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CUTTING EDGE |
*
Reproductive Immunology Laboratory, Swedish Medical Center, Denver CO 80110; and
Obstetrix Medical Group of Colorado, P.C. Denver, CO 80110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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14+ TCRint (NKT)
lymphocytes. Although these cells bear the above features in common
with other NKT cells populations in thymus, bone marrow, liver, and
spleen, they differ from these other populations in terms of an altered
V
repertoire and absence of a CD4+ component. In this
study, we demonstrate that the uterine population also differs from
other NKT cell populations because they recognize a class I/class
I-like molecule other than CD1, whereas most previously described
V14+ NKT cells are CD1-restricted. Moreover, the class
I/class I-like molecule leading to the uterine NKT cell expansion may
be supplied by the fetus. These data demonstrate a novel mechanism
whereby the fetus is capable of modulating the maternal immune
system. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we demonstrated a large increase in uterine
NK1.1+ V14+
CD4-/CD8- (NKT) cells in
the maternal decidua at peri-implantation. (7) Here, we
demonstrate that a fetal Ag regulates this increase. This Ag is most
likely a class I/Ib molecule since
2-microglobulin-deficient
(2m-/-)3
mice do not have increased numbers of NKT cells at peri-implantation.
However, the Ag does not appear to be CD1, the class I-like molecule
recognized by NKT cells in other tissues, as
CD1-/- mice have normal numbers of decidual NKT
cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathogen-free CD1+/- C57BL/6 and BALB/c
(8) mice were obtained from the laboratory of C.-R. Wang
at the University of Chicago (Chicago, IL), bred in the animal care
facility at Swedish Medical Center (Denver, CO), and screened, as
described, by PCR to obtain CD1+/+ and
CD1-/- mice of each strain for use in
additional experiments (8). Six- to 8-wk-old
2m-/- C57BL/6 and
BALB/c (9) mice were obtained commercially from The
Jackson Laboratory (Bar Harbor, ME). All experiments were approved by
the HealthONE Institutional Animal Care and Utilization Committee,
which acts for the Swedish Medical Center.
Cell preparation
Placental/decidual
tissues4 were obtained
from early pregnancies (days 6–8, day of plug detection = day 0),
as previously described (7). In all cases, (C57BL/6
(female) x BALB/c (male)) matings were used, with the animals
being wild-type (WT; CD1+/+,
2m+/+),
CD1-/- (CD1-/-,
2m+/+), or
2m-/-
(CD1+/+,
2m-/-). Timed pregnant
female mice were sacrificed using CO2 inhalation,
and placental/decidual tissues were collected, placed in a Cellector
tissue sieve (VMR Scientific Productions, Willard, OH), and
mechanically dispersed into balanced salt solution (BSS). Spleens,
livers, and thymi were obtained from the same pregnant mice and
similarly prepared. Placental/decidual cells, splenocytes, hepatocytes,
and thymocytes were centrifuged at 1000 rpm (250 x g)
for 5 min, cell pellets were resuspended in 1 ml of BSS and 3 ml of
Gey’s solution (0.155 M NH4Cl and 0.01 M
KHCO3) for 5 min at room temperature to lyse RBC,
washed with BSS, resuspended in 2 ml of BSS/5% FCS (Sigma, St. Louis,
MO), and lymphocytes were enriched over nylon wool, as previously
described (10). During each experiment, the phenotype of
each 2m-/- or
CD1-/- mouse was verified by cytofluorographic
analysis (data not shown).
mAbs and cytofluorographic analysis
H57.597 (anti-pan TCR-, FITC conjugated)
(11) and PK136 (anti-NK1.1, PE conjugated)
(12) were purchased commercially from BD PharMingen (San
Diego, CA). mAb S19.8 (anti-2m, FITC
conjugated) (13) was generated as cell culture supernatant
from an existing cell line, purified by affinity chromatography on
protein G -Sephadex G25 (Sigma), concentrated, dialyzed, and conjugated
with FITC (Sigma).
