thomas hviid, md, phd dept. of clinical biochemistry roskilde university hospital denmark...
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Thomas Hviid, MD, PhD
Dept. of Clinical Biochemistry
Roskilde University Hospital
Denmark
Reproductive Immunology
The HLA System in Pregnancy
’Why did your mother not reject you?’
Topics
Introduction: The semi-allogenic fetus
Fundamental aspects of MHC/HLA and HLA class Ib– ’Classical’ vs ’non-classical’ MHC/HLA genes– Gene structure, polymorphisms, expression, alternative
splicing, functions etc
Certain complications of pregnancy in relation to HLA/HLA-G– (Recurrent) spontaneous abortions, IVF, pre-eclampsia
Pregnancy and HLA diversity– Reproductive selection mechanisms (’mating preferences’)
and HLA
?The ’semi-allogenic fetus’
Medawar & Billingham, Nature, 1953
Four hypotheses:– The conceptus lacks immunogenicity
– Significant lowering of the immune response during pregnancy
– The uterus is an immunoprivileged site
– Immune barrier elaborated by the placenta:• Tolerance to the semi-allognic fetus by the maternal
immune system seems mainly an active mechanism:– Fetal tissue prevented from being recognized as foreign
tissue and/or being rejected by the maternal immune system
Hypotheses/concepts to explain maternal tolerance of the fetus
HLA/HLA-G expression by the trophoblast The Th1/Th2 balance Regulatory CD4+CD25+ T cells Others
– Leukemia inhibitory factor (LIF)
– Indoleamine 2,3-dioxygenase (IDO)
– Suppressor macrophages
– Hormones
– CD95 and its ligand
– Annexin II
– Lowered complement activity
– Hidden trophoblast antigens
(Thellin et al, review 2000)
HLA and the ’semi-allogenic fetus’
Human Leucocyte Antigen (HLA)Major Histocompatibility Complex (MHC)
Several classes of HLA genes:
– HLA class Ia (classical HLA class I antigens; on almost all cells)• HLA-A, HLA-B, HLA-C
– HLA class Ib (non-classical HLA class I antigens)• HLA-E, HLA-G, HLA-F
– HLA class II (expressed on antigen presenting cells, B cells)• HLA-DR, HLA-DP, HLA-DQ
Discovered / rejection of transplants (Jean Dausset 1952/1953) The function of HLA / MHC was elucidated in the 1970s
(Zinkernagel & Doherty 1974) MHC/HLA molecules present antigen peptides to T cells via the T cell receptor Antigen peptides (eg pathogenes) are recognized in combination with an
individual’s own variant of HLA
The classical HLA class Ia molecules are highly polymorphic
HLA-A10HLA-B12HLA-Cw5
HLA-A26HLA-B8
HLA-Cw5
HLA-A25HLA-B40HLA-Cw2
HLA-A11HLA-B16HLA-Cw8
HLA-A23HLA-B12HLA-Cw1
HLA-A3HLA-B5
HLA-Cw7
HLA-A2HLA-B27HLA-Cw6
HLA-A24HLA-B8
HLA-Cw4
HLA-A25HLA-B12HLA-Cw1
HLA-A19HLA-B15HLA-Cw2
HLA-A19HLA-B14HLA-Cw8
HLA-A28HLA-B17HLA-Cw5
The non-classical HLA class Ib molecules are
nearly monomorphic
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA-GHLA-EHLA-F
HLA in pregnancy
HLA-Am, -Bm, -Cm
HLA-Am, -Bm, -Cm
m = maternalp = paternal
HLA-Am, -Bm, -Cm
HLA-Ap, -Bp, -Cp
HLA-Gm, -Em, -Fm, -Cm
HLA-Gp, -Ep, -Fp, -Cp
Mother – HLA class Ia
Fetus – HLA class Ia
Placenta – HLA class Ib
class II class III class I
DP DQ DR B C E A G F
Human Leucocyte Antigen (HLA) systemMajor Histocompatibility Complex (MHC)
Chromosome 6
HLA class Ia and II (-A, -B, -C, -DR etc): highly polymorfic
HLA class Ib (-G, -E, -F): nearly monomorphic
HLA-G expression in the blastocyst
HLA-G expression can be detected already in the blastocyst
IVF = in vitro fertilization (= ”reagensglasbefrugtning”)’preimplantation human embryos’ (or blastocysts)
