Allergic women show reduced T helper type 1 alloresponses to fetal human leucocyte antigen mismatch during pregnancy.

Low-level alloreactivity between mother and fetus may provide stimulation for fetal T helper type 1 (Th1) cell immune maturation. This study explored the effects of human leucocyte antigen (HLA) mismatch on materno-fetal interactions detected as cytokine responses and lymphoproliferation in mixed lymphocyte reactions, and whether this was altered in allergic women (n = 62) who have a Th2 propensity compared with non-allergic women (n = 65). HLA-DRbeta1 mismatch was associated with significantly increased Th1 interferon (IFN)-gamma, Th2 interleukin (IL)-13 and lymphoproliferative responses by both mothers and fetuses. Allergic women showed significantly lower IFN-gamma Th1 production in response to HLA-DRbeta1 mismatch. The infants of these women also showed significantly lower IL-10 and lower IFN-gamma production relative to IL-13. Both HLA-DRbeta1 mismatch and maternal allergy had significant independent effects on maternal IFN-gamma Th1 responses. Maternal allergy modifies HLA-mediated alloreactivity between the mother and the fetus, reducing Th1 activation. This may affect the cytokine milieu at the materno-fetal interface and could be implicated in the attenuated Th1 responses observed commonly in infants of atopic mothers.

The evolution and diversity of TNF block haplotypes in European, Asian and Australian Aboriginal populations.

The region spanning the tumour necrosis factor (TNF) cluster in the human major histocompatibility complex is implicated in susceptibility to immunopathological disease, but ethnic differences and linkage disequilibrium have hampered identification of critical polymorphisms. Here, we investigate Europeans, Asians (Bidayuh, Chinese, Indian, Jehai, Malay, Temuan) and Australian Aborigines to provide a framework for disease-association studies. DNA from 999 unrelated healthy donors was genotyped at 38 loci, primarily in coding and promoter regions over a 60-kb region spanning seven genes near TNF. The PHASE algorithm was used to statistically infer TNF block haplotypes and estimate their frequencies in each population. The TNF block is carried as 31 haplotypes in all populations combined, with <19 in any single population. Only six haplotypes have a unique tag single nucleotide polymorphism (SNP) valid for all populations, but seven haplotypes could be tagged with individual SNPs in selected populations. Four to eight TNF block haplotypes exist across all ethnicities, and hence must pre-date the divergence of these populations from a common ancestor >160,000 years ago. Some haplotypes are unique to isolated populations, but they do not contain unique SNP. Hence, they reflect restricted migration and/or extinction of some families rather than de novo mutation.

TNF block haplotypes associated with conserved MHC haplotypes in European, Asian and Australian Aboriginal donors.

Associations between major histocompatibility complex (MHC) ancestral haplotypes (AHs) and immunopathological diseases are traditionally ascribed to human leukocyte antigen (HLA) class I or class II alleles. However, polymorphisms in TNF and nearby genes in the central MHC can influence risk. We have defined TNF block haplotypes in Asian, European and Australian Aboriginal donors and shown conservation of TNF block haplotypes in geographically distinct populations, consistent with a common evolutionary origin. Here we show that most TNF block haplotypes do not align with a single MHC AH and associations often vary with ethnicity. This suggests more recent recombination events between the TNF block and the HLA alleles.

Post-transplant HLA class II antibodies and high soluble CD30 levels are independently associated with poor kidney graft survival.

HLA-specific antibodies (HSA) and soluble CD30 (sCD30) were measured in 208 renal transplant recipients with functioning grafts at least 1 year after transplantation (median 8.2 years) to investigate the predictive value of HSA and sCD30 on subsequent graft outcome. HSA (class I and class II) were detected by both ELISA LAT-M and Luminex LabScreen assays. Data on graft outcome was collected with a median follow-up time of 3.5 years after antibody and sCD30 measurement. Recipients with post-transplant HLA class II antibodies had particularly poor graft outcome with a hazard ratio (HR) of 7.8 (p < 0.0001) when detected by ELISA, and a HR of 6.0 (p < 0.0001) when detected by Luminex. A high post-transplant sCD30 level >or=100 U/mL was associated with increased risk of subsequent graft failure (HR 2.7, p = 0.03). sCD30 and HSA had an independent and additive association with graft outcome. Recipients with HLA class II antibody and high sCD30 had the highest risk of subsequent graft failure (HR 43.4, p < 0.0001 and HR 18.1, p = 0.0008 for ELISA and Luminex, respectively). These data show that detection of HSA and serum sCD30 measured at least 1-year post-transplant provides valuable and predictive information regarding subsequent graft outcome.

