Second International Conference on Experimental and Clinical Reproductive Immunulogy, 15—18 November, 2000 Amsterdam, The Netherlands

Journal of Reproductive Immunology 51 (2001) 29 – 50 www.elsevier.com/locate/jreprimm

Second International Conference on Experimental and Clinical Reproductive Immunology, 15—18 November, 2000 Amsterdam, The Netherlands Abstracts of Oral Presentations Opening Session HIV in pregnancy Howard Minkoff, MD Maimonides Medical Center, Brooklyn, USA Over the last decade, there have been dramatic changes in the standards of care for HIV-infected pregnant women. The standard of care is now built upon powerful new therapeutic regimens and diagnostic and treatment protocols that are increasingly complex. Therapies that can dramatically reduce transmission of the virus to children and substantially improve the prognosis of women are now readily available in the US. In order to bring the recent therapeutic advances to bear in the care of women in the US and abroad, women’s health care providers must take an active role. Public health agencies and professional organizations have stressed the role of obstetricians and primary care providers, noting that it includes identifying and managing seropositive women. In this lecture I will discuss techniques to achieve the twin goals of perinatal management; prevention of transmission of HIV and optimizing maternal health. The cornerstone of care of the mother is viral load monitoring and aggressive pharmaceutical interventions. Any women whose viral load is higher than 5000– 10000 should be on highly active antiretroviral therapy (HAART). Since transmission to the infant may be linked to viral loads over 1000, it may be necessary to utilize HAART even for women whose counts are between 1000 and 5000 copies. HAART usually consists of two reverse transcriptase inhibitors and a protease or a non-nucleoside. Strict adherence to the regimens is required to minimize the risk of the development of viral resistance. Rates of mother-to-child transmission of HIV have been plummeting since the report in 1994 that ZDV given in the antepartum, intrapartum and neonatal periods could reduce 0165-0378/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0165-0378(00)00099-1

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rates by approximately 66%. Subsequent reports from around the world have demonstrated that shorter courses of therapy may also have a salutary effect and that at least one other agent, nevirapine, has some efficacy when used in a very abbreviated fashion. It has been suggested that HAART regimens may have particularly high success rates in preventing transmission, but definitive studies in that regard have not yet been produced. Recently ACOG has promulgated an opinion statement endorsing the finding of papers that have noted an association between elective cesarean section and lower rates of mother-to-child transmission of HIV. These finding held whether or not the mother received ZDV. When ZDV was used and elective cesarean section was performed transmission was in the 2% range. It is important to note that there is no evidence of benefit to cesarean section if the mother’s viral load is less than 1000 copies. Whether HAART can achieve similar outcomes without the need for surgery remains to be seen. Finally, the use of resistance testing may become an important addendum to the management of HIV-infection in general, and in pregnancy specifically. Although the prognosis for mother and child in the US has substantively improved over the last few years, the task for the care provider has become much more complex. Only by committing to bringing the new diagnostic and therapeutic interventions to bear in the care of his/her patients can the obstetrician assure the best outcome for HIV-infected women.

Interactions of SIV with the Immune System of the Female Genital Tract Christopher J. Miller Uni6ersity of California Da6is Despite recent insights into mucosal HIV transmission, the route used by primate lentiviruses to traverse the stratified squamous epithelium of mucosal surfaces remains undefined. In order to determine if dendritic cells (DC) are used by primate lentiviruses to traverse the epithelial barrier of the genital tract, rhesus macaque were intravaginally exposed to cell-free SIVmac251. We examined formalin-fixed tissues and HLA-DR+-enriched cell suspensions to identify the SIV RNA + cells in the genital tract and draining lymph nodes within the first 24 h of infection. Numerous SIV RNA+ DC were documented in cell suspensions from the vaginal epithelium 18 h after vaginal inoculation. In addition, we determined that SIV enters the vaginal mucosa within 60 min of intravaginal exposure, infecting primarily intraepithelial DCs and that SIV-infected cells are located in draining lymph nodes within 18 h of intravaginal SIV exposure. We also studied the range of SIV viral genotypes that are transmitted by different vaginal SIV transmission. The V1– V2 region of the SIV envelope gene was analyzed in viral RNA in plasma of the inoculated animals using variations of the heteroduplex mobility assay and selected cloning and sequencing. Fewer SIV variants were transmitted to IVAG inoculated animals compared to IV inoculated animals. However, a specific viral variant in the SIVmac251 stock was not consistently transmitted by vaginal inoculation. Thus, host factors or stochastic processes, and not viral determinants, determine the specific viral variants that infect an animal after IVAG SIV exposure. In addition, our results clearly demonstrate that the route of inoculation determines the complexity of the viral variant population in the earliest stages of systemic infection. The speed with which unique primate lentivirus variants penetrate mucosal surfaces, infect DC and disseminate to draining lymph nodes poses a serious challenge to HIV vaccine development.

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Autoimmunity & Immunogenetics Tryptophan Catabolism and T Cell Suppression: Lessons from the Maternal-Fetal Interface Andrew L. Mellor Uni6ersity of Georgia, USA Cells expressing the tryptophan catabolizing enzyme indoleamine 2,3,-dioxygenase (IDO) protect murine allogeneic fetuses from maternal T cell immunity during gestation. Inhibition of IDO activity during gestation elicits a rapid inflammatory response characterized by extensive complement deposition and hemorrhagic necrosis at the maternal-fetal interface prior to fetal rejection. Complement deposition occurs in the absence of maternal antibodies, suggesting that activation of maternal T cells when IDO is blocked leads to complement activation via the alternative pathway.

HLA-relationships of Pregnancy, Microchimerism and Autoimmune Disease J.L. Nelson Fred Hutchinson Cancer Res Ctr and Rheumatology, Uni6ersity of WA, Seattle, WA, USA Recent studies indicate bi-directional traffic of cells occurs during pregnancy. Moreover, long-term persistence of fetal cells has been described. Cells can also engraft and persist from a twin or after a blood transfusion. Graft-versus-host disease has clinical similarities to some autoimmune diseases especially systemic sclerosis (SSc). An important determinant of graft-versus-host disease is the HLA-relationship of donor and host. Considering this constellation of observations together led to the hypothesis that microchimerism and HLA relationships of cells may contribute to the pathogenesis of SSc and selected other autoimmune diseases (Nelson, 1996). In a prospective blinded study quantitatively greater levels of persistent fetal microchimerism was found in peripheral blood samples from women with SSc compared to controls. In other studies fetal microchimerism was detected in skin biopsy samples from SSc patients. Fetal microchimerism has also been found in skin biopsy samples of women who develop pruritic eruption of pregnancy. In patients with primary biliary cirrhosis fetal microchimerism was frequently detected in the liver but was also frequently found in liver biopsies from non-autoimmune liver diseases. Fetal microchimerism can be found among T and B lymphocytes, monocytes and NK cells. Persistent maternal cells have been described in men with SSc and also in some healthy individuals. Microchimerism may contribute to disease pathogenesis in the context of other risk factors, notably specific HLA alleles and the HLA relationship of mother and child. We found that prior birth of an HLA-DRB1 compatible child was associated with increased risk of subsequent SSc in the mother. Because women are recipients of cells from both their children and their own mother HLA relationships across the generations was also investigated. An increased risk of SSc was observed when the patient’s mother was HLA-DRB1 and/or DQA1 compatible from the patient’s child’s perspective. An HLA allele that is associated with risk of SSc was also associated with persistent microchimerism within the T cell population (DQA1*0501). These results lend support to the hypothesis that microchimerism may contribute to the pathogenesis of some autoimmune diseases within the context of other risk factors, notably HLA alleles. Supported by NIH grants AI 38583, AI 41721, AI 45659. Reference Nelson, J.L., 1996. Maternal-fetal immunology and autoimmune disease. Is some autoimmune disease auto-alloimmune or allo-autoimmune? Arthritis Rheum. 39, 191 – 194.