HLA-G expression in the blastocyst/embryo
Detection of HLA-G mRNA in around 40% of preimplantation human embryos (Jurisicova et al 1996, Cao et al 1999)
No detection of HLA-G mRNA in human embryos
(but only 11 embryos investigated) (Hiby et al 1999)
Detection of soluble HLA-G in some human embryo culture supernatants from IVF after 46-72 hrs (in total >1000 embryo cultures) (Fuzzi et al 2002, Sher et al 2004, Noci et al 2005, Yie et al 2005)
36% sHLA-G pos. of single embryo cultures (Noci et al 2005)
No detection of sHLA-G in human embryo cultures (Lierop et al 2002)
Expression of HLA-G mRNA and sHLA-G has been associated with an increased cleavage rate, as compared to embryos lacking HLA-G (Jurisicova et al 1996, Yie et al 2005)
Soluble HLA-G and success of IVF
The pregnancy rate in women who have embryos transferred from cultures where sHLA-G is detected is significantly higher than that in women who have only embryos transferred from sHLA-G negative cultures (Fuzzi et al 2002, Sher et al 2004, Noci et al 2005, Yie et al 2005)
Pregnancy and live births are observed in sHLA-G-neg. IVF cycles; however, the rate of spontaneous abortions is higher in the HLA-G-negative group (25%) vs. the HLA-G-positive group (11%)
(Yie et al
2005)
HLA-G expression
HLA-G positive (normal tissue):– Placenta extravillous cytotrophoblast (EVCT), dedicua
invading EVCT, syncytiotrophoblast (sHLA-G)
– Thymus, some monocytes and T-cells, sporadic in a few other cell types/tissues
Anchoring villous
DeciduaHLA-G HLA-G Cytokeratin
(From Emmer et al. Human Reproduction 2002; 17:1072)
(12.-13. weeks of gestation)
HLA-G gene, mRNA, protein, isoforms
1Exon
HLA-G gene
Signal peptide 1-domain 2-domain 3-domain
Trans-membraneregion
Cytoplasmicdomain
3’ UTR
0 1 2 3 4 kb
2 3 4 5 6 7 8
HLA-G protein
AATAAA
CCAAT
TCTAAA
TGA
5’-ATTTGTTCATGCCT-3’14-bp deletion polymorphism
(nt 3741)
e1 e2 e3 e4 e5 e6 e1 e2 e4 e5 e6 e1 e2 e5 e6 e1 e2 e3 e4 e5 e6e1 e2 e3 e5 e6 e1 e2 e4 e5 e6i4 i45’ 5’ 5’ 5’ 5’ 5’3’ 3’ 3’ 3’ 3’ 3’
HLA-G mRNA
Full-length Exon 3 spliced out Exons 3 and 4spliced out
Exon 4 spliced out Retains intron 4 Retains intron 4,exon 3 spliced out
12 1
3 1
1
23
12
3
1
3
HLA-G1
(membrane-bound isoforms)
HLA-G2 HLA-G3 HLA-G4
Soluble HLA-G5 HLA-G6
e8 e8 e8 e8 e8e8
codon 31: ACG/TCG (G*0103)codon 93: CAC/CAT (G*010102/0105N/0106)codon 107: GGA/GGT (G*010103)codon 110: CTC/ATC (G*0104)1597delC (G*0105N)codon 258: ACG/ATG (G*0106)
e1 i25’ 3’
Retains intron 2
1
HLA-G7
STOP codon STOP codon STOP codon
HLA-G1
1-domain
3-domain
2-domain
-2m
Other alternatively spliced HLA-G mRNA isoforms exist
(Harrison et al 1993)
+14 bp: 45 %14 bp deleted: 55 %
14 bp del polymorphism
HLA-G alleles (DNA sequences)
HLA-G 5’URR/Promotor*) Exon 2 Exon 3 Exon 4 3’UTR*)
alleles -725 -201 31 35 54 57 69 93 100 107 110 130 188 236 241 258 nt 3741
G*010101 C or G G ACG CGG CAG CCG GCC CAC GGC GGA CTC CTG CAC GCA TTC ACG -
G*010102 C A --- --- --- --A --- --T --- --- --- --- --- --- --- --- +14 bp
G*010103 C A --- --- --- --A --- --- --- --T --- --- --- --- -C- --- +14 bp
G*010104 C G --- --- --- --- --T --- --- --- --- --- n.d. n.d. n.d. n.d. n.d.
G*010105 n.d. n.d. --- --- --- --- --- --- --- --T --- --- --- --- --- --- -
G*010106 n.d. n.d. --- --- --- --- --- --- --- --- --- --- --T --- --- --- n.d.
G*010107 n.d. n.d. --- --- --- --A --- --T --- --T --- --- --- --- --- --- n.d.
