Search

Abacavir administration is associated with drug-induced hypersensitivity reactions in HIV+ individuals expressing the HLA-B*57:01 allele. However, the immunological effects of abacavir administration in an HLA-B57 mismatched transplantation setting have not been studied. We hypothesized that abacavir exposure could induce de novo HLA-B57-specific allorecognition. HIV-specific CD8 T cell clones were generated from HIV+ individuals, using single cell sorting based on HIV peptide/HLA tetramer staining. The T cell clones were assayed for alloreactivity against a panel of single HLA-expressing cell lines, in the presence or absence of abacavir. Cytokine assay, CD137 upregulation, and cytotoxicity were used as readout. Abacavir exposure can induce de novo HLA-B57 allorecognition by HIV-specific T cells. A HIV Gag RK9/HLA-A3-specific T cell did exhibit interferon-gamma production, CD137 upregulation, and cytolytic effector function against allogeneic HLA-B57, but only in the presence of abacavir. Allorecognition was specific to the virus specificity, HLA restriction, and T cell receptor TRBV use of the T cell. We provide proof-of-principle evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. We suggest that HIV-seropositive recipients of an HLA-B57 mismatched graft should not receive abacavir until further studies are completed.

We hypothesized that HIV-specific CD8 T cell clonotypes can be stimulated by allogeneic HLA molecules. Multiple HIV-specific CD8 T cell clones were derived from 12 individuals with chronic HIV infection, specific for 13 different HIV Gag antigens and restricted to 7 different HLA molecules. The generated T cell clones were assayed for alloreactivity against a panel of single HLA class I expressing cell lines (SALs). HIV-specific T cells recognising at least one allogeneic HLA molecule could be identified from 7 of 12 patients tested. Allorecognition was associated with IFNγ cytokine production, CD137 upregulation and cytotoxicity, suggesting high avidity allo-stimulation. Allo-HLA recognition by HIV-specific T cells was specific to the HIV target peptide/HLA restriction and TCR TRBV usage of the T cells. HIV-specific T cells do crossreact against allogeneic HLA molecules in an epitope and TRBV specific manner. Therefore allo-HLA stimulation could be exploited to induce or augment HIV-specific T cell responses.

Objective To establish gene copy number (GCN)–specific normal ranges for serum C4 genes and to determine their utility with respect to the interpretation of chronically low serum C4 concentrations in patients with clinically quiescent systemic lupus erythematosus (SLE). Methods C4 serum concentrations were estimated by automated turbidimetry, and C4 GCNs were determined using the TaqMan real-time polymerase chain reaction (PCR) analysis in 184 unselected individuals and in 10 patients with type 1 diabetes mellitus (DM) who were selected for the presence of only 2 copies of the C4 gene. C4 GCNs were also determined in 11 patients with clinically quiescent SLE who had chronically low serum C4 concentrations. Results A total of 33% of the variation in serum C4 concentrations could be accounted for by both C4A and C4B GCNs (R2 = 0.30, P ≤ 0.0001). There was a median of 2 gene copies at the C4A locus (53.8%) and 2 at the C4B locus (58.7%). The median total number of C4 genes was 4 (55.4%). C4 GCN-specific normal ranges were established. A chronically low serum C4 concentration was explained by a low C4 GCN in 3 of 11 patients tested. Conclusion This study establishes the feasibility of establishing C4 GCN-specific normal ranges using the TaqMan real-time PCR assay. Chronically low serum C4 concentrations in SLE patients are sometimes explained by low C4 GCNs.

The MHC class Ib molecule HLA-G has previously been reported to be the ligand for the NK cell receptor killer Ig-like receptor (KIR)2DL4, but this remains controversial. In this study, we investigated IFN-γ production by freshly isolated NK cells in response to both soluble and solid-phase ligands, including anti-KIR2DL4 mAbs and rHLA-G. Although freshly isolated CD56bright NK cells produced IFN-γ in response to soluble HLA-G preparations, the response was found to be absolutely dependent on the presence of small numbers of contaminating CD56−, CD14−, CD11c+ myeloid dendritic cells (mDCs). HLA-G tetramers bound only to the contaminating mDCs in the NK preparations, and Abs to KIR2DL4 and HLA-G did not block NK cell IFN-γ production. NK cells did not respond to plate-bound HLA-G. Freshly isolated NK cells also produced IFN-γ in response to unpurified soluble anti-KIR2DL4 mAb but not to low endotoxin affinity–purified Ab. The data suggest that previous reports of functional interactions between KIR2DL4 and HLA-G may have resulted from the use of purified NK cells that were contaminated with mDCs and HLA-G preparations that were contaminated with material capable of stimulating mDCs to produce cytokines that stimulate NK cells to produce IFN-γ.