MICA, MICB, and MHC beta block matching in bone marrow transplantation: relevance to transplantation outcome.

Genetic testing of the MHC is required for selection of donors for bone marrow transplantation. The outcome of related bone marrow transplantation is usually superior to that of unrelated bone marrow transplantation. This may be the result of inaccurate or incomplete genetic testing employed for selection of donor for transplantation. Based on MHC haplotype matching, MHC block matching has been developed for selection of potential unrelated donor. Block matching has been shown previously to improve outcome when added to the conventional method of human leukocyte antigen (HLA) typing for selection of donors. In this study, we have retrospectively analyzed 44 donor recipient pairs from the Australian Bone Marrow Donor Registry Repository with respect to matching of HLA-B and HLA-Cw by sequence-based typing and MICA and MICB by polymerase chain reaction-sequence specific primer and MHC beta block matching and correlated these results with survival. Beta block matching was correlated with MIC matching (p < 0.005) and with HLA-B and HLA-Cw matching. Patients who were HLA-B and -Cw matched had significantly improved survival when they were additionally matched for MHC beta block (p(c) = 0.04) or MIC (p(c) = 0.05).

The relevance of natural killer cell human leucocyte antigen epitopes and killer cell immunoglobulin-like receptors in bone marrow transplantation.

The discovery that killer cell immunoglobulin-like receptors (KIR) interact with genetically polymorphic epitopes on class I human leucocyte antigen (HLA) molecules and that the KIR receptor repertoire itself is genetically variable has led to investigation of the relevance of the KIR system to stem cell transplantation. A number of retrospective studies of transplant outcome have now demonstrated either beneficial or deleterious effects of mismatching for class I natural killer (NK) epitopes. A smaller number of studies have shown effects of the donor and/or patient KIR repertoire on outcome, irrespective of the patient and donor HLA type. The most parsimonious interpretation of the data, which are often conflicting, is that the effect of NK epitope matching is very much dependent on transplant protocols, with the extent of donor T-cell depletion possibly being the most important variable. A clearer picture of the role of matching for NK epitopes and the KIR-receptor repertoire of the donor is needed.

Menopausal hormone therapy and irregular endometrial bleeding: a potential role for uterine natural killer cells?

CONTEXT: Irregular bleeding affects many users of combined menopausal hormone therapy (HT) and commonly leads to invasive and expensive investigations to exclude underlying malignancy. In most cases no abnormality is found. OBJECTIVE: The main objective of this study was to explore the role of uterine natural killer (uNK) cells and their regulatory cytokine IL-15 in irregular bleeding in HT users. DESIGN: This was a prospective observational study conducted between 2002 and 2004. SETTING: The study was conducted in a tertiary referral menopause clinic at King Edward Memorial Hospital, Western Australia. PATIENTS: Patients included 117 postmenopausal women taking combined HT. INTERVENTIONS: Outpatient endometrial biopsies were taken during and outside bleeding episodes. MAIN OUTCOME MEASURES: The relationship between endometrial uNK cells (CD56+) and bleeding patterns was measured. We also addressed the impact of HT exposure on uNK cell populations, the relationship between endometrial IL-15 expression and uNK cell populations, and killer Ig like receptor genotype in subjects with irregular bleeding. RESULTS: Endometrial CD56+ uNK cells were significantly increased in biopsies obtained during bleeding episodes (P < 0.001), compared with HT users with no bleeding. The highest level of IL-15 expression was also seen in biopsies taken during bleeding. No clear relationship between killer Ig like receptor genotype and bleeding on HT was observed. CONCLUSIONS: Little is known about the mechanisms underlying irregular bleeding in HT users. This is the first report of uNK cells and their association with regulating cytokines in postmenopausal endometrium and demonstrates a possible mechanism by which HT may induce irregular bleeding.

