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Modulation of Allospecific CTL Responses
During Pregnancy in Equids: An
Immunological Barrier to Interspecies
Matings?
Jessica M. Baker, Anona I. Bamford and D. F. Antczak
J Immunol 1999; 162:4496-4501; ;
http://www.jimmunol.org/content/162/8/4496
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Copyright © 1999 by The American Association of
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References
Modulation of Allospecific CTL Responses During Pregnancy
in Equids: An Immunological Barrier to Interspecies
Matings?1
Jessica M. Baker,2 Anona I. Bamford,3 and D. F. Antczak
S
ince Peter Medawar (1) first posed the problem of the fetus-as-allograft nearly 50 years ago, “Nature’s transplant”
has provided a rich environment for theory and experimentation at the interfaces between mother and fetus, and between
immunology and reproduction. Three recent studies in mice have
provided evidence that maternal CTL responses to paternal MHC
class I Ags are disrupted by pregnancy (2–5). In the case of MHC
differences between mother and father, there appears to be downregulation of Ag-specific receptors and coreceptors on T cells,
which is reversible after pregnancy is completed. This may account for paternal Ag-specific tolerance to MHC alloantigens during pregnancy (2, 3). However, a study using transgenic mice with
TCRs specific for the H-Y minor histocompatibility Ag found evidence for two distinct types of immune regulation induced by
pregnancy. Some H-Y-specific T cells were specifically deleted,
while others became anergic, but without down-regulation of their
receptors (4). In the case of H-Y Ag, the pregnancy-induced tolerance lasted after pregnancy was completed, and may be persistent. Together, these studies provide evidence for multiple, Agspecific mechanisms that protect the fetal allograft from rejection.
In contrast, other research has demonstrated a generalized, nonspecific shift away from cell-mediated toward humoral immunity
during normal pregnancy (6 –9). Currently, there is active debate
over the relative importance of paternal Ag-specific tolerance
James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell
University, Ithaca, NY 14853
Received for publication October 9, 1998. Accepted for publication January 28, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by research Grants NICHD-15799 and NICHD-80436
from the National Institutes of Health and by the Dorothy Russell Havemeyer Foundation. J.M.B. was supported by a U.S. Department of Agriculture Training Grant in
Biotechnology, No. 96-38420-3061.
2
Address correspondence and reprint requests to Dr. Jessica M. Baker, James A.
Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. E-mail address: [email protected]
3
Current address: Bio-Rad Laboratories, Hertfordshire, United Kingdom.
Copyright © 1999 by The American Association of Immunologists
and/or the so-called Th13 Th2 shift in immune reactivity during
pregnancy.
The horse is a good species for studies of changes in immune
status during pregnancy by virtue of two striking phenomena observed after normal, histoincompatible matings. First, both primiparous and multiparous mares make strong paternal-specific cytotoxic alloantibody responses to paternal MHC class I Ags by day
60 of the 336-day horse gestation period (10, 11). Most species do
not make reproducible Ab responses to paternal alloantigens, and
in those species where pregnancy-induced alloantibody responses
have been detected, the responses are not consistent, and the Abs
do not arise as early relative to total gestation length as seen in the
horse (12–16). A recent report indicates that there is a partial deletion of B lymphocytes specific for paternal histocompatibility
Ags in pregnant mice, which may explain why alloantibody responses are rare in most species (17). The strong alloantibody responses observed in horses may be a reflection of differences in
placentation between rodents and equids. Rodents have an invasive, hemochorial placenta, whereas horses have a noninvasive
epitheliochorial placenta, with the exception of the endometrial
cups described below (18).
A second characteristic of equine pregnancy is that there is an
accumulation of CD41 and CD81 T lymphocytes around the
invasive trophoblast of the equine endometrial cups shortly
after they develop (19). The cups are formed by the migration
of the equine chorionic girdle into the endometrium to form
binucleate, equine chorionic gonadotrophin-secreting endometrial cup trophoblast cells (20). Interestingly, neither the alloantibody nor the cell-mediated immune response mounted by the
mother to the developing horse conceptus seems to compromise
pregnancy (21).