Historic red blood cell transfusion (RBCT) may induce anti-HLA antibody which, if donor specific (DSA), is associated with increased antibody-mediated rejection (AMR). Whether post-operative RBCT influences this risk is unknown.We examined the RBCT history in 258 renal transplant recipients stratified according to prevalent recipient HLA antibody (DSA, Non-DSA or No Antibody).AMR occurred more frequently in patients who received RBCT both pre and post transplant compared with all other groups (Pre+Post-RBCT 21%, Pre-RBCT 4%, Post-RBCT 6%, No-RBCT 6%, HR 4.1 p=0.004). In the 63 patients who received Pre+Post-RBCT, 65% (13/20) with DSA developed AMR compared with 0/6 in the Non-DSA group and 2/37 (5%) in the No-Antibody group (HR 13.9 p

The long term effect of donor specific antibodies (DSA) detected by Luminex Single Antigen Bead (SAB) assay in the absence of a positive complement-dependant cytotoxicity (CDC) crossmatch is unclear. DSA at the time of transplant were determined retrospectively in 258 renal transplant recipients from 2003 to 2007 and their relationship with rejection and graft function prospectively evaluated. After a median of 5.6 years follow-up 9% of patients had antibody mediated rejection (AMR) (DSA 11/37 (30%), DSA-Neg 13/221 (6%), HR 6.6, p0.001). Patients with anti-HLA class II (HR 6.1) or both class I+II (HR 10.1) DSA had the greatest risk for AMR. The Mean Fluorescent Intensity (MFI) of the DSA was significantly higher in patients with AMR than those with no rejection (p=0.006). Moreover, the strength of the antibody was shown to be important, with the risk of AMR significantly greater in those with DSA8000 MFI than those with DSA8000 MFI (HR 23, p0.001). eGFR progressively declined in patients with DSA but was stable in those without DSA (35.7 ± 20.4 mls/min vs 48.5 ± 22.7) and composite patient and graft survival was significantly worse in those with class II (HR 2.9) or both class I+II (HR 3.7) but not class I DSA. Class II DSA alone, or in combination with class I DSA had the strongest association with graft loss and patient death. Patients with DSA not only have increased rates of acute AMR, but also chronic graft dysfunction, graft loss and death. Antibody burden quantified by SAB assay may identify patients at highest immunological risk and therefore influence patient management and improve long-term patient outcome.

T-cell alloreactivity is generated via immune responsiveness directed against allogeneic (allo) human leucocyte antigen (HLA) molecules. Whilst the alloresponse is of extraordinary potency and frequency, it has often been assumed to be less peptide-specific than conventional T-cell reactivity. Recently, several human studies have shown that both alloreactive CD8(+) and CD4(+) T cells exhibit exquisite allo-HLA and endogenous peptide specificity that has also underpinned tissue-specific allorecognition. In this review, we summarize former and recent scientific evidence in support of endogenous peptide (self-peptide)-dependence of T-cell alloreactivity. The clinical implications of these findings will be discussed in the context of both solid organ transplantation and haematopoietic stem cell transplantation (HSCT). Insights into the understanding of the molecular basis of T-cell allorecognition will probably translate into improved allograft survival outcomes, lower frequencies of graft vs host disease and could potentially be exploited for selective graft vs leukaemia effect to improve clinical outcomes following HSCT.

We compared the carriage frequencies of HLA-DRB3 and its major alleles and of HLA-DRB4 and HLA-DRB5 in an Australian sIBM cohort and a population control group who had previously been genotyped for the HLA-DRB1*03:01 risk allele. There was a strong disease association with the carriage of the DRB3*01:01 allele which was accounted for by its linkage disequilibrium with DRB1*03:01. The carriage of HLA-DRB4 was found to be strongly protective and abrogated the risk effect of HLA-DRB1*03:01. The findings indicate that haplotypic combinations of alleles at the HLA-DRB1 and secondary HLA-DRB loci have important risk modifying effects in sIBM.

To identify susceptibility loci for visceral leishmaniasis, we undertook genome-wide association studies in two populations: 989 cases and 1,089 controls from India and 357 cases in 308 Brazilian families (1,970 individuals). The HLA-DRB1-HLA-DQA1 locus was the only region to show strong evidence of association in both populations. Replication at this region was undertaken in a second Indian population comprising 941 cases and 990 controls, and combined analysis across the three cohorts for rs9271858 at this locus showed P(combined) = 2.76 × 10(-17) and odds ratio (OR) = 1.41, 95% confidence interval (CI) = 1.30-1.52. A conditional analysis provided evidence for multiple associations within the HLA-DRB1-HLA-DQA1 region, and a model in which risk differed between three groups of haplotypes better explained the signal and was significant in the Indian discovery and replication cohorts. In conclusion, the HLA-DRB1-HLA-DQA1 HLA class II region contributes to visceral leishmaniasis susceptibility in India and Brazil, suggesting shared genetic risk factors for visceral leishmaniasis that cross the epidemiological divides of geography and parasite species.

