Male-specific Minor Histocompatibility Research Articles

Maternal carriage of H-Y restricting HLA class II alleles is a negative prognostic factor for women with recurrent pregnancy loss after birth of a boy

Immune system aberrations are suggested to be an important factor in the pathophysiology of unexplained secondary recurrent pregnancy loss (sRPL). The objective was to investigate if the sex ratio of the firstborn child in sRPL patients differs from the background population and whether the sex of the firstborn child has a negative impact on the pregnancy prognosis alone and/or in combination with carriage of male-specific minor histocompatibility (H-Y) restricting HLA class II alleles. From January 2016 to October 2022, 582 patients with unexplained RPL were admitted to the RPL Center of Western Denmark and continuously followed-up. HLA-DRB1 and -DQB1 typing was performed as part of the routine diagnostic work-up. In sRPL patients, a history of a firstborn boy was significantly more frequent than in the Danish background population and was associated with significantly lower odds of a successful reproductive outcome in the first pregnancy after admission compared to a firstborn girl (OR=0.41, 95% CI: 0.20–0.83, p = 0.014). The odds of a successful reproductive outcome were enhanced in patients carrying ≥ 1 H-Y-restricting HLA class II alleles with a first-born girl compared to a firstborn boy (OR=3.33, 95% CI: 1.40–7.88, p = 0.005), while no difference in successful reproductive outcome was seen in sRPL patients not carrying these alleles (OR=1.20, 95% CI: 0.33–4.43, p = 0.781). The sex ratio of children born after RPL was similar to the Danish background population. These findings confirm previous findings and suggests that a harmful immune response triggered by H-Y-antigen exposure during a previous pregnancy in preconditioned women may cause sRPL.

Anti-Thymocyte Globulin and Impact of Gender on Allogeneic Stem Cell Transplant

Background The female donor/male recipient combination increases the risks of graft-versus-host disease (GVHD) and non-relapse mortality (NRM) after allogeneic stem cell transplantation (allo-SCT), with a possible deleterious impact on overall survival. The mammalian Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that are recognized by female T cells. Some clinical data suggest a strong correlation between H-Y Antigen disparity and GVHD. In addition, female donors are notorious for bearing memory type lymphocytes induced by previous pregnancies; such allo-immune cells may provoke unwanted immune reactions such as GVHD in the recipient. Recently CIBMTR published GVHD data on patients who underwent T-cell replete SCT. Methods To explore the impact of gender on outcome of allo-SCT when using anti-thymocyte globulin in unrelated donors, we undertook a single center retrospective analysis of adult patients transplanted at our center in the last 10 years. Unrelated donors were matched at the allele level for HLA-A, B, C, DRB1, and DQB1. We excluded patients who received donor lymphocyte infusion post SCT. Results 157 patients were identified and included in this analysis. The median age at transplant was 57 years (19-73). Diseases were AML (n=66), ALL (n=30), MDS (n=59), and MPN (n=2). Donors were male- matched related donor (m- MRD) (n=32),female (f)-MRD (n=24) or unrelated donors (n=101). Source of stem cells was peripheral blood in all patients except 4. GVHD prophylaxis consisted of tacrolimus and methotrexate in MRD and tacrolimus, methotrexate and anti-thymocyte globulin in unrelated donors. Of the f-MRD donors, 54% had previous pregnancies before donation, 29% had no pregnancies and 17% were unknown. The median follow-up was 10.6 months. Rate of acute GVHD >=2 was 25% in patients who had m-MRD, 25% in patients who had f-MRD (42% in f-MRD without pregnancy, 23% in f-MRD with pregnancy), and 37.5% in unrelated donors. Conclusion In this small cohort of consecutive patients from a single center, we found only a small difference in acute GVHD between m-MRD, f-MRD and unrelated donors when adding anti-thymocyte globulin in unrelated SCT. These results should be interpreted with caution given the retrospective design of the study and because we cannot exclude a possible limiting effect of a small number of patients.

