonresponsive.
be
to
to render
an Third,
of oncogcnic
certain
oncogenic
vertical
manner,
the
of
a diversity
iso-
reactions. from
may
transmission
pregnancy
antigens
isolated
irnmuriizatiorr
in
attrac-
attempts
membrane
transplantation could
later
alteration
is particularly the
of
antigens
to prenatal the
sensitivpreventinr
of bypassing
response
view
in
The
graft
approach
and
than
useful
transplantation
to successful
late
rather
be
rejection.
tolerance barrier
Gill
tolerance
prove
transplantation could
and
individual
and
could
animal
rat,
induced
lymphoreticular
this
agent
tion
a prime
interrupting
the
the
and
offspring
against
The
fact
be
transmission
that
including
the
have
virally
neoplasms man.
to
in :t during
cat.
suspect
in
prove
animals.
transmitted
species,
mouse,
tumors may
x-e
protect
chicken,
vertical
In
immunization
of tumors.““,“’ of
the
agents.
viruses
development
similar cell
interrupt
for
the
makes etiology
Prenatal
immuniza-
a
method
useful
of thesr
of of
virusrs.
prenatal
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57. 58.
59.
60. 61. 62. 63. 64. 65. 66. 67. 68. 69.
70.
sensitization
prior
to birth
439
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