Objective: The identification of a vitamin D–responsive (VDRE) motif within the HLA-DRB1*15:01 promoter region provides an attractive explanation for the combined effects of HLA-DR inheritance and vitamin D exposure on multiple sclerosis (MS) risk. We therefore sought to incorporate HLA-DRB1 promoter variation, including the VDRE motif, in an assessment of HLA-DRB1–associated MS risk. Methods: We utilized 32 homozygous HLA cell lines (covering 17 DRB1 alleles) and 53 heterozygote MS samples (20 DRB1 alleles) for HLA-DRB1 promoter sequencing. The influence of HLA-DRB1 variation on MS risk was then assessed among 466 MS cases and 498 controls. Results: The majority of HLA*DRB1 alleles (including HLA-DRB1*15:01) express the functional VDRE motif, apart from HLA-DRB1*04, *07, and *09 alleles that comprise the HLA-DR53 serologic group. Allele-specific variation within functional X-box and Y-box motifs was also associated with serologically defined HLA-DR haplotypes. Incorporating these results in an analysis of MS risk, we identified a strong protective effect of HLA-DRB1*04, *07, and *09 (DR53) alleles ( p = 10 −12 ) and elevated risk associated with DRB1*15 and *16 (DR51) and *08 (DR8) alleles ( p −18 ). Conclusions: HLA-DRB1 groups corresponding to serologic HLA-DR profiles as well as promoter polymorphism haplotypes effectively stratified MS risk over an 11-fold range, suggesting functional relationships between risk-modifying HLA-DRB1 alleles. An independent contribution of VDRE motif variation to increase MS risk was not discernible, although vitamin D–dependent regulation of HLA-DR expression may still play an important role given that HLA-DRB1*04/*07/*09 (DR53) alleles that express the “nonresponsive” VDRE motif were associated with significantly reduced risk of MS.

The frequency of the killer-cell immunoglobulin-like receptor (KIR) genes and transmembrane alleles of KIR2DL4 were studied in coastal (Mugil community) and inland (Ilaita community) communities in Papua New Guinea. Linkage disequilibria between KIR genes and between alleles of KIR2DL4 and the KIR genes were similar to those found in other populations suggesting conservation of the usual gene order in Papua New Guinean haplotypes. Significant differences in the frequency of KIR genes were found between the two populations despite being separated by only 300 km. Examples of individuals who lacked the KIR2DL4 gene and others whose KIR2DL4 allele appeared to have 11 adenines in the polyadenine tract in exon 6 were identified. A relatively low frequency of the KIR A haplotype was found in both populations and particularly in the inland community. The KIR gene frequencies were consistent with the inland Ilaita community being closely related to Australian Aborigines and southern Indians, whereas the KIR gene frequencies of the coastal Mugil community appeared to have been influenced either by recent or ancient admixture from populations with a higher frequency of the KIR A haplotype.

Polymorphisms in cell surface receptors of natural killer cells and their ligands on target cells can affect susceptibility to viral infections including human immunodeficiency virus (HIV)-1. We found that the carriage of the human leukocyte antigen (HLA)-G minus 14-bp polymorphism, LILRB1 single nucleotide polymorphism rs1061680, and activating and inhibitory killer immunoglobulin-like receptors (KIRs) were different when data were compared between Caucasian, African Americans and Asian populations. However, carriage was similar when HIV-1 patients were compared with control donors, with the exception of the African American cohort.

Kawasaki disease is a systemic vasculitis of unknown etiology, with clinical observations suggesting a substantial genetic contribution to disease susceptibility. We conducted a genome-wide association study and replication analysis in 2,173 individuals with Kawasaki disease and 9,383 controls from five independent sample collections. Two loci exceeded the formal threshold for genome-wide significance. The first locus is a functional polymorphism in the IgG receptor gene FCGR2A (encoding an H131R substitution) (rs1801274; P = 7.35 × 10(-11), odds ratio (OR) = 1.32), with the A allele (coding for histadine) conferring elevated disease risk. The second locus is at 19q13, (P = 2.51 × 10(-9), OR = 1.42 for the rs2233152 SNP near MIA and RAB4B; P = 1.68 × 10(-12), OR = 1.52 for rs28493229 in ITPKC), which confirms previous findings(1). The involvement of the FCGR2A locus may have implications for understanding immune activation in Kawasaki disease pathogenesis and the mechanism of response to intravenous immunoglobulin, the only proven therapy for this disease.