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Antiphospholipids and Reproduction for the Millennium D. Ware Branch, M.D. The first published criteria for the antiphospholipid syndrome included a single reproductive feature-‘fetal loss’. During the late 1980s and early 1990s many investigators found that a significant proportion of women with recurrent pregnancy loss and evidence of severe placental insufficiency, especially that associated with preeclampsia, also had antiphospholipid antibodies. By 1998, international consensus held that the reproductive features of antiphospholipid syndrome include fetal loss ( ] 10 weeks’ gestation), recurrent early pregnancy loss ( B 10 weeks gestation), and early delivery because of severe preeclampsia or placental insufficiency. In the meantime, other investigators claimed an association between antiphospholipid antibodies and infertility or failure of IVF and embryo transfer, and some claimed a benefit to treatment with heparin or intravenous immune globulin. However, clinicians and scientists should understand that the exact relationships between antiphospholipid antibodies and infertility remains uncertain. Another area of controversy is that of antiphospholipid antibodies other than lupus anticoagulant and anticardiolipin. Some studies, but not all, found a significant proportion of women with recurrent pregnancy loss have antiphospholipid antibodies other than lupus anticoagulant or anticardiolipin. Finally, the problem of which assay is the best assay for pertinent antiphospholipid antibodies is remains recalcitrant. At the recent 9th International Symposium on Antiphospholipid Antibodies, many experts expressed an inclination for the use of anti-b2-glycoprotein I assays as the preferred antiphospholipid assay.

The Role of HLA Matching on Fertility and Mate Choice in Humans C. Ober The Uni6ersity of Chicago, Chicago, USA To better understand the reproductive effects of HLA region genes, we have conducted a prospective study of pregnancy outcome in a reproductively isolated human population, the Hutterites. This study is currently in its 14th year and includes nearly 200 women who are in their childbearing years. All study subjects have been genotyped for 16 HLA region genes, including non-classical class I genes, HLA-E and HLA-G, classical class I genes, HLA-A, -B, -C, and class II genes, HLA-DRB1, HLA-DQA1, HLA-DQB, and HLA-DPB1. We previously reported increased fetal loss rates among couples matching for HLA-B antigens (0.23 vs 0.10; odds ratio 2.54; P =0.019) or for the entire 16-locus haplotype (0.33 vs 0.13; odds ratio 4.39; P =0.002) (Ober et al., 1998. Hum. Reprod. 13, 33). Surprisingly, among the liveborn offspring of couples matching for HLA-B or matching for the haplotype there were not fewer than expected numbers of homozgyotes, indicating that our previous observation of homozygote deficiencies in the population was not due to the selective loss of HLA homozygous fetuses. To further explore this we examined the mating patterns in the population and showed that fewer couples matched for HLA than would be expected if mating was random with respect to the HLA haplotype (Ober et al., 1997. Am. J. Hum. Genet. 61, 497). A strategy of avoiding marriage partners with a similar HLA type would minimize the adverse effects of HLA matching on reproduction. Collectively, these data indicate that HLA region genes influence a variety of outcomes, including mate choice and fertility, that would enhance the genetic diversity at these polymorphic loci. (Supported by NIH grant HD21244).

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Reproductive Outcomes in Mice Genetically Deficient in b2-Glycoprotein I: a Major Antigen in Anti-Phospholipid Syndrome Sarah A. Robertsona, Yonghua Shengb, Eline van Beijeringa, Steven A. KrilisbaDepartment of Obstetrics and Gynaecology, The Uni6ersity of Adelaide, SA 5005, Australia b Department of Immunology, Allergy and Infectious Disease, Uni6ersity of New South Wales, St. George Hospital, NSW 2217, Australia b2 glycoprotein-1 (b2GP1) is a phospholipid binding protein and important target antigen for anti-phospholipid antibodies, which are associated with recurrent fetal loss. Mice with a null mutation in the b2GP1 gene have been generated using conventional gene targeting techniques in embryonic stem cells. In heterozygous ( + / −) intercrosses, only 8.9% of 336 surviving offspring were homozygous for the disrupted allele ( − / −), suggesting an effect of fetal b2GP1 deficiency on fetal survival in utero (Sheng et al., accepted for publication). To investigate the role of b2GP1 as a determinant of successful pregnancy, female mice ( − / − or +/+) were mated with males of the same genotype and sacrificed at day 18 of pregnancy. There was no effect of maternal b2GP1 deficiency on the proportion of mated mice pregnant at day 18 (21/25 and 17/20 + / + and − / − females respectively), or on litter size (mean 9S.D.=9.091.6 and 9.0 92.7 in + / + and − / − pregnancies respectively). Fetal and placental weights, and fetal:placental weight ratios in viable sites, were comparable in −/− and +/+ mice. In a second experiment, − / − females (n= 16) and + / + females (n=17) were mated with males of the same genotype and pregnancies were allowed to proceed to term. There was no effect of b2GP1 genotype on gestational length, litter size or weight of pups at birth. Growth trajectories were altered in female − / − but not male −/− pups [mean weights of −/− pups (n= 58) were 4.6, 6.2 and 9.2% greater than + / + pups (n=67) at 3, 6 and 16 weeks of age]. Together, these data suggest that while b2GP1 deficient conceptuses may be selectively disadvantaged in b2GP1 replete mothers, implantation and successful development to term is not compromised in the event of both fetal and maternal b2GP1 deficiency.

Maternally-derived ZP3 Autoantibodies Cause Neonatal Ovarian Injury Yulius Y. Setiady, Kenneth S.K. Tung Department of Pathology, Uni6ersity of Virginia, Charlottes6ille, VA 22908, USA Zona Pellucida 3 (ZP3), a glycoprotein of the zona pellucida, is the major sperm receptor. With two linear native B cell epitopes of murine ZP3, we have designed two chimeric peptides (CP), each containing a ZP3 B cell epitope (underlined) and a helper T cell epitope of bovine ribonuclease (94 – 104). CP2 (NCAYKTTQANK-QAQIHGPR) immunization generated a reversible anti-fertility effect without pathogenic T cell response to ZP3. CP3 (NCAYKTTQANK-FSLRLMEENW) induced antibodies to native ZP without anti-fertility effect. Herein, we report the occurrence of ovarian disease in the neonatal progeny of B6AF1 female mice immunized with CP2 or a mixture of CP2 +CP3 (in CFA). Ovarian pathology was not detected in the CP-immunized adult females, nor in the progeny of females injected with CP3 and CFA, or CFA alone. More than 70% of litters had variable degrees and incidences of neonatal ovarian disease, and the ovarian disease incidences in the progeny of CP2 and CP2+CP3 groups were 33 and 26%, respectively. Neonatal ovarian disease began as monocytic and granulocytic inflammation in the ovarian interstitium and ovarian follicles. Inflammation then regressed, and this was followed by either ovarian atrophy and complete depletion of primordial and growing oocytes, or apparent recovery. Maternal ZP antibody

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titers were not predictive of ovarian disease in the progeny, thus severe ovarian disease occurred in progeny of fertile mothers with low ZP antibody titers. However, neonatal mice injected with CP2 (not CP3) antiserum developed similar ovarian pathology. Thus, (1) maternally-derived antibodies to ZP transfer to progeny severe neonatal ovarian disease; (2) the responses of neonatal and adult mice (or their ovaries) to ZP antibodies are different; (3) the process appears to be ZP3 epitope specific, and (4) unless non-pathogenic contraceptive ZP3 epitopes are identified, this complication will preclude a safe ZP3 vaccine. (Supported by NIH HD29099 and a Fogarty training grant).Please consider this abstract for oral presentation.