The Divergence of Neandertal and Modern Human Y Chromosomes

Sequencing the genomes of extinct hominids has reshaped our understanding of modern human origins. Here, we analyze ∼120 kb of exome-captured Y-chromosome DNA from a Neandertal individual from El Sidrón, Spain. We investigate its divergence from orthologous chimpanzee and modern human sequences and find strong support for a model that places the Neandertal lineage as an outgroup to modern human Y chromosomes—including A00, the highly divergent basal haplogroup. We estimate that the time to the most recent common ancestor (TMRCA) of Neandertal and modern human Y chromosomes is ∼588 thousand years ago (kya) (95% confidence interval [CI]: 447–806 kya). This is ∼2.1 (95% CI: 1.7–2.9) times longer than the TMRCA of A00 and other extant modern human Y-chromosome lineages. This estimate suggests that the Y-chromosome divergence mirrors the population divergence of Neandertals and modern human ancestors, and it refutes alternative scenarios of a relatively recent or super-archaic origin of Neandertal Y chromosomes. The fact that the Neandertal Y we describe has never been observed in modern humans suggests that the lineage is most likely extinct. We identify protein-coding differences between Neandertal and modern human Y chromosomes, including potentially damaging changes to PCDH11Y, TMSB4Y, USP9Y, and KDM5D. Three of these changes are missense mutations in genes that produce male-specific minor histocompatibility (H-Y) antigens. Antigens derived from KDM5D, for example, are thought to elicit a maternal immune response during gestation. It is possible that incompatibilities at one or more of these genes played a role in the reproductive isolation of the two groups.

Impact of Gender: Female Related Donor Versus Male Matched Unrelated Donor on Peripheral Blood Allogeneic Stem Cell Transplant for Male Recipients

BackgroundThe female donor/male recipient combination increases the risks of graft-versus-host disease (GVHD) and non-relapse mortality (NRM) after allogeneic stem cell transplantation (allo-SCT), with a possible deleterious impact on overall survival (OS). The mammalian Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that are recognized by female T cells. Some clinical data suggest a strong correlation between H-Y Antigen disparity and GVHD. In addition, female donors are notorious for bearing memory type lymphocytes induced by previous pregnancies; such allo-immune cells may provoke unwanted immune reactions such as GVHD in the recipient. To explore the impact of Female related donor (F-MRD) versus male matched unrelated donor (M-MUD) on outcome of peripheral blood allo-SCT in male recipients, we undertook a single center retrospective analysis of male adult patients transplanted at our center in the last 10 years. MUDs were matched at the allele level for HLA-A, B, C, DRB1, and DQB1. We excluded patients who received donor lymphocyte infusion post SCT. MethodsDisease-free survival (DFS) and OS were calculated using the Kaplan-Meier estimate. Cumulative incidences (CI) were used for relapse (REL), and GVHD in a competing risks setting, NRM being a competing event for REL, and death for GVHD. The following variables were considered: recipient age (³ vs < 57 years, median age), F-MRD vs M-MUD, myeloablative (MAC) vs reduced-intensity ablative conditioning (Red ABL) vs non-myeloablative (NMA) regimen, early (CR1 or PR1 or chronic phase or untreated) vs advanced disease. Steroid-refractory (SR) GVHD was defined as acute GVHD progressing after 3 days of treatment, or unchanged after 7 days, or in incomplete response after 14 days, or chronic GVHD with no response after one month, no partial response after 2 months or progressive disease in 2 weeks of initiation of steroid treatment or during the methylprednisolone taper. RESULTSFifty-six patients were identified and included in this analysis. The median age at transplant was 57 years (19-73). Diseases were AML (n=21), ALL (n=7), NHL (n=9), Hodgkin’s disease (n=1), myeloma (n=5), MDS (n=11), CLL (n=1), and CML (n=1). Conditioning regimens were MAC (n=35), RED ABL (n=17) or NMA (n=4). Donors were F- MRD (n=24) or M-MUD (n=32). Source of stem cells was peripheral blood in all patients. GVHD prophylaxis consisted of tacrolimus and methotrexate in MRD and tacrolimus, methotrexate and anti-thymocyte globulin in MUD. Of the female donors, 63% had previous pregnancies before donation, 8% had no pregnancies and 29% were unknown. Mean age of female donors was 48 while the mean age for male donors was 35.The median follow-up was 8 months (0.8-54 months). The median OS for patients who received M-MUD was 8.1 months while it was 14.2 months for patients who received F-MRD (p= 0.72) Fig1. Median DFS for patients who received M-MUD was 6 months while it was 7.2 months for patients who received F-MRD (p= 0.94). The CI rates of aGVHD III-IV, extensive chronic GVHD, REL and NRM at 12 months in M-MUD vs F-MRD were 13% vs 16% (p=0.69), 21% vs 27% (p=0.61), 38% vs 44% (P=0.65), and 16% vs 14% (p=0.79), respectively. The CI of SR GVHD in M-MUD vs F-MRD was 24% vs 32% (The Hazard Ratio of srGVHD in M-MUD vs F-MRD is 0.711 (95% confidence interval is 0.29-1.72, p=0.45)) Fig2. Other variables considered in univariate analysis for SR GVHD were recipient age (p=0.55), disease status (p= 0.64), MAC vs other regimens (p=0.58). ConclusionIn this small cohort of consecutive patients from a single center, we found that compared to M-MUD, F-MRD had a trend for higher rates of SR GVHD after peripheral blood allo-SCT in adult male patients, although not statistically significant. There was no impact of F-MRD compared to M-MUD on OS, DFS, REL, NRM or rates of acute or chronic GVHD. However, the absence of statistical impact should be interpreted with caution given the retrospective design of the study and because we cannot exclude a possible limiting effect of a small number of patients.Fig 1 [Display omitted] Fig 2 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Advances of intravenous immunoglobulin G in modulation of anti-fetal immunity in selected at-risk populations: science and therapeutics.

