Vox Sang. 35: 234-240 (1978)
Fetal Human Hemolytic Plaque-Forming Cells Evidence of Reactivity to Maternal and Other Erythrocytes A . M . C . Koros, A . E . Szulman, E . C . Hamill and B . Merchant Department of Pathology, University of Pittsburgh, Magee-Womens Hospital, Pittsburgh, Pa., and lmmunohematology Branch, Division of Blood and Blood Products, Bureau of Biologics, NIH, Bethesda, Md.
Abstract. Lymphoid tissues from 24 human fetuses were assayed for hemolytic plaqueforming cells (PFC) against a variety of erythrocyte targets. PFC against maternal and other erythrocyte antigens were commonly detected in human fetal liver, lymph nodes, spleen, or thymus as early as 16 weeks gestation and were usually more abundant in liver than in spleen after I6 weeks gestation. These data corroborate studies from other laboratories which indicate that human fetuses develop some forms of immunocompetence very early during gestation.
Introduction Although maternal reactivity to certain antigens of a fetus has been well documented [ 11, evidence of fetal sensitization to maternal antigens is sparse [6, 181 despite data documenting passage of macromolecules from mother to fetus either during gestation or at parturition [ 5 ] . It might be expected a priori that a fetus should either be too immature to have reactivity against maternal antigens or that it might be rendered tolerant to them. The present study was undertaken to determine whether there is immune reactivity in human fetuses to maternal and other foreign antigens. It has been suggested that one such type of sensitization could occur in an Rh-negative fetus during gestation in a n Rh-
positive mother [ l , 61, but there is little evidence to this effect. Hemolytic plaque-forming cells (PFC) reactive against a variety of natural and synthetic erythrocyte epitopes have been described in both immunized and nonimmunized individuals in numerous species [3, 4, 8, 13, 141. Sensitive methods for enumerating individual PFC [8], each producing a particular class of immunoglobulin [20] have served to delineate early events in the immune response to erythrocyte-bound antigens [12]. Methods developed in animal models, and used recently [ S , 12, 201 to detect PFC among maternal peripheral blood leukocytes [9, 111 were employed in this investigation to survey fetal and neonatal tissues for immunocompetent cells directed against various erythrocyte-bound epitopes.
Fetal Reactivity to Erythrocytes
235 ~
Table I. Hemolytic PFC in tissues of deceased human fetuses of up to 16 weeks gestation Experiment No.
Method of delivery
Age of fetus, weeks after LMP
Size, cm CR/CH
Erythrocyte targets
suction
8-10 10
? 11/17
SRC SRC P D J N
0 0 0
K
0 0 0 0
12 '/*
prostaglandin
spontaneous 14 abortion prostaglandin 15
prostaglandin 16
spontaneous abortion saline hysterectomy (mole) saline saline saline
I6 'lZ 16 16 16 16 16
SRC 8/11 SRC P D J N K MRC O+ 6/8 SRC 7/9 (twins) SRC 10/14 P J N K MRC 12/17 N K MRC 9/12 SRC (macerated) 9/15 SRC 1I / ? SRC
P F C / I P viable cells in thymus
? 11/17
M RC SRC SRC P D J N K
spleen
lymph nodes
0 0 0
7!10
9/15
liver
0 0 0
0 0 0 0 0
0 0
0 0 0 0 1
0 0
0 0 0 0 0 0 0
0 0
0 0
0
I' 577' 0
0
0
0 0 0
0
0
0
3 2
6
14 5
0 19
0 36
0
7 8 0
0
0
0 0
0
0 0 0 0
0 0 0 0
0
0 0 74 ~
CR/CH =Crown rump/crown heel; SRC=sheep red cells from one animal; P=sheep red cells pooled from at least 12 animals; D, N, J, K = P cells coupled with the following haptens: dansyl 8-alanylglycylglycine, nitroiodophenyl-/?-alanylglycylglycine, dinitrophenyl-B-alanylglycylglycine, trinitrophenyl-B-alanylglycylglycine; MRC = maternal red cells; 0 + = 0 Rh(D)-positive red cells (from individual human donor). * Plaques counted on dried plates.
Koros/Szulman/Hamill/Merchant
236 ~~~
~
Table 11. Hemolytic PFC in tissues of deceased human neonates or fetuses older than 16 weeks gestation
Experiment No.