For cytofluorographic analysis, cells (0.1–1 x
106/well in 96-well plates) were preincubated
with rat anti-mouse Fc
R Ab (24G2, 1 µg/ml) (14)
or undiluted normal mouse serum to block nonspecific binding and one-
or two-color staining was performed. Cells were analyzed by flow
cytometry using a Becton Dickinson FACSort cytometer (Becton Dickinson,
San Jose, CA), and data plots were generated using the CellQuest
version 1.2 software package supplied by the manufacturer. In all
experiments, set up and calibration were performed with nylon wool
prepared splenocytes, and these cells were also used to set gates for
thymocytes and hepatic and placental/decidual lymphocytes. In all
cases, appropriate negative control experiments were performed to
verify staining specificity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We analyzed percentages of NKT cells in placental/decidual tissues
from (CD1+/+ x CD1+/+),
(CD1-/- x CD1-/-) and
(2m-/- x
2m-/-) matings. For
comparison, we also analyzed percentages for NKT cells in thymus,
spleen, and liver from the same pregnant female mice. In the
(CD1+/+ x CD1+/+) mice,
thymus, spleen, liver, and placenta/decidua contain 
1% (1.3 ±
0.2, mean ± SD, n = 7), 2% (2.0 ± 0.4,
mean ± SD, n = 6), 18% (18.3 ± 3.9,
mean ± SD, n = 5), and 4% (4.1 ± 1.2,
mean ± SD, n = 7) NKT cells among all nylon
wool-purified cells, respectively. Typical staining data are shown in
Fig. 1
a. Percentages of NKT
cells are greatly reduced in the (CD1-/- x
CD1-/-) females in thymus, liver, and spleen,
as previously reported (8, 15), but are unchanged in
placental/decidual tissues from these mice (Fig. 1b),
suggesting that CD1 is not required for the expansion of uterine NKT
cells. In (2m-/- x
2m-/-) matings,
percentages of NKT cells were also greatly reduced in thymus, liver,
and spleen, again as previously reported (16, 17, 18, 19) and were
also greatly reduced in placental/decidual tissues (Fig. 1c). Together with the data from Fig. 1b, these
data suggest that a 2m-associated molecule
other than CD1 regulates the expansion of uterine NKT cells.
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TCR and the percentages of T cells
coexpressing NK1.1 in these same organs. In
(CD1+/+ x CD1+/+) matings,
it was found that nearly 80% of thymus and liver NK1.1+ cells,
one-third of spleen NK1.1+ cells, and one-quarter
of uterine NK1.1+ cells coexpress -TCR on
their surface (Fig. 2, left).
Similarly, it was found that 5.2% of -TCR+
cells in thymus, 28% in liver, 3.4% in spleen, and 8.6% in
placenta/decidua coexpress NK1.1 (Fig. 2, right). By
contrast, these percentages were reduced in the
(CD1-/- x CD1-/-)
matings in thymus, spleen, and liver, but not in placenta/decidua.
Again, the percentages were reduced in all organs in the
(2m-/- x
2m-/-) matings,
suggesting regulation of uterine NKT cells by a
2m-associated molecule other than CD1. The
data from Fig. 2, c–f, suggest that there may be a small
pool of CD1-independent 2m-dependent NKT cells
in liver and spleen as well.
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2m-associated molecule regulates expansion
of placental/decidual NKT cells
The experiments performed indicate that the expansion of
placental/decidual NKT cells largely relies on the expression of a
2m-associated molecule other than CD1. The
next experiment was designed to assess whether fetal (placental)
expression of the 2m-associated molecule is
sufficient for expansion of placental/decidual NKT cells. To address
this question, we bred C57BL/6
2m-/- females with WT
BALB/c males and analyzed NKT cells from the resulting gestations.
Although thymic, splenic, and hepatic NKT cell numbers remained at the
very low levels seen in
(2m-/- x
2m-/-) gestations, the
numbers of NKT cells from these
(2m-/- x WT)
pregnancies were restored to those of WT matings. Typical staining data
are shown in Fig. 3. Thus, even in the
absence of maternal 2m, fetal
2m derived from the paternal genome is
sufficient to expand the placenta/decidual NKT cell population. These
data are summarized in Fig. 4.
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2m expression is
necessary for the expansion of placenta/decidua NKT cells, (WT x
2m -/-) gestations
were analyzed and normal numbers of NKT cells were found (data not
shown). Thus, paternal 2m expression is
sufficient, but not necessary, for NKT cells expansion. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-TCR, they uniformly express V14-J281 with a
canonical VJ junction, have a limited V repertoire (predominantly
V8.2 in addition to smaller proportions of V2 and V7), express
TCR at an intermediate level, secrete large amounts of IL-4 upon
engagement of the TCR (8, 22), and recognize the class
I-like CD1 molecule. (15, 23). In a recent study, we added
the peri-implantation pregnant murine uterus to the list of sites with
a large NKT cell population (7). In that study, we
speculated that NKT cells might play an important role at
peri-implantation by inducing a Th2-type environment via elaboration of
IL-4. Such a Th2 environment has been shown to be critical for
reproductive success (24, 25, 26, 27).
Ito et al. (28) have also recently confirmed the presence
of NKT cells in the pregnant uterus. These authors speculated that NKT
cells might mediate spontaneous abortion based upon experiments
involving in vivo stimulation of NKT cells with -galactosylceramide,
a known ligand of V14+ NKT cells. The
physiological significance of these findings remains to be
demonstrated.
In our description of NKT cells in the pregnant uterus and their
expansion at peri-implantation, we noted two features that suggested
these cells might develop independently from NKT cells in other organs.
First, unlike other populations of NKT cells that contain both
CD4+ and
CD4-/CD8- (DN)
components, uterine NKT cells are entirely CD4-.