Advances of intravenous immunoglobulin G in modulation of anti-fetal immunity in selected at-risk populations: science and therapeutics.

Immunity against male specific HY-antigens is prognostical important in secondary recurrent miscarriage

Immune reactions against male-specific minor histocompatibility (HY) antigens may be a causal factor in secondary recurrent miscarriage (SRM) defined as consecutive miscarriages after a birth. We wanted in a new cohort of SRM patients to confirm or reject previous findings that in those with a firstborn boy, HLA-class II allelles (DRB1*15 and DQB1*0501/0502 called risk HLA alleles) predisposing to anti-HY immunity have prognostically negative impact.1

High Non-Relapse Mortality and Low Relapse Incidence in Sex-Mismatched Allogeneic Hematopoietic Stem Cell Transplantation From Female Donor with Male Child

Abstract 1945Allogeneic hematopoietic stem cell transplantation (allo-HSCT) from female donor to male recipient (F to M) has been reported to be a risk factor of poor prognosis. Although immune responses to male specific minor histocompatibility antigens (H-Y) are suggested to be involved, this hypothesis has not yet been well validated. To clarify the significance of exposure to H-Y, we retrospectively surveyed the medical records of 292 patients with hematological malignancies who underwent allo-HSCT from a related donor at National Cancer Center Hospital from 2001 to 2010. Data about children’s sex and pregnancies of female donors were collected by chart review. The median follow-up of survivors was 69 months (range; 3–128) after HSCT. The median age of donors and recipients were 47 years (range; 12–69 years) and 51 years (range; 18–69 years), respectively. The distribution of sex combination of donors and recipients were as follows: 94 male to male, 57 female to female, 70 female to male and 71 male to female. About a half of female donors had pregnancy history (79 of 127; 62.2%, including abortion history). According to F to M sex-mismatch pattern and children’s sex, patients were divided into three groups: 31 female donor with male child to male recipient (F to M with MC), 39 female donor without male child to male recipient (F to M without MC) and 222 non-F to M sex combination (non-F to M). The F to M sex-mismatch pattern did not significantly affect OS regardless of children’s sex (5-year OS, F to M with MC; 52%, F to M without MC 40%; non-FtoM; 43%). In multivariate analysis, only disease status before HSCT significantly affected OS (hazard ratio [HR]; 1.64, 95% confidential interval [CI]; 1.17 to 2.28, P<0.01). However, non-relapse mortality (NRM) was significantly higher in F to M with MC than in the other groups (5-year NRM, F to M with MC; 34%, F to M without MC 24%; non-F to M; 20%, P=0.02), which was also confirmed in multivariate analysis (HR 2.00, 95% CI; 1.11 to 3.58, P=0.02). Furthermore, relapse incidence (RI) tended to be lower in F to M with MC than in the other groups (5-year RI, F to M with MC; 23%, F to M without MC 41%; non-F to M; 43%, P=0.12), which was of borderline significance in multivariate analysis (HR 0.48, 95% CI; 0.21 to 1.09, P=0.08). Because the cumulative incidences of acute and chronic GVHD in F to M with MC were higher than those of F to M without MC (26% v.s. 13%; P=0.12, 52% v.s. 37%; P=0.26, respectively), immune response to H-Y were suggested to be involved in the transplant outcome. In summary, F to M with MC had some special features, high NRM and low RI. These effects were counteracted by each other, resulting in similar OS to the other groups. These data suggest the possibility that poor outcome of allo-HSCT from F to M results from the immune response to H-Y and that the exposure of H-Y before donation is critical for the response. Further investigation in a large number of patients is warranted. Disclosures:No relevant conflicts of interest to declare.

