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HLA-G in immune tolerance in pregnancy

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The FASEB Journal • Review
HLA-G and immune tolerance in pregnancy
Joan S. Hunt,*,1 Margaret G. Petroff,* Ramsey H. McIntire,* and Carole Ober†
*University of Kansas Medical Center, Kansas City, Kansas, USA; and †The University
of Chicago, Chicago, Illinois, USA
ABSTRACT Multiple mechanisms underlie the sur-
prising willingness of mothers to tolerate genetically
different fetal tissues during pregnancy. Chief among
these is the choice of HLA-G, a gene with few alleles,
rather than the highly polymorphic HLA-A and -B genes,
for expression by the placental cells that interface
directly with maternal blood and tissues. Novel aspects
of this major histocompatibility complex class Ib gene
include alternative splicing to permit production of
membrane and soluble isoforms, deletions that
dampen responses to interferons, and a shortened
cytoplasmic tail that affects expression at the cell
surface. Placental cells migrating into the maternal
uterus synthesize both membrane and soluble iso-
forms, which interact with inhibitory receptors on leu-
kocytes such as ILT2 and ILT4. Cytotoxic T lympho-
cytes either die or reduce production of one of their
major coreceptor/activator cell surface molecules,
CD8; natural killer cells are immobilized and mononu-
clear phagocytes are programmed into suppressive
modes characterized by high production of anti-inflam-
matory cytokines. The idea that placental HLA-G pro-
teins facilitate semiallogeneic pregnancy by inhibiting
maternal immune responses to foreign (paternal) anti-
gens via these actions on immune cells is now well
established, and the postulate that the recombinant
counterparts of these proteins may be used as powerful
tools for preventing immune rejection of transplanted
organs is gaining in popularity.—Hunt, J. S., Petroff,
M. G., McIntire, R. H., Ober, C. HLA-G and immune
tolerance in pregnancy. FASEB J. 19, 681– 693 (2005)
Key Words: human 䡠 placenta 䡠 immune privilege
BACKGROUND
The success of human pregnancy, where the fetus
resides comfortably within the maternal uterus for 9
months, defies the precepts of immunology. Medawar
was the first to attempt sorting out the strategies used in
pregnancy to circumvent maternal rejection of the
embryo/fetus (1). The proposed mechanisms included
physical separation of maternal and fetal tissues, lack of
fetal antigens that could stimulate graft rejection, and
development of tolerance.
Today certain aspects of each of these strategies have
been identified: the blood circulations of the fetus and
the mother are entirely separate; in the fetus, transplan-
0892-6638/05/0019-0681 © FASEB
tation antigens are late appearing; tolerance and im-
mune privilege at the maternal-fetal interface are
readily identified. Three major principles emerging
from these studies are that 1) multiple mechanisms
provide protection, 2) both the fetus and the mother
contribute to development and maintenance of the
pregnant uterus as an immune privileged site, and 3)
fetal factors drive changes in maternal immune re-
sponses. In this article, we first briefly discuss a number
of conditions responsible for immune privilege and
maternal tolerance for which scientific evidence is
strong, then focus on a central feature—a unique
capacity of placental cells to select specific genes within
the major histocompatibility complex (MHC) for ex-
pression. We present evidence that this unique capacity
for selection of specific MHC antigens, which in hu-
mans are called human leukocyte antigens (HLA), may
be responsible in large part for the reprogramming of
local maternal immune responses that characterize
successful semiallogeneic pregnancy.
Strategies for protecting the semiallogeneic fetus
from maternal graft rejection responses
During pregnancy, the maternal immune system is
clearly active, and under certain conditions may con-
tribute to fetal damage/death. Well-defined pathologi-
cal processes include destruction of fetal erythrocytes
(Rh antigen, erythroblastosisis) and platelets (HPA-1
and -2, alloimmune thrombocytopenia) by maternal
antibodies and infections of pregnancy, where activated
macrophages secreting high levels of Th1-type cyto-
kines alter the delicate cytokine balance at the mater-
nal-fetal interface (2, 3). Yet even with a demonstrably
active maternal immune system, mothers usually seem
1
Correspondence: Department of Anatomy and Cell Biol-
ogy, University of Kansas Medical Center, Kansas City, Kansas,
66160-7400 USA. E-mail: jhunt@kumc.edu
Supported in part by grants from the National Institutes of
Health to J.S.H. (HD26429, HD35859, and HD39878), to
M.G.P. (HD045611), and to C.O. (HD21244). R.M. is sup-
ported by a fellowship from the Kansas University Medical
Center Biomedical Research Training Program. The authors
appreciate the assistance of S. Fernald, Kansas Reproductive
Sciences Center, in preparation of the figures. This publica-
tion was made possible by NIH grant number P20 RR016475
from the INBRE Program of the National Center for Re-
search Resources.
doi: 10.1096/fj.04-2078rev
681
to tolerate rather than reject their genetically disparate
fetuses. Ordinarily, the mother would be expected to
generate graft-attacking antibodies and cytotoxic T
lymphocytes (CTL) to foreign (paternal) HLA or other
antigens expressed by fetal cells. HLA antigens are
called “transplantation” antigens because they com-
prise the most powerful stimulators of graft rejection.
Thus, in organ transplantation the matching of certain
donor and patient alleles is an absolute requirement
for successful grafting.
Even though mothers and fathers are almost invari-
ably disparate at multiple HLA loci, rejection of the
fetus as a consequence of maternal recognition of
paternal HLA as foreign is essentially undocumented.
Although anti-paternal HLA antibodies are common in
pregnant women, they do no damage. Even novel HLA
antigens expressed in the fetal membranes are tolero-
genic rather than immunogenic (4). Attack by mater-
nal CTL is also effectively thwarted, as described in
detail below. Thus, mechanisms underlying maternal
tolerance are unusually effective and raise the critical
question of how immune privilege might be established
in the natural situation of pregnancy so as to assure
viability of the embryo/fetus.
