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Cellular immune aspects of the human fetal-maternal relationship
CELLULAR
Cellular
IMMUNOLOGY
11,
Immune III. Mixed
MARTIN Departments
332-341
Aspects
(1974)
of the Human
Fetal-Maternal
Relationship
Lymphocyte Reactivity Between Related Maternal and Cord Blood Lymphocytes 1
C. CARR, DANIEL
P. STITES,~ AND H. HUGH
FUDENBERG
of Obstetrics and Gynecology and Medicine, School of Medicine, University of California, San Francisco, California 94143 Received
July
30 1973
Gravida and related cord blood lymphocytes were studied in one-way mixed lymphocyte culture to find out if tolerance or sensitization existed between these organisms. Maternal lymphocytes were consistently stimulated by both related cord blood and adult allogeneic lymphocytes, and the kinetics of the reactions did not suggest that any specific tolerance or sensitization existed toward fetal histocompatibility antigens. Cord blood lymphocytes were stimulated by related maternal lymphocytes in approximately one-half the tests, and also seemed less reactive toward adu’t allogeneic cells. However, the temporal kinetics of the reaction did not suggest any sersitization or tolerance toward maternal lymphocytes. The findings suggest that the success of the human pregnancy in terms of cell-mediated immunity does not depend upon intrinsic cellular maternal-fetal tolerance.
INTRODUCTION Recent evidence suggests that there is a substantial exchange of antigenic material between mother and fetus during the normal human pregnancy (1-S). Fetal cellular and particulate matter entering ‘the maternal vascular system during pregnancy can result in sensitization (9-11). Conversely, maternal antigenic material may enter the fetal circulation and produce sensitization of #thefetus (12). Despite this reciprocal immunization, pregnancy usually progressesundeterred except when isoimmunization to erythrocyte antigens intervenes. What makes this intriguing is that the human pregnancy consists of two beings coexisting successfully, each invariably with distinctive histocompatibility antigens. Our best knowledge tells us that the fetus, analogous to an allogeneic graft, probably should be rejected as a result of activity of the cellular immune mechanisms available to the maternal host. Why does the fetus survive under such precarious conditions? Crucial parts of this question are whether mutual antigen exchange results in immunity or specific tolerance between the histocompatibility antigens of the participants, and whether Ithere is an altered state of cellular immunity of the mother toward the fetus. 1 Supported in part by United States Public Health HD-05894, and by training grant HL-05677. 2Reprint requests should be addressed to Dr. Stites of Medicine, University of California, San Francisco, 332 Copyri ht 0 1974 by Academic Press, All rig fl ts of reproduction in any form
Inc. reserved.
Service
research
at the Department California 94143.
grants
HD-03939
of Medicine,
and School
MLR
BETWEEN
MATERNAL
AND
CORD
LYMPHOCYTES
3.33
One way of examining these questions is to study the reactivity in vitro of the participants’ lymphocytes toward each other in a one-way mixed lymphocyte reaction (MLR). In the MLR, recognition of histocompatibility differences triggers blast transformation of the responding lymphocytes (13). In at least one animal system, the kinetics of this recognition and the subsequent proliferative response have been predictably altered by previously exposing ‘the animal to appropriate doses of the specific histocompatibility antigens, producing either tolerance (no lymphocyte proliferation results) or sensitization (the peak response is characterized by lolver isotope incorporation and an earlier appearance in time) (14). Tolerance and sensitization are both specific for the histocompatibility antigens used to induce the reaction. The MLR, therefore, seemedparticularly suitable to investigate whether specific modifications of cellular immunity are produced in the human gravida with regard to alien fetal histocompatibility antigens. Specifically, we asked if a form of tolerance or modified ‘tolerance might be produced in the mother to protect the fetus from a maternal cell-mediated allograft rejection phenomenon. Moreover, because there is evidence that the human fetus possesseslymphocytes having correlates in vitro of cell-mediated immunity (15-18)) the status of fetal cellular immunoreactivity towards the mother was also studied. The present studies show that human maternal lymphocytes satisfactorily respond in the MLR to related cord blood lymphocytes, and that the responses of cord blood lymphocytes in the MLR to related maternal lymphocytes and unrelated cells are either adequate or depressed. Kinetic studies of lymphocyte reactivity do not suggest the existence of either specific tolerance or specific sensitization between the participants. METHODS Blood from informed volunteers was collected into syringes containing phenolfree heparin (Lipo-Hepin, Riker Labs., Inc., Northridge, CA) at a concentration of 30 U/ml of blood. Blood was similarly collected from an umbilical cord vessel of the placenta immediately after normal term delivery. Lymphocytes were recovered from the blood by centrifugation on a Ficol-Hypaque gradient (19). To test their reactivity, we used the one-way MLR of Bach and Voynow (20) as modified by Hartzman et al. (21). Lymphocytes to be used at various doses as stimulator cells were incubated for 30 min at 37°C with mitomycin (Calbiochem, La Jolla, CA), 35-40 pg/ml of medium 199 containing 20% AB plasma. 2 x IO5 lymphocytes, not treated wi’th mitomycin, were used as the responding cell population in all experiments. Control cultures contained mitomycin-treated autologous lymphocytes as the stimulator cells. Triplicate cul,tures were prepared in medium 199 with Hepes buffer (Grand Island Biological Co., Grand Island, NY) and 20% pooled human AB plasma. Incorporation into DNA of an 1%hr pulse of 2 pCi of pH]thymidine (sp act > 18 Ci/mM) was used to measure response. Preparation of samples for scintillation counting was performed as previously described (22). Variability caused by quenching from tube to tube was less than 1% about the mean. A positive response in the MLR was assigned to cultures with at least twice the counts per minute (cpm) of the control cultures, i.e., stimulation index (SI) Z 2.
334
CARR,
STITES,
24
AND
FUDENBERG
. :’
x x z
20 @4 .
.