Altered sex ratio amongst women presenting with unexplained secondary infertility

Maternal immune reaction against male specific minor histocompatibility genes (HY antigens) may be initiated by carriage of a male fetus. Recently, a higher rate of prior male live birth has been reported amongst couples with secondary recurrent pregnancy loss. We investigated the sex ratio of prior live births in couples presenting with unexplained secondary infertility.

Sexing the Sciuridae: a simple and accurate set of molecular methods to determine sex in tree squirrels, ground squirrels and marmots

We determined the sequence of the male-specific minor histocompatibility complex antigen (Smcy) from the Y chromosome of seven squirrel species (Sciuridae, Rodentia). Based on conserved regions inside the Smcy intron sequence, we designed PCR primers for sex determination in these species that can be co-amplified with nuclear loci as controls. PCR co-amplification yields two products for males and one for females that are easily visualized as bands by agarose gel electrophoresis. Our method provides simple and reliable sex determination across a wide range of squirrel species.

Frequency and Intensity of Antibody Responses to Antigens Encoded by KDM5D (SMCY) and Other H-Y Genes After Allogeneic Hematopoietic Cell Transplantation,

Abstract 4028Male recipients of female hematopoietic cell grafts (F->M HCT), when compared with other donor/recipient gender combinations, face an increased risk for both acute and chronic graft-versus-host disease (GVHD), but also have a significantly decreased risk of posttransplant relapse. F->M HCT is characterized at the cellular level by female donor T cell responses against male-specific minor histocompatibility (H-Y) antigens, which can contribute to both GVHD and graft-versus-leukemia (GVL) activity. Data from both murine models and clinical studies of allogeneic HCT demonstrate that the protein product of the Y chromosome gene KDM5D (previously known as SMCY) is one of the major targets of H-Y-specific T cell responses. CD8+ T cell responses against KDM5D peptides have been associated with GVHD and GVL in male recipients of female grafts, and with rejection of male grafts by female recipients.A subset of Y chromosome-encoded proteins, including several that are targets for CD8+ and CD4+ T cell responses after F->M HCT, are also the targets of antibody responses in this setting (Miklos et al., Blood 2005; 105:2973). Antibody responses to the protein products of DDX3Y and UTY, in particular, have been detected in up to 50% of F->M HCT recipients. Moreover, antibody responses to these Y chromosome gene products have been associated with the development of chronic GVHD as well as the maintenance of remission. T cell responses to KDM5D in F->M HCT recipients have been extensively studied, but KDM5D-reactive antibody responses have not been thoroughly evaluated. We hypothesized that KDM5D -reactive antibodies would also develop in a significant fraction of F->M HCT recipients, and undertook an exploratory study to define the frequency and intensity of antibody responses to KDM5D in both allogeneic HCT patients and healthy adult male and female control subjects.Plasma samples were obtained from 39 male HCT patients with female donors, 16 HCT patients with other donor/recipient gender combinations, 12 healthy female controls, and 9 healthy male controls, and assessed for IgG responses against an array of 84 different protein targets by ELISA. The target panel included partial (SMCY and UTY) or whole (DDX3Y, ZFY RPS4Y, and E1F1AY) recombinant H-Y proteins and their X chromosome-encoded homologues that had been expressed in and purified from E. coli, as well as synthetic overlapping peptides of length 15–21 residues derived from the KDM5D and DDX3Y protein sequences or from their X chromosome-encoded homologues KDM5C (SMCX) and DDX3X. Of the 55 samples acquired from HCT recipients, 49 came from recipients who had previously been diagnosed with cGVHD and were receiving immunosuppressive therapy.Reactivity with KDM5D-derived peptides or recombinant KDM5D protein fragments was observed in HCT recipients of all four donor-recipient gender combinations and also in some healthy control males and females. Amongst male patients with female donors, 7 of 39 (18%) samples were reactive with more than 3 KDM5D peptides, and 2 of 16 (13%) samples from patients with other donor/recipient gender combinations were reactive with 3 or more KDM5D peptides. Significant differences in the frequency of reactivity with individual peptides were observed, with a high frequency of responses to peptides corresponding to sequences within the interval KDM5D281–375, a region of the protein where there is significant nonidentity with the sequence of its X chromosome-encoded homologue KDM5C. This region also contains the sequence FIDSYICQV, which comprises the epitope recognized by CD8+ HLA-A*0201-restricted T cell clones that have frequently been isolated from HLA-A*0201+ F->M HCT recipients and have been associated with both GVHD and GVL. Antibody reactivity to KDM5D was correlated with reactivity to several DDX3Y-derived peptides previously shown to be the targets of antibody responses in F->M HCT recipients, but weakly associated with responses to UTY protein fragments, which were observed in 11 of 39 (28%) F->M patients and 1 of 16 (6%) patients with other donor/recipient gender combinations. These results suggest that KDM5D is a frequent target of posttransplant antibody responses in allogeneic HCT recipients. Current studies are evaluating in a larger cohort of patients the relationship of KDM5D-reactive antibody responses to H-Y-specific T cell responses and to the development of acute and chronic GVHD. Disclosures:No relevant conflicts of interest to declare.