Method of delivery
Age of fetus or neonate, (weeks after LMP)
Size, cm (CR/CH)
saline
17
13/20
Ed1
saline
18
13/21
H3
saline
18
14/21
Hgl
saline
I8
15/21
H*
H9
H,*
F,,
saline
saline
saline
saline
18
18
19
20
prostaglandin 26*
PFC/IOHviable cells in
thymus
H,
Hl12
Erythrocyte targets
15/22
13/19
15/22
16/24
22/30
SRC P N D MRC SRC P J K M RC SRC MRC SRC P J K M RC SRC P J K M RC SRC P D N MRC SRC P D N MRC SRC P J K MRC P N
liver
20 2,125 957 1,788 9 0 0 0 28 0 0 7 90 I 198 89 0 476 999 430 0 0 0 0
spleen
1 5 7 I 1 88
9 2 5 2 2 0 98 233 195 0 204 159
II 0 21 5,443 3,272 3,147 0
I ,2** 0
J
o,o**
MRC
2**
0 0 0 0 8 0 0 31
0 31 52
36 0 1,038
40 0 0 20 113
lymph nodes
Fetal Reactivity to Erythrocytes
237
Table II. (continued)
Experiment No.
Method of delivery
HS
H,'
caesarean section
Age of fetus or neonate, (weeks after LMP)
Size, cm (CR/CH)
30 (deceased) neonate I day old)
29/39
40 (deceased neonate 2 days old)
40153
Erythrocyte targets
thymus
SRC
P D N O+ A+ SRC P D O+
A+ AB +
* **
PFC/lO* viable cells in
35 4
2 7 6 18 80 36 0 0 0
liver
0 395 629 496 0 0 94 188 94 0 0 0
spleen
lymph nodes
0 24 28 4 I 272 78
4,800 7.980
13 0 0
0 0 0
0+ , A + , AB + =Human red cells from individual donors. For other abbreviations see table I. Fetus had Krabbe's disease. Plaques counted on dried plates.
Methods Fetal Tissues
Tissues were obtained aseptically at autopsy from newborns or fetuses after therapeutic or spontaneous abortions. Entire spleens were assayed from small fetuses, whereas samples of 600mg o f liver or 200mg of spleen were used from larger fetuses or deceased neonates. Single cell suspensions were obtained by pressing tissues through a stainless steel wire mesh [12]. Cells were washed once in Eagle's medium, and resuspended to give final concentrations of 106 to 10s cells/ml depending on the original sample size. Cell viability was determined by trypan blue exclusion and was found to range between 25 and 60%. Erythrocyte Targets
The following erythrocytes served as targets for PFC assays: sheep red cells (SRC) from individual sheep (Flow Laboratories, Rockville, Md., or Sack's Farm, Evans City, Pa.), pooled sheep red cells (P), or sheep red cells coupled with tripeptideenlarged analogs of dansyl, dinitrophenyl, nitro-
iodophenyl, or trinitrophenyl haptens (D, J, N, and K, respectively) [7, 14, 171, maternal red cells (MRC) from the respective mothers of deceased fetuses or neonates, and other human (A) Rh(D)positive, or (B) Rh(D)-positive erythrocytes, all from individual donors. Hemolytic Plaque Assay
Target erythrocytes (SRC, MRC, and other human red cells) collected in Alsever's solution were washed and diluted to give a 20% suspension containing 4 x 109 red cells/ml 181. Pooled, plain sheep cells (P) or P cells coupled with haptens D, J, N, or K were washed and diluted to give a 50% suspension containing 1010 cells/ml [17]. Mixtures of 2 ml of 45 "C agar and Eagles' medium or modified Eagles' medium [17], 0.1 ml target erythrocyte suspension and 0.1 to 0.5 ml of fetal or neonatal cell suspension containing approximately lo5 viable lymphoid cells were plated in 15 x 100 mm Petri dishes. Many plates, each containing few lymphoid cells were required for each tissue tested, in order to be certain that each plaque contained a single lymphoid cell. Plaque counts in replicate
Koros/Szulman/Hamill/Merc hant
238
plates were reproducible within reasonable limits (i.e. they usually varied by no more than 20%). Only those plaques with an identifiable central cell (8) were counted. Control plates containing appropriate erythrocyte targets without fetal cells were included in each experiment. Other plates containing spleen cells from mice immunized with SKC were also included as positive controls in each experiment. Plates were incubated at 37 O C in a humidified atmosphere of 5% CO, and 95% air for 1 h; direct PFC were developed according to standard methods [8] by adding 10% guinea pig complement in Eagles' medium for an additional half hour. Complement was removed and PFC were counted i n wet preparations under a dissecting microscope. Plaques were circled; plates were dried at room temperature, fixed in 95% ethanol and stained with Giemsa. PFC were identified microscopically in stained preparations [12] at x 450 or 1,000 magnification, and results were expressed as PFC/lOR viable cells.