MFM
MAM
FMM
FAM
AMM
AFM
FIG. 1. Stimulation indexes (3) of one-way mixed lymphocyte cultures. M, maternal F, cord blood cells; M, mitomycin-treated (stimulator) cells ; A, adult allogeneic cells.
cells;
RESULTS Reactivity of Maternal Lywq%ocytes to Related Co+d Blood Lynzphocytes It was first necessary to determine whether maternal lymphocytes proliferated in culture with mitomycin-treated adult allogeneic lymphocytes. In 18 of 18 instances maternal lymphocytes responded positively (SI > 2) to adult allogeneic lymphocytes (Fig. 1). The mean SI was 13.2 f 9.4 (SD) with a range of 3.4-31.5. The mean cpm of the control cultures was 528 -C 203 (range, 230-1003) cpm. It was next necessary to establish that cord blood lymphocytes could stimulate adult allogeneic cells. In 17 of 18 instances cord blood lymphocytes stimulated adult allogeneic cells (Fig. 1). The mean SI was 14.9 I+ 12.4 (range, 2.2-42.4). The reactivity of maternal lymphocytes and the ability of cord blood lymphocytes Ito act as stimulator cells established, we looked at the responseof maternal lymphocytes to related cord blood lymphocytes. In 17 of 18 cases maternal lymphocytes were stimulated by related cord blood lymphocytes. The mean SI in those instances in which significant stimulation occurred was 8.3 * 6.0 (range, 2.6-18.8) (Fig. 1). As might be expected from genetic considerations, the SI of allogeneic adult lymphocytes responding to cord blood lymphocytes were usually greater than the SI of maternal cells responding to the same cord blood lymphocytes. Specifically, in 2 of 18 instances maternal cells had a larger SI than allogeneic cells, in 2 instances SI were similar, and in 14 SI were less. These results were based on a single dose of stimulator cells. We next varied the dose of stimulator cells to see whether one might obtain different results. In seven instances 2 X lo5 maternal lymphocytes were stimulated both by allogeneic adult cells and by cord blood cells at doses of 106, 2 X 105, 4 X 105, and 8 X lo5 cells, In five pairs, similar doses of both cord and allogeneic adult lymphocytes produced maximal stimulation in maternal lymphocytes ; in two instances maximal stimulation of maternal lymphocytes was seen at different doses of cord and adult cells (Fig. 2). Thus, no clear cut dose-responserelationship was established. We also examined the temporal nature of the response of maternal lymphocytes to related cord blood lymphocytes in four experiments; the general configuration of the time curve was similar whether maternal cells were stimulated by cord
MLR
BETWEEN
MATERNAL
AND
CORD
LYMPHOCYTES
FIG. 2. Reactivity of maternal lymphocytes to mitomycin-treated (0) and to mitomycin-treated related cord blood lymphocytes stimulator cells at 6 days of culture. The SI of adult allogeneic cord blood lymphocytes at a concentration of 2 X lo” was 11.3.
33.5
adult allogeneic lymphocytes ( 0) as a function of dose of cells stimulated by the same
blood cells or by allogeneic cells (Fig. 3). The stimulatory ability of the cord blood cells was also considered. A typical example (ITig. 3) shows that cord I~lootl cells produced parallel stimulation of both maternal and allogeneic cells from the fourth through the seventh day. Incorporation into maternal cells stinlulated by cord blood cells did not peak earlier- than incorporation into those stimulated l)b allogeneic cells within the time stutlietl. The possibility still existed that the pregnancy exerted some sort of subtle motlification on specific antigen recognition by maternal lymphocytes and on the subsequent proliferation pattern after stimulation by related cord blood lymphocytes ; such a modification might not be appreciated when totally allogeneic cells were used as controls. Therefore, the response of maternal lymphocytes and paternal lymphocytes to the common related cord blood lymphocytes was examined. Fiye
FIG. 3. Reactivity of lymphocytes in MLR as a function of time. Shown are maternal lymphocytes stimulated by related cord blood lymphocytes ( l ) and by adult allogeneic lymphocytes as well as maternal lymphocytes stimulated by mitomycin-treated autologous cells (01, (control (A), and adult allogeneic cells stimulated by mitomycin-treated cord blood cells ( n ).
336
CARR,
STITES,
AND
FUDENBERG
TABLE STIMULATORYABILITY
1
OF MITOMYCIN-TREATED
Expt. no.
CORD
Stimulation MFn”
13 23 64 68 75
LYMPHOCYTES
index
PFM
4.7 17.4 4.1 4.6 7.5
a MFn, related maternal lymphocytes paternal lymphocytes stimulated by cord lated by cord blood cells.
BLOOD
AFM
30.0 a.4 3.0 3.0 2.3
16.3 9.8 3.4 3.4 2.9
stimulated by cord blood lymphocytes; PFy, blood cells; AF M, adult allogeneic lymphocytes
related stimu-
sets were available for study, and in one of five stimulation of paternal cells was greater, while in four of five stimulation of maternal cells was greater (Table 1). We also compared the reactivity of maternal lymphocytes to paternal lymphocytes and to adult allogeneic lymphocytes. If modification of maternal lymphocytes was of biologic importance, response to paternal cells should be consistently less than to allogeneic cells. Response to paternal cells was clearly less than response to allogeneic cells only twice (Exps. 13 and 68) ; in the other cases it was either comparable or clearly higher (Table 2). Reactivity
of Cord Blood Lymphocytes
to Related Maternal
Lymphocytes
Human cord blood lymphocytes were reacted against mitomycin-treated adult allogeneic lymphocytes to test response of cord lymphocytes in the MLR. In 14 of 18 cases stimulation resuhed (Fig. 1). The mean stimulation index of these 14 was 6.9 -t- 3.1 (range, 2.2-12.5). In the remaining four cases the mean SI was 1.6 + 0.3 (range, 1.3-1.9). The mean cpm of all the control cultures (cord blood lymphocytes stimulated by mitomycin-treated autologous cells) was 8282 * 5526 (range, 191620,581). It was also necessary to establish #the stimulatory ability of mitomycin-treated maternal lymphocytes. In 17 of 18 cases maternal lympocytes TABLE
2
STIMULATION OF MATERNAL LYMPHOCYTES BY MITOMYCIN-TREATED PATERNAL ANDADULTALLOGENEICLYMPHOCYTES Stimulation
Expt. no.