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion related morbidity and mortality. Current concepts regarding the pathogenesis of this disorder imply a "two-hit" model in which neutrophils are sequestered in the pulmonary capillary bed, and subsequently activated by substances in the transfused blood product. We report a case of TRALI in a patient with neutropenia and discuss the possible factors contributing to the respiratory symptoms in this patient. We also emphasise the importance of recognising mild cases of TRALI in order to investigate the implicated donor/s appropriately, and to minimise the risk for more severe episodes in other patients.

SummaryLow-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-DRβ1 mismatch was associated with significantly increased Th1 interferon (IFN)-γ, Th2 interleukin (IL)-13 and lymphoproliferative responses by both mothers and fetuses. Allergic women showed significantly lower IFN-γ Th1 production in response to HLA-DRβ1 mismatch. The infants of these women also showed significantly lower IL-10 and lower IFN-γ production relative to IL-13. Both HLA-DRβ1 mismatch and maternal allergy had significant independent effects on maternal IFN-γ 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.

Numerous retrospective studies have been reported analysing the role of HLA mismatches that confer donor NK alloreactivity towards the recipient on the outcome of haematopoietic stem cell transplantation. A bewildering variety of findings have been observed with different studies showing either beneficial or deleterious effects on outcome. This review organises the reports into those that use similar definitions of NK alloreactivity, suggests possible reasons for such disparate results and assesses whether any recommendations can be made in relation to donor selection. There is growing evidence that the effect of human NK alloreactivity on outcome is transplant protocol dependent. Protocol variables most likely to affect outcome are those that influence donor T cell numbers. At this time, it is not possible to predict how the outcome of unrelated donor transplantation will be affected by HLA mismatches conferring potential donor NK alloreactivity.

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.

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, with19 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 ancestor160,000 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.

Natural killer (NK) cells are the predominant leukocyte in first trimester decidua and play a role in vascular remodelling through interferon gamma (IFNgamma) secretion. Membrane expression of the killer immunoglobulin-like receptor (KIR) KIR2DL4 on peripheral blood NK (pNK) cells is controlled by the 9A/10A transmembrane genetic polymorphism. On peripheral NK cells (pNK), KIR2DL4 can only be detected on the membrane of cells from individuals with at least one copy of the 10A allele and ligation of KIR2DL4 results in IFNgamma secretion. In this study, we assessed KIR2DL4 expression and IFNgamma secretion as a result of KIR2DL4 ligation, by decidual NK (dNK) cells. The 9A/10A transmembrane polymorphism was shown to control KIR2DL4 expression by dNK, as previously shown for pNK cells. Freshly isolated dNK cells from subjects with at least one 10A allele expressed KIR2DL4 whereas those from 9A homozygous subjects did not. Although freshly isolated dNK did not secrete IFNgamma in response to KIR2DL4 ligation regardless of KIR2DL4 genotype, activation by in vitro culture with IL-2 enabled dNK cells from individuals with at least one 10A allele, but not those without a 10A allele, to secrete IFNgamma in response to KIR2DL4 ligation. This study confirms that expression of KIR2DL4 by dNK is dependent on the 9A/10A polymorphism and that this polymorphism influences IFNgamma secretion by dNK cells.

NK cell cytotoxicity is controlled through a balance of both activating and inhibitory signals. The HLA specificity of alloreactive NK cells has been previously shown to be controlled by inhibitory killer immunoglobulin-like receptors (KIRs). Alloreactive NK cells lyse targets that lack the HLA ligand for their inhibitory KIR. We have characterized in detail an alloreactive NK clone in which the specificity is controlled by an activating receptor, KIR2DS1. Only target cells expressing the HLA-C group 2 (C2) epitope were lysed by this clone and homozygous C2 targets were lysed more strongly than heterozygous C1/C2 targets. Anti-CD158a (KIR2DS1) blocked lysis of targets confirming KIR2DS1 was responsible. Although this NK clone expressed NKG2A, an inhibitory receptor whose ligand is HLA-E, targets with ligands for both KIR2DS1 and NKG2A were lysed by this clone indicating that the KIR2DS1-mediated activation signal overrides the NKG2A-mediated inhibitory signal. KIR2DS1 activated NK clones in polyclonally expanded NK cultures from a donor that lacked the C2 epitope accounted for approximately 1% of all NK cells. This study highlights a potential role for NK cells controlled by activating KIR in mediating NK alloreactivity.

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).

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.

