Maria Therese Ahlen

T-cell responses associated with neonatal alloimmune thrombocytopenia: isolation of HPA-1a-specific, HLA-DRB3*0101-restricted CD4+ T cells.

T-cell responses have been implicated in the development of HPA-1a-induced neonatal alloimmune thrombocytopenia (NAIT). However, HPA-1a-specific T cells have neither been isolated nor characterized. Here, we aimed to determine whether HPA-1a-specific T cells could be isolated from HPA-1a-immunized women. In the present study, peripheral blood mononuclear cells (PBMCs) from an HPA-1a-alloimmunized woman were cultured for weeks in the presence of HPA-1a peptide, labeled with CFSE, and assayed for antigen-specific proliferation. Individual proliferating cells were isolated by fluorescence-activated cell sorting and expanded in culture. Antigen specificity and HLA restriction were determined by cytokine secretion (enzyme-linked immunospot [ELISPOT]) and proliferation assays. Several CD3(+)CD4(+) T-cell clones were isolated that proliferated and secreted cytokines in response to HPA-1a peptide. Two of these clones have been established in long-term culture in our laboratory. Both of these recognize synthetic as well as naturally processed HPA-1a antigen, and the recognition is restricted by the MHC molecule HLA-DRB3*0101 that is strongly associated with NAIT. These HPA-1a-specific T-cell clones represent unambiguous evidence for the association of T-cell responses with NAIT, and they will serve as unique tools to elucidate the cellular immune response that may result in NAIT.

Reconsidering fetal and neonatal alloimmune thrombocytopenia with a focus on screening and prevention

Uncertainty regarding the pathophysiology of fetal and neonatal alloimmune thrombocytopenia (FNAIT) has hampered the decision regarding how to identify, follow-up and treat the women and children with this potentially serious condition. Since knowledge of the condition is derived mainly from retrospective studies, understanding of the natural history of this condition remains incomplete. General screening programs for FNAIT have still not been introduced, mainly because of a lack of reliable risk factors and effective treatment. Now, several prospective screening studies involving up to 100,000 pregnant women have been published and the results have changed the understanding of the pathophysiology of FNAIT and, thereby, the approach toward diagnostics, prevention and treatment in a more appropriate way.

The development of severe Neonatal Alloimmune Thrombocytopenia due to Anti-HPA-1a Antibodies is correlated to maternal ABO genotypes

Background. Maternal alloantibodies against HPA-1a can cross placenta, opsonize foetal platelets, and induce neonatal alloimmune thrombocytopenia (NAIT). In a study of 100, 448 pregnant women in Norway during 1995–2004, 10.6% of HPA-1a negative women had detectable anti-HPA-1a antibodies. Design and Methods. A possible correlation between the maternal ABO blood group phenotype, or underlying genotype, and severe thrombocytopenia in the newborn was investigated. Results. We observed that immunized women with blood group O had a lower risk of having a child with severe NAIT than women with group A; 20% with blood group O gave birth to children with severe NAIT, compared to 47% among the blood group A mothers (relative risk 0.43; 95% CI 0.25–0.75). Conclusion. The risk of severe neonatal alloimmune thrombocytopenia due to anti-HPA-1a antibodies is correlated to maternal ABO types, and this study indicates that the observation is due to genetic properties on the maternal side.

The cellular immunobiology associated with fetal and neonatal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal antibodies that cross the placenta in connection with pregnancy and destroy fetal platelets. Recently, maternal T cell responses associated with FNAIT have been studied at the clonal level. These T cell clones recognize an integrin β3 epitope, which is anchored to the HLA-DRB3∗0101-encoded MHC molecule DR52a. The same MHC allele is strongly associated with FNAIT. As the production of pathological antibodies reactive with fetal platelets is likely dependent on these T cell responses, there exists a potential for preventing FNAIT by targeting these T cells.

Current perspectives on fetal and neonatal alloimmune thrombocytopenia – increasing clinical concerns and new treatment opportunities

Differences in platelet type between the fetus and the mother can lead to maternal immunization and destruction of the fetal platelets, a condition named fetal and neonatal alloimmune thrombocytopenia (FNAIT). FNAIT is reported to occur in ~1 per 1,000 live born neonates. The major risk is intracranial hemorrhage in the fetus or newborn, which is associated with severe neurological complications or death. Since no countries have yet implemented a screening program to detect pregnancies at risk, the diagnosis is typically established after the birth of a child with symptoms. Reports on broader clinical impact have increased clinical concern and awareness. Along with new treatment options for FNAIT, the debate around antenatal screening to detect pregnancies at risk of FNAIT has been revitalized.