Results The numbers of PFC in human fetal tissues up to 16 weeks of gestation are shown in table I. Hemolytic PFC against maternal and other erythrocyte antigens first appeared at 14-16 weeks of gestation. Such PFC were detected in variable numbers in human fetal liver, lymph node, spleen, or thymus samples, but most fetuses expressed PFC activity in one organ or another at about this time. Table I1 records the numbers of PFC detected in later fetal and neonatal tissues. These tissue samples were generally larger; however, the total numbers of fetal cells were still often insufficient and not every fetal tissue could be tested against all available target erythrocytes. Nevertheless, 4 of 9 fetuses from 17 to 26 weeks of age, whose livers were assayed, had PFC when plated
on maternal erythrocytes. 5 of 9 fetuses of the same age group, whose spleens were assayed, had PFC directed against MRC. Fetuses in this age group also usually had PFC to a single sheep's red cells (SRC), and against pooled sheep erythrocytes (P). They also had occasional reactivities against hapten-coupled sheep red cells. It is interesting that there were generally more PFC to pooled sheep cells than there were to SRC from an individual sheep. Because there are seven known blood types in sheep [16], a wider array of antigens would be expected in the pooled sheep cells (obtained from a minimum of 12 animals). Within the sensitivity of the present assay system, the reactivity of hapten-coupled sheep red cells was often not significantly greater than to plain red cells. Reactivity to SRC also appears earlier in mouse fetuses than does reactivity to haptens [ 141.
Discussion Data from the present study corroborate earlier work from other laboratories, which also indicate that human fetuses develop some forms of immunocompetence very early in gestation [2, 15, 191. Mixed lymphocyte reactivity (MLR) was detected as early as 10 weeks in fetal liver, and phytohemagglutinin (PHA) reactivity was detected at 13 weeks in the thymus [ 2 ] . PFC in human fetal tissues are by analogy probably of fetal origin rather than seedings from transplacental traffic of maternal PFC. In studies involving fetal mice there was no correlation between the numbers of PFC in maternal spleens compared with those detected in fetal spleens [14]. The time of appearance of PFC in fetal
Fetal Keactivity to Erythrocytes
mice directed against different specificities was also characteristic for each mouse strain that was tested [14]. The numbers of human fetal PFC observed in this study represent minimum values for the following reasons: ( I ) fetuses were obtained in most cases by saline induction and had died 1-16 h before tissues were assayed for PFC; cell viability was lower ( 2 5 6 0 % by trypan blue) than observed for standard suspensions of mouse cells (> 75% viable cells) at time of assay; (2) only direct PFC, presumably IgM producers, would have been observable under the conditions employed in this study; therefore, PFC producing other classes of immunoglobulins would not have been detected in the system used, and ( 3 ) in those cases in which only portions of liver were available, PFC could have been missed due to the nonrandom distribution of PFC in lymphoid tissue [8]. Nevertheless, of 13 cases tested against maternal erythrocytes, 8 had positive reactivity in at least one of the tissues assayed against MRC. The existence of background PFC to various erythrocyte targets at various stages in development of human and other species has been reported only sporadically [3, 4, 8-1 11. The biological significance of background PFC is unknown [lo]. Such cells may reflect the following: (1) prior exposure to cross-reacting antigens; (2) spontaneously arising clones of immunocompetent cells of restricted specificity, and ( 3 ) B cell activity generated in response to immunoregulatory cells belonging to either T or B lymphocyte populations [ 101. Although autoreactive background PFC have been described [lo, 111, their etiology is also a mystery. Further insight into their origins might help to provide some understanding of why a small
239
percentage of Rh-negative women appear to be sensitized as primagravidas to their Rh-positive fetuses. Despite the limited availability of tissues, over half of the fetuses studied which were older than 16 weeks had PFC against maternal erythrocyte antigens. This is significant because it represents either spontaneous, genetically-programmed antibody-producing PFC, or PFC arising from intrauterine immunization of the fetus. In the present study, adequate blood samples were not available for fetal blood types to be determined. Future studies should be directed at determining whether PFC reacting against MRC appear more often in ABO and in Rh incompatible pregnancies than in fetuses compatible with their mothers. It may well be relevant to the problem of apparently spontaneous anti-Rh activity in Rh-negative individuals [6] that reactivity to maternal red cell antigens can exist before birth. Evidence that such antenatal reactivity could be due to the Rh(D) antigens remains, however, to be determined.
Acknowledgements This work was supported by general research support grant RR0.5416 to A . M . C. Koros. The authors thank Helen Baginski for technical assistance, Lorraine Repasky for preparation of the manuscript, Drs. T. T . Hayashi, D . Medearis, T . J . Gill, 111, R . Sabbagha and G . Werner for their help in this project.