Anti‐HY Responses in Pregnancy Disorders

Cellular and humoral immune responses against male-specific minor histocompatibility (HY) antigens are important in the pathogenesis of graft-versus-host reactions and can be detected in women who have previously given birth to a boy. However, the importance of these responses for pregnancy outcome is unclear. Review of the current knowledge about the impact of anti-HY immunity on pregnancy outcome in terms of risk of miscarriage, placental abruption and low birth weight. Women with secondary recurrent miscarriage (RM) more often have given birth to a boy compared with a girl prior to a series of miscarriages (P<0.0001) and a firstborn boy seems significantly to reduce the chance of a subsequent live in these patients (P=0.0003). Human leukocyte antigen (HLA) class II alleles known to restrict CD4 T-cell-mediated anti-HY responses were investigated among 358 patients with secondary RM and 203 of their children born prior to the miscarriages. The chance of a subsequent live birth in secondary RM patients with firstborn boys compared with firstborn girls was significantly lower in women with HY-restricting HLA class II alleles [OR: 0.17 (0.1-0.4), P=0.0001]. In patients without these alleles, the chance of live birth was similar in those with firstborn boys and girls, respectively. In early pregnancy, both antibodies against HLA and HY antigens were found with increased prevalence in secondary patients with RM and in particular in those with a firstborn boy compared with controls (P=0.005). The presence of these antibodies was associated with a low subsequent live birth rate, and the presence of HY antibodies was associated with a low male/female ratio (12% boys versus 88% girls) in subsequent live births (P=0.03). Register-based population studies suggested that births of boys also are associated with subsequent adverse obstetric outcomes also in the background population. Findings strongly indicate that aberrant maternal immune reactions against fetal HY antigens playing a role in secondary RM and other pregnancy complications. We propose pathogenetic pathways for these conditions that in our view best explain the findings.

Fetus specific T cell modulation during fertilization, implantation and pregnancy

Recently there is an increasing interest in aspects of a more specific immunoregulation during pregnancy. Understanding these mechanism might have a broader application not only for reproductive immunology but also in general for biology and medicine. Especially the induction, already before conception, of feto-specific T cells with a possibly regulatory function gives a biological explanation of local immunotolerance at the maternal fetal interface, supporting the epidemiological evidence of a feto/paternal-specific immuneregulation. Understanding the expression of specific HLA-classes on trophoblast and the crosstalk of these antigens with various cell types, specifically modulated in the decidua, resulting in the secretion of cytokines and (angiogenic) chemokines has given us a more and more detailed understanding of this regulation. This regulation could be induced by fetal cells circulating in the mother (microchimerism) and from the interaction with fetal subcellular fractions as exosomes, but also from paternal antigens present in seminal fluid. Molecular interaction between paternal and fetal antigens and receptors in endometrium and the decidua are discussed.This review highlights besides uNK cells, especially the function of CD4+ and CD8+ T cells with a regulatory function in the context of recurrent miscarriage and pre-eclampsia. Besides HLA, also male-specific minor histocompatibility antigens and the genetic background for these pregnancy complications are discussed.