Foetal/neonatal alloimmune thrombocytopenia in Egypt; human platelet antigen genotype frequencies and antibody detection and follow-up in pregnancies

BACKGROUND AND OBJECTIVES Foetal and neonatal alloimmune thrombocytopenia (FNAIT) is studied mainly in Caucasian populations. Severe thrombocytopenia (<50×10(9)/L) gives risk of haemorrhage and the most feared complication is intracranial haemorrhage (ICH). In Caucasian populations anti-human platelet antigen (HPA)-1a antibodies are the cause of FNAIT in >80% of the cases. The aims of this project were to study the gene frequencies of HPA-1-5 and 15 alleles in an Egyptian population (Arabic), and to determine the frequency of HPA-1a and -5b immunisations in a cohort of Egyptian pregnant women. MATERIALS AND METHODS Altogether 6974 pregnant women were included in the study. Genotyping was performed by polymerase chain reaction and antibodies were detected by flow cytometry and enzyme-linked immunosorbent assay. HPA-1-5 and 15 alleles were studied in 367 individuals. RESULTS The HPA genotypes differed from genotypes published from different Caucasian and Chinese (Han) populations in HPA-1, -2, -3, and -5 systems with significant higher frequency of HPA-1b, -2b and -5b. The rate of HPA-1a alloimmunisation was found comparable to Caucasian populations. Severe thrombocytopenia was found in two newborns. No bleeding complication was reported. Anti-HPA-5b antibodies were detected in 4.4% of the pregnant women. Clinical consequences of these antibodies were not studied. CONCLUSION The HPA-1bb and -5bb genotypes are more frequent in the Egyptian Arabic population studied compared to Caucasian populations. FNAIT due to anti-HPA-1a and -5b antibodies must be suspected in cases of neonatal thrombocytopenia. Further large prospective studies are needed to increase the knowledge of clinical complications related to HPA alloantibodies in populations with different genetic backgrounds.

Unraveling the role of maternal anti-HLA class I antibodies in fetal and neonatal thrombocytopenia—Antibody specificity analysis using epitope data

Anti-HLA class I antibodies have been suggested as a possible cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT). The aim of this study was to characterize maternal anti-HLA class I alloantibodies in suspected cases of FNAIT. The study population consisted of all nationwide referrals of neonates with suspected FNAIT to the National Unit for Platelet Immunology in Tromsø, Norway, during 1998-2009 (cases), and 250 unselected pregnancies originally included in a prospective study (controls). Inclusion criterion was a positive screening for maternal anti-HLA class I antibodies. Neonates with other identifiable causes of thrombocytopenia, including maternal anti-human platelet antigens (HPA) antibodies, were excluded. Ultimately, 50 cases with suspected FNAIT were compared with 60 controls. The median neonatal platelet count nadir among cases was 24×109/L (range 4-98×109/L). Five children (10%) were reported to have intracranial hemorrhage. Maternal and neonatal HLA class I genotype was available for 33 mother/child pairs (66%). Immunization was not tied to any particular HLA class I antigen. Using epitope mapping, we could demonstrate that the maternal anti-HLA class I antibodies were specific towards mismatched paternally-inherited fetal epitopes, with little reactivity towards any third-party epitopes. Antibody reactivity patterns were similar to those found among controls, although the mean fluorescence intensities (MFI) among cases were significantly higher. This study demonstrates the value of using data on HLA epitope expression, instead of HLA antigens, to examine alloimmune responses in connection with neonatal thrombocytopenia. Our findings support the idea that maternal anti-HLA class I antibodies are involved in FNAIT.

T cell responses to human platelet antigen–1a involve a unique form of indirect allorecognition

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy-related condition caused by maternal antibodies binding an alloantigen on fetal platelets. In most cases the alloantigen is formed by a single amino acid, integrin β3 Leu33, referred to as human platelet antigen-1a (HPA-1a). Production of anti-HPA-1a antibodies likely depends on CD4+ T cells that recognize the same alloantigen in complex with the HLA-DRA/DRB3*01:01 molecule. While this complex is well characterized, T cell recognition of it is not. Here, to examine the nature of antigen recognition by HPA-1a-specific T cells, we assayed native and synthetic variants of the integrin β3 peptide antigen for binding to DRA/DRB3*01:01-positive antigen-presenting cells and for T cell activation. We found that HPA-1a-specific T cells recognize non-allogeneic integrin β3 residues anchored to DRA/DRB3*01:01 by the allogeneic Leu33, which itself is not directly recognized by these T cells. Furthermore, these T cell responses are diverse, with different T cells depending on different residues for recognition. This represents a unique form of indirect allorecognition in which a non-allogeneic peptide sequence becomes immunogenic by stable anchoring to MHC by an allogeneic residue.