MPn” 13 23 64 68 75 0 MPn, allogeneic
maternal cells.
cell stimulated
MAn
11.3 4.8 7.7 3.1 2.6 by paternal
cells;
index
13.2 2.8 7.8 7.2 3.4 MAn,
maternal
cells
stimulated
by adult
MLR
BETWEEN
MATERNAL
AND
CORD
4
-
337
LYMPHOCYTES
8
MITOMYCIN TREATED LYMPHOCYTE: ~viC-’
FIG.
of cord blood lymphocytes by mitomycin-treated related maternal lymphoand by mitomycin-treated adult allogeneic lymphocytes ( 0) as a function of
4. Stimulation
cytes (0)
stimulatory dose. The SI of adult allogeneic at a concentration of 2 X 10’ was 20.7.
cells
stimulated
by the same
maternal
lymphocytes
stimulated adult allogeneic cells (Fig. 1). The mean SI of the 17 was 10.1 * 8.6 (range, 2.0-23.8). The mean cpm in control cultures was 1666 -C 1855 (range, 267-6591) . We then #tested response of cord blood lymphocytes to related maternal
lymphocytes. In eight of eighteen instances fetal cells were stimulated by their mothers’ lymphocytes (Fig. 1). The mean SI of the eight was 4.6 -t 1.7 (range, 3.3-5.4). In the remaining 10 cases,the mean SI, 1.3 -t- 0.3 (range, 0.7-l.@, was within nonstimulatory limits. In these experiments the ratio of responding to stimulator cells was 1: 1. rn order to further examine the apparent depressed reactivity of some fetal lymphocytes #tomaternal cells, doses of mitomycin-treated maternal cells were varied. In six of eight instances, when the ratio of stimulator maternal cells was varied (2: 1, 1: 1, 1:2, 1 : 4), stimulation was detectable; in three of these the SI had been below 2 at the 1: 1 cell ratio. Figure 4 depicts an example wherein maternal cells stimulated cord blood cells at the lowest cell dose but not at the higher cell doses, whereas the SI of cord blood cells stimulated by allogeneic cells was still rising at the highest tested dose of stimulatory cells. The time course of the reaction of cord blood lymphocytes to mitomycin-treated maternal lymphocytes was examined for evidence of kinetic differences in four different experiments (Fig. 5). The shape of the temporal response curve was parallel both to that of cord blood cells stimulated by allogeneic cells and to that of allogeneic cells stimulated by maternal cells. They all appeared to peak at about the same time. Finally,
we looked
at the response
of cord
blood
lymphocytes
to maternal
and
paternal lymphocytes. If tolerance of the fetus to its mother is present, one would expect a consistent depression of reactivity of cord blood cells stimulated by maternal
cells as compared
to cord blood
cells stimulated
by paternal
cells
(Table
3).
In only one (Exp. 23) of the five casesstudied was stimulation of cord blood cells by paternal cells greater. In another case both were comparably stimulated. In the remaining cases, significant stimulation of cord cells did not occur with either cell population.
338
CARR,
STITES,
AND
FUDENBERG
FIG. 5. Reactivity of cord blood lymphocytes as a function of time. l , Cord stimulated by mitomycin-treated related maternal lymphocytes; 0, cord blood cells by mitomycin-treated adult allogeneic lymphocytes ; cord blood cells stimulated by cells (control) (A). Shown also are stimulation of adult allogeneic lymphocytes by treated maternal lymphocytes (w) and by autologous cells (control) (0).
blood cells stimulated autologous mitomycin-
DISCUSSION Maternal lymphocytes at the conclusion of human pregnancy can be stimulated in the MLR by both related cord blood lymphocytes and adult allogeneic lymphocytes. The temporal kinetics of these reactions have the appearance of the usual allogeneic lymphocyte response, and do not suggest ‘that any specific tolerance or specific sensitization exists toward fetal histocompatibility antigens. Although stimulation was less with related cord blood lymphocytes, this was not unexpected since at least one-half of their histocompatibility antigens are the same, whereas with allogeneic cells all the histocompatibility antigens very likely are different and more stimulation would be expected (23). The data also clearly show that pregnant women’s lymphocytes react to their husbands’ lymphocytes consistently and at a level comparable to their reaction to adult allogeneic lymphocytes. This contradicts a prior report which may have been influenced by the autologous plasma that was TABLE STIMULATION
3
OF CORD BLOOD LYMPHOCYTES BY MITOMYCIN-TKEATED RELATED AND PATERNAL, AND ADULT ALLOGENEIC LYMPHOCYTES
Stimulation
Expt.
MATERNAL
index
110. FM
13 23 64 68 75
Ma
3.7 1.2 1.0 1.3 1.2
FPaa
3.8 4.4 1.5 1.4 1.8
FAM
6.1 5.6 1.8 1.9 2.2
0 FMn, cord blood cells stimulated by related maternal cells; FPM, cord blood cells stimulated by related paternal cells; FAM, cord blood cells stimulated by adult allogeneic cells.