Figure 1 illustrates the principle that maternal and
fetal processes contribute to the generation of a safe
environment. Within the uterus, a dramatic change in
endometrial leukocyte subpopulations occurs as a con-
sequence of implantation. After a brief inflammatory
reaction caused by blastocyst breaching of the uterine
epithelium, a reaction best documented in rodents, the
altered endometrium (now termed decidua) settles
into a pattern where local protection is provided by the
innate immune system. From this point onward, the
major players in acquired immunity, T and B lympho-
Figure 1. Multiple mechanisms underlie maternal tolerance
of the fetus. Mothers, via changes that occur in the uterus,
and embryo/fetuses, via special adaptations of the placenta,
contribute to the establishment of an immune privileged
environment within which the semiallogeneic fetus resides
safely until termination. NK cells, natural killer cells; Treg,
CD4⫹ regulatory T cells; TNF superfamily, tumor necrosis
factor superfamily.
682 Vol. 19 May 2005 The FASEB Journal
cytes, are identified mainly in the myometrium distal to
fetal tissues whereas members of the innate immune
system, natural killer (NK) cells and macrophages,
predominate in the decidua (5, 6). An exception is the
Treg subset of CD4⫹/CD25⫹ cells, which produce inter-
leukin-10 (IL-10) and transforming growth factor-␤1
(TGF-␤1), and are believed to be critical to mainte-
nance of tolerance (7, 8). These cells, whose prolifera-
tion is stimulated by estrogen (9) comprise ⬃14% of
CD4⫹ cells in early decidua (10). Uterine cytokine
networks, while of great complexity and containing
many examples of Th1-type reactivities (11, 12), were
first shown in the mouse to be dominated by these and
other Th2-type cytokines (13). Similar reports have
appeared in human pregnancy (14). Immunomodula-
tory hormones such as prolactin, chorionic gonadotro-
pin, and progesterone are abundant; chemokines op-
erate to control immune cell numbers and types.
The fetal contributions are unique. Figure 2 shows
that the fetus, which is derived from the inner cell mass
of the blastocyst, is secluded within a protective shell
composed of trophoblast cells that arise from the
trophectoderm layer of the blastocyst. It is therefore the
heavy responsibility of the trophoblast cells to interact
appropriately with the maternal environment and pro-
tect the fetus from maternal immune attack. As illus-
trated in Fig. 1, the trophoblast cells circumvent anti-
body-mediated damage by exhibiting high levels of the
complement regulatory proteins (15), and reduce cell-
mediated immunity by expressing inhibitory members
of the B7 family (16), and apoptosis-inducing members
of the tumor necrosis factor (TNF) family of ligands
(17). As with cells in the decidua, fetal cells produce
immunosuppressive cytokines, chemokines, and prosta-
glandins that dampen T lymphocyte proliferation and
export high levels of immune suppressive hormones
such as progesterone.
Most important, trophoblast cells strictly regulate
their expression of HLA genes and the production of
their proteins. It is these proteins that, if recognized as
foreign by maternal immune cells, would stimulate
maternal anti-fetal CTL capable of destroying HLA-
expressing fetal cells (18). Instead, the antigens ex-
pressed in trophoblast cells program maternal leuko-
cytes into pathways consistent with tolerance.
HLA-G: A NOVEL GENE EXPRESSED IN
PLACENTAS
The genes encoding HLA antigens are clustered on
chromosome 6p21 at the telomeric end of the HLA
region. Although this region contains ⬃20 to 25 HLA
class I genes, relatively few are transcribed or translated;
most are pseudogenes or gene fragments. The ex-
pressed class I genes are subdivided into class Ia, which
includes HLA-A, -B, and -C, and class Ib, which includes
HLA-E, -F, and -G. HLA class II (HLA-D) genes, if
transcribed, are not translated in human trophoblast
HUNT ET AL.

Figure 2. A schematic illustration of the human fetus, pla-
centa and extraplacental membranes, and modified endome-
trium known as decidua. The fetus developing within the
amniotic sac is surrounded and encased by trophoblast cells
in the placenta and chorion membrane (red). Upper insert:
Cytotrophoblast (CTB) cells within the placental villi serve as
the progenitors for all differentiated trophoblast cell sub-
populations, including the syncytiotrophoblast (sTB) layer,
which is continuously exposed to maternal blood. This single
cell layer is responsible for transfer of maternal-fetal nutrients
and wastes, synthesis of placental hormones, and providing a
physical barrier to maternal cell traffic into the fetus. CTB
cells proliferate and migrate into the decidua, attaching the
placenta to the mother and facilitating certain crucial physi-
ological events required for successful pregnancy. Lower
insert: The amnion membrane comprised of a single layer of
epithelial cells is a strong sac holding the fetus in amnionic
fluid. The chorion membrane CTB cells, derived from the
migrating extravillous CTB cells, interface directly with ma-
ternal decidual cells.
cells even under inducing conditions (19) and will not
be discussed further in this review.
One remarkable difference between the HLA class Ia
and Ib genes is that the former are highly polymorphic,
with many alleles, and the latter have few variants.
Major differences have been observed in glycoproteins
associated with these two subsets of class I antigens. In
general, class Ia antigens are membrane bound. By
contrast, one member of the class Ib group, HLA-G, is
alternatively spliced. Seven alternatively spliced tran-
scripts have been identified, of which four are pre-
dicted to encode membrane bound and three are
predicted to encode soluble proteins. A final difference
is that expression of class Ia antigens is ubiquitous
whereas expression of class Ib antigens may be tissue/
organ-specific and/or conditional.
Human trophoblast cells express one class Ia mole-
cule (HLA-C) and all three class Ib molecules. The
HLA-C gene is moderately polymorphic, and could
stimulate maternal anti-fetal acquired immunity if pa-
ternal alleles differed from maternal. Yet allelic dispar-
HLA-G AND PREGNANCY IMMUNE PRIVILEGE
ity at the HLA-C locus does not seem to be a causal
factor in infertility or termination of pregnancy. The
other HLA class I antigens expressed by trophoblast
cells are HLA-E, -F, and -G. These class Ib antigens are
distinguished by low numbers of alleles that differ at
the protein level. For example HLA-E has 2 alleles (20)
and HLA-G has five alleles (reviewed in ref 21). There
are no allelic variants of HLA-F in the published
literature, although two nonsynonymous (amino acid)
substitutions are reported in online databases (http://
genome.ucsc.edu), which are likely rare in the general
population. Moreover, most polymorphisms in the
HLA-G gene do not alter the amino acid sequence; the
few that do are not predicted to change secondary
structures of the heavy chains (4, 21).