G*010108 C G --- --- --- --A --- --- --- --- --- --- --- --- --- --- -
G*0102 n.d. n.d. --- --- -G- --- --- --- --- --- --- --- --- --C --- --- n.d.
G*0103 C (or T) G T-- --- --- --- --- --- --- --- --- --- --T n.d. n.d. --- +14 bp
G*010401 C A --- --- --- --A --- --- --- --- A-- --- --- --- --- --- -
G*010402 n.d. n.d. --- --- --- --C --- --- --- --- A-- --- --T --- --- --- n.d.
G*010403 n.d. n.d. --- --- --- --- --- --- --- --- A-- --- --- --- --- --- -
G*0105N C A --- --- --- --A --- --T --- --- --- TG --- n.d. n.d. --- +14 bp
G*0106 C A --- --- --- --A --- --T --- --- --- --- --- --- --- -T- +14 bp
G*0101g**) n.d. n.d. --- --A --- --- --- --- --- --- --- --- --- n.d. n.d. --- +14 bp
G*G3d5**) n.d. n.d. --- --- --- --A --- --T --T --- --- --- --- n.d. n.d. --- +14 bp
Amino acid substitution
HLA-G polymorphismsConsensus: Only a handful of HLA-G alleles with amino acid substitutions
Around 15 HLA-G alleles at the DNA level(Bodmer et al. Hum Immunol 1999; 60:361)
Deletion of a cytosine (codon 129/130)frameshift
HLA-A HLA-G
(After Ober & Aldrich, J Reprod Immunol 1997; 36:1-21 and Parham, Eur J Immunogenetics 1992; 19:347-359.Based on work by Bjorkman et al, Nature 1987; 329:512-518).
Threonin Serin
Leucin Isoleucin
HLA-G*0105N HLA-G*0105N is a so-called null allele
– One base pair is deleted in exon 3 of the HLA-G gene
(Most likely) non-functional HLA-G1 and HLA-G5 (full membrane and soluble isoforms)
Clinical data on HLA-G*0105N homozygotes shows that HLA-G1 and –G5 are not essential for fetal survival
However, normal HLA-G2 – G4 and G6/G7 are encoded and these isoforms seem to be functional in much the same way as G1/G5
(Sala et al 2004, Le Discorde et al 2005)
HLA-G functions
Possible contributions of HLA-G in the implantation process:
1) Attachment of the blastocyst to the endometrium• HLA-G has been found to be involved in cellular adhesion
(Ødum et al 1991)
2) Trophoblast invasion of uterine tissue and maternal spiral arteries• HLA-G is expressed by endovascular trophoblast cells and may
be a modulator of angiogenesis (Le Bouteiller et al)
3) Trophoblast interaction with maternal immune effector cells• HLA-G interacts with receptors on immune cells
responder T cellT cellreceptor
stimulator cell
HLA-DR4
HLA-DR4
HLA-DR1
HLA-G
Inhibition of allo-CTL response
IL-10 TNF- INF-
Allo-cytotoxic T lymphocyte (CTL) response
HLA-G
Augmentation of theallo-CTL response
IL-10 TNF- INF-
UNCOMPLICATED PREGNANCYRECURRENT MISCARRIAGE AND PRE-ECLAMPSIA
Upregulation of the Th1 response,downregulation of Th2: IL-2 INF- (TNF-)
(Kapasi et al 2000)
Th2 cytokine production:IL-4IL-5IL-10IL-13
The Th1/Th2 balance
Successful pregnancy more often correlated with a Th2-type response than Th1
However, the Th1/Th2 concept may be too simplistic
HLA-G/sHLA-G???
Functions of HLA-G Several in vitro studies have shown that HLA-G and
HLA-E protect against Natural Killer-mediated cell lysis
Functions of HLA-GSuppression of allo-reactive cytotoxic T
cells
responder T cellT cellreceptor
stimulator cell
HLA-DR4
HLA-DR4
HLA-DR1
K562
HLA-G1
Inhibition of T cellallo-proliferation
inhibitory receptor(ILT-2, p49 ?)
(Carosella et al. Immunol Today 1999; 20:60 / Riteau et al. J Reprod Immunol 1999; 43:203)
‘Mixed Lymphocyte Reaction’ (MLR) CD4+ responder T cell
Secretion of soluble HLA-G5
(Lila et alPNAS 2001;98:12150)
FETUS
Maternal NK cell
ILT-2/(-4)*)
KIR2DL4
Cell lysis
Trophoblast cell
HLA-E
HLA-G1HLA-C
HLA-F (?)