In the course of developing an assay to determine whether allospecific CTL generated from horse PBL could lyse equine trophoblast cells carrying paternal MHC class I Ags (22), we found
that PBL from pregnant mares showed lower levels of CTL activity than lymphocytes from nonpregnant control mares. We have
explored this phenomenon and compared it with the recent data
from other species described above.
0022-1767/99/$02.00
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Maternal immune recognition of the developing conceptus in equine pregnancy is characterized by the strongest and most
consistent alloantibody response described in any species, a response directed almost exclusively against paternal MHC class I Ags.
This work investigated the cellular immune response to paternal MHC Ags in pregnant and nonpregnant horses and donkeys, and
in horses carrying interspecies hybrid mule conceptuses. We observed profound decreases in classical, MHC-restricted, CTL
activity to allogeneic paternal cells in peripheral blood lymphocytes from both horse mares and donkey jennets carrying intraspecies pregnancies, compared with cells from nonpregnant controls. This is the first evidence in a randomly bred species for a
generalized systemic shift of immune reactivity away from cellular and toward humoral immunity during pregnancy. Surprisingly,
mares carrying interspecies hybrid mule conceptuses did not exhibit this transient, pregnancy-associated decrease in CTL activity.
The failure of interspecies pregnancy to down-regulate cellular immune responses may be a heretofore-unrecognized, subtle
barrier to reproductive success between species. The Journal of Immunology, 1999, 162: 4496 – 4501.
The Journal of Immunology
Materials and Methods
Animals
Horses (Equus caballus) and donkeys (Equus asinus) of the Equine Genetics Center at Cornell University College of Veterinary Medicine (Ithaca,
NY) were used for these experiments, and all animal care was performed
in accordance with institutional care and use guidelines. The horses were
comprised of several breeds, and the animals ranged in age from 4 to 18 yr.
Pregnancy was established using artificial insemination, and the day of
ovulation was determined by daily transrectal ultrasound examination,
from which gestational ages were deduced. The MHC class I phenotype for
each animal was determined by serological typing of lymphocytes based on
the equine leukocyte Ag system established by the Third International
Workshop on Lymphocyte Alloantigens of the Horse (23). MHC typing of
donkeys was performed using the lymphocyte microcytotoxicity assay with
pregnancy sera, mixed lymphocyte culture assays, and CTL assays (J.M.B.
and D.F.A., manuscript in preparation). Stallions and jack donkeys homozygous for MHC haplotypes were used as semen donors in this study.
For horses, MHC class I haplotypes are indicated by the letter A followed by a number (e.g., MHC type A2/A4, a heterozygote). For donkeys,
the haplotypes are abbreviated Ag followed by a number (e.g., MHC type
Ag1/Ag1, a homozygote).
PBMC were isolated as previously described (24) to be responder or stimulator cells. Briefly, venous whole blood was collected through jugular
venepuncture into tubes or bottles containing the anticoagulant sodium
heparin (Sigma, St. Louis, MO) at a final concentration of 15 IU/ml of
blood. Lymphocytes were isolated by centrifugation over a Ficoll density
gradient and washed twice in DAB-1-FCS: PBS containing 10% FCS, 1
IU/ml heparin, and 0.4% DAB-B salts (25 mg/ml CaCl2 z 2H2O and 25
mg/ml MgCl2 z 6H2O). Following the last wash, the pellet was resuspended in 1 ml per every 5 ml of blood originally collected, in ARM, a 1:1
(v/v) mixture of AIM V and RPMI 1640 media (Life Technologies, Grand
Island, NY), containing sodium bicarbonate (Life Technologies), 10%
heat-inactivated normal horse serum (pooled, Ab negative serum collected
from five male horses), and supplemented with 13 penicillin-streptomycin
(Life Technologies), 13 MEM nonessential amino acids (Life Technologies), 0.29 mg/ml L-glutamine (Sigma), 0.05 mg/ml sodium pyruvate (Life
Technologies), and 0.025 ml/ml molecular biology grade 2-ME (Sigma).
Stimulator cells were irradiated using a cesium source (Gammacell 40;
Nordion International, Kanata, Ontario, Canada), at a dose of 950 rads.