Of the HLA class Ib molecules expressed by tropho-
blast cells, HLA-G was the first to be identified and
remains an antigen of great interest and a focus of
experimental evaluation (reviewed in refs 22–24).
Structural features of HLA-G
Although the genomic structure of HLA-G is similar to
other class I genes, it is unique in most other respects.
The HLA-G gene has eight exons encoding a signal
peptide (exon 1), the ␣1, ␣2, and ␣3 domains (exons 2,
3, and 4, respectively), the transmembrane domain
(exon 5), and the intracellular domain (exons 6 and 7),
similar to other class I genes (Fig. 3). However, a
premature stop codon in exon 6 results in a truncated
cytoplasmic tail that reveals a cryptic retrieval motif
(25). This results in the slower turnover and prolonged
expression of HLA-G at the cell surface, and possibly
the inefficient presentation of exogenous peptides.
Park and colleagues interpret this as evidence that the
primary function of HLA-G is not antigen presentation
but as an inhibitory ligand for NK cells (25).
A second unique feature of HLA-G is that it encodes
multiple isoforms as a result of alternative splicing. Five
of the seven transcripts that result from alternative
splicing are shown in Fig. 3. The full-length isoform
HLA-G1 is structurally similar to other class I genes,
except for the truncated cytoplasmic tail. The G2
isoform results from the removal of exon 3 and ho-
modimerizes to form an HLA class II-like structure (26,
27). These two isoforms are expressed as soluble pro-
teins (HLA-G5 and -G6, respectively) due to the inclu-
sion of intron 4 sequences in the mature mRNA,
resulting in secreted proteins with an additional 21
amino acids (encoded by intron 4 sequences) following
the ␣3 domain (28). HLA-G3 results from the removal
of exons 3 and 4. HLA-G4 and -G7 (not shown in Fig. 3)
mRNAs are not abundant in placentas. Exon 4 (encod-
ing the ␣3 domain) is spliced out of the HLA-G4
transcript; the HLA-G7 transcript includes exon 2 and
part of intron 2 and is predicted to encode a small
soluble isoform.
Compared with the classical class I genes, the most
polymorphic genes in the human genome, HLA-G has
relatively little polymorphism in its coding region.
683
Figure 3. Multiple HLA-G proteins re-
sult from alternative mRNA splicing.
Upper: The HLA gene is composed of 8
exons arranged in the same sequence as
other HLA class I genes. The gene is
alternatively spliced to yield 7 tran-
scripts. In two of these, a stop sequence
in intron 4 results in soluble isoforms.
Alleles encoded by the polymorphisms
and their amino acid substitutions or
deletion are shown: *0103 (Thr31Ser),
*0104 (Leu110ILe), *0105 (1597deltaC),
*0106 (Thr258Met). A 14 bp insertion/
deletion is present in exon 8 in the 3⬘
UTR. ␣1, ␣2, ␣3 extracellular domains.
Lower: Three messages encode mem-
brane isoforms (HLA-G1, -G2, -G3) and
two encode soluble isoforms (HLA-G5
and -G6, also known as sG1 and sG2,
respectively). HLA-G1 and -G5 associate
with light chain, ␤2m; the other three
do not. Isoforms HLA-G4 and -G7 re-
main poorly defined and are not illus-
trated.
Figure 3 shows the location of the 13 polymorphisms in
exons 1– 4 that have been identified to date and one in
the 3⬘UTR. Polymorphisms at codon 31 in the ␣1
domain (Thr3 Ser), at codon 110 in the ␣2 domain
(Leu3 Ile), and at codon 258 in the ␣3 domain
(Thr3 Met) result in an amino acid substitution; poly-
morphisms at nucleotide ⫹15 and ⫹36 in exon 1, at
codons 35, 57, and 69 in exon 2, at codons 93, 100, and
107 in exon 3, and at codon 188 in exon 4 do not alter
the amino acid sequence of the protein. A single base
pair (bp) deletion at nucleotide 1597 causes a frame-
shift at amino acid 130 (29), resulting in nonfunctional
HLA-G1 and -G5 proteins (30). The polymorphisms
that alter the protein sequence define five alleles, called
G*0101, G*0103, G*0104, G*0105N, and G*0106. Si-
lent variation within these allelic classes defines sub-
types, referred to as G*010101, G*010102, etc. A 14 bp
insertion/deletion polymorphism is present in the un-
translated exon 8.
In contrast to the class Ia HLA loci, amino acid
substitutions at codons 31 and 110 in the ␣1 and ␣2
domains, respectively, are conservative changes that
occur in residues that are not predicted to interact with
bound peptide or T cell receptor (21). The third amino
acid polymorphism at codon 258 is a nonconservative
substitution in the ␣3 domain that is highly conserved
in the class Ia genes (31). It is located in the pleated
sheet structure of the ␣3 domain, where it might affect
recognition of CD8 in the HLA-G1 and -G5 isoforms, or
binding to CD4 in the HLA-G2 and -G6 isoforms (4).
Last, a polymorphic 1 bp deletion of a cytosine (C)
residue at codon 130 results in a null allele (called
G*0105N), which does not encode functional HLA-G1
or -G5 protein isoforms (30). This mutation, called
1597⌬C, occurs in the homozygous form in ostensibly
healthy individuals, indicating that HLA-G1 and -G5
isoforms are not essential for fetal survival (30, 32). In
these situations other isoforms presumably suffice (33).
684 Vol. 19 May 2005 The FASEB Journal
However, this null allele has been associated with
increased risk for recurrent miscarriage (34, 35), sug-
gesting that HLA-G1 and/or -G5 proteins do indeed
play an important role in the maintenance of preg-
nancy and that reduced levels of one or both is a risk
factor for recurrent miscarriage.