CD94/NKG2
Maternal monocyte/macrophage/lymphocyte
sHLA-G?
HLA-G (influenced by
HLA-G genotype)HLA-G
Augmentation of allo-CTL response ?IL-10 ??TNF- INF-
IL-10
Inhibition of allo-CTL response ?IL-10 ??TNF- INF-TGF-1 VEGF
Influence, interact with or modulateSecretion of the specific factor
Summary/ Acceptance of the semi-allogenic fetus…
No expression of polymorfic HLA class Ia and II on fetal trophoblast cells in the placenta
NB! Natural Killer cell-mediated lysis
Expression of non-polymorfic HLA class Ib molecules by trophoblast: HLA-G (and HLA-E and –F)
This expression profile may influence the cytokine profile in favour of maintaining pregnancy
HLA-Am, -Bm, -Cm
HLA-Am, -Bm, -Cm
m = maternalp = paternal
HLA-Am, -Bm, -Cm
HLA-Ap, -Bp, -Cp
HLA-Gm, -Em, -Fm, -Cm
HLA-Gp, -Ep, -Fp, -Cp
Mother – HLA class Ia
Fetus – HLA class Ia
Placenta – HLA class Ib
Regulatory T cells (Tregs)
CD4+CD25bright(FoxP3+)– CD4: co-receptor binds to
MHC class II– CD25: alpha-chain of IL-2
receptor– FoxP3: transcription factor
essential for differentiation into CD4+CD25+ Tregs
Tregs important for their potential to prevent autoimmune diseases
May also play important roles in tolerance induction in organ transplantations
(Sasaki et al 2004)
Regulatory T cells in reproduction
Mice:– Transfer of CD4+CD25+ Tregs from normal pregnant mice to
abortion-prone mice prevented spontaneous abortion– Decidual TGF-beta and LIF were upregulated in Treg-
treated mice (Zenclussen et al 2006)
Humans:– Pregnancy is associated with an increase in circulating
CD4+CD25+ Tregs, and also an increase in decidua, during early pregnancy (Somerset et al 2004; Tilburgs et al 2006)
– Proportion of decidual CD4+CD25bright Tregs has been shown to be significantly lower in cases of spontaneous abortion compared to induced abortion
– Decreased CD4+CD25+ Tregs in spontaneous abortion might induce maternal lymphocyte activation to the semi-allogenic fetus (Sasaki et al 2004)
HLA and certain complications of pregnancy…
Recurrent spontaneous abortions =
Recurrent miscarriage
Early pregnancy lossNB Chromosomal abnormalities are the most frequent cause of spontaneous abortions – however, many are ’unexplained’ and some may be due to immunological dysfunction
HLA and recurrent miscarriage (RM)
Prospective studies in inbred populations clearly show an influence of HLA genes or closely linked loci on reproductive processes, (studies in the Hutterites by Ober and co-workers)
Many studies have focused on a possible increased sharing of HLA alleles/haplotypes between the mother and the father(/the fetus) in RM. However, ’HLA sharing’ is a controversial issue and lacks evidence.
Specific HLA-DR alleles are associated with increased risk of RM– Meta-analysis (18 published/unpublished case-control studies):
HLA-DRB1*01 risk factor (OR 1.3; 95%CI 1.1-1.6) (Christiansen et al
1999)
– HLA-DRB1*03 risk factor in patients with 4 or more miscarriages and a significantly increasing trend with increasing number of previous miscarriages (OR 1.4; 95%CI 1.1-1.9) (Kruse et al 2004)
Soluble HLA-G assays (plasma/serum)
Some confusion exists regarding the detection of sHLA-G in blood samples
It seems that sHLA-G can be detected in all plasma samples from pregnant and non-pregnant women, while sHLA-G can only be detected in some serum samples from at least non-pregnant women (and from men)
Low amounts of sHLA-G may be ’trapped’ in the clot formation in serum samples. Therefore, the serum sHLA-G levels may be lower than the plasma sHLA-G level, and blood with low amounts of sHLA-G might be sHLA-G negative when serum samples are investigated
Levels of sHLA-G in maternal blood (plasma)
Maternal sHLA-G levels do not change substantially during a normal course of pregnancy
Soluble HLA-G levels of non-pregnant and pregnant women seems to be very similar
Therefore, a substantial part of the sHLA-G detected in maternal circulation may be produced by immunocompetent cells of the mother
Reduced levels of sHLA-G in maternal plasma may be associated with pre-eclampsia, spontaneous abortion and
placental abruption (sHLA-G < 9.95 ng/ml RR 7.1; 3 trim)
(Steinborn et al 2003)
Pregnancy after IVF and soluble HLA-G
In 20 women who experienced an early spontaneous abortion, the preovulatory sHLA-G conc. was significant reduced compared to women with an intact pregnancy.