MLR
BETWEEK
MATERNAL
AND
CORD
LYMI’JTOCYT1~S
330
employed (24). Our findings provide evidence that intrinsic alterations of maternal lymphocytes with respect to known fetal histocompatibility antigens are not a regular occurrence in the human pregnancy and, therefore, are not essential to fetal survival. On the other hand, when cord blood lymphocytes were culmtured with related maternal lymphocytes stimulation occurred in only about half the cases, although the quantitative experiments lead us to believe this percentage of nonreactivity is considerably lower. But cord lymphocytes also seemed to be less reactive against adult allogeneic cells. Where stimulation by maternal and adult unrelated cells was seen, the time course of the reaction did not show any alteration suggestive of specific tolerance or sensitization. Cord blood lymphocytes have high cpm in control cultures; this coultl mask :I significant response after calculation of stimulation ratios. After allowances are made for these artificially low SI, it still appears that in many instances human term cord blood lymphocytes are nonspecifically depressed in ‘their ability to recognize and proliferate in vitro in response to allogeneic cells. However, since many cord lymphocytes seem to react as expected in the MI,R, and there appears to be no difference in the outcome of the pregnancy, it is doubtful if the occasional deproud response is biologically meaningful in terms of overall fetal survival. The finding of an intact mixed lymphocyte reaction in gravida lymphocytes is additional evidence of the cellular immunocompctence of the maternal organism. Earlier, TVC found that the general cellular immune state. as measured by Iymphoc!.tr stimulation over a dose range of phytohemagglutiIlil1. was not depressed, and was probably in a state of low level antigenic stimulation (25). The data here suggest that this stimulation of maternal lymphoc\.tes is not due to sensitization by fetal histocompatibility antigens. Stimulation oi cord blood Iymphocyt~s with phytohemagglutinin also revealed no impairment in reactivity and suggested low level antigenic stimulation (26). The data here likewise suggest that this stimulation is not due to sensitization by gravida histocompatibility antigens. Noreovcr. since cord blood lymphocytes stimulated by phytohemagglutinin seemed to havr an intact proliferative function, the reduced MLR response found in some cord lymphocytes might result from a nonspecifically depressed recognition function for allogeneic cells. Since we have found no apparent alteration in the gravida’s cellular immune system toward the histocompatibility antigens of her fetus, the reportrd cellular interchange between organisms (1-S) appears tlot to 1~ of sufficient magnitude in terms of histocompatibility antigens to affect the relationship. Specificall!.. this suggests that the numbers of fetal lymphocytes crossing to the mother are cluanticatively insignificant, and that chorionic syncytial trophoblasts, which are fetally derived and deposited daily in large amounts into the mother’s vascular svstrm (27, 28), contain no significant amount of inz~zzzl~?ogcGc histocompatibility antigens. The suggestion that the termination of pregnancy tnay represent au allograft rejection phenomenon (29) is not entirely contradicted by our data. The data would argue that such a phenomenon is not dependent upon histocompatibility antigens common to placenta and fetus, since no sensitization was demonstrated by these tests. However, a specific placental antigen not found in other fetal tissues has been postulated. Such an antigen could provoke a maternal rejection phenome-
340
CARR,
STITES,
AND
FUDENBERG
non against the placenta, thus precipitating labor and delivery, without necessarily modifying maternal-fetal lymphocyte relationships. Our results are compatible with such a hypothesis. Finally, our studies, in agreement with other work (30), indicate that survival of the pregnancy is not dependent upon elimination of those lymphocyte clones in the gravida that recognize the alien histocompatibility antigens of the term fetus. By the same token, our studies show that the term fetus possesses lymphocyte clones capable of recognizing and responding Ito those maternal histocompatibility antigens that are foreign to it, and that the cord lymphocytes possess another faculty reflective of functioning cellular immunity, in addition to phytohemagglutininresponsiveness ( 15-H). Overall, our findings suggest that the success of the human pregnancy in terms of cellular immunity does not depend upon maternal-fetal tolerance. Instead, it is more likely to depend either upon humoral modification (31) of the antigenicity of the syncytial trophoblast, the intervening ‘tissue between the mother and fetus, or upon the absence of histocompatibility antigens on this tissue (32). ACKNOWLEDGMENTS The competent technical assistance of Janice Perlman knowledged.
and Joanne Rush is gratefully
ac-
REFERENCES 1. Desai, R. G., and Creger, W. P., Blood 21, 665, 1963. 2. Kadowski, J., Thompson, R. I., Zuelzer, W. W., Woolley, P. V., Jr., Brough, A. J., and Gruber, D., Lancet 2, 1152, 1965. 3. Bellanti, J. A., and Jackson, A. L., J. Pediut. 71, 783, 1967. 4. Githens, J. H., Muschenheim, F., Fulginiti, V. A., Robinson, A., and Kay, H. E. M., I. Pediat. 75, 87, 1969. 5. Walknowska, J., Come, F. A., and Grumbach, M. M., Lancet 1, 1119, 1969. 6. Leiken, S., and Oppenheim, J. J., Lancet 2, 876, 1971. 7. Schriider, J., and de la Chapelle, A., Blood 39, 153, 1972. 8. Whang-Peng, J., Leiken, S., Harris, C., Lee, E., and Sites, J., Proc. Sot. Exp. Biol. Med. 142, 50, 1973. 9. Cohen, F., and Zuelzer, W. W., Yox. Sasg. 9, 75, 1964. 10. ‘Fudenberg, H. H., and Fudenberg, B. R., Science 145, 170, 1964. 11. Rocklin, R. E., Zuckerman, J. E., Alpert, E., and David, J. R., Nature (London) 241, 130, 1973. 12. Vyas, G. N., Levin, A. S., and Fudenberg, H. H., Nature (London) 225, 275, 1970. 13. Bach, F. H., and Amos, D. B., Science 156, 1506, 1967. 14. Wilson, D. B., Silvers, W. K., and Nowell, P. C., J. Exp. Med. 126, 655, 1967. 15. Papiernik, M., Blood 36, 470, 1970. 16. Kay, H. E. M., Doe, J., and Hockley, A., Immunology 18, 393, 1970. 17. August, C. S., Berkel, A. I., Driscoll, S., and Merler, E., Pediat. Res. 5, 539, 1971. 18. Carr, M. C., Stites, D. P., and Fudenberg, H. H., Nature New Biol. 241, 279, 1973. 19. Boyurn, A., SC&. J. c&s. Lab. Invesst. Suppl. 97, 9, 1968. 20. Bach, F. H., and Voynow, N. K., Science 153, 545, 1966. 21. Hartzman, R. J., Segall, M., Bach, M. L., and Bach, F. H., Transplanfatiolz 11, 268, 1971. 22. Stites, D. P., Carr, M. C., and Fudenberg, H. H., Proc. Nat. Acad. Sci. USA 69, 1440, 1972. 23. Albertini, R. J., and Bach, F. H., J. Exp. Med. 128, 639, 1968. 24. Lewis, J., Jr., Whang, J., Nagel, B., Oppenheim, J. J., and Perry, S., Amer. J. Obstet. Gynecol. 96, 287, 1966. 25. Carr, M. C., Stites, D. P., and Fudenberg, H. H., Cell. Zmmunol. 8, 448, 1973.