Secondary recurrent miscarriage and H-Y immunity

Approximately half recurrent miscarriage (RM) cases remain unexplained after standard investigations. Secondary RM (SRM) is, in contrast to primary RM, preceded by a birth, which increases the transfer of fetal cells into the maternal circulation. Mothers of boys are often immunized against male-specific minor histocompatibility (H-Y) antigens, and H-Y immunity can cause graft-versus-host disease after stem-cell transplantation. We proposed the H-Y hypothesis that aberrant H-Y immunity is a causal factor for SRM. This is a critical review of the H-Y hypothesis based on own publications and papers identified by systematic PubMed and EMBASE searches. SRM is more common after the birth of a boy and the subsequent live birth rate is reduced for SRM patients with a firstborn boy. The male:female ratio of children born prior and subsequent to SRM is 1.49 and 0.76 respectively. Maternal carriage of HLA-class II alleles presenting H-Y antigens to immune cells is associated with a reduced live birth rate and increased risk of obstetric complications in surviving pregnancies in SRM patients with a firstborn boy. In early pregnancy, both antibodies against HLA and H-Y antigens are increased in SRM patients compared with controls. Presence of these antibodies in early pregnancy is associated with a lower live birth rate and a low male:female ratio in subsequent live births, respectively. Births of boys are also associated with subsequent obstetric complications in the background population. Epidemiological, immunogenetic and immunological studies support the hypothesis that aberrant maternal H-Y immune responses have a pathogenic role in SRM.

Gene Therapy with IgG-HY Fusion Proteins to Reduce Male-Specific T-Cell ReactivityIn Vitro

Graft-versus-host disease is still a major complication in stem-cell transplantation. In HLA-identical stem-cell transplantation, mismatches in minor histocompatibility antigens contribute to the development of graft-versus-host disease. As treatment of graft-versus-host disease with immunosuppressive drugs potentially also reduces the graft-versus-leukemia responses, antigen-specific tolerance induction may be used to reduce graft-versus-host disease while leaving the graft-versus-leukemia responses intact. In mouse models, induction of antigen-specific tolerance using IgG-antigen-transduced B cells can prevent and ameliorate autoimmunity. In this study, the principle of the in vivo animal model has been applied to a human in vitro model wherein induction of tolerance against the HLA class II-restricted male-specific minor histocompatibility antigen RPS4Y1 has been analyzed. T cells precultured in the presence of B cells expressing the IgG-coupled HY antigen showed reduced responsiveness selectively against the minor HY antigen RPS4Y1 in different in vitro protocols. These first observations serve as a basis for further studying and understanding the tolerizing potential of IgG constructs with human T cells.

Secondary recurrent miscarriage is associated with previous male birth

Secondary recurrent miscarriage (RM) is defined as three or more consecutive pregnancy losses after delivery of a viable infant. Previous reports suggest that a firstborn male child is associated with less favourable subsequent reproductive potential, possibly due to maternal immunisation against male-specific minor histocompatibility antigens. In a retrospective cohort study of 85 cases of secondary RM we aimed to determine if secondary RM was associated with (i) gender of previous child, maternal age, or duration of miscarriage history, and (ii) increased risk of pregnancy complications. Fifty-three women (62.0%; 53/85) gave birth to a male child prior to RM compared to 32 (38.0%; 32/85) who gave birth to a female child (p=0.002). The majority (91.7%; 78/85) had uncomplicated, term deliveries and normal birth weight neonates, with one quarter of the women previously delivered by Caesarean section. All had routine RM investigations and 19.0% (16/85) had an abnormal result. Fifty-seven women conceived again and 33.3% (19/57) miscarried, but there was no significant difference in failure rates between those with a previous male or female child (13/32 vs. 6/25, p=0.2). When patients with abnormal results were excluded, or when women with only one previous child were considered, there was still no difference in these rates. A previous male birth may be associated with an increased risk of secondary RM but numbers preclude concluding whether this increases recurrence risk. The suggested association with previous male birth provides a basis for further investigations at a molecular level.