The same difference was observed during monitorering of sHLA-G levels in intact pregnancy vs early spontaneous abortion until 9th week of gestation (p < 0.0001).
(Pfeiffer et al 2000)
Study (listed by type and chronology)
Type of study
Technical comments
Cases Controls Results
HLA-G polymorphism
Penzes et al. (1999) Case-control
PCR-RFLP of exons 2 and 3; low resolution of alleles
21 RSA couples
72 healthy, unrelated individuals
Negative. However, a trend towards a higher frequency of the G*01012 allele in the controls
Yamashita et al. (1999)
Case-control
PCR-SSCP of exons 2, 3 and 4. DNA sequencing of intron 4
20 RSA couples
54 healthy fertile controls (27 females/27 males)
Negative; no implication of polymorphism in intron 4
Pfeiffer et al. (2001) Case-control
DNA sequencing of exons 2 and 3
78 RSA women (3 abortions; 28% secondary aborters)
52 women (1 successful pregnancies)
G*010103 and G*0105N associated with RSA
Aldrich et al. (2001) Cohort PCR-SSOP, exons 2 and 3
113 couples (3 abortions; one live born child)
G*0104 or G*0105N in either partner associated with increased risk for abortion
Ober et al. (2003) Cohort (15 years)
DNA sequencing of 5’URR; 18 SNPs
42 Hutterite non-RSA women
Increased risk for abortion in couples both carrying a –725G allele (OR 2.7; 95%CI 1.1-7.1)
Hviid et al. (2002, 2004a)
Case-control
DNA sequencing of exons 2 and 3, polymorphisms in exons 4 and 8
61 RSA couples (3 abortions; 38% secondary aborters)
47 fertile couples/93 fertile women (2 normal pregnancies)
+14/+14 bp HLA-G genotype of female associated with RSA (OR 2.7; 95%CI 1.1-6.5). More RSA women carried the G*0106 allele (15%) compared to controls (2%) (n.s.)
Tripathi et al. (2004) Case-control
-14/+14 bp HLA-G genotype of female associated with RSA
Abbas et al. (2004) Case-control
ACLA/lupus anticoagulant positive RSA women? 300 included; 180 lost? PCR-SSOP analysis of exons 2 and 3; typing of G*0106?
120 RSA women (3 abortions; primary aborters)
120 fertile women (3 live births)
Higher frequency of G*010103 in RSA women (n.s.)
HLA-G genetics and women with RM
Negative
Negative
G*010103 and G*0105N
G*0104 and G*0105N
-725G in 5’URR
+14/+14-bp genotypeTrend for G*0106
-14/+14-bp genotypeTrend for G*010103
HLA-G 14-bp genotypes in in vitro fertilisation (IVF)
- a pilot study (Hviid et al 2004) Association of the 14-bp HLA-G polymorphism to the
outcome of IVF treatments ?
Two groups of couples attending IVF:– ”Uncomplicated” pregnancy with twins after first IVF
treatment (n = 15) 3 IVF treatments without pregnancy/implantation
(n = 14)
HLA-G genotyping Clinical and laboratory data / eg. embryo grade,
inseminated oocytes etc
14-bp HLA-G genotype of women in in vitro fertilization (IVF) treatments or with recurrent
miscarriage
Mantel-Haenszel statistics (combined 2x2 tables) : P < 0.01
(Hviid et al 2004)
HLA-G and RM: Odds ratio 2.7 [95% CI 1.1-6.5]
Membrane-bound and soluble HLA-G mRNA levels in relation to the 14 bp sequence
polymorphism in trophoblast cells
(Hviid et al 2003)
0
5
10
15
20
25
30
35
40
Re
lati
ve
Flu
ore
sc
en
ce
Un
its
(R
FU
)
HLA-G RT-PCR product 545 bp
HLA-G1/G2/G3 (-14 bp) mRNA
HLA-G RT-PCR product 559 bp
HLA-G1/G2/G3 (+14 bp) mRNA
S9S10
S1
S4
S7S3
S8
Membrane-bound HLA-GComparison of HLA-G1/G2/G3 (-14 bp) and HLA-G1/G2/G3 (+14 bp)
mRNA levels in heterozygous samples
p = 0.0156
0
10
20
30
40
50
60
70
Re
lati
ve F
luo
resc
en
ce U
nit
s (R
FU
)
HLA-G RT-PCR product 417 bp
HLA-G5/G6 (-14 bp) mRNA
HLA-G RT-PCR product 431 bp
HLA-G5/G6 (+14 bp) mRNA
S1
S3
S7
S8-
S10
S4