Additionally, although uterine NKT cells are
V14+, their V repertoire is different from
other organs, with an apparent predominance of
V3+ cells and few V8+
cells.5
We did note expression of CD1 within the pregnant uterus and speculated that these cells might nonetheless recognize CD1. Although experiments have not yet been done directly assessing cognate recognition of CD1 by uterine NKT cells, the experiments reported herein suggest that development of this uterine population is regulated by a class I/class I-like molecule other than CD1.
Additionally, these findings indicate that the relevant
2m-associated molecule can be supplied by the
paternal genome, presumably via placental expression. In the HLA-G
experiments discussed in the introduction, HLA-G likely mediates its
effects via placental expression, as its expression is highest in the
trophoblasts (29). It seems likely that the relevant fetal
Ags (H-Y, Kb) in the transgenic models also
mediate the observed effects via placental expression, as the placental
trophoblast are the cells are most likely to be encountered by maternal
immune cells. In this regard, it is interesting to note that the
paternal genome is preferentially expressed in the placental tissues,
presumably due to imprinting (30). Since we have not
specifically identified the 2m-associated
molecule that regulates the expansion of uterine NKT cells, we cannot
yet assess whether it is imprinted or not. We did observe that maternal
2m expression is capable of regulating uterine
NKT cell expansion. Whether this effect is mediated by placental
expression of the relevant 2m-associated
maternal allele or by expression by adjacent maternal tissues is
unknown at present.
In these experiments, as in previous reports, thymus, liver, and spleen
of 2m-/- females
contain very few NKT cells (16, 17, 18, 19, 23). Similarly, the
uteri of pregnant
2m-/- females contain
very few NKT cells when crossed with
2m-/- males. In
contrast, when crossed with
2m+/+ males, normal
numbers of uterine NKT cells are found, while numbers of NKT cells in
the other organs remain very low. Current data support both thymic and
extrathymic development of NKT cells (reviewed in Ref.
23). It is unclear from these data whether the local
expansion of uterine NKT cells represents de novo development of
uterine NKT cells from a resident progenitor or a local expansion of a
small resident pool. The data do not support thymic development and
migration. This model may provide an interesting system for the study
of NKT cell development.
Interactions between the mother and fetus have long been known to occur via a variety of steroid and peptide hormones. These data document another mechanism whereby maternal-fetal interaction occurs via their immune systems. Unlike similar situations that occur in tumor and transplant models, for example, these phenomena take place within the physiologic setting of the hemochorial placenta and emphasize the specialized nature of maternal-fetal immunology.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kent D. Heyborne, Maternal-Fetal Medicine, Swedish Medical Center, 501 East Hampden Avenue, Denver CO 80110.
3 Abbreviations used in this paper: 2m, 2-microglobulin; WT, wild type; BSS, balanced salt solution.
4 Because the embryo cannot easily be separated from the other tissues at this early gestation, it was included in the tissue preparations. Because the embryo does not contain lymphoid cells at this time, its inclusion will not alter the results. Due to the intimate apposition of the fetal trophoblasts and maternal tissues, they cannot be easily separated. For simplicity, we refer to the fetal and maternal tissues thus obtained as "placenta/decidua."
5 K. Ito, M. Karasawa, T. Kawano, T. Akasaka, H. Koseki, Y. Akutsu, E. Kondo, S. Sekiya, K. Sekikawa, M. Harada, et al., who characterized NKT cells at a later gestation, found a predominance of V7+ cells in their report. We did note a high degree of staining with anti-V7 mAb in lymphocyte preparations from uterine tissues, but found this staining to be nonspecific.
Received for publication December 27, 2000. Accepted for publication January 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells in the periimplantation uterus. Immunology 101:484.[Medline]
2-microglobulin locus in mouse embryonic stem cells by homologous recombination. Proc. Natl. Acad. Sci. USA 86:8932. T cell receptors. J. Immunol. 142:2736.[Abstract]
/+ cells in the liver of mice. J. Exp. Med. 180:699.8+ CD4-8- thymocytes by class I molecules expressed by hematopoietic cells. J. Exp. Med. 178:901. TCR+NK1.1+ cells. J. Immunol. 145:3209.[Abstract]
chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180:1097. and TNF and reduced IL-10 production by placental cells. J. Immunol. 156:653.[Abstract]
14 NKT cells in abortion. Proc. Natl. Acad. Sci. USA 97:740.This article has been cited by other articles:
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 T. Michimata, H. Tsuda, M. Sakai, M. Fujimura, K. Nagata, M. Nakamura, and S. Saito Accumulation of CRTH2-positive T-helper 2 and T-cytotoxic 2 cells at implantation sites of human decidua in a prostaglandin D2-mediated manner Mol. Hum. Reprod., February 1, 2002; 8(2): 181 - 187. [Abstract] [Full Text] [PDF]
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