1. 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?’

2. 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

3. ? The ’semi-allogenic fetus’

4. 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

5. 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)

6. HLA and the ’semi-allogenic fetus’

7. 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

8. The classical HLA class Ia molecules are highly polymorphic HLA-A10 HLA-B12 HLA-Cw5 HLA-A26 HLA-B8 HLA-Cw5 HLA-A25 HLA-B40 HLA-Cw2 HLA-A11 HLA-B16 HLA-Cw8 HLA-A23 HLA-B12 HLA-Cw1 HLA-A3 HLA-B5 HLA-Cw7 HLA-A2 HLA-B27 HLA-Cw6 HLA-A24 HLA-B8 HLA-Cw4 HLA-A25 HLA-B12 HLA-Cw1 HLA-A19 HLA-B15 HLA-Cw2 HLA-A19 HLA-B14 HLA-Cw8 HLA-A28 HLA-B17 HLA-Cw5

9. The non-classical HLA class Ib molecules are nearly monomorphic HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F HLA-G HLA-E HLA-F

10. HLA in pregnancy HLA-A m, -B m, -C m m =maternal p =paternal HLA-A m, -B m, -C m HLA-A p, -B p, -C p HLA-G m, -E m, -F m, -C m HLA-G p, -E p, -F p, -C p Mother – HLA class Ia Fetus – HLA class Ia Placenta – HLA class Ib Human Leucocyte Antigen (HLA) system Major 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

11. 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)

12. 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)

13. 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)

14. 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 Decidua HLA-G Cytokeratin (From Emmer et al. Human Reproduction 2002; 17:1072) (12.-13. weeks of gestation)

15. HLA-G gene, mRNA, protein, isoforms

16. Other alternatively spliced HLA-G mRNA isoforms exist (Harrison et al 1993) +14 bp: 45 % 14 bp deleted: 55 % 14 bp del polymorphism

17. HLA-G alleles (DNA sequences) Amino acid substitution

18. HLA-G polymorphisms C onsensus: 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-AHLA-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

19. 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)

20. 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

21. responder T cell T cell receptor 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 the allo-CTL response IL-10  TNF-   INF-   UNCOMPLICATED PREGNANCY RECURRENT MISCARRIAGE AND PRE- ECLAMPSIA Upregulation of the Th1 response, downregulation of Th2: IL-2 INF-  (TNF-  ) (Kapasi et al 2000) Th2 cytokine production: IL-4 IL-5 IL-10 IL-13

22. 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???

23. Functions of HLA-G  Several in vitro studies have shown that HLA-G and HLA-E protect against Natural Killer-mediated cell lysis

24. Functions of HLA-G Suppression of allo-reactive cytotoxic T cells responder T cell T cell receptor stimulator cell HLA-DR4 HLA- DR4 HLA- DR1 K562 HLA-G1 Inhibition of T cell allo-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 al PNAS 2001; 98:12150)

26. 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

27. Regulatory T cells (Tregs)  CD4 + CD25 bright (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)

28. 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 + CD25 bright 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)

29. HLA and certain complications of pregnancy… Recurrent spontaneous abortions = Recurrent miscarriage Early pregnancy loss NB Chromosomal abnormalities are the most frequent cause of spontaneous abortions – however, many are ’unexplained’ and some may be due to immunological dysfunction

30. 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)

31. 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

32. 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)

33. 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)

34. HLA-G genetics and women with RM Negative G*010103 and G*0105N G*0104 and G*0105N -725G in 5’URR +14/+14-bp genotype Trend for G*0106 -14/+14-bp genotype Trend for G*010103

35. 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

36. 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]

37. Membrane-bound and soluble HLA-G mRNA levels in relation to the 14 bp sequence polymorphism in trophoblast cells (Hviid et al 2003)

38. HLA-G alleles / alternative splicing (Hviid et al 2003, Rousseau et al 2003)

39. 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

40. Soluble HLA-G in serum and the HLA-G genotype Italian serum samplesDanish serum samplesAll samples *) HLA-G genotype Total HLA-G5/sG1 detected Total HLA-G5/sG1 detected Total HLA-G5/sG1 detected  14/  14 55122357817  14/+14 6611481311424 +14/+14280140420 Total14923851823441 *)  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)

41. 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)

42. 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…..

43. HLA and certain complications of pregnancy… Pre-eclampsia and HLA-G (pre-eclampsia = ”svangerskabsforgiftning”)

44. 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

45. 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.

46. (From Khong et al. British Journal of Obstetrics and Gynaecology 1986; 93:1049-1059) Placental pathoanatomy / pre-eclampsia

47. Step one (1. and 2. trimester?) Step two (3. trimester) (From Rubin & Farber, ”Pathology”; 1988)

48. 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)

49. 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)

50. 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

51. 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)

52. 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

53. 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?

54. (Hylenius et al 2004)

55. HLA-G expression in pre-eclamptic placentas  GeneChip data on HLA-G mRNA expression: HLA-G mRNA expression reduced in pre-eclamptic placentas Control_AControl_BControl_Cbaseline mean PE_APE_BPE_Cexperiment mean fold change lower bound of FC upper bound of FC 1517,42969,89445,3974,69598,46744,79556,99632,91-1,54-0,72-2,5 (Hviid et al 2004)

56. 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

57. Subfecundity correlates with pre-eclampsia…

58. 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)

59. 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)

60. 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

61. 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)

62. 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)

63. MHC/HLA and mating preferences

64. 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

65. 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).

66. 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….

67. 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)

68. Summary MHC/HLA in reproduction

69.  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