The impact of anti-HY responses on outcome in current and subsequent pregnancies of patients with recurrent pregnancy losses

Women pregnant with a male fetus often generate cellular and humoral immune responses against male-specific minor histocompatibility (HY) antigens-however, the importance of these responses for pregnancy outcome is unclear. Epidemiologic studies have shown that the birth of a boy compared with a girl prior to a series of miscarriages significantly reduces the chance of a subsequent live birth and pregnancies with boys have an increased risk of placental abruption. This paper aims to review the current knowledge about the impact of anti-HY immunity on pregnancy outcome in terms of miscarriage and placental abruption. Our knowledge primarily comes from studies of the impact on pregnancy outcome of HLA class II alleles known to restrict CD4 T cell mediated anti-HY responses among 358 secondary recurrent miscarriage (SRM) patients and 203 of their children born prior to the miscarriages and investigation of these HLA alleles in 8 patients with recurrent severe placental abruptions. The chance of a subsequent live birth in SRM patients with firstborn boys compared to firstborn girls was significantly lower in women with HY-restricting HLA class II alleles [OR: 0.17 (0.1-0.4), p=0.0001]. Most patients with recurrent placental abruptions had firstborn boys and significantly more of these patients carried HLA haplotypes with HY-restricting class II alleles compared with controls (p=0.009). These findings are strongly indicative of aberrant maternal immune reactions against fetal HY antigens playing a role in recurrent miscarriage and placental abruption. We propose pathogenetic pathways for these conditions that in our view best explain the findings.

Sex of Prior Children and Risk of Stillbirth in Subsequent Pregnancies

Delivery of a boy has been reported to increase a woman's risk of recurrent miscarriage in subsequent pregnancies. We explored whether delivery of boys similarly increases the risk of a subsequent stillbirth. We identified all Danish women delivering their first child (singleton) between 1980 and 1998 (n = 499,731) using the Danish Birth Registry. These women had subsequent singleton births through 2004 (n = 558,314). We assessed the risk of stillbirth conditional on sex of prior children. The risk of stillbirth was increased by 12% after deliver of boys compared with girls (relative risk = 1.12 [95% confidence interval = 1.02-1.23]). This association did not appear to be explained by maternal confounders. Stillbirth risk appears to be slightly higher among the pregnancies of women who have previously delivered a boy. One possible mechanism is maternal immune response to male-specific minor histocompatibility antigens initiated during pregnancies with boys.

Identification of Novel MHC Class II-Restricted Male-Specific mHAg Encoded bySMCY (JARID1D)..