Natural killer cell HLA-C epitopes and killer cell immunoglobulin-like receptors both influence outcome of mismatched unrelated donor bone marrow transplants.

Matching of donor and recipient for the class I human leukocyte antigen-C (HLA-C)-encoded natural killer (NK) epitopes has been reported to influence stem-cell (SC) graft outcome, but a consistent picture has not yet emerged. We have analyzed transplant outcome in 104 unrelated SC grafts in relation to NK epitope (C1 and C2) matching and donor killer cell immunoglobulin-like receptor (KIR) genotype. NK epitope mismatching in the rejection direction was strongly associated with an increased probability of rejection subsequent to engraftment. The prevalence of grades III-IV acute graft-vs-host disease (GVHD) was significantly higher and occurred significantly earlier when there was NK epitope mismatching in the GVH direction. Higher transplant-related mortality and lower disease-free survival rates were associated with epitope mismatching regardless of the mismatch direction. A greater number of KIR receptors, both activating and inhibitory, in the donor protected against grades III-IV GVHD and improved survival.

Population frequencies and putative haplotypes of the killer cell immunoglobulin-like receptor sequences and evidence for recombination.

BACKGROUND: The killer cell immunoglobulin-like receptors (KIR) are a family of receptors expressed on natural killer (NK) cells and some T cells. Class I HLA molecules on target cells are the ligands for the KIR receptors. The number of KIR genes has been reported to vary between individuals, resulting in different KIR haplotypes. There is little published data on the frequency of each KIR gene and the linkage disequilibrium between the genes. Because there is evidence that NK cells may be involved in bone marrow transplant rejection, we have determined the KIR gene frequencies and possible haplotypic arrangements by linkage disequilibrium analysis in an Australian population. METHODS: Controls, patients with leukemia, and unrelated bone marrow donor-recipient pairs were typed for the presence of 11 KIR genes by polymerase chain reaction-sequence specific priming. RESULTS: Ninety percent of the population was found to have a sufficient number and variety of KIR genes to detect any mismatch of HLA-A, -B, and -C alleles on NK target cells. The 11 KIR genes could be divided into two groups based on linkage disequilibrium between pairs of genes. Evidence for a recombination within the KIR gene complex is presented. CONCLUSION: Typing for the presence of particular KIR genes may be indicated for bone marrow donor-recipient pairs for whom a class I HLA mismatch is unavoidable.

Periodontal attachment loss in HIV-infected patients is associated with the major histocompatibility complex 8.1 haplotype (HLA-A1,B8,DR3).

Periodontal attachment loss is mediated by overproduction of tumour necrosis factor (TNF) and interleukin (IL)-1, and appears to have a genetic component. The 8.1 major histocompatibility complex (MHC) ancestral haplotype (HLA-A1,B8,TNFA-308(2),DR3) is associated with elevated TNF production and predisposes carriers to several autoimmune/immunopathological disorders, including rapid progression of HIV disease, but not early onset periodontal disease in healthy individuals. Rather a high proportion of subjects with severe periodontal disease carry allele 2 at IL-1A-889 and IL-1B+3953. We predicted that genetic associations may be different or clearer in HIV patients, as they often show elevated production of TNF and IL-1 and periodontal attachment loss. Hence periodontal parameters and IL-1 polymorphisms were assessed in HIV-positive subjects expressing HLA-B8 with or without other markers of the 8.1 haplotype. Of 16 HLA-B8 subjects, 13 demonstrated elevated probing pocket depth and clinical attachment loss. The difference was statistically significant and did not correlate with smoking, age, CD4 T-cell counts, HIV viral load or levels of dental plaque. As TNFA-308 (allele 2) was present in four non-B8 subjects who had minimal attachment loss, it may not mediate the effect of the 8.1 haplotype. Moreover, polymorphisms at IL-1A-889 and IL-1B+3953 did not significantly affect periodontal parameters. Thus a central MHC gene characteristic of the 8.1 haplotype was the clearest determinant of periodontal attachment loss in HIV-infected individuals.

Mapping of a candidate region for susceptibility to inclusion body myositis in the human major histocompatibility complex.