One hundred million responder cells were placed into a T75 flask (Corning, Corning, NY) with 50 3 106 stimulator cells, and ARM was added for
a final concentration of 3 3 106cells/ml. The flasks were incubated upright
at 37°C in 5% CO2 for 7 days, and the cells were resuspended every other
day. On day 7, the cultures were restimulated with freshly prepared, irradiated stimulator lymphocytes that were plated at half the density of the
surviving responder cells. Additionally, half of the medium was replaced
with fresh ARM. All cultures were stimulated for a total of 10 days.
Target preparation
Three days before the assay, lymphocytes (3 3 106 cells/ml in 10 ml) from
each target animal were incubated with 2.5 mg/ml pokeweed mitogen (Sigma) in T25 tissue culture flasks (Corning) at 37°C in 5% CO2 for 48 h. The
day before the assay, the cell concentrations were adjusted to 2.5 3 106
cells/ml and plated in one well each of a 24-well, flat-bottom tissue culture
plate (Costar, Cambridge, MA). To each target well, 125 mCi of 51Cr
(Na251CrO2, Dupont/NEN Research Products, Boston, MA) were added,
and the plate was incubated overnight in a 37°C incubator with a CO2
concentration of 5%. The next morning, the labeled cells were transferred
to tubes and washed six times in DAB-1-FCS at 1300 rpm for 5 min at 4°C.
51
Cr release assay
The 51Cr release assay we used in this study was adapted from previous
studies (25, 26). In most assays, killer cells were plated at four concentrations: 1 3 106, 5 3 105, 2.5 3 105, and 1.25 3 105 cells/well, in a volume
of 100 ml/well in round-bottom tissue culture plates (Fisher Scientific,
Pittsburgh, PA). Targets were always plated at 1 3 104 cells/well in 25 ml,
giving killer:target ratios of 100:1, 50:1, 25:1, and 12.5:1.
Spontaneous release wells contained only target cells and medium. Total release was obtained by adding 50 ml of 10% Triton X-100 (Sigma) to
wells that contained only targets and medium. The average spontaneous
release did not exceed 30% in any assay. All test dilutions and controls
were done in triplicate.
The plates were incubated at 37°C in 5% CO2 for 4 h. Afterwards, the
supernatants from each well were harvested using Skatron (Sterling, VA)
harvesting frames and the 51Cr activity was measured in a gamma counter.
Counts generally ranged from 500 to 12,000 cpm. The percent cytotoxicity
was normalized to the spontaneous and total release controls using the
following formula: % cytotoxicity 5 [mean sample release 2 mean spontaneous release]/[mean total release 2 mean spontaneous release].
Monoclonal Abs
The mAb WS#57 (MHC class I) (27) was created in our laboratory. Monoclonal Abs WS#72 (equine CD4) (28) and WS#12 (equine CD8) (28) were
obtained from Dr. William Davis of Washington State University (Pullman, WA). The specificity of these Abs were verified in two International
equine leukocyte Ag workshops (27, 28).
When Ab blocking was attempted, 100 ml of the mAbs were preincubated with either the targets (aMHC class I, neat supernatant) or the effectors (aCD4 or aCD8, ascites product diluted to 1:80) for 1 h. The cells
were washed and then plated as described above.
Statistical analyses
Student’s t tests were used to determine significance of pregnant vs nonpregnant data (see Fig. 3); a value of p # 0.05 was considered significant.
For mule vs horse pregnancy comparisons, ANOVA ruled out an effect
of the species, the mating stallion or jack, the day of pregnancy, histocompatibility Ags expressed, and the mare. With the exception of one mare
who showed significance based on her histocompatibility type (and who
was subsequently removed from the analysis so that the model could be
generalized, and thus independent of MHC effects), none of these variables
was significant (all with p # 0.1). Testing the data by the general linear
model ensured that the day of pregnancy did not have an effect ( p # 0.4),
and nested ANOVA results showed that there was no effect of parity ( p #
0.09) on the outcome. Because none of the other tests resulted in significance, the data from each treatment group (intraspecies pregnancy or interspecies pregnancy) were pooled and subjected to a Student’s t test. A
value of p # 0.05 was considered statistically significant.