References 1 Beer, A. E., and Billingham, K.E.: Irnrnunobiology of mammalian reproduction. Adv. Irnrnunol. 14: 1-84 (1971).
240
2 Carr, M. C.; Sites, D. P., and Fudenberg, H. H.: Dissociation of responses to phytohaemagglutinin and adult allogeneic lymphocytes in human foetal lymphoid tissues. Nature new Biol. 241: 279-281 (1973). 3 Diener, E. and MacKay, I. R.: Cells in human spleen forming antibody to sheep erythrocytes. Lancet i: 820-821 (1967). 4 Friedman, H.: Distribution of antibody plaque forming cells in various tissues of several strains of mice injected with sheep erythrocytes. Proc. SOC.exp. Biol. Med. 117: 526-530 (1964). 5 Gitlin, J. D. and Gitlin, D.: Protein binding by cell membranes and the selective transfer of proteins from mother to young across tissue barriers; in Hemmings, Maternofoetal transmission of immunoglobulins, pp. 113-122 (Cambridge University Press, Cambridge 1976). 6 Hindemann, P.: Maternofoetal transfusion during delivery and Rh-sensitization of the newborn. Lancet i : 46 (1973). 7 Inman, J.K.; Merchant, B., and Tacey, S.E.: Synthesis of large haptenic compounds having a common functional group that permits covalent linkage to proteins, cell surfaces, and absorbents. lrnmunochemistry 10: 153-163 (1973). 8 Jerne, N. K.; Henry, C.; Nordin, A. A.; Fuji, H.; Koros, A.M. C., and Lefkovits, I.: Plaque forming cells. Methodology and theory. Transplantn Rev. 18: 130-191 (1974). 9 Katz, J. and Marcus, R. G.: Incidence of Rh immunization following abortion. Possible detection of lymphocyte priming to Rh antigen. Am. J. Obstet. Gynec. 117: 261-267 (1973). 10 Koros, A. M . C.; Axelrod, A. E.; Hamill, E. C., and South, D. J.: Immunoregulatory consequences of vitamin deficiencies on background plaque-forming cells in rats. Proc. SOC. exp. Biol. Med. 152: 322-326 (1976). 11 Koros, A. M. C.; Hamill, E. C., and Depp, 0. R.: Anti-autologous erythrocyte plaque-forming cells in pregnancy. Vox Sang. 35: 277-287 (1978). 12 Koros, A . M . C.; Mazur, J. M., and Mowery, M. J.: Radioautographic studies of plaqueforming cells. 1. Antigen-stimulated proliferation of plaque-forming cells. J. exp. Med. 128: 235-257 (1968).
Koros/Szulman/Hamill/Merchant
13 Leiper, J. B.: Ontogeny of haemolytic plaqueforming cells in newborn rabbit's spleen in response to different erythrocyte antigens. Immunology 26: 1061-1067 (1974). 14 Merchant, B.; Inman, J. K., and Claflin, L.: Ir genes linked to Ig allotypes; in McDevitt and Landy, Genetic control of immune responsiveness. Relationship to disease susceptibility, pp. 195-203 (Academic Press, New York 1972). 15 Pirofsky, B.; Davies, G. H.; Kamirez-Mateos, J. C., and Newton, B. W.: Cellular immune competence in the human fetus. Cell. Immunol. 6: 324-328 (1973). 16 Rasmusen, B. A.: Blood groups in sheep. Ann. N.Y. Acad. Sci. 97: 306-319 (1962). 17 Rouquirs, R.; Inman, J. K., and Merchant, B.: Detection of cells secreting antibodies more reactive with an alternate structure than with immunizing hapten. Int. Archs Allergy appl. Immun. 42: 852-870 (1972). 18 Speiser, P.; Mickerts, D.; Pausch, V., and Mayr, W. R.: The transient nature of anti-Gm; in Grubb and Samuelsson, Human anti-human gammaglobulins. Their specificity and function, pp. 151-160 (Pergamon Press, New York 1971). 19 Stites, D. P.; Caldwell, J.; Carr, M. C., and Fudenberg, H.H.: Ontogeny of immunity in man. Acta endocr., suppl. 194, pp. 306-317 (1975). 20 Wortis, H. H.; Dresser, D. W., and Anderson, H. R.: Antibody production studied by means of the localized haemolysis in gel (LHG) assay. 111. Mouse cells producing five different classes of antibody. Immunology 17: 93-110 (1969).