Immune Infertility The Effect of TGFb1 Deficiency on Female Reproductive Performance in Mice W.V. Ingman, S.A. Robertson Department Obstetrics and Gynaecology, Uni6ersity of Adelaide 5005, Australia Transforming growth factor b1 (TGFb1) is a pleiotropic cytokine implicated in regulation of proliferation and function in many cell lineages. Mice deficient in TGFb1 must be rendered deficient in the adaptive immune compartment to prevent lethal autoimmunity. TGFb1 is produced in the ovary, and in vitro studies have suggested a role for this cytokine in regulation of folliculogenesis. In the implantation site, TGFb1 is synthesised by uterine epithelial cells and placental trophoblast cells. To investigate the role of TGFb1 in female reproductive function, we have studied the effect of TGFb1 deficiency on ovarian cycling, mating frequency and pregnancy outcome in scid mice. Estrous cycles were tracked in 6 week old TGFb1−/− and control (TGFb1+ / + or + / −) littermates (n= 6 per group) for 28 days by histological analysis of vaginal smears. The average number of cycles completed over 28 days differed significantly (P B0.01) between TGFb1− / − and control mice, averaging 2.891.0 and 5.3 91.0 cycles, respectively. Mean cycle length was 9.4 92.3 days in TGFb1−/− mice and 5.5 9 1.1 days in control mice (PB 0.01). The proportion of days spent in each phase of the estrous cycle over the study period did not vary significantly between genotypes. TGFb1−/− females (8 –10 week old) tended to mate less frequently when placed with TGFb1+/+ males (interval between co-caging and mating event 11.7 9 9.1 versus 5.2 9 3.1 days in TGFb1− / − and control females respectively, P=0.06). Pregnancy was rarely maintained to parturition in TGFb1− / − females (pups were born from 3/14 versus 8/8 mating events in TGFb1− / − and control mice respectively). These data show that genetic TGFb1 deficiency causes considerable disruption in ovarian function and support of conceptus development. The mechanisms underlying estrous cycle perturbation and pregnancy failure in TGFb1 deficient mice are now being investigated.

NK and T-Cell Subpopulations in Follicular Fluid in Relation to Cause of Infertility H.G.M. Lukassena, A. van der Meerb, E. Lindemana, A. Wetzelsa, F. Wijnands, M.J.C. van Lieropc, S. Mosselmanc, I. Joostenb, D.D.M. Braata a Department of Obstetrics and Gynaecology, Uni6ersity Medical Center Nijmegen, The Netherlands b Department of Bloodtransfusion and Transplantation Immunology, Uni6ersity Medical Center Nijmegen, The Netherlands c NV Organon Oss, The Netherlands

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Leukocytes and cytokines play a key role in ovarian physiology alongside gonadotrophins and traditional growth factors. Many reports describe cytokine levels in follicular fluid (FF), but only a few studies focused on the cellular content. The question arises whether distribution of immune cell populations in follicular fluid varies depending on the cause of infertility and whether this is influencing the success rate of IVF treatment. We evaluated leukocyte subpopulations present in FF of patients undergoing IVFET treatment for idiopathic infertility or tubal factor infertility in comparison to andrological infertility (control group). Triple colour flow cytometry was used to discriminate T-cells and NK cell subsets. So far 121 follicles from 47 patients were analysed. Only erythrocyte contaminated FF (n =59) contained sufficient leukocytes for analyses. The data show that the immune cell composition of FF differed significantly from that of peripheral blood leukocytes from the same patients. Interestingly, FF from patients with idiopathic infertility contained a higher proportion of NK cells, and a lower proportion of T cells as compared to the tubal factor or andrological infertility groups. This difference was not observed in peripheral blood. Remarkably, also the composition of the NK cell subset was different, with a relatively high percentage of immunoregulatory CD16 – CD56+ + NK cells. These results show striking differences in distribution of lymphocyte subsets in FF between idiopathic infertility and other causes of infertility, possibly implicating an immunological basis for infertility. We take an interest in the functional capacity of these cells with respect to fertilization rate, oocyt maturation and embryo quality.

Cloning and Characterization of C58, a Novel, Hydrophobic, Putative Human Sperm Membrane Antigen Related to the Ly-6 Family of Proteins J. Shetty, M.J. Wolkowicz, A. Diekman, F.L. Jayes, C.J. Flickinger, J.C. Herr Department of Cell Biology, Uni6ersity of Virginia, Charlottes6ille, VA 22908, USA The search for candidate sperm immunocontraceptive proteins requires the identification and cloning of molecules exposed on the sperm surface. 2-D gel electrophoretic studies of surface exposed and autoantigenic sperm proteins were carried out earlier to identify immunocontraceptive candidate molecules (Naaby-Hansen et al., 1997. Biol. Reprod. 56, 771– 787; Shetty et al., 1999. Biol. Reprod. 61, 61 –69). In the present study, we have enriched and resolved on 2-D gels potential membrane proteins using surface labeling with sulfo-NHS-LC-biotin and subsequent TX-114 phase partitioning of hydrophobic proteins. A biotinylated 13 kDa (pI 4.8) protein was microsequenced by tandem mass spectrometry yielding sequence of ATSCGLEEPVSYR. Utilizing this sequence, a cDNA was cloned and sequenced containing a 372 bp ORF encoding 124 amino acids with a predicted mass of 13 kDa and a pI of 5.5, which included the embedded microsequence noted above. Notable features of C58 are: (1) a 19 aa amino terminal signal peptide; (2) a Ly-6/urokinase plasminogen activator receptor like domain (aa 22 –112); (3) a potential transmembrane domain near the carboxy terminus (aa 101 –124); and (4) a carboxy terminal cleavage site for transamidase, (aa 97 –99) suggesting C58 is possibly glycosylphosphatidylinositol (GPI) anchored. Transcripts for C58 were expressed only in testis as studied by Northern blot of eight human tissues and by dot blot analysis of 67 human tissue RNAs. Recombinant C58 was produced in E. coli using the pET 28b vector and antiserum production is underway. Supported by: D43 TW/HD 00654 from the Fogarty International Center, NIH HD U54 29099, the Andrew W. Mellon Foundation, and Schering A.G.

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Genetic Analysis for Y Chromosome Microdeletions in Japanese Infertile Males K. Sakataa, S. Komoria,b, Y. Nakatab, H. Katoa, H. Sawaib, K. Koyamaa,b a Department of Obstetrics and Gynaecology, Hyogo College of Medicine, Nishinomiya, Japan b Laboratory of De6elopmental Biology and Reproduction, Institute for Ad6anced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan Introduction: The genetic basis of infertility remains unclear in a majority of infertile men. In this study, Y chromosome long arm involving DAZ (Deleted in Azoospermia) was screened for evaluating the incidence of microdeletion in Japanese infertile men. Methods and results: 157 infertile Japanese men with azoospermia and severe oligozoospermia were analyzed for microdeletions of the interval 6 of the Y chromosome by using polymerase chain reaction with sequence-tagged site markers. The eight sets of oligonucleotides primer were synthesized for polymerase chain reaction, and southern blot analysis were also performed. All analyzed infertile men were divided into five categories on the basis of sperm concentration: functional azoospermia (A) 24, azoospermia due to obstruction (AO) 20, oligozoospermia-I (OI) (less than 1 × 105/ml) 33, oligozoospermia II (OII) (less than 1 ×106/ ml) 30 and oligozoospermia III (OIII) (less than 1 × 107/ml) 50. Thirty fertile men with sperm concentration of more than 2 × 107/ml were also analyzed as control. Results: In 12 (7.6%) of 157 infertile men, microdeletions were identified as follows; one in category A, one in category AO, five in category OI, four in category OII and one in category OIII. On the other hand, no deletion was identified in fertile men. One common region around sY240 was identified in 11 infertile men with microdeletions. This locus might contain specific genes for spermatogenesis. Sperm concentration of nine oligozoospermic men with microdeletions were below 1×106/ml. There were no correlation between the severity of spermatogenesis and the extent of microdeletions. Conclusion: These results suggested that genes in the interval 6 of the Y chromosome might be important for spermatogenesis.