Variation in the host response to infection by pathogens including HIV-1 may be conferred by polymorphic genetic factors such as HLA and killer immunoglobulin-like receptors (KIR) genes. Here, we examined KIR and HLA genotype effects on pretreatment viral load, rate of CD4(+) T-cell decline and progression to AIDS among adult HIV-1-infected patients within the Western Australian HIV Study Cohort. In this study, carriage of KIR genes within the 'B' haplotype (eg KIR2DS2) was specifically associated with a more rapid CD4(+) T-cell decline over time and progression to AIDS. In contrast, KIR gene repertoire had no effect on pretreatment viral load while selected HLA alleles (eg HLA-B*5701, HLA-B*2705) demonstrated significant protective effects on viremia. Furthermore, interactions between specific HLA and KIR genes did appear to influence HIV disease progression. The results suggest that host genetic variation within the HLA and KIR gene complexes have clinically relevant effects on the course of HIV-1/AIDS, acting independently as well as synergistically to modify disease progression at multiple levels.

The Killer Ig-like receptors (KIRs) on NK cells regulate NK activity via the recognition of specific HLA class I products on the surface of target cells. To investigate the level at which these genetically polymorphic receptors and ligands influence HIV-1 disease progression, we examined the effect of KIR and HLA genotype on HIV outcome. We observed a significant association between particular combinations of KIR epitopes expressed by HLA-B/-C haplotypes and propose that the repertoire of KIR epitopes expressed by HLA-B and HLA-C alleles within the context of particular haplotypes may be an important component of the NK mediated immune response to HIV and/or other infectious pathogens.

BACKGROUND: In view of evidence suggesting an immunological cause of recurrent spontaneous abortions (RSA) and the large number of maternal natural killer (NK) cells present in the pregnant uterus, we investigated the genetic polymorphism of the killer cell immunoglobulin-like receptors (KIR) in women with RSA. METHODS: KIR gene repertoire and KIR2DL4 (a receptor for HLA-G) genotyping were determined by SSP and SSCP respectively, in women experiencing RSA and controls. RESULTS: The KIR repertoire did not differ between RSA patients and controls in terms of: (i) the number of inhibitory receptors; (ii) the number of activating receptors; (iii) the ratio of inhibitory to activating receptors. KIR2DL4, a receptor for HLA-G, has different transmembrane alleles, which produce functionally different phenotypes. The frequency of KIR2DL4 transmembrane genotypes differed significantly between RSA patients and controls (P = 0.03). However, although homozygosity for a membrane-bound receptor was more frequent in patients (25%) than controls (10%), other genotypes that would produce the same phenotype were not more frequent in patients than controls. CONCLUSIONS: The data provide little evidence that KIR polymorphism plays a role in predisposition to RSA.

Members of the killer immunoglobulin (Ig)-like receptor (KIR) gene family are tightly clustered on human chromosome 19q13.4. Despite considerable variation in KIR gene content and allelic polymorphism, most KIR haplotypes belong to one of two broad groups termed A and B. The availability of contiguous genomic sequences for these haplotypes has allowed us to compare their genomic organization, nucleotide (nt) diversity and reconstruct their evolutionary history. The haplotypes have a framework of three conserved blocks containing (i) KIR3DL3, (ii) KIR3DP1, 2DL4, and (iii) KIR3DL2 that are interrupted by two variable segments that differ in the number and type of KIR genes. Low (0.05%) nucleotide diversity was detected across the centromeric and telomeric boundaries of the KIR gene cluster while higher SNP density (0.2%) occurred within the central region containing the KIR2DL4 gene. Phylogenetic and genomic analyses have permitted the reconstruction of a hypothetical ancestral haplotype that has revealed common groupings and differences between the KIR genes of the two haplotypes. The present phylogenetic and genomic comparison of the two sequenced KIR haplotypes provides a framework for a more thorough examination of KIR haplotype variations, diversity and evolution in human populations and between humans and non-human primates.

During discussion at the WHO Nomenclature Committee for Factors of the HLA System meeting in Victoria, Canada in May 2002, it was decided to form a subcommittee to coordinate the naming of alleles of the genes encoding the killer-cell immunoglobulin-like receptors (KIRs) (Marsh et al., 2002). These genes are encoded on chromosome 19 (19q13.4) and have varying degrees of polymorphism. The receptors encoded by the KIR genes are expressed by natural killer (NK) cells and a subset of T cells, and some of them have been shown to have specificity for determinants of HLA class I molecules. The extracellular ligand-binding part of KIR consists of two or three immunoglobulin (Ig)-like domains. The discussions which took place in Victoria are further to earlier discussions on KIR nomenclature at the NK Polymorphism meeting (27-29 July 2001) in Cambridge, UK. In addition, a request has been made by the International Union of Immunological Societies (IUIS) to provide a standardized nomenclature for the expressed protein products of the KIR genes.