Received: April 8, 1977 Accepted: December 8, 1978 Dr. Aurelia Koros, Clinical Radiation Therapy, Research Center, Division of Radiation Oncology, Allegheny General Hospital, 320 East North Avenue, Pittsburgh, Pa. 15212 (USA)
Fetal Human Hemolytic Plaque-Forming Cells Evidence of Reactivity to Maternal and Other Erythrocytes A . M . C . Koros, A . E . Szulman, E . C . Hamill and B . Merchant Department of Pathology, University of Pittsburgh, Magee-Womens Hospital, Pittsburgh, Pa., and lmmunohematology Branch, Division of Blood and Blood Products, Bureau of Biologics, NIH, Bethesda, Md.
Abstract. Lymphoid tissues from 24 human fetuses were assayed for hemolytic plaqueforming cells (PFC) against a variety of erythrocyte targets. PFC against maternal and other erythrocyte antigens were commonly detected in human fetal liver, lymph nodes, spleen, or thymus as early as 16 weeks gestation and were usually more abundant in liver than in spleen after I6 weeks gestation. These data corroborate studies from other laboratories which indicate that human fetuses develop some forms of immunocompetence very early during gestation.
Introduction Although maternal reactivity to certain antigens of a fetus has been well documented [ 11, evidence of fetal sensitization to maternal antigens is sparse [6, 181 despite data documenting passage of macromolecules from mother to fetus either during gestation or at parturition [ 5 ] . It might be expected a priori that a fetus should either be too immature to have reactivity against maternal antigens or that it might be rendered tolerant to them. The present study was undertaken to determine whether there is immune reactivity in human fetuses to maternal and other foreign antigens. It has been suggested that one such type of sensitization could occur in an Rh-negative fetus during gestation in a n Rh-
positive mother [ l , 61, but there is little evidence to this effect. Hemolytic plaque-forming cells (PFC) reactive against a variety of natural and synthetic erythrocyte epitopes have been described in both immunized and nonimmunized individuals in numerous species [3, 4, 8, 13, 141. Sensitive methods for enumerating individual PFC [8], each producing a particular class of immunoglobulin [20] have served to delineate early events in the immune response to erythrocyte-bound antigens [12]. Methods developed in animal models, and used recently [ S , 12, 201 to detect PFC among maternal peripheral blood leukocytes [9, 111 were employed in this investigation to survey fetal and neonatal tissues for immunocompetent cells directed against various erythrocyte-bound epitopes.
Fetal Reactivity to Erythrocytes
235 ~
Table I. Hemolytic PFC in tissues of deceased human fetuses of up to 16 weeks gestation Experiment No.
Method of delivery
Age of fetus, weeks after LMP
Size, cm CR/CH
Erythrocyte targets
suction
8-10 10
? 11/17
SRC SRC P D J N
0 0 0
K
0 0 0 0
12 '/*
prostaglandin
spontaneous 14 abortion prostaglandin 15
prostaglandin 16
spontaneous abortion saline hysterectomy (mole) saline saline saline
I6 'lZ 16 16 16 16 16
SRC 8/11 SRC P D J N K MRC O+ 6/8 SRC 7/9 (twins) SRC 10/14 P J N K MRC 12/17 N K MRC 9/12 SRC (macerated) 9/15 SRC 1I / ? SRC
P F C / I P viable cells in thymus
? 11/17
M RC SRC SRC P D J N K
spleen
lymph nodes
0 0 0
7!10
9/15
liver
0 0 0
0 0 0 0 0
0 0
0 0 0 0 1
0 0
0 0 0 0 0 0 0
0 0
0 0
0
I' 577' 0
0
0
0 0 0
0
0
0
3 2
6
14 5
0 19
0 36
0
7 8 0
0
0
0 0
0
0 0 0 0
0 0 0 0
0
0 0 74 ~
CR/CH =Crown rump/crown heel; SRC=sheep red cells from one animal; P=sheep red cells pooled from at least 12 animals; D, N, J, K = P cells coupled with the following haptens: dansyl 8-alanylglycylglycine, nitroiodophenyl-/?-alanylglycylglycine, dinitrophenyl-B-alanylglycylglycine, trinitrophenyl-B-alanylglycylglycine; MRC = maternal red cells; 0 + = 0 Rh(D)-positive red cells (from individual human donor). * Plaques counted on dried plates.
Koros/Szulman/Hamill/Merchant
236 ~~~
~
Table 11. Hemolytic PFC in tissues of deceased human neonates or fetuses older than 16 weeks gestation
Experiment No.