Results
MHC specificity of equine CTL activity
PBL from nonpregnant female horses and ponies were used as
responder cells and stimulated with irradiated lymphocytes from
an MHC homozygous stallion. The CTL activity in this system
was specific for polymorphic class I Ags of the equine MHC (Fig.
1). The in vitro primed CTL lysed targets from the stimulator of
the 10-day culture and target lymphocytes from a genetically unrelated animal that carried the same MHC class I type as the stimulator, but did not lyse the autologous control lymphocytes or target cells from an animal carrying a MHC type different from that
of the stimulator. Fig. 1 shows data representative of that obtained
in .85 similar experiments, in which we tested animals of various
ages and genetic backgrounds. A sample of the MHC specificities
of different responder and stimulator combinations tested is shown
in Table I.
For Ab blocking experiments, CTL were primed in vitro as described above, but only lymphocytes from the stimulating stallion
were used as targets. Where Ab blocking was attempted, mAbs
were added to the effector or target cells as described in Materials
and Methods. The E:T ratio for these assays was 50:1. Cytolysis of
the primary target was blocked by mAbs to equine CD8 (added to
the effector cells) and equine MHC class I molecules (added to the
targets), but not by a mAb specific for equine CD4 (also added to
the effector cells) (Fig. 2).
Effect of intraspecies pregnancy on CTL activity
Normal, intraspecies horse and donkey pregnancies were established using artificial insemination. In vitro mixed lymphocyte cultures were set up between days 7 and 325 of gestation to prime
CTL from the pregnant mares to MHC class I Ags of the sire of the
mating. The CTL were tested in the 4-h, 51Cr release assay. After
10 days of culture, CTL activity was dramatically decreased when
the responder female from which lymphocytes were isolated was
pregnant at the time of blood collection (Fig. 3). This effect was
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CTL priming
4497
4498
MODULATION OF CTL RESPONSES DURING PREGNANCY IN EQUIDS
observed in lymphocyte cultures from both horse mares and donkey jennets. There was a significant depression in the CTL responses to the mating males by the females while they were pregnant, when compared with the cytotoxicity generated against the
same male while the animals were nonpregnant. The CTL activity
to autologous and third party targets was very low in these assays
(data not shown in Fig. 3 for clarity; see Fig. 1 and Table II for
examples). CTL activity returned to normal levels after pregnancy
was terminated.
Interspecies hybrid pregnancy did not cause a shift in maternal
antipaternal CTL activity
Four mares were inseminated using semen from either a jack donkey (for interspecies hybrid mule pregnancy) or a horse stallion
(for intraspecies horse pregnancy) and tested for their ability to
mount CTL against PBL from the respective sire (Table II). For
Table I. MHC specificity of equine CTL assays MHC types a
Stimulator
Responder
Lysedb
Not Lysedb
A2/A2
A1/A6
A2/A2
A1/A6
A3/A3
A3/A3
A2/A2
A2/A2
A2/A19
A2/A6
A2/A10
A6/A7
A3/A3
A9/A9
A9/A19
A10/A10
A9/A9
A2/A2
A9/A9
A3/A3
A1/A6
A2/A2
A2/A6
A1/A6
A3/A3
A6/A7
A3/A3
A6/A7
A9/A9
A10/A15
A9/A9
A3/A3
A6/A7
A10/A10
A6/A7
A2/A6
A3/A3
A10/A10
A3/A3
A1/A6
A6/A7
A9/A9
A2/A2
A6/A7
A2/A2
A3/A3
a
MHC types were determined by serological typing of lymphocytes (22). See
nomenclature information in Materials and Methods.
b
Lysed MHC types of lymphocyte targets that were specifically lysed by the in
vitro-primed CTL from a specific responder and stimulator combination; not lysed
lymphocyte targets not recognized by the CTL from the same responder and stimulator combination. See Fig. 1 for a representative assay.
FIGURE 3. The ability to make cytotoxic T cells against paternal histocompatibility Ags is decreased in horse mares and jennet donkeys carrying intraspecies pregnancies at the time of blood collection. A, Horse
mare 2470 (MHC type A6/A7). B, Donkey jennet 2175 (Ag2/Ag4). Each
point for pregnancy data is representative of at least five experiments and
at least two different pregnancies. The mare was mated with stallion 0834
(A2/A2), and the jennet was mated with jack 1992 (Ag1/Ag1). Each point
for the nonpregnant data is representative of at least nine experiments, with
data collected both before and after pregnancy. The letters above symbols
indicate values are significantly different, p , 0.05.