Abstract 1344Poster Board I-366Male recipients of female hematopoietic cell grafts, when compared with all other donor/recipient gender combinations, have an increased risk for both acute and chronic GVHD, but also have a significantly decreased risk of posttransplant relapse. F→M HCT is also characterized at the cellular level by donor (female) T cell responses against male-specific minor histocompatibility (H-Y) antigens, which can contribute to both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) activity. SMCY is a Y-chromosome gene that has previously been shown to encode at least two distinct MHC class I-restricted H-Y antigens presented by HLA-A*0201 and HLA-B*0702, respectively. Also, association between CD8+ T cell responses specific for the SMCY311-319 FIDSYICQV epitope and GVHD or GVL has been reported. A CD8+ FIDSYICQV-specific T cell clone was also reported to induce histological signs of GVHD reaction in an in vitro skin-explant assay. To date, however, only two MHC class I-restricted, and no MHC class II-restricted, H-Y antigens encoded by SMCY have been characterized. Given the large size of the SMCY and the homologous SMCX proteins and the fact that they are only 85% identical at the amino acid sequence level, we hypothesized that SMCY encodes other MHC class I- and class II-restricted H-Y antigens, and that T cell responses against these epitopes may likewise contribute to GVHD and GVL activity after F→M HCT. Arrays of pentadecapeptides with eleven-residue overlap were designed to tile regions of the SMCY protein that are non-identical to the corresponding regions of its X chromosome-encoded homologue SMCX, and then used to generate SMCY-specific T cell lines recognizing novel SMCY-encoded MHC class I- and class II-restricted H-Y antigens. Peripheral blood mononuclear cells (PBMC) were obtained on posttransplant day +126 from a 46 year-old male patient with monosomy 7 AML who had received a hematopoietic cell graft from his MHC-identical sister, and were stimulated in vitro with dendritic cells derived from his pretransplant PBMC that had been pulsed with the SMCY pentadecapeptides. After three stimulations, a SMCY peptide-specific CD4+ T cell line as well as a SMCY311-319 (FIDSYICQV)-specific CD8+ T cell line were obtained. After cloning by limiting dilution, we further characterized the SMCY-specific CD4+ T cell clone, 13H3. The 13H3 T cell clone recognizes the SMCY232-246 15-mer peptide, ELKKLQIYGPGPKMM, presented by HLA-DRB1*1501, and has a CD3+, CD4+, CD8−, CD45RA−, CD45RO+ surface phenotype. The cytokine release profile of this clone when assessed with SMCY232-246-loaded donor-derived EBV-LCL, as measured by the Luminex assay, is characterized mainly by Th1 cytokines (IFN-g and IL-2), but the clone also produced low to moderate levels of the Th2 cytokines IL-4, IL-10, and TGF-β. A minigene encoding SMCY232-246 was recognized by the 13H3 clone in a HLA-DRB1*1501-dependent fashion when transfected into COS-7 cells, but a minigene encoding the homologous SMCX-derived ELKKLQIYGAGPKMM peptide was not recognized, demonstrating that the clone is SMCY-specific. The 13H3 clone recognized 3 of 5 HLA-DRB1*1501+ male primary leukemia cells, but did not recognize either of 2 HLA-DRB1*1501− male or either of 2 HLA-DRB1*1501+ female primary leukemia cells. These results suggest that CD4+ T cell responses against the SMCY232-246 epitope could potentially contribute to GVL activity after F→M HCT. A SMCY232-246/HLA-DRB1*1501 tetramer has been constructed which specifically marks the 13H3 T cell clone, and future studies will use this reagent to determine whether CD4+ T cells specific for this epitope can be detected directly ex vivo in posttransplant blood samples from HLA-DRB1*1501+ F→M HCT recipients. DisclosuresNo relevant conflicts of interest to declare.

Association of HY-restricting HLA class II alleles with pregnancy outcome in patients with recurrent miscarriage subsequent to a firstborn boy

Healthy females, pregnant with a boy, generate immune responses against male-specific minor histocompatibility (HY) antigens. The clinical importance of these responses is evident in stem cell transplantation. Birth of a boy prior to a series of miscarriages reduces the chance of a subsequent live birth. This study explores the putative impact of known HY-presenting HLA alleles on future pregnancy outcome in women with at least three consecutive miscarriages following a birth [secondary recurrent miscarriage (SRM)]. HLA-A, -B, -DRB1, DRB3-5 and DQB1 genotyping was performed in 358 SRM patients and in 203 of their children born prior to the miscarriages. The subsequent live birth in women with boys prior to the miscarriages compared with girls is lower in women with HY-restricting HLA class II alleles [odds ratio (OR): 0.17 (0.1-0.4), P = 0.0001]. One HY-restricting HLA class II allele in women with firstborn boys significantly reduces the chances of a live birth [OR: 0.46 (0.2-0.9), P = 0.02]. Two HY-restricting HLA class II alleles further reduced this chance [OR: 0.21 (0.1-0.7), P = 0.02]. HY-restricting HLA class II did not reduce the chances of a live birth in SRM women with firstborn girls. HY-restricting HLA class II alleles are associated with a decreased chance of a live birth in SRM women with firstborn boys. These findings strongly indicate an aberrant maternal immune reaction against fetal HY-antigens in SRM. The results may shed light on the as-yet unknown immunological causes of SRM and may help understand the successful maternal acceptance of the fetal semi-allograft.

DDX3Y encodes a class I MHC–restricted H-Y antigen that is expressed in leukemic stem cells

AbstractThe Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8+ cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705–restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)–identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705+ patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8+ T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.