MLR
BETWEEN
MATERNAL
26. Carr, M. C., Stites, D. P., and Fudenberg, 27. Thomas, L., Douglas, G. W., and Carr,
AND
CORD
H. H., Ceil. M. C., l‘rans.
341
LYMPHOCYTES
Immunol.
5, 21, 1972.
Assoc. Amer.
f’hysiciczn.r 72. 140,
1959. 28. Carr, M. C., Calif. Med. 107, 338, 1967. 29. Thomas, L., Ir, “Cellular and Humoral Aspects of the Hypersensitive States” Lawrence, Ed.), pp. 529-532. Paul B. Hoeber, Xew York, 1959. 30. Bonnard, G. D., and Lemos, L., Trc~nsplant. Proc. 4, 177, 1972. 31. Hellstrijm, K. E., Hellstram, I., and Brawn, J., Nature (hzdon) 224, 914, 1969. 32. Simmons, R. L., and Russell, P. S., Amer. J. Oh.\-trot. G.~nrcol. 85, 583, 1963.
iFI.
5.
Cellular
IMMUNOLOGY
11,
Immune III. Mixed
MARTIN Departments
332-341
Aspects
(1974)
of the Human
Fetal-Maternal
Relationship
Lymphocyte Reactivity Between Related Maternal and Cord Blood Lymphocytes 1
C. CARR, DANIEL
P. STITES,~ AND H. HUGH
FUDENBERG
of Obstetrics and Gynecology and Medicine, School of Medicine, University of California, San Francisco, California 94143 Received
July
30 1973
Gravida and related cord blood lymphocytes were studied in one-way mixed lymphocyte culture to find out if tolerance or sensitization existed between these organisms. Maternal lymphocytes were consistently stimulated by both related cord blood and adult allogeneic lymphocytes, and the kinetics of the reactions did not suggest that any specific tolerance or sensitization existed toward fetal histocompatibility antigens. Cord blood lymphocytes were stimulated by related maternal lymphocytes in approximately one-half the tests, and also seemed less reactive toward adu’t allogeneic cells. However, the temporal kinetics of the reaction did not suggest any sersitization or tolerance toward maternal lymphocytes. The findings suggest that the success of the human pregnancy in terms of cell-mediated immunity does not depend upon intrinsic cellular maternal-fetal tolerance.
INTRODUCTION Recent evidence suggests that there is a substantial exchange of antigenic material between mother and fetus during the normal human pregnancy (1-S). Fetal cellular and particulate matter entering ‘the maternal vascular system during pregnancy can result in sensitization (9-11). Conversely, maternal antigenic material may enter the fetal circulation and produce sensitization of #thefetus (12). Despite this reciprocal immunization, pregnancy usually progressesundeterred except when isoimmunization to erythrocyte antigens intervenes. What makes this intriguing is that the human pregnancy consists of two beings coexisting successfully, each invariably with distinctive histocompatibility antigens. Our best knowledge tells us that the fetus, analogous to an allogeneic graft, probably should be rejected as a result of activity of the cellular immune mechanisms available to the maternal host. Why does the fetus survive under such precarious conditions? Crucial parts of this question are whether mutual antigen exchange results in immunity or specific tolerance between the histocompatibility antigens of the participants, and whether Ithere is an altered state of cellular immunity of the mother toward the fetus. 1 Supported in part by United States Public Health HD-05894, and by training grant HL-05677. 2Reprint requests should be addressed to Dr. Stites of Medicine, University of California, San Francisco, 332 Copyri ht 0 1974 by Academic Press, All rig fl ts of reproduction in any form
Inc. reserved.
Service
research
at the Department California 94143.
grants
HD-03939
of Medicine,
and School
MLR
BETWEEN
MATERNAL
AND
CORD
LYMPHOCYTES
3.33
One way of examining these questions is to study the reactivity in vitro of the participants’ lymphocytes toward each other in a one-way mixed lymphocyte reaction (MLR). In the MLR, recognition of histocompatibility differences triggers blast transformation of the responding lymphocytes (13). In at least one animal system, the kinetics of this recognition and the subsequent proliferative response have been predictably altered by previously exposing ‘the animal to appropriate doses of the specific histocompatibility antigens, producing either tolerance (no lymphocyte proliferation results) or sensitization (the peak response is characterized by lolver isotope incorporation and an earlier appearance in time) (14). Tolerance and sensitization are both specific for the histocompatibility antigens used to induce the reaction. The MLR, therefore, seemedparticularly suitable to investigate whether specific modifications of cellular immunity are produced in the human gravida with regard to alien fetal histocompatibility antigens. Specifically, we asked if a form of tolerance or modified ‘tolerance might be produced in the mother to protect the fetus from a maternal cell-mediated allograft rejection phenomenon. Moreover, because there is evidence that the human fetus possesseslymphocytes having correlates in vitro of cell-mediated immunity (15-18)) the status of fetal cellular immunoreactivity towards the mother was also studied. The present studies show that human maternal lymphocytes satisfactorily respond in the MLR to related cord blood lymphocytes, and that the responses of cord blood lymphocytes in the MLR to related maternal lymphocytes and unrelated cells are either adequate or depressed. Kinetic studies of lymphocyte reactivity do not suggest the existence of either specific tolerance or specific sensitization between the participants. METHODS Blood from informed volunteers was collected into syringes containing phenolfree heparin (Lipo-Hepin, Riker Labs., Inc., Northridge, CA) at a concentration of 30 U/ml of blood. Blood was similarly collected from an umbilical cord vessel of the placenta immediately after normal term delivery. Lymphocytes were recovered from the blood by centrifugation on a Ficol-Hypaque gradient (19). To test their reactivity, we used the one-way MLR of Bach and Voynow (20) as modified by Hartzman et al. (21). Lymphocytes to be used at various doses as stimulator cells were incubated for 30 min at 37°C with mitomycin (Calbiochem, La Jolla, CA), 35-40 pg/ml of medium 199 containing 20% AB plasma. 2 x IO5 lymphocytes, not treated wi’th mitomycin, were used as the responding cell population in all experiments. Control cultures contained mitomycin-treated autologous lymphocytes as the stimulator cells. Triplicate cul,tures were prepared in medium 199 with Hepes buffer (Grand Island Biological Co., Grand Island, NY) and 20% pooled human AB plasma. Incorporation into DNA of an 1%hr pulse of 2 pCi of pH]thymidine (sp act > 18 Ci/mM) was used to measure response. Preparation of samples for scintillation counting was performed as previously described (22). Variability caused by quenching from tube to tube was less than 1% about the mean. A positive response in the MLR was assigned to cultures with at least twice the counts per minute (cpm) of the control cultures, i.e., stimulation index (SI) Z 2.