Inclusion body myositis (IBM) is a form of idiopathic inflammatory myopathy of unknown aetiology. A strong association with HLA class II (HLA-DR3) suggested a role for genes in the human major histocompatibility complex (MHC) in the predisposition to this disease. In this study, we have taken advantage of the ancestral haplotype (AH) concept and historical recombinations to map for a possible susceptibility gene(s) in the MHC. We performed detailed typing of three MHC-related HSP70 genes and defined allelic combinations in the context of MHC AH. We also modified existing methods to give a simple and accurate method for typing two TNF microsatellites. Using the HSP70 and TNF markers and HLA-DR, -B, and C4 typing of our patients with IBM, we defined a potential site for the MHC-associated susceptibility gene(s) in the region between HLA-DR and C4.

The significance of HLA matching in cardiac transplantation.

It is argued that HLA matching is not worthwhile in heart transplantation. However, transplanting HLA compatible hearts enhances graft survival and should significantly reduce infection and malignancies related to aggressive immunosuppression. It is our view that the problem is technical and we offer a potential solution.

Characterisation of the human central MHC gene, BAT1: genomic structure and expression.

The BAT1 gene (D6S81E) encodes a member of the DEAD-box family of RNA-binding proteins, and lies in the central MHC. This region contains genes which affect susceptibility to immunopathological diseases. A 14-kb section of the human MHC containing the BAT1 gene and a further 5-kb telomeric of BAT1 was sequenced using DNA from individuals homozygous for HLA-A1, B8, DR3 and HLA- A1, B57, DR7. Analysis of our sequences and the previously reported human cDNA sequence showed that the expressed sequence of the 8.1 and 57.1 haplotypes is identical with only minor substitutions in the introns. Phylogenetic analysis suggests BAT1 may be a translation initiation factor. Screening of cells and tissues for BAT1 mRNA suggests an abundant member of a family of proteins expressed in multiple cell types, notably macrophages and hepatocytes. Expression was independent of MHC haplotype, consistent with the lack of sequence polymorphism.

Conservation of ancestral haplotypes telomeric of HLA-A.

Genes that predispose to haemochromatosis are though to be located within the several megabases telomeric of HLA-A. Further recombinant mapping has been used previously to map susceptibility genes for diseases such as insulin-dependent diabetes mellitus, myasthenia gravis and cystic fibrosis, and should be useful in relation to haemochromatosis. However, this method requires the recognition of ancestral haplotypes within the susceptibility region. Using a panel of six microsatellite markers from this region (MOG A, MOG B, MOG C, D6S464, D6S306 and D6S105), we show that ancestral haplotypes extend telomeric of HLA-A, at least as far as D6S105. Nine of 14 haplotypes carrying HLA-B7 and HLA-A3 shared the same microsatellite alleles between HLA-A and at least D6S105. Similarly, nine of 10 haplotypes sharing HLA-B8 and HLA-A1 shared the same microsatellite alleles, although a different set to those with HLA-B7 and HLA-A3. Haplotypes representing historical recombination events were also identified. These two findings demonstrate that recombinant mapping may be applicable to the mapping of disease genes in this region.

The identification of MHC identical siblings without HLA typing.

By comparing genomic sequences of different MHC haplotypes, we defined highly polymorphic markers. After amplification, electrophoresis, and scanning with a laser, we have identified profiles that serve as signatures of the haplotype and its component alleles. One set of markers can be used to define the block that includes HLA-B and HLA-C, among other loci. Another set provides signatures for the entire HLA-DR and -DQ multigene cluster. By profile overlay, it is possible to identify siblings who share both haplotypes from HLA-C to HLA-DQ. Here we demonstrate the value of genomic analysis ("block matching") in selecting genotypically identical siblings prior to transplantation. Forty-six siblings from 10 families were genotyped by family analysis after meticulous HLA, C4, and Bf typing including molecular methods for HLA-DRB1. In 43 siblings, the haplotype assignments were unequivocal. Twenty-two identical sibling pairs could then be compared with 77 nonidentical pairs. Independent genomic analysis yielded entirely concordant results. In three siblings, the possibility of parental recombination was considered but could not be defined by the conventional typing. By genomic analysis, however, it was clear that recombination had indeed occurred in one case. In the remaining two cases, additional, more telomeric markers will be necessary to resolve the issue. This simple, cost-effective method has immediate application to the identification of matched pairs (HLA-C to HLA-DQ) for bone marrow and renal transplantation.