The Role of Pantophysin(h-Sp-1) in Human Sperm-Egg Interaction Shinji Komoria,b, Kazuko Sakataa, Hiroyuki Tanakaa, Ri-ichiro Kanazawaa, Yoshiyuki Tsujia, Koji Koyamaa,b a Department of Obstetrics and Gynaecology, Hyogo College of Medicine, 1 -1 Mukogawa-cho, Nishinomiya, Hyogo, 663 -8501, Japan b Laboratory of De6elopmental Biology and Reproduction, Institute for Ad6anced Medical Sciences, Hyogo College of Medicine, 1 -1 Mukogawa-cho, Nishinomiya, Hyogo, 663 -8501, Japan Introduction: Previously we isolated a human sperm antigen gene (h-Sp-1; EMBL accession no. X68194) by screening from human testis cDNA library with human sperm specific antisera. Since the sequence analysis of the h-Sp-1 gene indicated that it had 45% homology to human synaptophysin gene. The product of the h-Sp-1 gene was termed as pantophysin. In this study, we analyzed the function of pantophysin in sperm-egg interaction. Methods and results: Northern blot analysis indicated that h-Sp-1 gene had two kind of transcripts (2.3 and 1.1 kb). 2.3 kb transcript expressed ubiquitously whereas 1.1 kb transcript expressed abundantly in human testis. In order to characterize the biological function of pantophysin in sperm, antisera were generated by immunizing rabbits with synthetic polypeptides (amino acid No 174 –198) based on the h-Sp-1 gene. The antisera stained fixed human sperm head by indirect immunofluorescence staining. The purified antisera showed an inhibitory effect on

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human sperm penetration assay into zona-free hamster eggs. However, they did not show any sperm immobilizing and agglutinating activities. Since pantophysin is homologous to synaptophysin, we are now analyzing the interaction between pantophysin and sperm membrane molecules. Conclusion: These results suggested that pantophysin might play an important role in the sperm-egg interaction.

The Immune Response of Male Baboons to Testis-specific LDH-C4 Reduces Fertility But Does Not Cause Orchitis E. Goldberga, J.L. VandeBergb, M. Mahonyc, G.F. DoncelcaNorthwestern Uni6ersity, E6anston, IL, USA b Southwest Foundation for Biomedical Research, San Antonio, TX, USA c Eastern Virginia Medical School, Norfolk, VA, USA Immunocontraception based on sperm antigens has been proposed for use by women but not men because of potential autoimmune disease. Nevertheless immunologic infertility in males with circulating anti-sperm antibodies has been documented and there is no evidence of testicular pathology. The present study was designed to examine the effect in the male baboon, of immunization with the sperm specific antigen, LDH-C4. This vaccine suppresses fertility reversibly in female baboons. Four sexually mature male baboons were immunized with a chimeric peptide containing the B-cell epitope of LDH-C4 and a promiscuous T-cell epitope of Tetanus toxin (hC519:TT). The animals received booster injections, at 29, 61 and 344 days after priming. A robust immune response developed in three of the four males and was maintained for approximately a year. There was no evidence of testicular histopathology in biopsies taken at days 61, 127, and 183 of the study. Spermatogenesis appeared to be normal. To assess function, sperm-zona binding was assayed. Sperm from three of the four males had a reduced hemizona index compared to non-immune controls. Therefore, a breeding trial with three males of proven fertility was undertaken. The fertility of these males was reduced by immunization with hC5-19:TT. The results suggest that immunocontraception of males with sperm antigens is not compromised by autoimmune disease and may provide an additional contraceptive choice. Supported by CONRAD (CICCR) Grant c 640510 to Northwestern University and by NIH Grant P51RR13986 to the Southwest Regional Primate Research Center. The views expressed by the authors do not necessarily reflect those of the CONRAD Program.

Inhibition of Fertility in Mice Immunized with Recombinant Mouse ZP2 I.A. Lea, E.E. Widgren, M.G. O’Rand Department of Cell Biology and Anatomy, Uni6ersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA The immunological inhibition of fertility by targeting antibodies to the zona pellucida has received much interest in recent years. Most studies have focused on ZP3 as the antigen target for the antibody; ZP2 has evoked less interest. We now present studies to demonstrate the immunogenicity and fertility rates of mice immunized with near full-length recombinant ZP2 (rZP2) and ZP2 N- and C-terminal fragments. In contrast to immunization with either N- or C-terminal fragments, which produced low antibody responses, rZP2 was highly immunogenic in female mice. In fertility trials the pregnancy rate of mice immunized with

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rZP2 was reduced to 9% from 100% in adjuvant controls. However, this effect on fertility was not reversible within 32 weeks post immunization. Histological examination of the ovaries revealed normal follicular development and no apparent pathology. To understand the mechanism causing infertility, female mice were passively immunized with antibodies to rZP2. Fertility testing showed these mice to be infertile (0% pregnancy rate). Moreover, when sections of ovary were probed with antibodies to mouse immunoglobulin, antibody was detectable bound to the zona pellucida of developing oocytes. Likewise, induced ovulation in mice actively immunized with rZP2 produced normal numbers of eggs but with antibody bound to the zona pellucida. In sperm binding assays, these eggs demonstrated a 75% reduction in the number of sperm bound compared to adjuvant control eggs. We conclude that immunization with rZP2 can bring about infertility in mice solely through the binding of antibody to the zona pellucida of the egg and without significant ovarian pathology. Supported by NIH U54HD29099.

Immunocontraception in Macropods 2: Immunisation of Tammar Wallabies (Macropus eugenii) and Eastern Grey Kangaroos (Macropus giganteus) with Porcine ZP and Recombinant Brushtail Possum ZP3A. Kitchener, L. Edds, A. Harmon, D.J. Kay, C.R.C. Marsupial Department Biological Sciences, The Uni6ersity of Newcastle, Callaghan, NSW 2308, Australia As has been indicated, a main aim of the Marsupial CRC is the development of field deliverable fertility control for overabundant macropods in Australia and New Zealand. In this study the potential for immunocontraception using the zona pellucida (ZP) as a target was examined by immunising eight female tammar wallabies with porcine zona pellucida (PZP) and eight controls with saline in Freunds adjuvant. Antibodies to PZP were assayed in serum, follicular and oviduct fluids by ELISA and in tissue by immunofluorescence. Following establishment of a response the wallabies were placed in a natural mating trial followed by a further examination of fertility using artificial insemination. At the end of the trial serial sections of ovarian tissue were examined. Antibodies to PZP were detected in all fluids studied and to wallaby ZP in situ in immunised animals. Antibodies were directed at all three glycoproteins of PZP. The natural mating and AI trial demonstrated absolute infertility. Examination of ovarian tissue showed reduced ovarian weight and reduced numbers of large pre-antral and antral follicles. A pilot study immunising eastern grey kangaroos with PZP resulted in sustained levels of antibody to PZP3 (only) 14 months after a single immunisation, however there was no antibody binding to ZP in ovarian tissue of these animals. An immunisation and natural mating trial using brushtail possum recombinant ZP3 in eastern greys has commenced and results will be discussed at the presentation of this paper. However the potential of the ZP as an immunocontraceptive target in macropods has been established.

Infection & Host Defence Mechanisms of HIV Infection and Host Defence in the Genital Tract: Insights for Vaccine Development Deborah J. Anderson Har6ard Uni6ersity, Boston, USA

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Considerable recent molecular evidence suggests that HIV-1 infection of the male and female genital tracts is compartmentalized. We are performing experiments to determine immunological factors that may distinguish different HIV-1 profiles in the genital tract from the systemic compartment. Our evidence indicates that genital tract populations of cells that govern HIV-specific humoral and cellular immunity differ dramatically between the two compartments. The genital tract antibody profile often contains species that are not detectable in blood, whereas the genital tract T cell populations are severely oligoclonal. These observations may contribute to the development of vaccines against HIV-1 and other genital tract pathogens.