334
CARR,
STITES,
24
AND
FUDENBERG
. :’
x x z
20 @4 .
.
MFM
MAM
FMM
FAM
AMM
AFM
FIG. 1. Stimulation indexes (3) of one-way mixed lymphocyte cultures. M, maternal F, cord blood cells; M, mitomycin-treated (stimulator) cells ; A, adult allogeneic cells.
cells;
RESULTS Reactivity of Maternal Lywq%ocytes to Related Co+d Blood Lynzphocytes It was first necessary to determine whether maternal lymphocytes proliferated in culture with mitomycin-treated adult allogeneic lymphocytes. In 18 of 18 instances maternal lymphocytes responded positively (SI > 2) to adult allogeneic lymphocytes (Fig. 1). The mean SI was 13.2 f 9.4 (SD) with a range of 3.4-31.5. The mean cpm of the control cultures was 528 -C 203 (range, 230-1003) cpm. It was next necessary to establish that cord blood lymphocytes could stimulate adult allogeneic cells. In 17 of 18 instances cord blood lymphocytes stimulated adult allogeneic cells (Fig. 1). The mean SI was 14.9 I+ 12.4 (range, 2.2-42.4). The reactivity of maternal lymphocytes and the ability of cord blood lymphocytes Ito act as stimulator cells established, we looked at the responseof maternal lymphocytes to related cord blood lymphocytes. In 17 of 18 cases maternal lymphocytes were stimulated by related cord blood lymphocytes. The mean SI in those instances in which significant stimulation occurred was 8.3 * 6.0 (range, 2.6-18.8) (Fig. 1). As might be expected from genetic considerations, the SI of allogeneic adult lymphocytes responding to cord blood lymphocytes were usually greater than the SI of maternal cells responding to the same cord blood lymphocytes. Specifically, in 2 of 18 instances maternal cells had a larger SI than allogeneic cells, in 2 instances SI were similar, and in 14 SI were less. These results were based on a single dose of stimulator cells. We next varied the dose of stimulator cells to see whether one might obtain different results. In seven instances 2 X lo5 maternal lymphocytes were stimulated both by allogeneic adult cells and by cord blood cells at doses of 106, 2 X 105, 4 X 105, and 8 X lo5 cells, In five pairs, similar doses of both cord and allogeneic adult lymphocytes produced maximal stimulation in maternal lymphocytes ; in two instances maximal stimulation of maternal lymphocytes was seen at different doses of cord and adult cells (Fig. 2). Thus, no clear cut dose-responserelationship was established. We also examined the temporal nature of the response of maternal lymphocytes to related cord blood lymphocytes in four experiments; the general configuration of the time curve was similar whether maternal cells were stimulated by cord
MLR
BETWEEN
MATERNAL
AND
CORD
LYMPHOCYTES
FIG. 2. Reactivity of maternal lymphocytes to mitomycin-treated (0) and to mitomycin-treated related cord blood lymphocytes stimulator cells at 6 days of culture. The SI of adult allogeneic cord blood lymphocytes at a concentration of 2 X lo” was 11.3.
33.5
adult allogeneic lymphocytes ( 0) as a function of dose of cells stimulated by the same
blood cells or by allogeneic cells (Fig. 3). The stimulatory ability of the cord blood cells was also considered. A typical example (ITig. 3) shows that cord I~lootl cells produced parallel stimulation of both maternal and allogeneic cells from the fourth through the seventh day. Incorporation into maternal cells stinlulated by cord blood cells did not peak earlier- than incorporation into those stimulated l)b allogeneic cells within the time stutlietl. The possibility still existed that the pregnancy exerted some sort of subtle motlification on specific antigen recognition by maternal lymphocytes and on the subsequent proliferation pattern after stimulation by related cord blood lymphocytes ; such a modification might not be appreciated when totally allogeneic cells were used as controls. Therefore, the response of maternal lymphocytes and paternal lymphocytes to the common related cord blood lymphocytes was examined. Fiye
FIG. 3. Reactivity of lymphocytes in MLR as a function of time. Shown are maternal lymphocytes stimulated by related cord blood lymphocytes ( l ) and by adult allogeneic lymphocytes as well as maternal lymphocytes stimulated by mitomycin-treated autologous cells (01, (control (A), and adult allogeneic cells stimulated by mitomycin-treated cord blood cells ( n ).
336
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TABLE STIMULATORYABILITY
1
OF MITOMYCIN-TREATED
Expt. no.
CORD
Stimulation MFn”
13 23 64 68 75
LYMPHOCYTES
index
PFM
4.7 17.4 4.1 4.6 7.5
a MFn, related maternal lymphocytes paternal lymphocytes stimulated by cord lated by cord blood cells.