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FIGURE 1. MHC specificity of equine CTL activity. PBL were collected from a nonpregnant mare (MHC type A6/A7) and cultured in vitro
for 10 days with irradiated lymphocytes from a genetically unrelated horse
(A2/A2) to prime CTL. On day 10, these CTL effectors were incubated for
4 h with 51Cr-labeled lymphocyte targets, and cell lysis was determined by
the formula shown in Materials and Methods. Lysis was specific for the
MHC type of the priming lymphocytes (A2/A2), as seen by the strong
killing against the primary lymphocytes and MHC-matched lymphocytes
(A2/A10). Lysis was negligible against the autologous control (A6/A7) and
MHC-mismatched (A10/?) targets. Data are shown as mean of triplicate
wells 6 SD. See Table I for a list of other genetic combinations tested.
FIGURE 2. Monoclonal Ab blocking of equine CTL killing. Ab blocking was performed just before the start of the 4-h 51Cr release assay. Monoclonal Abs directed against MHC class I, CD8, or CD4 (26, 27) were added
to the target wells (anti-MHC class I) or to effector cells (anti-CD8 or
anti-CD4) and incubated for 1 h. The data demonstrate percent change in
cytotoxicity from control (no Ab blocking). Data are shown as mean 6 SD
of five observations. p, p , 0.001 by a Student’s t test; significant inhibition of cytotoxicity compared with control or anti-CD4 cytotoxicity levels.
The Journal of Immunology
4499
Table II. Antipaternal CTL activity of mares carrying either intraspecies horse or interspecies hybrid mule
pregnancies a
/?
H3H
% Cytotoxicity
E:T Ratio
Mare
(MHC type)
50:1
25:1
12:1
2446
(A10/A10)
Control
Test
Control
Test
10
15
1
3
7
11
1
5
1406
(A5/A5)
Control
Test
Control
Test
3
0
7
10
2996
(?/?)
Control
Test
Control
Test
2470
(A6/A7)
6:1
50:1
25:1
12:1
6:1
8
10
0
7
3
1
3
6
0
20
3
24
0
13
1
24
0
9
1
19
0
3
3
18
1
12
8
6
0
0
14
6
0
0
14
3
3
20
0
39
5
15
0
40
6
5
0
37
3
2
0
35
3
2
8
8
4
4
1
1
3
3
1
4
3
2
0
4
5
38
0
34
6
31
5
29
4
32
12
25
6
26
0
18
Control
Test
Control
Test
3
15
16
17
0
13
20
16
0
9
12
13
0
4
10
9
7
61
7
74
9
68
9
65
4
67
5
65
2
67
2
67
SD(Test-Control)
MeanD(Test-Control)b
19
2
26
3
11
1
24
21
339
42
310
39
286
36
282
35
a
One intraspecies (horse) and one interspecies (mule) pregnancy was established in each mare. The sire of each horse
pregnancy was stallion 0834 (MHC type A2/A2), and the sire of each mule pregnancy was jack 1992 (Ag1/Ag1). At two time
points in each pregnancy, between days 14 and 33, the ability of mares to make a CTL response to paternal alloantigens was
determined by the 51Cr release assay. “Control” is cytotoxicity against autologous lymphocytes; “test” is cytotoxicity against
lymphocytes from the sire of the mating.
b
Intraspecies vs interspecies pregnancy, t 5 4.3; p # 0.01.
mares 2446 and 1406, the order was interspecies followed by intraspecies pregnancy. For mares 2996 and 2470, intraspecies pregnancy was established first, followed by the interspecies pregnancy. PBL were isolated from each mare and the breeding male
between days 14 and 30 after the establishment of pregnancy, and
in vitro cultures were established as described. Each mare was
tested twice in each pregnancy. Between days 33 and 35 of pregnancy, conceptuses were removed by means of nonsurgical uterine
lavage (29), and mares were allowed to remain nonpregnant for
one or more estrous cycles. The mares were then inseminated with
semen from either a jack donkey or horse stallion for the reciprocal
mating. Mares pregnant with horse conceptuses (intraspecies pregnancy) had decreased ability to make CTL responses against paternal alloantigens (mean [test-control] values approached
zero). In contrast, the same mares, when carrying interspecies
mule conceptuses, made strong CTL responses against paternal
alloantigens.