The Immune Response to Genital Chlamydial Infection Michael Ward, PhD Southampton Uni6ersity Medical School, UK Chlamydia trachomatis infections are a major cause of sexually transmitted disease and its sequelae: pelvic inflammatory disease, infertility and ectopic pregnancy. They are also the cause of preventable blindness, due to trachoma. In general, these infections tend to be chronic, insidious, and characterized by intermittent periods of activity and the severe sequelae are caused by scarring and fibrotic tissue damage. Repeated infections are common, suggesting that immunity is, at best, incomplete. Nevertheless there is evidence for protective immunity. Thus, isolation of C. trachomatis from the genital tract decreases significantly with age independently of diminished sexual activity, suggesting that exposure leads to moderate protection, perhaps explaining why chlamydial genital tract infection is relatively uncommon in high risk groups repeatedly exposed to sexually transmitted infection. However, this limited protection appears short lived, as chlamydiae are reportedly isolated more often at a second isolation attempt if the interval between cultures is longer than 6 months. Limited protection has been achieved in experimental or farm animals and in human volunteers following immunization with crude, whole organism vaccines and it is clear that antibody to the immunodominant chlamydial outer membrane protein (MOMP) is capable of neutralizing chlamydial infection. The corresponding neutralizing epitopes on MOMP have been defined to single amino acid resolution, but attempts over many years to develop chlamydial vaccines based on MOMP have been unsuccessful. Problems include the facts that the most relevant B cell epitopes probably have a complex and critical topography difficult to mimic with synthetic or recombinant antigens and that it is difficult to sustain an adequate immune response in the absence of replicating agent. However it has become clear from work in knock out mice that the T-helper 1 response and its key effector, IFNg, is crucial for adaptive immunity to chlamydial and other intracellular bacterial infections. IL-12, a key regulatory cytokine supporting T-helper 1 responses, is protective in mice. Thus while neutralizing antibody might provided protection against the initial infection of genital tract epithelia, T-helper 1 responses are probably essential for eliminating the intracellular infection. Recognition of the potential importance of Chlamydophila pneumoniae infection in coronary artery disease in humans and the availability of whole genomic sequences for six different chlamydiae has just recently provided an important stimulus for chlamydial vaccine research. DNA vaccine constructs have been prepared for over 200 C. pneumoniae open reading frames and systematically tested for their protective activity in mouse models. A number of new vaccine candidates have thereby emerged and, interestingly, some of these constructs protect against both C. pneumoniae and C. trachomatis infection. However

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important and fundamental problems remain as to how best to sustain the immune response in the genital tract.

Immunobiology of Papillomavirus Infections Margaret A. Stanley Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK HPV infection in the genital tract is common in young, sexually active individuals, the majority of whom clear the infection without overt clinical disease. Those who develop lesions, also in most cases, mount an effective cell mediated immune response and the lesions regress. Lesions in which productive viral infection is occurring (ano-genital warts and LGSIL) are not associated with inflammation or histological evidence of immune activity. Regression of ano-genital warts is accompanied histologically by a response characteristic of delayed type hypersensitivity, animal models support this and provide evidence that the response is regulated by CD4 T cell dependent mechanisms. The increased prevalence of HPV infections in individuals immunosuppressed either as a consequence of organ transplantation or HIV infection demonstrates the central importance of the CD4 T cell population in the control of established HPV infections. Although it seems clear that the CD4 T cell subset is critical for the induction and regulation of the host response to HPV the nature of the effector response remains unclear. There is increasing evidence that both natural killer cells and antigen specific cytotoxic T lymphocytes (CTL) are important effectors but these responses are still poorly understood. Antibody responses to the major virus capsid protein L1 accompany the induction of successful cell mediated immunity and these responses are certainly protective against subsequent viral challenge in natural infections in animals suggesting that prophylactic immunisation will be effective in controlling HPV induced genital disease. If the cell mediated response fails to induce lesion regression and viral clearance then a persistent viral infection results which is in part due, to operational immune tolerance. This seems to be reflected by the detection in vitro of enhanced cellular and humoral responses to early viral proteins but the failure of these responses to clear virus in vivo. The importance of the MHC in susceptibility to, or protection from, papillomavirus infection and disease remains a crucial one for investigation.

Mucosal Antibody Responses in HIV-Infected Individuals J. Mestecky, P.A. Goepfert, Z. Moldoveanu, L.R. Brewer, R. Kulhavy, S.J. Prince, M.J. Mulligan, S. Jackson Uni6ersity of Alabama at Birmingham, Birmingham, AL, USA External secretions (tears, saliva, urine, nasal, seminal ejaculate, vaginal and rectal washes) and sera from 50 HIV-1-infected, and 20 uninfected individuals; 60 concordant and discordant African couples; and ten HIV-1 exposed but seronegative women were examined for the levels of total and antigen-specific IgG and IgA antibodies by ELISA and chemiluminescence-enhanced Western blots. Despite the pronounced dominance of total IgA in most external secretions (e.g. saliva, nasal, and rectal washes), the levels of HIV-specific IgG antibodies were higher than those of IgA in all external secretions. Furthermore, we did not detect anti-HIV antibodies in any external secretions of seronegative individuals, and we did not identify any individual who displayed the exclusive presence of IgA antibodies in

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secretions or sera among HIV-infected, exposed but seronegative, or uninfected individuals as reported by others. The highest levels of mucosal HIV antibodies were detected in nasal and genital tract secretions, and urine; unexpectedly, intestinal secretions contained the lowest levels. The specificity of IgG HIV antibodies was the same for all external secretions sampled. IgA HIV antibodies (most frequently present m genital tract secretions, and very rarely in rectal washes) were specific mostly for gp160. Although high numbers of specific antibody-secreting cells are readily detectable in the peripheral blood of individuals infected with other viruses (e.g. influenza), a relatively low number of specific antibody-secreting cells were seen in HIV-infected individuals. Thus, in contrast to other mucosally acquired viral and bacterial infections in which antigen-specific IgA antibodies are dominant, HIV induces preferentially IgG responses in sera as well as in external secretions.

ICAM-1 and VCAM-1 Recruit Lymphocytes into the Vagina of Immune Mice Challenged with Herpes Simplex Virus-Type 2 M.B. Parr, E.L. Parr Southern Illinois Uni6ersity, Carbondale, IL 62901, USA Previous studies showed a rapid secretion of interferon gamma in the vagina of immune mice after vaginal challenge with herpes simplex virus-type 2. This interferon gamma secretion was correlated with a 20-fold increase in the number of lymphocytes in the vagina. Neutralization of the interferon gamma in vivo with monoclonal antibody eliminated the cytokine from vaginal secretions and blocked recruitment of lymphocytes. The expression of four endothelial addressins in the vagina was studied in these mice, since lymphocyte recruitment into tissues involves interactions between adhesion molecules on vascular endothelial cells and corresponding ligands on the lymphocyte surface. The adhesion molecules ICAM-1 and VCAM-1 were minimally expressed in the vagina of nonimmune mice with or without vaginal challenge and in immune mice before challenge, but both were upregulated by interferon gamma in immune mice after challenge. MAdCAM-1 was detected in most vaginae but was not upregulated by interferon gamma, and E-selectin was not detected in any vaginae. The results suggested that ICAM-1 and VCAM-1 may be involved in the rapid, interferon gamma-mediated recruitment of lymphocytes to the vagina. In the present study we neutralized ICAM-1 and/or VCAM-1 in vivo with monoclonal antibodies and counted the number of lymphocytes in the vagina after vaginal challenge with virus. Neutralization of these adhesion molecules significantly reduced the numbers of CD4+ and CD8+ lymphocytes (M 9S.E.M. per high power field) from 6.4 90.8 and 5.8 90.6 to 1.0 9 0.2 and 0.7 90.2, respectively. B lymphocytes were reduced from 13.4 91.1 to 4.1 90.5. Collectively these results indicate that ICAM-1 and VCAM-1 are upregulated by interferon gamma in vivo and participate in the recruitment of lymphocytes to the vaginal mucosa of immune mice after challenge with herpes simplex virus-type 2.

Enhanced Fetal Loss as a Result of Allogeneic Mating in Mice Lacking the IL-4 Signaling Molecule, STAT6 Surendra Sharmaa, Nazeeh Hannaa, Loren Fastb a Women & Infants’ Hospital, Brown Uni6ersity, Pro6idence, Rhode Island 02905, USA b Rhode Island Hospital, Brown Uni6ersity, Pro6idence, Rhode Island 02905, USA

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Bidirectional maternal-fetal immune tolerance is an important part of the microenvironment of the developing fetus. Previous studies have shown that infection prior to or during pregnancy which biases the maternal immune system toward production of inflammatory Th1 cytokines results in increased abortions. Another approach to generate a bias toward Th1 cytokines is to inhibit part of a series of signals that are required for the induction of Th2 cytokines. The IL-4 signaling molecule Stat6 has been shown to be a requirement to induce Th2 cytokines. Thus, the in vivo role of enhanced Th1 immunity in pregnancy was assessed by studying the outcome of allogeneic matings using Stat6-deficient (Stat6 − / − ) mice. When 7 weeks old Stat6 − / − BALB/c (H-2d) female mice were mated with Stat6 − / − C57B6/129 (H-2b) male mice, fetal resorption at a significantly high frequency (80 –100%) was observed when evaluated at day 11 of gestation. On the other hand, matings between 7 weeks old wild-type BALB/c and C57B6/129 or syngeneic Stat6 − / − BALB/c mice resulted in normal litter size with minimum fetal resorption (10 –30%). Fetal resorption in allogeneic Stat6 − / − matings did not appear to be due to failed implantation as demonstrated by hemosiderin staining in the uteri. Our data provide evidence that Stat6 deficiency imparts deleterious effects on murine pregnancy and will be further discussed to highlight the role of supportive or harmful cytokines.