A 14 bp insertion/deletion polymorphism in the
untranslated exon 8 was first described by Harrison and
colleagues (36), but has recently been shown to influ-
ence mRNA transcript size and stability. The presence
of the 14 bp insertion allele generates a 92 bp deletion
in the 3⬘UTR of the G*01012 and G*01013 mRNAs,
possibly because it acts as a cryptic splice site (37).
Transcripts with the 92 bp deletion were associated with
more stable mRNA in JEG-3 cells (which are homozy-
gous for G*01013) and in an M8 cell line transfected
with G*01012, perhaps because they were less suscepti-
ble to degradation (38). Further, the relative abun-
dance of the alternatively spliced transcripts may be
influenced by polymorphisms in HLA-G (39). For ex-
ample, studying mRNA from term trophoblast cells,
Hviid et al. (39) showed that heterozygotes for the
G*01012 allele (but not the G*01013 allele) had re-
duced levels of transcripts encoding membrane-bound
isoforms (G1, G2, G3) with the 14 bp insertion, whereas
heterozygotes for the G*01013 allele (but not for the
G*01012 allele) had higher levels of the G2/G4 tran-
scripts than the G*01011 allele (39). Although the
significance of these findings is not clear, it is notewor-
thy that a number of studies have demonstrated re-
duced expression of HLA-G mRNA or protein in term
placentas of pre-eclamptic pregnancies (40 – 43). The
lack of association between the G*0105N null allele and
pre-eclampsia in one study suggested that deficiencies
of the HLA-G1 and/or -G5 isoforms were not a risk
factor for pre-eclampsia (44), but reduced expression
of other isoforms, such as the short G3 transcript, may
influence risk (45). Indeed, the relative abundance of
HUNT ET AL.
transcripts and noncoding polymorphisms in HLA-G
have been associated with pre-eclampsia (45), suggest-
ing that the regulation of expression of HLA-G is
influenced by genetic variation.
This suggestion is further supported by the recent
discovery of variation in the 5⬘-upstream regulatory
region of HLA-G (46, 47). In contrast to the limited
polymorphism in the exons, the upstream region con-
taining all of the known promoter and regulatory
elements is extraordinarily polymorphic (Fig. 4). To
date there have been 18 polymorphisms identified in
the ⬃1300 bp upstream from exon 1. Most are very
common polymorphisms with minor allele frequencies
(⬎20%), and many reside within or close to known
transcription factor binding sites (Fig. 4). These poly-
morphisms define at least eight unique haplotypes
(47). Some promoter region haplotypes are shared
among alleles that differ in their coding regions (for
example, G*01012, G*0105N, and G*01061 have iden-
tical promoter region sequences), while some alleles
with identical coding regions have different promoter
region haplotypes (e.g., the common G*01011 allele is
associated with at least three different promoter region
sequences). One variant, –725G, changes the methyl-
ation status of a CpG dinucleotide in the promoter
region and has been associated with an increased risk
for sporadic miscarriage in an unselected sample of
healthy women, which suggest at least some of these
polymorphisms may influence transcription and mRNA
abundance (47).
Patterns of expression of HLA-G
Figure 2 illustrates that placentas contain several dis-
tinct subpopulations of trophoblast cells that arise from
progenitor cells within the trophectoderm layer of the
blastocyst. These progenitor cells may merge to form a
syncytialized single cell layer that interfaces directly
with maternal blood or proliferate to form columns of
HLA-G AND PREGNANCY IMMUNE PRIVILEGE
extravillous cytotrophoblast cells that contact and infil-
trate the decidua (upper insert, Fig. 2). The migrating
cells anchor the placenta to the decidua and infiltrate
maternal spiral arteries, thus facilitating blood flow to
the placenta. The extravillous cytotrophoblast cells
ultimately regress to form the chorion membrane
(lower insert, Fig. 2).
HLA class I antigen expression was first identified
(and usually is described) as being restricted to the
extravillous trophoblast cell population, with proteins
particularly prominent in cells immediately adjacent to
the decidua in both early and late gestation placentas
(reviewed in refs 22–24). However, the reagent used
most frequently to identify these antigens in tissue
sections or isolated cells, the mouse monoclonal anti-
body W6/32, requires light chain (␤2m)/heavy chain
association, so neither free heavy chains nor isoforms
such as HLA-G2 and -G6 that do not associate with
␤2-microglobulin (␤2m) were detected. More recent
studies using monoclonal antibodies that identify the
amino acid sequence derived from intron 4 nucleo-
tides—16G1 generated by D. Geraghty being a good
example— have supplied localization data on HLA-G5
and -G6 heavy chains. New isoform-specific antibodies
show that HLA-G5 is present throughout the placenta and
within the chorion membrane, decidua, and maternal
blood (48). In villous CTB cells, HLA-G5 is likely to be
mainly free heavy chain (J. S. Hunt and P. Morales,
unpublished results) as ␤2m is missing. An antibody
recognizing HLA-G2 and -G6 shows that one or another
of these isoforms is prominent in/on extravillous cytotro-
phoblast cells distal to the placental villi, cytotrophoblast
cells infiltrating the decidua, and some chorion mem-
brane cytotrophoblast cells (48) (Fig. 5). HLA-G2/G6 is
located in the same cells as HLA-G1 (43), suggesting that
in women carrying the null allele (G*0105N), which does
not encode functional HLA-G1 or -G5 protein isoforms,
HLA-G2/G6 may comprise an adequate substitute (30).
Figure 4. Variation in the 5⬘-upstream
regulatory region of HLA-G. Eight
unique haplotypes are defined by the
polymorphisms. Note that polymor-
phisms are frequently associated with
transcription factor binding sites and
could affect the efficiency of transcrip-
tion of HLA-G.
685
 rosine phosphorylation, SHP-1 association, and calcium
regulation, may be translated into expression of specific
genes in decidual leukocytes as required for program-
ming the cells into pregnancy-appropriate behavior.

FUNCTIONS OF HLA-G

Although it has been proposed that HLA-G may be an

evolutionary artifact without function (53), recent stud-
ies using HLA-G proteins from transfected cells indi-
cate that these proteins may regulate immune cells and
thus be integral to immune privilege in pregnancy.