Soluble HLA-GComparison of HLA-G5/G6 (-14 bp) and HLA-G5/G6 (+14 bp)
mRNA levels in heterozygous samples
p = 0.0313
HLA-G alleles / alternative splicing
HLA-G*010101 HLA-G*010102 HLA-G*010103
G1 (-92 bp)G5/G6 (-92 bp)
G2(/G4) (-92 bp)
G1G2/G4G3G5G6
G1 (+14 bp)G2/G4 (+14 bp)G3 (+14 bp)G5 (+14 bp)G6 (+14 bp)
G1 (+14 bp)G2/G4 (+14 bp)G3 (+14 bp)G5 (+14 bp)G6 (+14 bp)
Genomic DNA
mRNA isoforms
-14 bp +14 bp 3’UTR polymorphism
(Hviid et al 2003, Rousseau et al 2003)
HLA-G / alleles / mRNA
Conclusions…
HLA-G alleles are associated with different HLA-G mRNA isoform expression profiles
The HLA-G mRNAs including the 14 bp sequence in exon 8 are processed further than HLA-G mRNAs with the sequence deleted. This may influence HLA-G mRNA stability
Soluble HLA-G in serum and the HLA-G genotype
Italian serum samples Danish serum samples All samples *)
HLA-G genotype
TotalHLA-G5/sG1 detected
TotalHLA-G5/sG1 detected
TotalHLA-G5/sG1 detected
14/14 55 12 23 5 78 17
14/+14 66 11 48 13 114 24
+14/+14 28 0 14 0 42 0
Total 149 23 85 18 234 41
*) 2 test for observed distribution of serum samples with HLA-G5/sHLA-G1 detected in relation to HLA-G genotype and the expected independent distribution according to the overall HLA-G genotype frequencies/proportions (14/14: 13.7; 14/+14: 20.0; +14/+14: 7.4): 2 = 9.04; df = 2; P = 0.011
(Hviid et al 2004, Rizzo et al 2005)
Soluble HLA-G levels in plasma Associations of soluble HLA-G (sHLA-G) plasma levels
and HLA-G alleles For example, in four healthy individuals:
In comparison to HLA-G*01011:– ”Low secretors”: G*01013 and G*0105N
– ”High secretors”: G*0104
(Rebmann/van der Ven and co-workers 2001)
Functional significance
HLA-G gene sequence variation influences individual HLA-G expression
Low or aberrant expression of membrane-bound and soluble HLA-G may have implications for NK-cell and T-cell interactions and cytokine profiles during pregnancy
And hence - may influence the outcome of the pregnancy…..
HLA and certain complications of pregnancy…
Pre-eclampsia and HLA-G
(pre-eclampsia = ”svangerskabsforgiftning”)
Pre-eclampsia- ’a disease of theories’
Second half of pregnancy:– hypertension – proteinuria – (oedema)
2-7% of all pregnancies World-wide still a prominent cause of maternal and fetal
mortality The fetus may also be compromised
– Intrauterine growth retardation, low birth-wight, prematurity, and intrauterine asphyxia
The etiology involves probably a combination of genetic and environmental risk factors
Pre-eclampsia – patogenesis ?
The presence of a placenta is both necessary and sufficient to cause the disorder. A fetus is not required as pre-eclampsia can occur with hydatidiform mole
(Chun et al 1964)
• Pre-eclampsia may develop with abdominal pregnancy (Piering et al 1993)
• Central to management, is delivery, which removes the causative organ, the placenta.
(From Khong et al. British Journal of Obstetrics and Gynaecology 1986; 93:1049-1059)
Placental pathoanatomy / pre-eclampsia
Step one(1. and 2. trimester?)
Step two(3. trimester)
(From Rubin & Farber, ”Pathology”; 1988)
Development of the clinical syndrome(described by Roberts 1989)
Factors shed from the placenta to the maternal blood circulation (cytokines and trophoblast cell elements) may result in endothelial cell dysfunction
• This results in vasoconstriction, and activation of the coagulation system
• The clinical symptoms can then be explained:
hypertension (vasoconstriction), proteinuria (endothelial cell dysfunction in the glomeruli) and oedema (increased vascular permeability)
Focal ulceration of the syncytium. Scanning electronmikroskopi.