Method of delivery
Age of fetus or neonate, (weeks after LMP)
Size, cm (CR/CH)
saline
17
13/20
Ed1
saline
18
13/21
H3
saline
18
14/21
Hgl
saline
I8
15/21
H*
H9
H,*
F,,
saline
saline
saline
saline
18
18
19
20
prostaglandin 26*
PFC/IOHviable cells in
thymus
H,
Hl12
Erythrocyte targets
15/22
13/19
15/22
16/24
22/30
SRC P N D MRC SRC P J K M RC SRC MRC SRC P J K M RC SRC P J K M RC SRC P D N MRC SRC P D N MRC SRC P J K MRC P N
liver
20 2,125 957 1,788 9 0 0 0 28 0 0 7 90 I 198 89 0 476 999 430 0 0 0 0
spleen
1 5 7 I 1 88
9 2 5 2 2 0 98 233 195 0 204 159
II 0 21 5,443 3,272 3,147 0
I ,2** 0
J
o,o**
MRC
2**
0 0 0 0 8 0 0 31
0 31 52
36 0 1,038
40 0 0 20 113
lymph nodes
Fetal Reactivity to Erythrocytes
237
Table II. (continued)
Experiment No.
Method of delivery
HS
H,'
caesarean section
Age of fetus or neonate, (weeks after LMP)
Size, cm (CR/CH)
30 (deceased) neonate I day old)
29/39
40 (deceased neonate 2 days old)
40153
Erythrocyte targets
thymus
SRC
P D N O+ A+ SRC P D O+
A+ AB +
* **
PFC/lO* viable cells in
35 4
2 7 6 18 80 36 0 0 0
liver
0 395 629 496 0 0 94 188 94 0 0 0
spleen
lymph nodes
0 24 28 4 I 272 78
4,800 7.980
13 0 0
0 0 0
0+ , A + , AB + =Human red cells from individual donors. For other abbreviations see table I. Fetus had Krabbe's disease. Plaques counted on dried plates.
Methods Fetal Tissues
Tissues were obtained aseptically at autopsy from newborns or fetuses after therapeutic or spontaneous abortions. Entire spleens were assayed from small fetuses, whereas samples of 600mg o f liver or 200mg of spleen were used from larger fetuses or deceased neonates. Single cell suspensions were obtained by pressing tissues through a stainless steel wire mesh [12]. Cells were washed once in Eagle's medium, and resuspended to give final concentrations of 106 to 10s cells/ml depending on the original sample size. Cell viability was determined by trypan blue exclusion and was found to range between 25 and 60%. Erythrocyte Targets
The following erythrocytes served as targets for PFC assays: sheep red cells (SRC) from individual sheep (Flow Laboratories, Rockville, Md., or Sack's Farm, Evans City, Pa.), pooled sheep red cells (P), or sheep red cells coupled with tripeptideenlarged analogs of dansyl, dinitrophenyl, nitro-
iodophenyl, or trinitrophenyl haptens (D, J, N, and K, respectively) [7, 14, 171, maternal red cells (MRC) from the respective mothers of deceased fetuses or neonates, and other human (A) Rh(D)positive, or (B) Rh(D)-positive erythrocytes, all from individual donors. Hemolytic Plaque Assay
Target erythrocytes (SRC, MRC, and other human red cells) collected in Alsever's solution were washed and diluted to give a 20% suspension containing 4 x 109 red cells/ml 181. Pooled, plain sheep cells (P) or P cells coupled with haptens D, J, N, or K were washed and diluted to give a 50% suspension containing 1010 cells/ml [17]. Mixtures of 2 ml of 45 "C agar and Eagles' medium or modified Eagles' medium [17], 0.1 ml target erythrocyte suspension and 0.1 to 0.5 ml of fetal or neonatal cell suspension containing approximately lo5 viable lymphoid cells were plated in 15 x 100 mm Petri dishes. Many plates, each containing few lymphoid cells were required for each tissue tested, in order to be certain that each plaque contained a single lymphoid cell. Plaque counts in replicate
Koros/Szulman/Hamill/Merc hant
238
plates were reproducible within reasonable limits (i.e. they usually varied by no more than 20%). Only those plaques with an identifiable central cell (8) were counted. Control plates containing appropriate erythrocyte targets without fetal cells were included in each experiment. Other plates containing spleen cells from mice immunized with SKC were also included as positive controls in each experiment. Plates were incubated at 37 O C in a humidified atmosphere of 5% CO, and 95% air for 1 h; direct PFC were developed according to standard methods [8] by adding 10% guinea pig complement in Eagles' medium for an additional half hour. Complement was removed and PFC were counted i n wet preparations under a dissecting microscope. Plaques were circled; plates were dried at room temperature, fixed in 95% ethanol and stained with Giemsa. PFC were identified microscopically in stained preparations [12] at x 450 or 1,000 magnification, and results were expressed as PFC/lOR viable cells.