Chronic natural killer (NK) lymphocytosis is a rare disorder characterized by an indolent clinical course. Despite high NK cell numbers, many patients present with only mild clinical symptoms, and are often asymptomatic. NK cells are equipped with a range of receptors that bind human leucocyte antigen (HLA)-class I molecules. The killer immunoglobulin-like receptors (KIR, CD158) bind groups of HLA alleles, the CD94/NKG2 receptors bind HLA-E, and the CD85j (ILT2, LIR-1) receptor binds to the relatively non-polymorphic alpha3 domain of HLA molecules. Inhibitory HLA class I receptors silence NK cells against cells expressing normal levels of HLA class I. Analysis of NK cells in six patients with chronic NK lymphocytosis revealed a high level of the inhibitory CD94/NKG2A receptor on all NK cells. In four patients, KIR were absent, in one patient a single KIR was expressed in the absence of self-ligand, and in one patient CD85j and multiple KIR were expressed. Cytotoxicity assays demonstrated that all HLA class I receptors were functional. The ability of monoclonal antibodies to block the receptors and allow killing of autologous target cells established that both receptor and ligand expression were adequate for inhibitory function. We propose that the silent behaviour of NK cells in patients with chronic NK lymphocytosis is due to effective inhibitory HLA class I receptors.

The aim of this study was to determine whether a common diabetic haplotype, including human leukocyte antigen (HLA)-B8 and HLA-DR3, in Northern India is the same haplotype as the European HLA-B8-DR3 haplotype. DNA samples from Northern Indian subjects selected on the basis of HLA-B8 and HLA-DR3 were tested for microsatellite and single nucleotide polymorphism alleles throughout the major histocompatibility complex (MHC). It was found that the Indian samples represent a conserved haplotype in which all alleles were shared by Indian subjects with HLA-B8 and HLA-DR3, but were different to those that are characteristic of the European 8.1 ancestral haplotype. The Indian and European haplotypes share HLA-B*0801, HLA-DRB1*0301 and HLA-DQB1*02 but differ for subtypes of HLA-Cw*07 and HLA-DRB3 and all central MHC alleles tested. In contrast, Indian subjects selected on the basis of HLA-B58 ( 1–17) and HLA-DR3 shared the same alleles at other MHC loci as have been described in the common Chinese haplotype with HLA-B58/17 and HLA-DR3. A third haplotype, HLA-B50/21 and HLA-DR3, was also found to be highly conserved but shares little in common with the other two HLA-DR3-containing Indian haplotypes.

Here, we compare the architecture of membrane receptors with extracellular Ig-like domains located within the leukocyte Ig-like receptor complex (LRC) of humans and mice. The receptors can be classified broadly into four groups, based on the homology of their Ig-like domains and gene architecture. Receptors in the first group are characterized by the presence of the Ig constant type 2-1 (IgC2-1) and variant Ig (vIg) domains, and include the leukocyte Ig-like receptors (LILRs) and murine paired Ig-activating receptors (PIRs). The second group of receptors possess an IgC2-2 domain and comprise the killer-cell Ig-like receptors (KIRs) and platelet collagen receptor glycoprotein VI (GPVI). The third group consists of receptors with IgC2-1, and IgC2-3 or IgC2-4 domains, and includes the receptor for IgA Fc (FCAR), NKp46 and murine Ly94. The fourth group, with a single extracellular IgC2-1 domain, consists of the leukocyte-associated Ig-like receptors (LAIRs). The genomic organization of and evolutionary associations between these receptors and their domains are examined.

A survey of KIR2DL4 polymorphism revealed seven common sequences in the Australian population. The seven sequences encode three different amino acid sequences of the immunoglobulin domains. Two of the sequences encoding different amino acid sequences in the immunoglobulin domains also occur on some chromosomes with a single nucleotide deletion at the end of exon 6, which encodes the transmembrane domain (ΔTM mutation), resulting in exon 6 skipping during mRNA production. Due to alternate splicing, a fraction of the mRNA produced by these alleles includes the transmembrane region but is missing the cytoplasmic region. The remaining two sequences differed only by synonymous substitutions. All of the exonic polymorphisms of KIR2DL4 could be detected by single-stranded conformational polymorphism of individually amplified exons. The ΔTM mutation is in linkage disequilibrium with the killer cell immunoglobulin-like receptor (KIR) A haplotype, and the wild-type sequence is in linkage disequilibrium with the B haplotype. The frequencies of alleles with the ΔTM mutation or Ig-domain polymorphisms did not differ between women who experienced pre-eclampsia and normotensive controls. Similarly there was no difference in the KIR gene repertoire in pre-eclampsia and normotensive controls.