(From Fox: ”Pathology of the placenta” 2ed)
Studies of genotypes in family trios mother-father-offspring
Large epidemiological study concluded, that both the mother and the fetus contribute to the development of pre-eclampsia, and the fetus’ contribution is under influence of paternal genes
(Lie et al 1998)
Pre-eclampsia and HLA-G
A role for HLA-G ? An obvious candidate gene
Pre-eclampsia might be a consequence of an immunological maladaptation of the pregnant woman to the semi-allogenic fetus
HLA-G and pre-eclampsia
Several studies have found an aberrant or absent HLA-G expression in pre-eclamptic placentas both at the mRNA and protein level compared to control placentae
– Colbern et al 1994 (mRNA) NB! Inconclusive– Hara et al 1996 (immunohistochemistry)– Troeger et al 1999 [abstract] (immunohistochemistry)– Lim et al 1997 (in vitro trophoblast cultures
mRNA og protein)– Goldman-Wohl et al 2000 (in situ hybridisation)
– O’Brien et al 2001 (mRNA and polymorphisms)– Association to +14 / +14 HLA-G genotypes
– Yie et al 2004 (serum and placental HLA-G)
Pre-eclampsia trios (58) Control trios (98)
HLA-G polymorphism in case control study of family-trios
HLA-G genotyping: - DNA sequencing of exons 2 and 3- specific analysis of polymorphisms in exons 4 and 8
Mother Father
Child
Hypotheses
The aberrant HLA-G expression in pre-eclampsia might be influenced by HLA-G genetics
A higher number of +14/+14 exon 8 HLA-G genotypes in the pre-eclamptic offspring compared to the control group?
(O’Brien et al 2001)
HLA-G histo-incompatibility between mother and fetus?
Frequencies of the 14 bp deletion polymorphism in exon 8 of the HLA-G gene in primipara
family triads with a history of preeclampsia and controls
PREECLAMPSIA CONTROLS Mothera Fatherb Offspringc Mothera Fatherb Offspringc NO. (%) NO. (%) NO. (%) NO. (%) NO. (%) NO. (%) Allele: 14 bp 44 55.0 41 51.3 38 47.5 86 61.4 92 65.7 96 68.6 +14 bp 36 45.0 39 48.8 42 52.5 54 38.6 48 34.3 44 31.4 n = 80 80 80 140 140 140 Genotype: 14 bp/ 14 bp 12 30.0 10 25.0 10 25.0 29 41.4 30 42.9 31 44.3 14 bp/+14 b 20 50.0 21 52.5 18 45.0 28 40.0 32 45.7 34 48.6 +14 bp/+14 bp 8 20.0 9 22.5 12 30.0 13 18.6 8 11.4 5 7.1 n = 40 40 40 70 70 70
a Alleles: P = 0.393; Fisher’s exact test. Genotypes: P = 0.472; 2 = 1.50, df 2.
b Alleles: P = 0.045; Fisher’s exact test. Genotypes: P = 0.106; 2 = 4.49, df 2.
c Alleles: P = 0.003; Fisher’s exact test. Genotypes: P = 0.004; 2 = 11.21, df 2. The +14 bp/+14
bp genotype vs others: P = 0.002; Fisher’s exact test. Odds ratio = 5.57 [95% CI 1.79-17.31].
(Hylenius et al 2004)
HLA-G expression in pre-eclamptic placentas
GeneChip data on HLA-G mRNA expression:
HLA-G mRNA expression reduced in pre-eclamptic placentas
Control_A Control_B Control_C baseline mean
PE_A PE_B PE_C experiment mean
fold change
lower bound of FC
upper bound of FC
1517,42 969,89 445,3 974,69 598,46 744,79 556,99 632,91 -1,54 -0,72 -2,5
(Hviid et al 2004)
Conclusions / HLA-G and pre-eclampsia
The HLA-G genotype of the fetus and the HLA-G expression in the placenta seems to be involved in the pathogenesis of pre-eclampsia
Subfecundity correlates with pre-eclampsia…
HLA-G and organ transplantation
Expression of soluble HLA-G in serum and HLA-G by heart and liver/kidney grafts have been associated with significant better prognosis and fewer rejection episodes – (…remember…only in some serum samples can HLA-G be
detected; and only some grafts are positive for HLA-G expression)
(Lila et al 2002, Creput et al 2003)
HLA-G and organ transplantation
Recent independent study / heart transplants:– Two groups of heart transplant patients (n=9 and n=10)
• One group displayed a significant increase (p>0.001) in sHLA-G during the first month after transplantation (>50 ng/ml)
• The other group maintained low levels of sHLA-G (<30 ng/ml)
– 50% of the patients with low levels of sHLA-G had recurrent severe rejection episodes, whereas patients with high levels of sHLA-G did not have any episodes of recurrent severe rejection
(Luque et al 2006;Hum Immunol 67,257)
HLA-G and organ transplatation Ongoing study / Transplantation Unit, Rigshospitalet, Cph:
– Monitoring sHLA-G with different assays, HLA-G genetics and regulatory T cells in lung and heart transplantations
After all there might be some parallels in mechanisms inducing tolerance during pregnancy and during organ transplantation
sHLA-G might have potential as a therapeutic agent in transplantation
MHC class Ib in other species…….