Matching for MHC haplotypes results in improved survival following unrelated bone marrow transplantation.

Unrelated bone marrow donor-recipient pairs were assessed retrospectively for matching of the HLA-B, -C region (beta-block) and HLA-DR, DQ region (delta block) of the major histocompatibility complex (MHC) using a new DNA-based method referred to as MHC-block typing. The method utilises non-HLA DNA polymorphisms in the MHC as markers of blocks of ancestral haplotypes. Kaplan-Meier analysis of recipients who were matched at both the beta- and delta-blocks revealed a 6 months survival of 54%. Survival was better than for patients who were matched only by conventional criteria, including SSO-typing for class II.

Immunogenetic analysis of successful and rejected bone marrow grafts within one family.

We report the case of an Indonesian patient who required urgent bone marrow transplantation for acute leukaemia and who received successive transplants from two siblings. The first transplant failed while the second was successful. There were some uncertainties in serological typing due to the presence of cross-reacting HLA-B alleles, lack of paternal typing and the use of Caucasoid sera for Indonesian patients. Distinction between the two donors was also difficult. Interestingly, the use of a new DNA technique identified the presence of differences between the patient and the first unsuccessful donor but not the second successful donor.

Proliferation of alloreactive human natural killer cells independent of specific allogeneic stimulation.

This study establishes that natural killer (NK) cells cytolytic for B-lymphoblastoid cell lines (B-LCL) expressing NK-defined alloantigens can be stimulated to proliferate in culture independently of allogeneic stimulation. NK cells proliferate following co-culture with a gamma-irradiated malignant melanoma cell line (MM-170) and IL-2-conditioned medium. The cultured NK cells from some donors showed a high level of cytotoxicity against NK-1+ B-LCL and this corresponded with a high precursor frequency (46-64%) determined from limiting dilution analysis. Alloreactive NK cells proliferated in cultures containing autologous activated T cells, demonstrating that alloantigens were not essential to stimulate proliferation. B-LCL expressing NK-1 or NK-2 or neither of these alloantigens stimulated proliferation of NK cells cytolytic for NK-1+ B-LCL. Studies using metabolically inactivated B-LCL confirmed that stimulation was not alloantigen dependent. The results demonstrate that recognition of alloantigens by NK cells, sufficient to trigger their lytic program, is not required and indeed is not sufficient to confer a stimulatory signal for proliferation of alloreactive NK cells.

The MHC influences acute graft versus host disease in MHC matched adults undergoing allogeneic bone marrow transplantation.

To determine whether the MHC plays an antigen non-specific role in the development of acute GVHD, the prevalence of acute GVHD in relation to MHC genotype was examined in 51 adult patients undergoing allogeneic BM grafting. The majority of patients received grafts from HLA-identical siblings. HLA-B7 haplotypes were associated with a decreased risk of acute GVHD (2 of 15, p = 0.005) whereas HLA-B44 haplotypes were associated with a higher risk of acute GVHD (11 of 14, p = 0.02). As these alleles have been reported previously as having opposite effects in relation to inflammatory mediators, these findings may have important implications with respect to donor selection and patient management.

Characterization of 4AOHW cell line panel including new data for the 10IHW panel.

There will be a continuing need for well characterized panels of EBV-transformed lymphoblastoid cell lines. Selection of the 4AOH panel was based on prior MHC typing and was intended to ensure representation of ancestral haplotypes from various racial groups. Cells from nonhuman primates, bone marrow donor-recipient pairs, and patients with IDDM were included. Selected cells from the 10IHW were included to enable further characterization. Cells were distributed to participants in the 4AOHW and were typed at multiple loci by a variety of procedures. Non-HLA genes such as TNF were included. Since the cells were distributed "blind" with hidden replicates, it was possible to evaluate the quality of the typing data. An approach to data management is described. The best current estimates of the typing of these cells are presented. The panel will be useful since it provides standards for most alleles at most loci. Since the cells are so well characterized, they represent a useful resource for MHC sequencing and for the evaluation of new typing procedures.