These results were robust, and they were detected in animals of
diverse genetic backgrounds (Fig. 4). When mares were pregnant
with interspecies mule conceptuses, their antipaternal CTL activity
was greater than the CTL activity generated by nonpregnant control females challenged in vitro with the same histocompatibility
Ag barrier ( p , 0.05). In contrast, when mares or jennets were
pregnant with intraspecies horse or donkey conceptuses, respectively, the antipaternal CTL activity was less than that generated in
lymphocyte cultures from nonpregnant control females tested in
the same experiments ( p , 0.001).
Discussion
These data demonstrate a dramatic decrease in antipaternal alloantigenic CTL activity in lymphocytes from mares or jennets carrying intraspecies pregnancies (Figs. 3 and 4). This effect appears
similar to the hyporesponsiveness of T cells to paternal alloantigens described in mice (2, 3). Furthermore, to our knowledge, it is
the first demonstration of systemic decreases in antipaternal T cell
reactivity during pregnancy in any randomly bred species. It is
important that these alterations in maternal immune reactivity occur in species with very different placental structure. The mare has
an epitheliochorial (noninvasive) placenta, and the mouse has a
hemochorial (invasive) placenta (18). Moreover, the type of immune cells that are present in the uterus do not appear to affect the
described effects. In mice, the predominant immune cell in the
uterus is the NK-like granulated metrial gland cell (30), while the
uterus of the mare primarily contains classical CD41 and CD81 T
cells (19).
Contrary to the finding by Jiang and Vacchio (4) for the H-Y
minor histocompatibility Ag, the described hyporesponsiveness in
our experiments is transient rather than permanent, and full responsiveness to paternal alloantigens returned postpartum (Fig. 3).
Thus, the horse results correspond to research in both transgenic
and nontransgenic mice that demonstrated that T cell responses to
paternal MHC alloantigens were restored shortly after parturition
(2, 3). There may be many reasons for the discrepancies between
the different studies; one simple explanation is that there are multiple, overlapping mechanisms of fetal survival, and not all mechanisms are utilized by every species. However, Jiang and Vacchio
(4) argue that their results differ from those previously described
because H-Y is present at more physiological levels than the MHC
Ags used in other experiments (5). Our work, and that done by
Robertson et al. (3), also demonstrate physiological systems, since
the antigenic stimuli are fetal and paternal alloantigens, and the
strong alloreactions permit the use of nontransgenic T cells for the
experiments. Although it appears that the hyporesponsiveness in
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Variable
/?
H3D
% Cytotoxicity
E:T Ratio
4500
MODULATION OF CTL RESPONSES DURING PREGNANCY IN EQUIDS
our model is reversible postpartum, it is possible that there is permanent deletion of some T cells clones that is impossible to measure in our system.
Other evidence from equids indicates that pregnancy may also
induce a generalized shift away from cell-mediated immunity, and
toward Ab-mediated responses, as Wegmann and his colleagues
have proposed for mice (6, 8, 9) and humans (7). Our conclusion
was drawn from a comparison of immunological aspects of horse
and mule pregnancy.
Mares make high-titered Ab responses to paternal MHC class I
Ags that are detected early in gestation in normal horse pregnancy
(10). By contrast, in mares carrying hybrid mule conceptuses, the
antipaternal Ab responses arise later and are of lower titer (11, 31).
These responses are directed against allelic epitopes of paternal
MHC Ags of the mating donkey (J.M.B. and D.F.A., unpublished
observations).