Dr. Long’s assertion that killer Ig-like receptor (KIR)2DL4 is not detectable at the cell surface is misleading. KIR2DL4 is not detectable on the membrane of resting CD56dim NK cells. However, we and others ([1][1]) have demonstrated surface expression of KIR2DL4 on freshly isolated CD56bright NK

Numerous studies have associated carriage of HLA-DRB1*1501, DQA1*0102 and DQB1*0602 (DR15, DQ6) with dominant resistance to type 1 diabetes and have concluded that one or more of the component HLA class II molecules mediate this effect. Mechanisms for MHC class II-mediated resistance to diabetes have been proposed from studies of transgenic mice, usually using the diabetes-prone non-obese diabetic (NOD) strain. However, these studies have not reached any consensus on a plausible mechanism. In this study we question why the role of central MHC genes in resistance to diabetes has not been addressed, as the central MHC carries markers of susceptibility to diabetes in linkage disequilibrium with several genes with known or putative immunoregulatory functions. To illustrate the type of studies required to address this issue, we selected diabetes patients and control subjects for carriage of HLA-DR15 and the C allele at position +738 in the inhibitor of kappa B-like gene (IKBL). These alleles mark the 7.1 haplotype (HLA-A3, B7, IKBL738*C, DR15, DQ6). HLA-DR15 was the most effective marker of resistance, but an effect may be evident with IKBL738*C in a larger study. Moreover, carriage of the entire haplotype was particularly rare in patients. The best explanation for this is that the critical gene lies between IKBL and HLA-DRB1, and is more closely linked to HLA-DRB1. Candidate genes at the centromeric end of the central MHC are reviewed, highlighting the need for further study.

The HLA-DRB1 sequencing based typing strategies reported to date require separate amplifications of each sample with a series of group-specific primers followed by sequencing of any resulting polymerase chain reaction (PCR) products. Whilst this results in high resolution typing in the majority of cases, a number of unnecessary amplifications are performed. We report here a novel approach where amplification of the second exon of HLA-DRB1 is performed in a single tube for all alleles. Retrospective analysis of 642 consecutive Western Australian unrelated bone marrow registry donors has shown that this approach results in unambiguous typings in 71.1% of cases. Ambiguities can be readily resolved if necessary with a single additional sequencing reaction on the original PCR product.

Single-stranded conformation polymorphism (SSCP) by capillary electrophoresis was assessed as a screening and typing method for alleles of KIR2DL4. Exon 6 was investigated as this exon was reported to include three polymorphic nucleotides. Exon 6, intron 6 and exon 7 were amplified as a single polymerase chain reaction (PCR) product of 650 bp from genomic DNA. The PCR product was sequenced and analysed by SSCP. Exon 7 was found to be invariant. Only two nucleotides were found to be polymorphic in exon 6 and another three were found in intron 6. Strong linkage disequilibrium was found between the polymorphic nucleotides resulting in the presence of three alleles in a panel of 20 cell lines. Two alleles differed within intron 6 while the third allele differed at two nucleotides in exon 6. All six possible genotypes were distinguishable by SSCP providing information from both the forward and reverse primers was used. Exon 6 of one allele was one nucleotide shorter than that of the other alleles and the resulting frame shift is predicted to produce a truncated cytoplasmic tail due to a premature stop codon four codons into exon 7. SSCP was found to be an efficient method of typing exons 6 and 7 in a panel of 46 bone marrow donors. All three alleles were found to be common and one was in strong linkage disequilibrium with the presence of another KIR sequence KIR3DS1.

Natural killer (NK) immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors which interact with specific motifs on HLA class I molecules and modulate NK cytolytic activity. In this study, we analyzed a recently sequenced subgenomic region on chromosome 19q13.4 containing eight members of the KIR receptor repertoire. Six members are clustered within a 100-kb continuous sequence. These genes include a previously unpublished member of the KIR gene family 2DS6, as well as 2DL1, 2DL4, 3DL1, 2DS4, 3DL2, from centromere to telomere. Two additional KIR genes, KIRCI and 2DL3, which may be located centromeric of this cluster were also analyzed. We show that the KIR genes have undergone repeated gene duplications. Diversification between the genes has occurred postduplication primarily as a result of retroelement indels and gene truncation. Using pre- and postduplication Alu sequences identified within these genes as evolutionary molecular clocks, the evolution and duplication of this gene cluster is estimated to have occurred 30-45 million years ago, during primate evolution. A proposed model of the duplication history of the KIR gene family leading to their present organization is presented.

Susceptibility to sIBM is strongly associated with the HLA-DRB1*03 allele and the 8.1 MHC ancestral haplotype (HLA-A1, B8, DRB1*03) but little is known about the effects of allelic interactions at the DRB1 locus or disease-modifying effects of HLA alleles. HLA-A, B and DRB1 genotyping was performed in 80 Australian sIBM cases and the frequencies of different alleles and allele combinations were compared with those in a group of 190 healthy controls. Genotype-phenotype correlations were also investigated. Amongst carriers of the HLA-DRB1*03 allele, DRB1*03/*01 heterozygotes were over-represented in the sIBM group (p

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

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.