Interkleukin-11 and its Receptor (IL-11Ra) in Human Endometrium and the effects of Cytokines on IL-11 Production In Vitro B.A. Corka,b, T.C. Lib, M.A. Warrenc, S.M. Lairda,b a Biomedical Research Centre, Sheffield Hallam Uni6ersity, Sheffield, UK b Biomedical Research Unit, Jessop Hospital for Women, Sheffield, UK c Department of Biomedical Sciences, Uni6ersity of Sheffield, Sheffield, UK Interleukin-11 is a member of the IL-6 family of cytokines. Its presence in mouse decidua has been shown and female mice with either an inactive or null mutation for IL-11Ra undergo an abnormal stromal cell decidualization reaction and subsequent pregnancy loss. Immunocytochemical staining for both IL-11 and IL-11Ra was carried out on sections cut from 19 endometrial biopsies obtained throughout the menstrual cycle. IL-11 was expressed in both human endometrial epithelial and stromal cells, with epithelial staining being more intense than that seen in the stromal cells, at all times except the late secretory phase when the intensity was similar. IL-11Ra was also expressed in both epithelial and stromal cells. Expression in stromal cells was less intense than that seen in epithelial cells. Staining in epithelial cell did not change significantly throughout the menstrual cycle, but stromal expression did increase slightly during the early-mid secretory phase. The production of IL-11 by cultured epithelial and stromal cells was also studied. Basal IL-11 production by cultured epithelial cells was greater than basal production by stromal cells. IL-1a, TNFa and TGFb (0.1–10 ng/ml) all caused a concentration-dependent increase in IL-11 production by both epithelial and stromal cells, but stimulated: basal values were greater for stromal than epithelial cells for all three cytokines. This work shows, for the first time, the presence of IL-11 within the human endometrium and that its production is controlled by other cytokines, known to be important in implantation. Thus IL-11 may also play an important role in human endometrial function and embryo implantation.

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MHC and Reproduction Homozygous HLA-G*0105N Healthy Individuals Indicate that Membrane Anchored HLA-G1 Molecule is not Necessary for Survival Pablo Morales, Maria Jose Castro, Ricardo Rojo-Amigo, Mario Gonzalez-Hevilla, Jorge Martinez-Laso, Pilar Varela, Miguel Garcia-Berciano, Jesus Guillen-Perales, Antonio Arnaiz-Villena Department of Immunology and Molecular Biology, Hospital 12 de Octubre, Uni6ersidad Complutense, Carretera de Andalucia, 28041 Madrid, Spain HLA-G is expected to play an important role during fetal development. Recently, a healthy individual homozygous for the HLA-G*0105N allele has been described, suggesting that HLA-G expression was not essential for fetal survival. We now report studies of one family with five healthy siblings homozygous for HLA-G*0105N, who had been normally delivered; three of these siblings were females who also had normal deliveries. In addition, HLA-G*0105N cDNA has been fully sequenced, and normal G1 membrane anchored protein cannot be translated since after the codon 130 cytosine deletion (exon 3) a reading frameshift is observed, leading to the existence of premature stop codon at position 189 (beginning of exon 4). Other protein isoforms (G2, G3 and G6), all containing the leader peptide and the a1 domain, are possible and their messenger RNAs were found; any of these may undertake the necessary HLA-G functions. Our data show that the membrane anchored HLA-G molecule is not necessary in either mother or fetus for a normal pregnancy and survival. Also, individuals homozygous for HLA-G*0105N are healthy and with no indications of immunodeficiency or autoimmunity. Keywords: Alternative splicing; Evolution; HLA-G; Null allele

Natural Killer Cell Receptors (NKRs) in Couples with Recurrent Spontaneous Abortions (RSAs) M. Varla-Leftheriotia,b, M. Spyropoulou-Vlachouc, D. Niokouc, C. Tsekouraa,c, R. Darlamitsouc, Th. Keramitsogloua, M. Papadimitropoulosc, Ch. Balafoutasb, C. Stavropoulos-Giokasc a Department of Immunology, Athens, Greece b RSA Clinic ‘Helena Venizelou’ Maternity Hospital, Athens, Greece c National Histocompatibility Center ‘G. Gennimatas’ Hospital, Athens, Greece It is known that the lytic activity of Natural Killer (NK) cells is controlled by a balance between activating/inhibitory signals transduced by distinct receptors (NKRs), which recognize specific MHC-I molecules on targets. In this study, NKR polymorphisms were determined in RSA couples to investigate the possibility that trophoblast is more likely to be targeted for lysis if women lack inhibitory NKRs expressed by their husbands. The study included 20 childless couples with ] 3 unexplained RSAs, 19 couples with ] 2 abortions after childbearing and 18 fertile couples without abortions. All subjects were genotyped by PCR-SSP and the use of 13 pairs of primers specific for NKRs known to have as ligands HLA-I molecules with trophoblast expression: (a) inhibitory 2DL1, 2DL2, 2DL3 and activating 2DS1, 2DS4 NKRs of the KIR family which recognize Cw; and (b) inhibitory NKG2A and activating NKG2C NKRs of the CD94/NKG family which recognize HLA-E (or alleles regulating its surface expression). In each group, the NKR repertoire was compared between partners.

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The results revealed that, in 50% (10/20) of the group A couples, the women lack inhibitory KIR-NKR/s expressed by their husbands, against 31.1% (6/19) in group B and 16.6% (3/18) in group C (A vs C: P B0.05). No differences were found in the activating KIR-NKR or the NKG repertoires. According to the above results it seems possible that, in RSAs, paternal MHC-I on trophoblast are recognized by NK maternal cells lacking appropriate inhibitory KIRs that would prevent lysis, a finding that may be an evidence for an NKR allorecognition system in pregnancy.

Evidence for an Altered Interaction between HLA-G Expressing Trophoblast and Decidual NK Cells in Pathological Pregnancies Peter M. Emmera, Eric A.P. Steegersb, Harold M.J. Kerstensc, Johan Bultenc, Willianne L.D.M. Nelenb, Irma Joostena a Department of Bloodtransfusion and Transplantation Immunology, Uni6ersity Medical Center Nijmegen, Nijmegen, The Netherlands b Department of Obstetrics and Gynaecology, Uni6ersity Medical Center Nijmegen, Nijmegen, The Netherlands c Department of Pathology, Uni6ersity Medical Center Nijmegen, Nijmegen, The Netherlands. [email protected] The interaction between decidual NK cells and allo-antigens expressed on fetal trophoblast cells is thought to be essential for successful implantation and placentation. Consequently, a disturbed interaction during the first trimester of pregnancy might well lead to a subsequent pregnancy failure. We investigated the expression of HLA-G and NK cell markers in tissue sections from recurrent miscarriage (n= 9), ectopic tubal pregnancies (n= 5) and a single hysterectomy specimen of a healthy pregnancy, used as control. We show in normal pregnancy not only a decrease, but also a morphological change in CD56pos NK cells upon interaction with HLA-G expressing trophoblasts. The cells appear to be transitioning from a blast-like (activation) state into a state of apoptosis. The number of CD16pos NK cells was low. In contrast, in recurrrent miscarriage tissue a sustained NK cell marker expression of both CD56 and CD16 was paralleled by a decreased expression of HLA-G. No morphological changes from the blast-like stage were apparent. Finally, in ectopic pregnancies HLA-G expression in the absence of decidual NK cells was associated with a disturbed trophoblast differentiation. In conclusion, in pathological pregnancies we show an in situ altered interaction between trophoblast and NK cells.