Results The numbers of PFC in human fetal tissues up to 16 weeks of gestation are shown in table I. Hemolytic PFC against maternal and other erythrocyte antigens first appeared at 14-16 weeks of gestation. Such PFC were detected in variable numbers in human fetal liver, lymph node, spleen, or thymus samples, but most fetuses expressed PFC activity in one organ or another at about this time. Table I1 records the numbers of PFC detected in later fetal and neonatal tissues. These tissue samples were generally larger; however, the total numbers of fetal cells were still often insufficient and not every fetal tissue could be tested against all available target erythrocytes. Nevertheless, 4 of 9 fetuses from 17 to 26 weeks of age, whose livers were assayed, had PFC when plated
on maternal erythrocytes. 5 of 9 fetuses of the same age group, whose spleens were assayed, had PFC directed against MRC. Fetuses in this age group also usually had PFC to a single sheep's red cells (SRC), and against pooled sheep erythrocytes (P). They also had occasional reactivities against hapten-coupled sheep red cells. It is interesting that there were generally more PFC to pooled sheep cells than there were to SRC from an individual sheep. Because there are seven known blood types in sheep [16], a wider array of antigens would be expected in the pooled sheep cells (obtained from a minimum of 12 animals). Within the sensitivity of the present assay system, the reactivity of hapten-coupled sheep red cells was often not significantly greater than to plain red cells. Reactivity to SRC also appears earlier in mouse fetuses than does reactivity to haptens [ 141.
Discussion Data from the present study corroborate earlier work from other laboratories, which also indicate that human fetuses develop some forms of immunocompetence very early in gestation [2, 15, 191. Mixed lymphocyte reactivity (MLR) was detected as early as 10 weeks in fetal liver, and phytohemagglutinin (PHA) reactivity was detected at 13 weeks in the thymus [ 2 ] . PFC in human fetal tissues are by analogy probably of fetal origin rather than seedings from transplacental traffic of maternal PFC. In studies involving fetal mice there was no correlation between the numbers of PFC in maternal spleens compared with those detected in fetal spleens [14]. The time of appearance of PFC in fetal
Fetal Keactivity to Erythrocytes
mice directed against different specificities was also characteristic for each mouse strain that was tested [14]. The numbers of human fetal PFC observed in this study represent minimum values for the following reasons: ( I ) fetuses were obtained in most cases by saline induction and had died 1-16 h before tissues were assayed for PFC; cell viability was lower ( 2 5 6 0 % by trypan blue) than observed for standard suspensions of mouse cells (> 75% viable cells) at time of assay; (2) only direct PFC, presumably IgM producers, would have been observable under the conditions employed in this study; therefore, PFC producing other classes of immunoglobulins would not have been detected in the system used, and ( 3 ) in those cases in which only portions of liver were available, PFC could have been missed due to the nonrandom distribution of PFC in lymphoid tissue [8]. Nevertheless, of 13 cases tested against maternal erythrocytes, 8 had positive reactivity in at least one of the tissues assayed against MRC. The existence of background PFC to various erythrocyte targets at various stages in development of human and other species has been reported only sporadically [3, 4, 8-1 11. The biological significance of background PFC is unknown [lo]. Such cells may reflect the following: (1) prior exposure to cross-reacting antigens; (2) spontaneously arising clones of immunocompetent cells of restricted specificity, and ( 3 ) B cell activity generated in response to immunoregulatory cells belonging to either T or B lymphocyte populations [ 101. Although autoreactive background PFC have been described [lo, 111, their etiology is also a mystery. Further insight into their origins might help to provide some understanding of why a small
239
percentage of Rh-negative women appear to be sensitized as primagravidas to their Rh-positive fetuses. Despite the limited availability of tissues, over half of the fetuses studied which were older than 16 weeks had PFC against maternal erythrocyte antigens. This is significant because it represents either spontaneous, genetically-programmed antibody-producing PFC, or PFC arising from intrauterine immunization of the fetus. In the present study, adequate blood samples were not available for fetal blood types to be determined. Future studies should be directed at determining whether PFC reacting against MRC appear more often in ABO and in Rh incompatible pregnancies than in fetuses compatible with their mothers. It may well be relevant to the problem of apparently spontaneous anti-Rh activity in Rh-negative individuals [6] that reactivity to maternal red cell antigens can exist before birth. Evidence that such antenatal reactivity could be due to the Rh(D) antigens remains, however, to be determined.
Acknowledgements This work was supported by general research support grant RR0.5416 to A . M . C. Koros. The authors thank Helen Baginski for technical assistance, Lorraine Repasky for preparation of the manuscript, Drs. T. T . Hayashi, D . Medearis, T . J . Gill, 111, R . Sabbagha and G . Werner for their help in this project.