BLOOD
AFM
30.0 a.4 3.0 3.0 2.3
16.3 9.8 3.4 3.4 2.9
stimulated by cord blood lymphocytes; PFy, blood cells; AF M, adult allogeneic lymphocytes
related stimu-
sets were available for study, and in one of five stimulation of paternal cells was greater, while in four of five stimulation of maternal cells was greater (Table 1). We also compared the reactivity of maternal lymphocytes to paternal lymphocytes and to adult allogeneic lymphocytes. If modification of maternal lymphocytes was of biologic importance, response to paternal cells should be consistently less than to allogeneic cells. Response to paternal cells was clearly less than response to allogeneic cells only twice (Exps. 13 and 68) ; in the other cases it was either comparable or clearly higher (Table 2). Reactivity
of Cord Blood Lymphocytes
to Related Maternal
Lymphocytes
Human cord blood lymphocytes were reacted against mitomycin-treated adult allogeneic lymphocytes to test response of cord lymphocytes in the MLR. In 14 of 18 cases stimulation resuhed (Fig. 1). The mean stimulation index of these 14 was 6.9 -t- 3.1 (range, 2.2-12.5). In the remaining four cases the mean SI was 1.6 + 0.3 (range, 1.3-1.9). The mean cpm of all the control cultures (cord blood lymphocytes stimulated by mitomycin-treated autologous cells) was 8282 * 5526 (range, 191620,581). It was also necessary to establish #the stimulatory ability of mitomycin-treated maternal lymphocytes. In 17 of 18 cases maternal lympocytes TABLE
2
STIMULATION OF MATERNAL LYMPHOCYTES BY MITOMYCIN-TREATED PATERNAL ANDADULTALLOGENEICLYMPHOCYTES Stimulation
Expt. no.
MPn” 13 23 64 68 75 0 MPn, allogeneic
maternal cells.
cell stimulated
MAn
11.3 4.8 7.7 3.1 2.6 by paternal
cells;
index
13.2 2.8 7.8 7.2 3.4 MAn,
maternal
cells
stimulated
by adult
MLR
BETWEEN
MATERNAL
AND
CORD
4
-
337
LYMPHOCYTES
8
MITOMYCIN TREATED LYMPHOCYTE: ~viC-’
FIG.
of cord blood lymphocytes by mitomycin-treated related maternal lymphoand by mitomycin-treated adult allogeneic lymphocytes ( 0) as a function of
4. Stimulation
cytes (0)
stimulatory dose. The SI of adult allogeneic at a concentration of 2 X 10’ was 20.7.
cells
stimulated
by the same
maternal
lymphocytes
stimulated adult allogeneic cells (Fig. 1). The mean SI of the 17 was 10.1 * 8.6 (range, 2.0-23.8). The mean cpm in control cultures was 1666 -C 1855 (range, 267-6591) . We then #tested response of cord blood lymphocytes to related maternal
lymphocytes. In eight of eighteen instances fetal cells were stimulated by their mothers’ lymphocytes (Fig. 1). The mean SI of the eight was 4.6 -t 1.7 (range, 3.3-5.4). In the remaining 10 cases,the mean SI, 1.3 -t- 0.3 (range, 0.7-l.@, was within nonstimulatory limits. In these experiments the ratio of responding to stimulator cells was 1: 1. rn order to further examine the apparent depressed reactivity of some fetal lymphocytes #tomaternal cells, doses of mitomycin-treated maternal cells were varied. In six of eight instances, when the ratio of stimulator maternal cells was varied (2: 1, 1: 1, 1:2, 1 : 4), stimulation was detectable; in three of these the SI had been below 2 at the 1: 1 cell ratio. Figure 4 depicts an example wherein maternal cells stimulated cord blood cells at the lowest cell dose but not at the higher cell doses, whereas the SI of cord blood cells stimulated by allogeneic cells was still rising at the highest tested dose of stimulatory cells. The time course of the reaction of cord blood lymphocytes to mitomycin-treated maternal lymphocytes was examined for evidence of kinetic differences in four different experiments (Fig. 5). The shape of the temporal response curve was parallel both to that of cord blood cells stimulated by allogeneic cells and to that of allogeneic cells stimulated by maternal cells. They all appeared to peak at about the same time. Finally,
we looked
at the response
of cord
blood
lymphocytes
to maternal
and
paternal lymphocytes. If tolerance of the fetus to its mother is present, one would expect a consistent depression of reactivity of cord blood cells stimulated by maternal
cells as compared
to cord blood
cells stimulated
by paternal
cells
(Table
3).
In only one (Exp. 23) of the five casesstudied was stimulation of cord blood cells by paternal cells greater. In another case both were comparably stimulated. In the remaining cases, significant stimulation of cord cells did not occur with either cell population.
338
CARR,
STITES,
AND
FUDENBERG
FIG. 5. Reactivity of cord blood lymphocytes as a function of time. l , Cord stimulated by mitomycin-treated related maternal lymphocytes; 0, cord blood cells by mitomycin-treated adult allogeneic lymphocytes ; cord blood cells stimulated by cells (control) (A). Shown also are stimulation of adult allogeneic lymphocytes by treated maternal lymphocytes (w) and by autologous cells (control) (0).
blood cells stimulated autologous mitomycin-
DISCUSSION Maternal lymphocytes at the conclusion of human pregnancy can be stimulated in the MLR by both related cord blood lymphocytes and adult allogeneic lymphocytes. The temporal kinetics of these reactions have the appearance of the usual allogeneic lymphocyte response, and do not suggest ‘that any specific tolerance or specific sensitization exists toward fetal histocompatibility antigens. Although stimulation was less with related cord blood lymphocytes, this was not unexpected since at least one-half of their histocompatibility antigens are the same, whereas with allogeneic cells all the histocompatibility antigens very likely are different and more stimulation would be expected (23). The data also clearly show that pregnant women’s lymphocytes react to their husbands’ lymphocytes consistently and at a level comparable to their reaction to adult allogeneic lymphocytes. This contradicts a prior report which may have been influenced by the autologous plasma that was TABLE STIMULATION
3
OF CORD BLOOD LYMPHOCYTES BY MITOMYCIN-TKEATED RELATED AND PATERNAL, AND ADULT ALLOGENEIC LYMPHOCYTES
Stimulation
Expt.