Cocultures of Choriocarcinoma Cells and Isolated Decidual Cells: the Influence of MHC Class I Expression Lorenz Rieger, Marc Su¨ tterlin, Johannes Dietl, Ulrike Ka¨ mmerer Department of Obstetrics and Gynaecology, The Uni6ersity of Wu¨ rzburg Problem: In the present study we examined how MHC class I expression on choriocarcinoma cells affects the production of TNF-a, IFN-g, IL-10, IL-4 and GM-CSF by isolated CD56+ + human decidual LGL and LGL-depleted decidual cells (WASH) in cocultures. Method of study: Decidual tissue was obtained by legal therapeutic abortions from 11 women undergoing legal therapeutic abortion at 7 –11 weeks gestation. Anti-CD56 labeled LGL were isolated in a magnetic cell separator. LGL and WASH were each cocultured with

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either JEG-3 or JAR cells. The cytokines in the supernatant were measured by ELISA. Differences were analyzed for significance by Wilcoxons’s test. Results: None of the cytokines could be detected in the supernatant of JEG-3 or JAR alone. We found a significant increase of IL-10 in LGL/JEG-3 and of TNF-a in LGL/ JAR cocultures. There was a significant increase of IL-10 and TNF-a and a significant decrease of GM-CSF in WASH/JEG-3 and WASH/JAR cocultures compared to WASH. IFN-g was only found in three cases of LGL/JAR. No IL-4 was found in any experiment. Conclusions: The cytokine release of decidual LGL can be modulated by the MHC class I expression of cocultured choriocarcinoma cells. They seem to preferably produce Th2 cytokines in contact with MHC class I positive cells, whereas they tend to produce Th1 cytokines in cocultures with MHC class I negative cells. In contrast, the cytokine production of the remaining decidual cells seems to be modulated independently of the MHC class I expression of the cocultured cells. Further investigations with HLA-G positive/negative transfectants are needed to ascertain if this phenomenon is mediated by HLA-G.

HLA-G Modulates Cytokine Production by Uterine NK Cells A. van der Meera, H.G.M. Lukassenb, M.J.C. van Lieropc, F. Wijnandsc, P.M. Emmera, S. Mosselmanc, D.D.M. Braatb, I. Joostena a Department of Bloodtransfusion and Transplantation Immunology of the Uni6ersity Medical Center Nijmegen, Nijmegen, the Netherlands b Department of Gynaecology and Obstetrics of the Uni6ersity Medical Center Nijmegen, Nijmegen, the Netherlands c NV. Organon, the Netherlands, E-mail:[email protected] The unique expression of HLA-G by invading trophoblasts suggests that this molecule plays a role in the induction of tolerance during embryo implantation. Putative targets for HLA-G in the endometrium are the uterine NK cells (uNK). HLA-G might inhibit the killing activity and/or at the same time activate the cytokine production of these uNK cells to favour implantation. To assess whether HLA-G can modulate uNK cell responses, uNK cells were co-cultured in the presence of K562 or 721.221 cells transfected with HLA-G. Cytotoxicity, proliferation and cytokine production were determined. uNK cells were purified from endometrium biopsies taken from non-pregnant women 7 days after the peak of luteinizing hormone, the day that implantation occurs. To assess whether uNK cells act differently from peripheral NK cells, NK cells were also isolated from peripheral blood of healthy donors. The data show that uNK cells exhibit increased proliferation in response to HLA-G transfected 721.221 cells as compared to untransfected cells. Furthermore, co-culture with 721.221-HLA-G cells surprisingly leads to an increased production of IFN-k, a pro-inflammatory Th1 type cytokine. The latter effect was also observed with peripheral NK cells. However, co-culture with HLA-G transfected K562 cells did not induce proliferation nor IFN-k production in these cells. This difference between 721.221 cells and K562 cells was also apparent in the cytotoxicity assay where HLA-G expressed on 721.221 cells was more effective in inhibiting the cytotoxic activity as were the K562-HLA-G cells. Studies are in progress to determine whether this is caused by co-expression of HLA-E by the 721.221-HLA-G cells or by other factors and whether this is also observed with uNK cells.

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HLA-G Present HCMV-derived Peptides to Specific HLA-G Restricted Cytotoxic T Cells Franc¸ oise Lenfanta, Anatolij Horuzskob, Christian Davrinchea, Philippe Le Bouteillera a INSERM U395 - BP 3028 - 31024 Toulouse cedex, France b IMMAG, Medical College of Georgia, Augusta, GA 30912 -2660, USA During pregnancy, there is a balance between the mother’s need to fight infection and the fetus to avoid allo-reactive response of the mother. Whereas expression of HLA class Ia molecules is almost absent of placental tissues, the HLA-G class Ib is specifically expressed in extravillous trophoblast cells which are in direct contact with maternal tissues. Such patterns of expression have led to the hypothesis that HLA-G molecules could play important functions during pregnancy. It has recently been shown that HLA-G binds endogenously processed nonameric peptides, suggesting that HLA-G could exert a peptide presentation function to effector CTL. In this context, the aim of our project was to investigate whether HLA-G could bind and present viral peptides in vivo and then function as restriction elements for a CTL response to viruses which could infect trophoblast tissues. Using the HLA-G transgenic mice, Horuzsko et al. (USA) have demonstrated that HLA-G molecules function as a restriction element capable of eliciting a CTL response. Consequently, the triple transgenic mice (HLA-G, hb2m, CD8) have been immunized with recombinant viruses expressing matrix protein from hCMV in order to induce HLA-G restricted CTL responses in vivo. HCMV-derived peptides that bind efficiently HLA-G molecules have been identified and used to load HLA-G expressing dendritic cells which have been further used as immunizing materials. Preliminary results show that such immunizations may induce a HLA-G restricted-HCMV derived peptide specific CD8+ T cell response, as demonstrated by the killing of target cells expressing both HLA-G and appropriate peptides. These results demonstrate that in vivo induction of a primary CTL response is feasible using HLA-G as the restriction element for HCMV-derived peptides. Consequently, HLA-G can present viral peptides and induce HLA-G restricted CTL responses against HCMV proteins. These data may indicate that HLA-G have a function in protecting the fetus against pathogens during pregnancy.

Transcriptional Control of Antigen Presentation Functions in Fetal Trophoblast Cells Peter J. van den Elsena, Nienke van der Stoepa, Henk E. Vie¨ torb, Louis Wilsona, Marlijn van Zutphena, Sam J.P. Gobina a Department of Immunohematology and Blood Transfusion, Building 1, E3 -Q, Leiden Uni6ersity Medical Center, 2300 RC Leiden, The Netherlands b Department of Clinical Genetics, Uni6ersity Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. Our studies demonstrate that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of CIITA expression due to hypermethylation of its IFN-g responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induces cell surface expression of HLA-DR, -DP and -DQ, leading to an acquired capacity to present antigen to antigen-specific T-cells. Transfection of CIITA in JEG-3 cells also up-regulates functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-g-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorion villus biopsies Together, these observations demonstrate that

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lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells during fetal development.

Murine Female Reproductive Tract Intraepithelial Lymphocytes Display Both Peripheral and Muscosal Selection Characteristics Dina S. Goulda, Hidde Ploegha, Danny J. Schustb a Department of Pathology, Har6ard Medical School, Boston, MA, USA b Department of Obstetrics, Gynaecology, and Reproducti6e Biology, Har6ard Medical School, Boston, MA, USA Introduction: Despite immense effort, the development of vaccines effective at mucosal sites has proceeded at a faltering pace. Efforts concentrating on humoral immunity but neglecting cellular immunity may be misdirected by ignoring many viral mucosal pathogens. Improved understanding of the development and maintenance of lymphocytes populating the reproductive tract (rtIELs) may inform advances in vaccination strategies for sexually transmitted diseases. Recent studies highlight tissue-specific differences in the development of mucosal immunity, suggesting that the local milieu may play a role in selection, maintenance and function of resident lymphoctes. Here we describe MHC class I and thymus dependence of subpopulations of rtIELs. Materials and methods: Vaginas, uteri, and fallopian tubes were excised from 6 – 8 week, virginal classical MHC class I deficient (KbDb − / −), athymic or control mice. Intraepithelial lymphocytes were isolated from these organs using standard protocols, and pooled (n= 3– 4 animals) for FACS analysis. Results: TCRab+ CD8ab+ T cells in the periphery, intestine and female reproductive tract are all developmentally dependent on classical class I MHC and the thymus. TCRab+ CD8aa+ are absent from the periphery and the rtIELs but are present, and classical MHC class I- and thymus-independent, in the intestine. TCRgd+ rtIELs display a cannonical Vd and are thymus dependent. This is in contrast to intestinal TCRgd+ cells. Conclusions: Lymphocytes isolated from the murine female reproductive tract have characteristics of both peripheral T cells and of T cells found in other mucosal sites. Therefore, in approaching vaccination strategies, the female reproductive tract should regarded neither as peripheral nor mucosal, but rather as a unique tissue with distinctive immunological characteristics.