References 1 Beer, A. E., and Billingham, K.E.: Irnrnunobiology of mammalian reproduction. Adv. Irnrnunol. 14: 1-84 (1971).
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2 Carr, M. C.; Sites, D. P., and Fudenberg, H. H.: Dissociation of responses to phytohaemagglutinin and adult allogeneic lymphocytes in human foetal lymphoid tissues. Nature new Biol. 241: 279-281 (1973). 3 Diener, E. and MacKay, I. R.: Cells in human spleen forming antibody to sheep erythrocytes. Lancet i: 820-821 (1967). 4 Friedman, H.: Distribution of antibody plaque forming cells in various tissues of several strains of mice injected with sheep erythrocytes. Proc. SOC.exp. Biol. Med. 117: 526-530 (1964). 5 Gitlin, J. D. and Gitlin, D.: Protein binding by cell membranes and the selective transfer of proteins from mother to young across tissue barriers; in Hemmings, Maternofoetal transmission of immunoglobulins, pp. 113-122 (Cambridge University Press, Cambridge 1976). 6 Hindemann, P.: Maternofoetal transfusion during delivery and Rh-sensitization of the newborn. Lancet i : 46 (1973). 7 Inman, J.K.; Merchant, B., and Tacey, S.E.: Synthesis of large haptenic compounds having a common functional group that permits covalent linkage to proteins, cell surfaces, and absorbents. lrnmunochemistry 10: 153-163 (1973). 8 Jerne, N. K.; Henry, C.; Nordin, A. A.; Fuji, H.; Koros, A.M. C., and Lefkovits, I.: Plaque forming cells. Methodology and theory. Transplantn Rev. 18: 130-191 (1974). 9 Katz, J. and Marcus, R. G.: Incidence of Rh immunization following abortion. Possible detection of lymphocyte priming to Rh antigen. Am. J. Obstet. Gynec. 117: 261-267 (1973). 10 Koros, A. M . C.; Axelrod, A. E.; Hamill, E. C., and South, D. J.: Immunoregulatory consequences of vitamin deficiencies on background plaque-forming cells in rats. Proc. SOC. exp. Biol. Med. 152: 322-326 (1976). 11 Koros, A. M. C.; Hamill, E. C., and Depp, 0. R.: Anti-autologous erythrocyte plaque-forming cells in pregnancy. Vox Sang. 35: 277-287 (1978). 12 Koros, A . M . C.; Mazur, J. M., and Mowery, M. J.: Radioautographic studies of plaqueforming cells. 1. Antigen-stimulated proliferation of plaque-forming cells. J. exp. Med. 128: 235-257 (1968).
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13 Leiper, J. B.: Ontogeny of haemolytic plaqueforming cells in newborn rabbit's spleen in response to different erythrocyte antigens. Immunology 26: 1061-1067 (1974). 14 Merchant, B.; Inman, J. K., and Claflin, L.: Ir genes linked to Ig allotypes; in McDevitt and Landy, Genetic control of immune responsiveness. Relationship to disease susceptibility, pp. 195-203 (Academic Press, New York 1972). 15 Pirofsky, B.; Davies, G. H.; Kamirez-Mateos, J. C., and Newton, B. W.: Cellular immune competence in the human fetus. Cell. Immunol. 6: 324-328 (1973). 16 Rasmusen, B. A.: Blood groups in sheep. Ann. N.Y. Acad. Sci. 97: 306-319 (1962). 17 Rouquirs, R.; Inman, J. K., and Merchant, B.: Detection of cells secreting antibodies more reactive with an alternate structure than with immunizing hapten. Int. Archs Allergy appl. Immun. 42: 852-870 (1972). 18 Speiser, P.; Mickerts, D.; Pausch, V., and Mayr, W. R.: The transient nature of anti-Gm; in Grubb and Samuelsson, Human anti-human gammaglobulins. Their specificity and function, pp. 151-160 (Pergamon Press, New York 1971). 19 Stites, D. P.; Caldwell, J.; Carr, M. C., and Fudenberg, H.H.: Ontogeny of immunity in man. Acta endocr., suppl. 194, pp. 306-317 (1975). 20 Wortis, H. H.; Dresser, D. W., and Anderson, H. R.: Antibody production studied by means of the localized haemolysis in gel (LHG) assay. 111. Mouse cells producing five different classes of antibody. Immunology 17: 93-110 (1969).
Received: April 8, 1977 Accepted: December 8, 1978 Dr. Aurelia Koros, Clinical Radiation Therapy, Research Center, Division of Radiation Oncology, Allegheny General Hospital, 320 East North Avenue, Pittsburgh, Pa. 15212 (USA)