Figure 6 shows that HLA-G proteins probably target all
of the major immune cell subsets. In the following
paragraphs we describe activities related to T and B
lymphocytes, NK cells and antigen-presenting cells
Figure 5. HLA-G2/G6 isoforms are induced during cytotro-
phoblast migration and invasion. First trimester decidua is (APC).
shown in the left panel; a cytotrophoblast column is shown in
the right panel. Immunostaining with the anti-HLA-G5 mAb HLA-G interactions with T lymphocytes
1-2C3 identifies positive cells in the maternal decidua, the
proximal cytotrophoblast column and trophoblast cells in the Wegmann and co-workers were the first to report that
villus (pink label, small arrows). Double staining with the
in pregnant mice, Th2 cytokine-producing lymphocytes
anti-HLA-G2/G6 mAb 26-2H11 identifies positive cells in the
decidua and at the leading edge of the cytotrophoblast cell flourish in preference to those producing Th1 (13).
column (brown label, large arrows). Original magnifications, Subsequent research in women has shown that failure
⫻200. to achieve this preference for Th2-type, anti-inflamma-
tory cytokines may lead to unsuccessful pregnancy (54).
Receptors for HLA-G The idea that trophoblast cell signals drive T cells into
this anti-inflammatory profile has emerged as a popular
explanation for maternal tolerance to the semialloge-
The original view of how regulation of the HLA class I
neic fetus.
antigens in fetal trophoblast cells might contribute to
Convincing evidence for an ability of HLA-G to
maternal tolerance during pregnancy was restricted to
influence T cells was first presented by Sanders et al.
the negative: a failure to express these antigens meant
(55), who showed that HLA-G-expressing cells bind to
that T lymphocytes would not recognize trophoblast
CD8␣-expressing cells. This finding has recently been
cells as foreign, thus allowing them to survive (49). The
confirmed by Shiroishi et al. (56). In both studies,
finding that subpopulations of trophoblast cells invad-
HLA-G binding to the ␣␣ CD8 homodimer, the molec-
ing the decidua express HLA class I antigens (50) drove
ular form expressed by a subset of T cells in the
investigators to revise their thinking and explore new
explanations for placental immune privilege.
Binding of trophoblast cell HLA-G to receptors that
inhibit activating signals on decidual leukocytes may be
the answer (Fig. 6). HLA-G appears to be recognized
mainly by immunoglobulin-like transcript (ILT) recep-
tors, which are expressed by T and B lymphocytes, as
well as by NK cells and mononuclear phagocytes (51),
and abrogate activating signals received by these cells.
Whether these ILT receptors or others, such as TcR
and the CD94/NKG2A HLA-E receptors on uterine
CD56bright NK cells, are more important to lymphocyte
recognition remains to be established. Early studies
suggested that ILT4 may be the main receptor for
HLA-G exhibited by monocyte/macrophages, the sec-
ond most populous leukocyte population in the human
decidua (6). However, ILT2 has not been formally
excluded as a mononuclear phagocyte recognition
entity, and recent experiments in a macrophage cell
Figure 6. Potential receptors on immune cells targeted by
line suggest these two receptors may exhibit isoform- HLA-G. The six subsets of leukocytes believed to be targeted
specific binding (52). This novel finding illustrates the by HLA-G and potential receptors for HLA-G on each cell
point that little is known of how HLA-G-activated type are shown. TcR, T cell receptor; ILT, immunoglobulin-
signaling pathways, which for ILT2/ILT4 include ty- like transcript; KIR, killer inhibitory receptor.

686 Vol. 19 May 2005 The FASEB Journal HUNT ET AL.

intestine and by NK cells (57), was evaluated. Yet most
T cells express the CD8␣␤ heterodimer, which acts as a
coreceptor to the T cell receptor (TcR) and an essential
signal transduction molecule during T cell activation.
Interaction between the TcR and HLA-G has not been
demonstrated. Many investigators question whether
binding to the TcR would be a primary function of
HLA-G because of its limited polymorphism (58). How-
ever, it was recently shown that cytomegalovirus-derived
peptides can stabilize surface HLA-G expression, lead-
ing to HLA-G-restricted, cytomegalovirus-specific cyto-
toxic T cell response in transgenic mice (59). This
strongly suggests that HLA-G can indeed act as a
classical presenter of foreign peptide. In solving the
debate of whether there is any role for cooperative
physiological binding between HLA-G and the
␣␤TcR, it will be important to analyze HLA-G-derived
peptides from women with known human cytomega-
lovirus infection.
Functional studies have yielded even stronger evi-
dence. In vitro studies have clearly demonstrated an
ability of soluble and membrane-associated HLA-G to
modulate cytokine release from human allogeneic
peripheral blood mononuclear cells (60) and to have
a concentration-dependent effect on generation of
an allogeneic CTL response (61). Regarding initia-
tion of the acquired immune response, Le Maoult et
al. (62) have shown that APC transfected with
HLA-G1 prevent proliferation of CD4⫹ T cells and
direct them toward an immunosuppressive pheno-
type. Although Le Maoult et al. did not test CD4⫹
cells to ascertain whether they might be related to
the CD4⫹CD25bright T regulatory phenotype appar-
ently essential for pregnancy in mice (63) and possi-
bly in women (64), it is tempting to speculate this
might be the case. The idea that this phenotype
might be the consequence of exposure to a murine
Qa-2, which bears structural similarities to HLA-G
(65), is intriguing.
Membrane-associated and/or soluble HLA-G may
play a critical role in regulating CD8⫹ T cells during
pregnancy by eliminating alloreactive (antipaternal) T
cells. Two independent groups have reported that
HLA-G induces CD8⫹ T cell apoptosis (66 – 68). In both
models, exposure of CD8⫹ T cells to soluble HLA-G was
shown to trigger surface expression and secretion of Fas
ligand, resulting in death of the activated T cells
through the Fas/FasL pathway. These experiments
were based on the well-documented ability of other
HLA antigens to induce Fas/FasL-related cell death, a
phenomenon that has been identified in patients un-
dergoing transplantation and is believed to dispose of
alloreactive CD8⫹ cells (69). In pregnancy, soluble
HLA-G might induce apoptosis in CD8⫹ cells that react
to paternal antigens; soluble HLA-G has been reported
in maternal sera by several groups (70, 71).