Rat– Transcripts for a soluble form of the RT1-E MHC class Ib molecule
have been detected in placenta; could have a regulatory role on NK-cell function
(Solier et al 2001)
Non-human primates– Rhesus monkey: expression in placenta of a Mamu-AG gene with
a soluble isoform (Ryan et al 2002)– Baboon: placental expression of soluble/membrane-bound Paan-
AG MHC class Ib proteins (Langat et al 2002)
MHC class Ib in other species…….
Mouse: Qa-2 antigen / Preimplantation embryo development
(Ped) gene phenotype Present on oocytes/blastocysts Major influence on preimplantation embryonic cleavage
rate and division (Warner et al 1993, Exley & Warner 1999)
Presence of the Ped gene phenotype correlates to heavier birth weight and larger litters
(Warner et al 1991) During development there is a selection in favour of
presence of the Ped gene phenotype in the offspring (Exley & Warner 1999)
MHC/HLA and mating preferences
HLA / mating preferences / body odors
T-shirts experiments………………….
Studies of inbred mouse strains and humans have revealed avoidance of mates with a very high number of matching MHC alleles. (In humans, female students dislike the smell of T-shirts worn by males with identical HLA alleles…)
(e.g. Potts et al 1991, Wedekind et al 1995, Ober et al 1997)
MHC homozygote deficiency reported in small, isolated populations (e.g. Ober et al)
In human populations the frequency of MHC heterozygotes are typically higher than expected by chance
HLA / mating preferences / body odors
Possible mechanisms…..
– Avoids inbreeding(e.g. Bateson
1983)
– MHC heterozygosity confers a selective advantage against multiple-strain infections
(Penn et al 2002)
– In mice, the MHC seems to influence fertilization and pregnancy outcome; maybe even oocyte/sperm selection (e.g. Wedekind et al 1996, Rühlicke et al 1998, Exley & Warner 1999).
Can I buy you a drink?
In the pub……
Buzz off – I think you’re my (HLA) type….Can I buy you a drink?
Sorry – I don’t think you’re my type….
HLA / mating preferences / body odors
Detection of MHC-mediated body odor may result from the close linkage between the MHC loci and olfactory receptor genes (Fan et al 1996, Amadou et al 1999)
MHC/HLA-specific odors may be soluble MHC proteins, odor molecules bound selectively to MHC proteins, or by-products of MHC-specific bacteria colonization in skin or axillae (Yamazaki et al 1999, Vincent & Revillard 1979, Pearse-Pratt et al 1999) (Ehlers et al 2000. Genome research 10:1968-1978)
Mating preferences seem to be influenced by MHC/HLA diversity
FertilizationWeak evidence for MHC/HLA-mediated effects on spermatogenesis
Early embryo development and implantationHLA-G expression associated with cleavage rate and implantation success
Balance between foetal/paternal and maternal interests?Some HLA-G/MHC polymorphisms may work in favour of the foetus, others in favour of maternal interests?
Foetal growth and survivalSome evidence that HLA haplotypes and HLA-G polymorphism are associated with birth weight, risk of abortion and immuneadaptation
Maternal genome Paternal genome
Deficiency of MHC/HLA homozygotes in isolated populations: frequency of MHC heterozygotes in human populations higher than expected
Heterozygote advantage Heterozygotes at the MHC/HLA loci may provide a broader immune response
SummaryMHC/HLA in reproduction
Anne-Marie Nybo Andersen Alessandra Balboni Olavio R. Baricordi Ole B. Christiansen Maria Teresa Grappa Sine Hylenius Anne Mette Høgh Christina Kruse Anette Lindhard Mads Melbye Loredana Melchiorri Nils Milman Lone G. Nielsen Marina Stignani Roberta Rizzo Christina Rørbye
Collaborators