A second difference between horse and mule pregnancy is seen
in the leukocyte infiltration around the invasive trophoblast of the
endometrial cups. In pregnant horse mares, there is a triphasic
leukocytic response to the endometrial cup cells (19). The first
phase occurs immediately following the invasion of the chorionic
girdle trophoblast cells to form the endometrial cups and is characterized by a striking infiltration of CD41 and CD81 T lymphocytes around the cups at about day 40 of gestation. The second
phase occurs in the midlife of the cups (around day 60), where the
lymphocytes are greatly diminished in number. In the third phase,
a more complex leukocytic infiltration is observed that may bring
about the demise of the cups between days 80 and 120. In mule
pregnancy, the leukocyte response to the endometrial cups seems
to progress uniformly, with no reduction in leukocyte numbers
around day 60 (31, 32). In fact, the mule endometrial cups are
usually destroyed by this stage of pregnancy.
Considered together with the differences in maternal antipaternal CTL activity between intraspecies and interspecies pregnancy
described in this paper, these observations suggest a unifying hypothesis: in normal intraspecies horse pregnancy there may be a
shift away from cell-mediated immunity (Figs. 3 and 4), toward
humoral immunity (or from Th1 to Th2 type immune responses)
that does not occur in interspecies mule pregnancy (Table II, Fig.
4). These differences may reflect a subtle immunological barrier to
interspecies mating that in itself does not prevent interspecies
pregnancy, but which might compromise the ability of females to
carry to term hybrids made between closely related species.
In 1949, Coombs (33) reviewed data obtained by Caroli and
Bessis (34) demonstrating an 8% mortality in newborn mules
caused by hemolytic disease of the newborn resulting from isoimmunization during pregnancy. Moreover, an immunological basis
for pregnancy failure has been proposed in interspecies hybrid
(sheep 3 goat) pregnancies, based on the discovery of antifetal
hemolytic activity in serum from does after hybrid pregnancy
and a faster rejection of second hybrid pregnancies than first
pregnancies (35, 36).
Thus, there is strong evidence from both pre- and postnatal studies indicating pathological consequences of maternal immune reactivity to hybrid interspecies conceptuses. We propose that such
untoward consequences result in part from abnormal signaling between mother and hybrid fetus early in pregnancy. This fails to
establish an immunological relationship emphasizing recognition
(as opposed to destruction) of the developing fetus by the maternal
immune system. In both the horse and donkey, two very closely
related species, similar mechanisms appear to operate to ensure
intraspecies pregnancy success (a shift away from cell-mediated
immunity), yet this protective mechanism seems to break down
when the two species are mated to create an interspecies hybrid
conceptus. Perhaps it is correct, as hypothesized by Mourant (33)
of hemolytic disease of the newborn, that its effect “will thus lead
to the development of new species” by perpetuating immunogenetic differences arising in reproductively separate populations.
The subtle differences between intraspecies horse and interspecies
mule pregnancy described here may be a newly recognized manifestation of mechanisms with similar outcome.
Acknowledgments
We thank James Hardy for assistance with animal breeding; Jane Miller,
Melissa Carlson, Laurie Lantagne, and Sarah Deacon for their technical
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FIGURE 4. Differences in CTL activity during intraspecies (horse and donkey) vs interspecies (mule) pregnancy. For horse pregnancies, mares were
inseminated with semen from either stallion 0834 (MHC type A2/A2) or stallion 2505 (A3/A3). In some cases, data are from two or more pregnancies in
one mare, with both stallions used as sires. Mule and donkey pregnancies were established by inseminating mares and jennets with semen from either jack
1989 (Ag3/Ag3) or jack 1992 (Ag1/Ag1). Again, in some cases, data are representative of two or more pregnancies. The MHC types of each female are
shown in the . “?” indicates that alloantibodies were not available for the MHC type of that animal, so its haplotype was undetermined. Each bar indicates
the difference between the mean CTL activity of lymphocytes from a pregnant animal and a control, nonpregnant animal tested at the same time. Open bars
represent interspecies hybrid mule pregnancy. Hatched bars represent intraspecies horse or donkey pregnancies. Data represent varying numbers of tests
and pregnancies. CTL activity from mares or jennets carrying horse or donkey intraspecies conceptuses, respectively, was always less than the control (p ,
0.001), and CTL activity from mares carrying interspecies hybrid mule conceptuses was always greater than or equal to the control values (p , 0.05).
The Journal of Immunology
help; and Dr. Charles McCulloch from the Department of Biometrics at
Cornell University for statistical consultation.
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