A second mechanism by which HLA-G may induce
maternal tolerance to fetal antigens is to reduce or
prevent cytotoxic activity of CD8⫹ T cells against target
cells, possibly independent of inducing T cell apopto-
HLA-G AND PREGNANCY IMMUNE PRIVILEGE
sis. The specific receptor capable of binding HLA-G is
not clearly defined, but could be either the CD8
molecule itself, as proposed by Contini et al. (66) and
Shiroishi et al. (56), or the immunoglobulin-like tran-
script receptor 2 (ILT2), which transduces an inhibi-
tory signal (61) (Fig. 6). HLA-G1 and its soluble
counterpart HLA-G5 protect potential target cells from
lysis by antigen-specific cytotoxic T cells (61, 66, 72).
The same protection by the smaller isoforms of mem-
brane HLA-G, HLA-G2, -G3, and -G4 has now been
shown (73), which is consistent with the idea that
smaller isoforms compensate when HLA-G1 and -G5
are absent in mothers who are homozygous for the
G*0105N allele (74).
Intriguingly, HLA-G down-regulates expression of
CD8␣ mRNA and protein in IFN-␥-treated blood mono-
nuclear cells without inducing apoptosis or altering
CD3 expression (48). Although T cells and NK cells
express CD8␣/␤, signals for CD8␣ specific message and
protein in Northern blots and immunoblots were
strong, suggesting it is the more numerous T cell
affected. Because the effects of HLA-G are highly
concentration dependent and HLA-G-producing cells
are in the placental bed, these results imply that soluble
isoforms of placental HLA-G might reduce the ability of
T cells to function effectively in the pregnant uterus but
be less potent in the periphery (48, 71).
When considering the impact of HLA-G on T cells, it
is critical to bear in mind two facts: 1) T cells are not
numerous in the decidua (6), and 2) trophoblast cells
in normal, healthy placentas are unlikely to elicit a CTL
cell response. The inability of trophoblast cells to
stimulate CTL is due to the fact that polymorphic HLA
class I antigens are absent on syncytiotrophoblast and
are scarce on other subpopulations. However, an ex-
ception might occur in the case of virally infected
extravillous trophoblast cells, where trophoblast cell-
associated HLAs might present antigen to CD8⫹ cells,
thus becoming potential targets themselves.
HLA-G interactions with B lymphocytes
ILTs appear to be major receptors on T lymphocytes
and antigen-presenting cells that interact with HLA-G,
as described above. They are also present on B lympho-
cytes, which express ILT2 inhibitory receptors (75). So
far there is no evidence for binding of HLA-G to these
receptors on B cells or of B cells responding directly to
HLA-G; yet the possibility exists that this occurs, be-
cause production of antibodies to placental HLA-G
occasionally occurs in pregnant women.
A recent study in our laboratories demonstrated that
maternal tolerance to HLA-G in terms of antibody
production is the usual condition (4). Ninety-one per-
cent of mothers, as well as all women who had never
been pregnant and all men, lack antibodies to HLA-G
in their sera. Yet tolerance is not absolute: ⬃9% of
women who had undergone at least one pregnancy
generated anti-HLA-G antibodies that were readily
identified in maternal sera by ELISA and immunoblot-
687
ting. Whether a “mimic” antigen on microbes or other
environmental molecules might stimulate these anti-
bodies is not known, but since the anti-HLA-G antibod-
ies were found only in women who had been pregnant,
some association with the state of pregnancy would be
required. Not unexpectedly, maternal anti-HLA-G an-
tibodies have no deleterious effect; all the women who
developed these antibodies had multiple successful
pregnancies. In this respect, circulating anti-HLA-G
antibodies resemble other anti-HLA antibodies, which
are common in pregnancy and do not damage the
developing fetus. Because several HLA-G alleles have
been identified, we tested the hypothesis that mothers
would generate antibodies to foreign (paternal) alleles,
but found no relationship between exposure to a
foreign (paternal) HLA-G protein on placental cells
and maternal production of anti-HLA-G (4).
How tolerance is achieved remains to be elucidated.
Potential routes include 1) activation of B cell ILT2
receptors by circulating HLA-G5 or -G6, an idea that
remains untested, and 2) activation of ILT2 receptors
on Th lymphocytes, as suggested by Le Maoult and
colleagues (62). The potential role of APC driven into
a B lymphocyte-inhibiting immunosuppressive profile
should not be overlooked, as these cells are abundant at
the maternal-fetal interface where HLA-G isoforms are
prominent. Alternatively, HLA-G produced in the pla-
centa during fetal life might well induce lifelong toler-
ance in the adult.
In any event, data accumulated to date, though
scarce, strongly support the idea that inhibition is
specific to HLA-G and that the tolerogenic mechanisms
underlying specific repression of production of mater-
nal antibodies are immensely effective. The deficiency
of anti-HLA-G antibodies is not due to maternal, preg-
nancy-associated reduction in B cell function. Mothers
produce high levels of other types of antibodies in
order to provide defense against pathogens during
pregnancy and assure transfer of protective antibodies
to the fetus. The placenta supports this hyperproduc-
tion. A recent study from our laboratory shows that
human placentas produce two non-apoptosis-inducing
TNF superfamily ligands: BAFF (also known as BlyS, or
B lymphocyte stimulator, as well as TALL-1, THANK,
and zTNF4) and APRIL (a proliferation inducing li-
gand), both of which sustain B lymphocytes (76).
Placental BAFF and APRIL therefore may bear a degree
of responsibility for maternal host defense and provi-
sion of antibodies to the fetus required in the postpar-
tum period.