MATERNAL
index
110. FM
13 23 64 68 75
Ma
3.7 1.2 1.0 1.3 1.2
FPaa
3.8 4.4 1.5 1.4 1.8
FAM
6.1 5.6 1.8 1.9 2.2
0 FMn, cord blood cells stimulated by related maternal cells; FPM, cord blood cells stimulated by related paternal cells; FAM, cord blood cells stimulated by adult allogeneic cells.
MLR
BETWEEK
MATERNAL
AND
CORD
LYMI’JTOCYT1~S
330
employed (24). Our findings provide evidence that intrinsic alterations of maternal lymphocytes with respect to known fetal histocompatibility antigens are not a regular occurrence in the human pregnancy and, therefore, are not essential to fetal survival. On the other hand, when cord blood lymphocytes were culmtured with related maternal lymphocytes stimulation occurred in only about half the cases, although the quantitative experiments lead us to believe this percentage of nonreactivity is considerably lower. But cord lymphocytes also seemed to be less reactive against adult allogeneic cells. Where stimulation by maternal and adult unrelated cells was seen, the time course of the reaction did not show any alteration suggestive of specific tolerance or sensitization. Cord blood lymphocytes have high cpm in control cultures; this coultl mask :I significant response after calculation of stimulation ratios. After allowances are made for these artificially low SI, it still appears that in many instances human term cord blood lymphocytes are nonspecifically depressed in ‘their ability to recognize and proliferate in vitro in response to allogeneic cells. However, since many cord lymphocytes seem to react as expected in the MI,R, and there appears to be no difference in the outcome of the pregnancy, it is doubtful if the occasional deproud response is biologically meaningful in terms of overall fetal survival. The finding of an intact mixed lymphocyte reaction in gravida lymphocytes is additional evidence of the cellular immunocompctence of the maternal organism. Earlier, TVC found that the general cellular immune state. as measured by Iymphoc!.tr stimulation over a dose range of phytohemagglutiIlil1. was not depressed, and was probably in a state of low level antigenic stimulation (25). The data here suggest that this stimulation of maternal lymphoc\.tes is not due to sensitization by fetal histocompatibility antigens. Stimulation oi cord blood Iymphocyt~s with phytohemagglutinin also revealed no impairment in reactivity and suggested low level antigenic stimulation (26). The data here likewise suggest that this stimulation is not due to sensitization by gravida histocompatibility antigens. Noreovcr. since cord blood lymphocytes stimulated by phytohemagglutinin seemed to havr an intact proliferative function, the reduced MLR response found in some cord lymphocytes might result from a nonspecifically depressed recognition function for allogeneic cells. Since we have found no apparent alteration in the gravida’s cellular immune system toward the histocompatibility antigens of her fetus, the reportrd cellular interchange between organisms (1-S) appears tlot to 1~ of sufficient magnitude in terms of histocompatibility antigens to affect the relationship. Specificall!.. this suggests that the numbers of fetal lymphocytes crossing to the mother are cluanticatively insignificant, and that chorionic syncytial trophoblasts, which are fetally derived and deposited daily in large amounts into the mother’s vascular svstrm (27, 28), contain no significant amount of inz~zzzl~?ogcGc histocompatibility antigens. The suggestion that the termination of pregnancy tnay represent au allograft rejection phenomenon (29) is not entirely contradicted by our data. The data would argue that such a phenomenon is not dependent upon histocompatibility antigens common to placenta and fetus, since no sensitization was demonstrated by these tests. However, a specific placental antigen not found in other fetal tissues has been postulated. Such an antigen could provoke a maternal rejection phenome-
340
CARR,
STITES,
AND
FUDENBERG
non against the placenta, thus precipitating labor and delivery, without necessarily modifying maternal-fetal lymphocyte relationships. Our results are compatible with such a hypothesis. Finally, our studies, in agreement with other work (30), indicate that survival of the pregnancy is not dependent upon elimination of those lymphocyte clones in the gravida that recognize the alien histocompatibility antigens of the term fetus. By the same token, our studies show that the term fetus possesses lymphocyte clones capable of recognizing and responding Ito those maternal histocompatibility antigens that are foreign to it, and that the cord lymphocytes possess another faculty reflective of functioning cellular immunity, in addition to phytohemagglutininresponsiveness ( 15-H). Overall, our findings suggest that the success of the human pregnancy in terms of cellular immunity does not depend upon maternal-fetal tolerance. Instead, it is more likely to depend either upon humoral modification (31) of the antigenicity of the syncytial trophoblast, the intervening ‘tissue between the mother and fetus, or upon the absence of histocompatibility antigens on this tissue (32). ACKNOWLEDGMENTS The competent technical assistance of Janice Perlman knowledged.
and Joanne Rush is gratefully
ac-
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MLR
BETWEEN
MATERNAL
26. Carr, M. C., Stites, D. P., and Fudenberg, 27. Thomas, L., Douglas, G. W., and Carr,
AND
CORD
H. H., Ceil. M. C., l‘rans.
341
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1959. 28. Carr, M. C., Calif. Med. 107, 338, 1967. 29. Thomas, L., Ir, “Cellular and Humoral Aspects of the Hypersensitive States” Lawrence, Ed.), pp. 529-532. Paul B. Hoeber, Xew York, 1959. 30. Bonnard, G. D., and Lemos, L., Trc~nsplant. Proc. 4, 177, 1972. 31. Hellstrijm, K. E., Hellstram, I., and Brawn, J., Nature (hzdon) 224, 914, 1969. 32. Simmons, R. L., and Russell, P. S., Amer. J. Oh.\-trot. G.~nrcol. 85, 583, 1963.
iFI.
5.