Paracrine and Cellular Events Cytokine Cross-Talk Between the Human Trophoblasts and Decidual Leukocytes Shigeru Saito Department of Obstetrics and Gynaecology, Toyama Medical and Pharmaceutical Uni6ersity, Toyama Cytokines are regulatory peptides or glycoproteins that can be produced by virtually every cell type in the body and have pleiotropic regulatory effects on hematopoietic, endocrine, nervous and immune systems. Unlike hormones, cytokines usually act as intercellular (paracrine) and/or intracellular (autocrine) signals.

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There is much evidence that cytokines play a very important role in the reproduction, i.e. ovarian follicular development, embryo implantation, endometrial development, and trophoblast growth and differentiation by modulating immune and endocrine systems. Cytokines mediate the endocrine and immune systems of the maternal body, fetus and placenta. For example, Th2 type cytokines such as IL-4, IL-6, and IL-10, which seems to be of prime importance in the maintenance of pregnancy, are produced by trophoblasts and decidual Th2 and Tc2 cells. Th2 and Tc2 derived cytokines induce hCG release from trophoblasts. As a result, hCG induces the production of progesterone by the corpus luteum so that Th2 cells can be stimulated to differentiate from Th0 cells by progesterone. Thus, cytokines and hormones cross-talk each other. Recently, we demonstrated that decidual Th2and Tc2-cells gathered around the trophoblasts, suggesting that trophoblasts secreted a kind of chemotactic factor of Th2 and Tc2 cells. Thus, cytokines or chemokines cross-talk between the human trophoblasts and decidual leukocytes. From the early stage of implantation until the expulsive phase of delivery, the cross-talk between immune system and endocrine system via cytokines is critical for successful gestation. In this study, the author review the characteristics of cytokines in the immune and endocrine systems in the human reproduction.

Regulation of Pregnancy by Hormone Controlled TH1 and TH2-Type Cytokines Marie-Pierre Piccinni Department of Internal Medicine. Immunoallergology Unit, Uni6ersity of Florence-Viale Morgagni 85, 50134 Florence, Italy The factors involved in the successful implantation of blastocyst are not yet completely understood. However, we showed that the concomitant production of leukemia inhibitory factor (LIF), interleukin (IL)-4, and IL-10 by the decidual T cells could favor the maintenance of pregnancy during the first trimester of pregnancy (M.-P. Piccinni et al., 1998. Nat. Med. 4, 1020 –1024). We also suggest that relaxin, which upregulates the development of IFN-g producing T cells (Piccinni et al., 1999. Eur. J. Immunol. 29, 2241 – 2247) and progesterone, which favors the development of IL-4 producing T cells (M.-P. Piccinni et al., 1995. J. Immunol. 155, 128 –133), may contribute to the regulation of the immune homeostasis during pregnancy. LIF expression could not be confined exclusively to the blastocyst implantation and to the maintenance of pregnancy but could also be necessary for preimplantation blastocyst development. Indeed, LIF improves the development of ovine and murine embryos in culture and enhance blastocyst formation. Recently, we found that T cells and macrophages are present in the human cumulus oophorus, which is a mass of cells surrounding the human oocyte. It has been reported that high levels of progesterone are produced by the oocyte-cumulus oophorus complex. We found that cumulus oophorus T cells produce levels of IL-4 and LIF that are higher than those produced by the peripheral blood T cells and the ovarian T cells of women, suffering from blocked fallopian tubes and undergoing an assisted reproduction programme. The role of cumulus oophorus T cells in the regulation and in the pre-implantation development of the embryo could be suggested.

Mucosal Immunity in the Human Female Reproductive Tract William H. Kutteh, M.D., Ph.D. Uni6ersity of Tennessee, USA

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In almost all human external secretions (e.g. saliva and milk) secretory IgA (S-IgA) is the dominant isotype. In contrast, secretions of the human female genital tract contain both IgA and IgG antibodies derived mostly from local antibody-secreting cells (ASC). Understanding the source and properties of specific antibodies in the human genital secretions, the origin of ASC and the selective induction of specific antibodies in genital secretions may have significant relevance to understanding and preventing sexually transmitted diseases. ASC that populate mucosal tissues, such as the gut and salivary and mammary glands, originate from mucosal inductive sites, including Peyer’s patches in the small intestine and rectal, nasal and bronchial lymphoepithelial tissues. Furthermore, recent studies indicate that the homing of activated lymphocytes from inductive sites to specific effector sites (e.g. salivary and mammary glands) is dependent on the expression of specific lymphocyte surface homing receptors that recognize corresponding addressins on endothelial cell walls. Thus, the a4b7 receptor guides the lymphocytes to home to the intestinal mucosa, while L-selectin is recognized as a peripheral lymph node homing receptor. Consequently, administration of antigens at mucosal inductive sites results in the appearance of specific antibodies in distant mucosal effector sites due to the selective dissemination of ASC. To determine if the female genital tract is a component of this common mucosal immune system, immunization routes effective in the induction of specific humoral and cellular responses in women have only recently been explored. Our recent studies investigated the efficacy of gastrointestinal tract immunization in the induction of specific antibodies in human female genital tract secretions. As primary immunization, volunteers received the live attenuated Salmonella Typhi Ty 21a vaccine orally. We also evaluated the effect of rectal boosting on the induction of a mucosal immune response, because the rectal tonsils have been considered a possible source of lymphocytes destined to migrate to and produce antibodies at the mucosal surface of the female reproductive tract. Salmonella-specific antibodies of the IgG and IgA isotypes were measured in vaginal lavage and cervical mucus collected at mid-cycle for 4 months. Salmonella-specific antibodies measured 1 month after rectal vaccination demonstrated larger increases in vaginal fluids and cervical mucus and were dominated by IgA. The results indicate that specific antibodies in the human female genital tract induced by oral vaccination can be enhanced by subsequent rectal administration of vaccine. Moreover, this study is of importance to female reproductive tract immunity since recombinant Salmonella have been used as live vectors for the expression of gene products from unrelated microorganisms (e.g. HIV) suitable for intestinal immunization.

What are the Possible Functions of Uterine NK Cells? Ashley King Research Group in Human Reproducti6e Immunobiology, Uni6ersity of Cambridge, UK The human uterine mucosa is populated by a population of Natural Killer (NK) cells with an unusual phenotype of CD3-, CD16-, CD56bright. These cells are sparse during the proliferative phase of the menstrual cycle, increase significantly in number at the luteal phase and ultimately show signs of death a few days before menstruation. If pregnancy occurs, these cells remain viable and in large numbers in decidua for the early part of pregnancy. We believe these NK cells have two major roles to play in relation to pregnancy. One is in the initiation and maintenance of decidualisation. The other is in the control of trophoblast invasion during implantation. The mechanism for the first of these functions is probably the production of relevant cytokines and growth factors (e.g. VEGF-C) acting predominantly on the stromal cells and uterine blood vessels. For the control of trophoblast

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Abstracts

invasion, NK cells express several families of receptors that are capable of recognising the HLA class I antigens expressed by trophoblast. The resultant interaction will generate a combination of positive and negative signals which will influence trophoblast behaviour. Thus, our proposal is that uterine NK cells are crucial to reproduction during both the pre-implantation as well as the post-implantation stages of the process. Key words: NK cells; Decidua; Trophoblast; Placentation

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