HLA-G interactions with NK cells
A complete absence of HLA class I molecules on
trophoblast cells could provide protection against CTL
but could target the trophoblast subpopulation migrat-
ing into the decidua (Fig. 2, upper insert) for destruc-
tion by resident NK cells, a leukocyte programmed to
recognize and destroy HLA null cells. Expression of
HLA class Ib genes HLA-E, -F and -G may be an
688 Vol. 19 May 2005 The FASEB Journal
evolutionary move to avoid this risk. As noted above,
these genes have few alleles and their products are
unlikely to be recognized as foreign by the mother.
NK cells of an unusual phenotype, CD16–CD56bright,
are abundant in first and second trimester decidua but
decline in number thereafter (6). These cells are poor
killers of the usual NK targets, suggesting that environ-
mental conditions in the pregnant uterus affect their
functions. Decidual NK cell cytotoxicity could be by-
passed by ligation of HLA class I antigens to one or
more NK cell surface inhibitory receptor. Transfection-
based assays initially suggested that interaction between
HLA-G and the CD94/NKG2A heterodimer on the
surface of NK cells prevented cytolysis induced by NK
cells (77–79). Further investigation, however, led to the
realization that class Ib gene HLA-E utilizes a leader
peptide derived from other class I proteins, including
HLA-G, to stabilize its own surface expression. Thus,
initial findings led to the erroneous conclusion that
HLA-G was responsible for reducing NK-mediated cy-
tolysis via CD94/NKG2A, when in fact HLA-G expres-
sion only permitted expression of HLA-E, the true
ligand for this inhibitory receptor (80, 81).
Nonetheless, the idea that HLA-G serves as the major
inhibitor of NK toxicity at the maternal fetal interface
remains popular because experiments using HLA-G-
specific antibodies show that trophoblast cell HLA-G
inhibits NK-mediated cell death in the absence of
HLA-E (82, 83). These effects could be mediated
through the killer inhibitory receptor (KIR) 2DL4,
through ILT2, or both (75, 84, 85). ILT2 has been
shown to bind HLA-G and is expressed by decidual NK
cells, albeit the quantity of ILT2 on decidual NK cells
may be low (86 – 89). More controversial is a role for
KIR2DL4, which in soluble form binds to HLA-G-
transfected cells (84, 85). More recently, however,
biochemical analysis of KIR2DL4 binding to HLA-G
monomers and dimers yielded only negative results,
raising the question of validity of HLA-G-KIR2DL4
interaction (90). Furthermore, decidual NK cells ap-
pear to express an abundance of CD94/NKG2A, the
receptor for HLA-E, suggesting this interaction may
supercede that of HLA-G and its NK-expressed recep-
tors (91). A final confounding observation against a
role for HLA-G-mediated inhibition of NK cytolytic
activity is that immortalized and primary trophoblast
cells are resistant to NK cell-mediated lysis independent
of HLA class I expression (91–94), suggesting these
cells use other strategies for self protection.
New information has recently surfaced regarding a
potential role for HLA-G/NK cell interactions at the
maternal-fetal interface. KIR2DL4 ligation and treat-
ment of NK cells with HLA-G result in production of
the cytokine interferon-␥ (IFN-␥) (95–97) but, as sug-
gested above, it remains to be determined whether this
commonality is causally linked. Even so, stimulation of
NK cell production of IFN-␥ at the maternal-fetal
interface by HLA-G is an intriguing possibility. IFN-␥,
usually considered a proinflammatory cytokine, can
drive cells into immunosuppressive profiles when
HUNT ET AL.
linked with other modulators, and could therefore
paradoxically serve as an anti-inflammatory cytokine.
Whether HLA-G might stimulate IFN-␥-associated path-
ways of vascular and decidual remodeling (98 –100)
remains an intriguing possibility.
Interactions with antigen-presenting cells
Two populations of APC, macrophages and dendritic
cells, reside in the human decidualized endometrium
throughout pregnancy. These powerful, multifunctional
leukocytes are located in close proximity to invasive cy-
totrophoblast cells, uterine glandular epithelium, and
uterine blood vessels (6, 101–104), and are proposed to
play central roles in uterine and placental homeostasis as
well as immune modulation (2, 6, 101–105).
APC in pregnant endometrium include 1) CD14⫹
macrophages (106, 107), 2) CD83⫹ mature dendritic
cells (103, 104, 108), and 3) CD83– immature macro-
phage/dendritic cells. The immature cell type appears
to be a family of related cell types, some of which are
dendritic cell (DC)-SIGN⫹CD14⫹ (109) and others are
DC-SIGN–CD14–DEC-205⫹ (104). Alternatively, the im-
mature phenotype cluster may be composed of similar
cells in different transitional stages. It has been sug-
gested that DC-SIGN⫹ cells are a precursor population
capable of differentiating into macrophages or den-
dritic cells (110), an idea supported by the finding that
macrophages and immature dendritic cells have been
shown to trans-differentiate in vitro under the influ-
ence of various cytokines (111).
Decidual macrophages, which are activated in the
pregnant uterus as evidenced by expression of HLA
class II, CD11c, and CD86 antigens (112), appear to be
programmed for immunosuppression. These cells pro-
duce the lymphocyte inhibitory molecule, prostaglan-
din E2 (113, 114), elicit reduced allogeneic and autol-
ogous T cell responses in comparison with monocytes
(105, 115, 116), and spontaneously secrete anti-inflam-
matory cytokines such as interleukin (IL)-10 and trans-
forming growth factor (TGF)-␤1 (52, 114, 117). More
recently, decidual macrophages have been reported to
express B7-H1 (16), ILT3 (117), DC-SIGN (104, 110,
118), MS-1, and factor 13 (119), all of which are
markers associated with immune evasion and activation
of macrophages into a suppressive profile. Mature
CD83⫹ decidual dendritic cells appear to exhibit an
immune cell inhibitory profile. These cells secrete less
IL-12 than monocyte-derived dendritic cells and induce
Th2 cells when cocultured with naı̈ve CD4⫹ T cells. The
C-type lectin, DC-SIGN, is used for immune evasion by
several viral and bacterial pathogens, with ligand bind-
ing to DC-SIGN altering cytokine production and anti-
gen presentation to the benefit of the pathogen (120).