Noninvasive prenatal HPA-1 typing in HPA-1a negative pregnancies selected in the Polish PREVFNAIT screening program: Non-invasive prenatal HPA-1a typing

Anti‐HPA‐1a alloantibodies in HPA‐1a negative mothers can lead to fetal/neonatal alloimmune thrombocytopenia (FNAIT). Noninvasive prenatal testing (NIPT) of HPA‐1a determines fetuses at risk and the course of maternal antenatal treatment.

The DR7‐DQ2 Haplotype in a Native Norwegian Population

To the Editor Non-random association of HLA-DR and DQ alleles in haplotypes has become important in dissecting the genetic influence in different diseases. Recently, it was shown that DRB1*07 and DQB1*02:01 represent the most common DR7-DQ2 association in one defined Asian immigrant population in Norway [1], while DRB1*07 and DQB1*02:02 are more common in European Americans [2]. The association between these alleles in native Norwegians is not known. Because DR7-DQ2 associations have direct relevance to our research related to foetal and neonatal alloimmune thrombocytopenia, we set out to address this question. In the Norwegian population, 24 different HLA-DRDQ haplotypes are considered common, with frequencies 1% in the population [3, 4]. Spurkland et al. [3] reported in 1992, that the DR7-DQ2 haplotype consists of DRB1*07:01 and DQB1*02:01. Only later was the distinction made between the DQB1*02:01 allele and a second DQB1*02 allele, DQB1*02:02 [5] (assigned in 1994; IMGT/HLA database). Thus, because the original paper on HLA class II haplotypes in Norwegians only typed for polymorphisms in exon 2 [3] and the polymorphism distinguishing DQB1*02:01 and DQB1*02:02 lies in exon 3, the reported DR7-DQ2 haplotype in Norway could have included both DRB1*07:01-DQB1*02:01 and DRB1*07:01-DQB1*02:02. In 2003, Klitz et al. [2] reported haplotype frequencies of the MHC class II loci DRB1, DQA1 and DQB1 (high-resolution typing) in European Americans. In this population, the DRB1*07:01 allele was in linkage disequilibrium with DQB1*02:02 (11.1%), DQB1*03:03 (3.7%) and DQB1*02:01 (1.1%). We therefore expected this to be the case also in the Norwegian population, and this was our assumption until the recent report on DR-DQ haplotypes in Pakistani immigrants in Norway [1], where the DRB1*07:01:01-DQB1*02:01:01 is clearly more frequent (9.9%) than the DRB1*07:01:01-DQB1*02:02 haplotype (1.1%). The finding by Rønningen et al. [1] prompted us to determine the nature of this association in a native Norwegian population. To assess the occurrence of the DRB1*07DQB1*02:01:01 haplotype, 202 random native Norwegian blood donors were typed for DRB1*07, DQB1*02 and DRB1*03 by group-specific primers [6–8]. All DQB1*02-, DRB1*03and/or DRB1*07-positive individuals (n = 61) were further analysed by sequence-based typing of DQB1 for exons 2 and 3 [9]. Haplotypes were assigned to individuals according to the identified alleles. In 21 of the DR3or DR7-positive individuals, additional typing for DRB1 allowed identification of both haplotypes (data not shown), providing confidence to the assignment. The DR and DQ alleles in the Norwegian population are reported to have a good fit to the Hardy–Weinberg proportion model [10]. We found that DRB1*07 was seen in combination with DQB1*02:02 and DQB1*03:03:02, and in one individual, in a rare combination with either DQB1*04:02:01 or *05:01:01 (Table 1). In contrast, DQB1*02:01:01 was only found in combination with DRB1*03. Two donors were positive for both DQB1*02:01:01, DQB1*02:02, DRB1*07 and DRB1*03; which corresponds to haplotypes DRB1*03-DQB1*02:01:01 and DRB1*07-DQB1*02:02. Frequencies of the different haplotypes in our material are in agreement with the haplotype frequencies given in the sample of European Americans in 2003 [2]. In conclusion, we have determined that in a native Norwegian population, the DR7-DQ2 haplotype consists mainly or only of DRB1*07-DQB1*02:02, and not DRB1*07-DQB1*02:01:01.