The Jewish population in Israel comprises of inhabitants of heterogeneous ethnic backgrounds. Genetic studies classify the Israeli Jewish population into two major groups: Ashkenazi from Central and Eastern Europe and Sephardic or non Ashkenazi, from the Mediterranean and North Africa. The present study was aimed at elucidating the differential influx of HLA class II alleles in Ashkenazi, in various non-Ashkenazi subgroups and in Israeli Moslem Arabs. Using the PCR-SSOP technique, a large number of alleles were detected at each of the loci examined (DRB1, DQA1 and DQB1). In addition, gene frequencies, characteristic DR/DQ linkage disequilibria, population distances and their corresponding dendogram, were used to study the relationship between Israelis as a group, non Jewish Caucasians and Blacks. These populations could be grouped into three main clusters: the first consists of all the Israeli groups with the exception of the Ethiopian Jews; the second consists of non Jewish Caucasians, with a clear distinction seen between Israelis and non Jewish Europeans and U.S. Caucasians; the third, composed of Blacks, is distinctly different from the other populations. Ethiopian Jews were found to be closer to the Blacks than to any of the Israeli Jewish groups. We have shown that Jews share common features, a fact that points to a common ancestry. A certain degree of admixture with their pre-immigration neighbors exists despite the cultural and religious constraints against intermarriage.

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.

An individual's major histocompatibility complex (MHC) ancestral haplotype (AH) is the clearest single determinant of susceptibility to MHC associated immunopathological disease, as it defines the alleles carried at all loci in the MHC. However, the direct effects of any of the 150-200 genes that constitute the MHC are difficult to determine since recombination only occurs at defined hotspots. This review concerns the 8.1 AH (HLA-A1, C7, B8, C4AQ0, C4B1, DR3, DQ2), which is carried by most Caucasians with HLA-B8. It is associated with accelerated human immunodeficiency virus (HIV) disease, and susceptibility to insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus, dermatitis herpetiformis, common variable immunodeficiency and IgA deficiency, myasthenia gravis and several other conditions. We have mapped susceptibility genes for HIV, IDDM and myasthenia gravis to the central MHC between HLA-B and the tumour necrosis factor or complement genes. Here we consider which of the remaining 8.1-associated diseases are more closely associated with HLA-DR3 and/or DQ2. Several candidate genes in the central MHC have the potential to modulate immune or inflammatory responses in an antigen-independent manner, as is seen in studies of cultured cells from healthy carriers of the 8.1 AH. Hence these genes may act as a common co-factor in the diverse immunopathological conditions associated with the 8.1 AH.

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.

Conventional matching is based on numbers of alleles shared between donor and recipient. This approach, however, ignores the degree of relationship between alleles and haplotypes, and therefore the actual degree of difference. To address this problem, we have compared family members using a block matching technique which reflects differences in genomic sequences. All parents and siblings had been genotyped using conventional MHC typing so that haplotypes could be assigned and relatives could be classified as sharing 0, 1 or 2 haplotypes. We trained an Artificial Neural Network (ANN) with subjects from 6 families (85 comparisons) to distinguish between relatives. Using the outputs of the ANN, we developed a score, the Histocompatibility Index (HI), as a measure of the degree of difference. Subjects from a further 3 families (106 profile comparisons) were tested. The HI score for each comparison was plotted. We show that the HI score is trimodal allowing the definition of three populations corresponding to approximately 0, 1 or 2 haplotype sharing. The means and standard deviations of the three populations were found. As expected, comparisons between family members sharing 2 haplotypes resulted in high HI scores with one exception. More interestingly, this approach distinguishes between the 1 and 0 haplotype groups, with some informative exceptions. This distinction was considered too difficult to attempt visually. The approach provides promise in the quantification of degrees of histocompatibility.

Life-threatening graft-versus-host disease (GVHD) limits the use of HLA-C-mismatched unrelated donors in transplantation. Clinicians lack criteria for donor selection when HLA-C-mismatched donors are a patient's only option for cure. We examined the role for HLA-C expression levels to identify permissible HLA-C mismatches. The median fluorescence intensity, a proxy of HLA-C expression, was assigned to each HLA-C allotype in 1975 patients and their HLA-C-mismatched unrelated transplant donors. The association of outcome with the level of expression of patients' and donors' HLA-C allotypes was evaluated in multivariable models. Increasing expression level of the patient's mismatched HLA-C allotype was associated with increased risks of grades III to IV acute GVHD, nonrelapse mortality, and mortality. Increasing expression level among HLA-C mismatches with residue 116 or residue 77/80 mismatching was associated with increased nonrelapse mortality. The immunogenicity of HLA-C mismatches in unrelated donor transplantation is influenced by the expression level of the patient's mismatched HLA-C allotype. HLA-C expression levels provide new information on mismatches that should be avoided and extend understanding of HLA-C-mediated immune responses in human disease.

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.