Thus, this molecule may be of importance to immuno-
suppression by the decidual CD83–/DC-SIGN⫹ macro-
phage/dendritic cell-like cells.
The question then arises as to how decidual APC are
driven into immune inhibitory profiles. Placental HLA-G
is a reasonable possibility. Although decidual dendritic
HLA-G AND PREGNANCY IMMUNE PRIVILEGE
cells have not been tested, mononuclear phagocytes and
decidual macrophages express the two well-described true
receptors for HLA-G: ILT2 and ILT4 (75, 121, 122). The
decidual dendritic cells are likely to exhibit these recep-
tors; studies in mice show that HLA-G tetramers bind only
to dendritic cells (123, 124). The ability of the mice to
reject grafts is strikingly diminished in the presence of
HLA-G tetramers. In humans, an immune suppressive
effect of HLA-G on APC or other immune cell function is
implied by the finding that high levels of HLA-G proteins
in the sera of heart transplant recipients is positively
associated with prolonged graft survival (125).
The development of recombinant HLA-G5 (rG5)
and rG6 from eukaryotic cells (48) has permitted us to
test the hypothesis that HLA-G drives mononuclear
phagocytes into an immune cell inhibitory profile. In
contrast to results of another group who reported that
G5 killed T cells via a Fas/FasL pathway (68), we found
that our rG5 and rG6 did not negatively affect blood
mononuclear cell viability (48) or the viability of APC
(52). This result is in accord with a study of monocyte-
derived dendritic cells indicating that HLA-G does not
decrease the viability of immature or mature dendritic
cells, alter differentiation of dendritic cells from blood
monocytes, or influence maturation of dendritic cells
(126). Recombinant G5 and rG6 drive mononuclear
phagocytes into suppressive pathways. In particular,
both strongly stimulate TGF-␤1 production by activated
APC. The condition of activation is extremely impor-
tant, as clearly illustrated in the study by Morales et al.
(48), where rG5 and rG6 reduced CD8␣ mRNA and
protein in cells treated with the monocyte-activating
factor IFN-␥ to levels the same as, not below, those of
CD8 in unstimulated cells.
Do APC themselves, when activated, express HLA-G?
This possibility was first suggested in a report from our
laboratory indicating that IFN-␥-activated, but not rest-
ing, mononuclear phagocytes contain HLA-G mRNA
and protein (127). The idea that inflammatory condi-
tions stimulate APC production of HLA-G has been
supported by numerous reports of HLA-G expression
in macrophages and dendritic cells during cytomegalo-
virus infection, lung carcinoma, nontumoral pulmo-
nary disease, and HIV infection (128 –130). Although
immunohistochemical studies may be compromised by
the use of antibodies that lack specificity, soluble iso-
forms of HLA-G have been identified by Western blot in
the sera of male heart transplant patients (125). There
are indications that APC-derived HLA-G is functional.
LeMaoult et al. (62) showed that HLA-G1-transfected
myelomonocytic cell lines (KG1a and U937) suppress
CD4⫹ T cell proliferation in a mixed lymphocyte
reaction, with the resultant T cells driven into an
immune-suppressive phenotype. The converse idea that
resting APC are not producers of HLA-G is supported
by a report from Laupeze et al., who were unable to
detect HLA-G expression in/on resting mononuclear
phagocytes or dendritic cells (131). It is therefore
puzzling that activated APC in the decidua reportedly
fail to exhibit membrane or soluble HLA-G and do not
689
contain a specific message (132). More work is needed
to understand local tissue conditions that might direct
APC into production of HLA-G, as activation is clearly
not the only important factor.
PROSPECTS AND PREDICTIONS
Human and nonhuman primate pregnancies provide
natural models for studying mechanisms of immune
tolerance and features of immune privileged sites. Studies
conducted on pregnancy over the past half century have
provided immunologists with definitive proof that in
successful transplants, which include the fetus, “foreign”
tissue will have devised overlapping and complementary
mechanisms to avoid rejection. Of these, selection of a
gene with limited polymorphism, HLA-G, for expression,
and derivation of an entire family of immunomodulatory
glycoproteins by alternative splicing of the gene’s single
message are among the cleverest.
Studying HLA-G structure-function relationships re-
minds us of several unifying principles of immunity,
including 1) the genetic variation provided by alleles, 2)
the potential of polymorphisms outside the coding region
to influence rates of transcription and regulate produc-
tion consistent with varying conditions and specific needs,
3) the power of alternative splicing, which has been
revealed in other systems to be directly related to func-
tional diversity (133), 4) the central relationship between
the concentration of an immunomodulatory molecule
and its final effects, and 5) the precise, possibly cell
type-specific conditions such as activation and inflamma-
tion that may be required for stimulating production of
the molecule and achieving desired effects.
The major HLA-G function that has emerged to date
is its ability to program cells into immunosuppressive
phenotypes. Since HLA-G is produced in great amounts
at the maternal-fetal interface, the implication is that
the immunosuppressive function must be important.
Yet definitive proof that HLA-G is required remains
elusive, as in vivo experiments are difficult to design.
Mice do not demonstrate a clear correlate of HLA-G,
although Qa-2 appears to have some of the same
structural characteristics as HLA-G and is strongly ex-
pressed in placentas (65). Nonhuman primate placen-
tal cells transcribe and translate proteins from HLA-G-
like genes, but the products are not identical to those
described above for HLA-G. For example, in baboons,
no HLA-G6-like isoform is detectable (134). Thus,
although all indications are that HLA-G serves as a key
effector of the changes that occur in maternal immune
cells with the onset of pregnancy, definitive in vivo
studies remain to be done.
Assessment of levels of HLA-G isoforms may well be
useful in predicting the ability of mothers to establish
and maintain pregnancy (71, 135, 136) as well as
determining the likelihood that patients will accept
allografts (125). If such assessments are ultimately
found to be reliable, recombinant HLA-G might be a
powerful tool for inducing a desirable state of local
690 Vol. 19 May 2005 The FASEB Journal
immune suppression so as to facilitate pregnancy or
graft acceptance, as has been shown in the porcine
model (137).
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Received for publication September 7, 2004.
Accepted for publication December 22, 2004.
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