Distinct distribution of killer-cell immunoglobulin-like receptor genes in the Mugil and Ilaita areas of Papua New Guinea.

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.

Virtual crossmatch approach to maximize matching in paired kidney donation.

We developed and tested a new computer program to match maximal sets of incompatible live donor/recipient pairs from a national paired kidney donation (PKD) registry. Data of 32 incompatible pairs included ABO and 4 digit-high-resolution donor and recipient HLA antigens and recipient's HLA antibodies. Three test runs were compared, in which donors were excluded from matching to recipients with either donor-specific antibodies (DSA) >8000MFI (mean fluorescent intensity) at low-resolution (Run 1) or >8000MFI at high-resolution (Run 2) or >2000MFI and high-resolution (Run 3). Run 1 identified 22 703 possible combinations, with 20 pairs in the top ranked, Run 2 identified 24 113 combinations, with 19 pairs in the top ranked and Run 3 identified 8843 combinations, with 17 pairs in the top ranked. Review of DSA in Run 1 revealed that six recipients had DSA 2000-8000MFI causing a possible positive crossmatch resulting in breakdown of two 3-way and three 2-way chains. In Run 2, four recipients had DSA 2000-8000MFI, also potentially causing breakdown of three 2-way chains. The more prudent approach of excluding from matching recipients with DSA with >2000MFI reduces the probability of matched pairs having a positive crossmatch without significantly decreasing the number of possible transplants.

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 genotype of the NK cell receptor, KIR2DL4, influences INFgamma secretion by decidual natural killer cells.

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.

Sequencing-based typing identifies novel alleles due to single nucleotide polymorphisms in 'conserved' regions.

The Royal Perth Hospital laboratory has been using sequencing-based typing for all HLA loci since 2002. In the period to October 2005, approximately 12,000 HLA A and HLA B, 5000 HLA C and DQB1, and 17,000 DRB1 requests have been processed. Twenty nine novel alleles have been identified in that time. These comprise 10 HLA-A (including one null allele), five HLA-B, six HLA-C, six DRB1 (including a null allele), and one DQB1 novel allele. (At the time of identifying the DRB1 null allele, there were no other reported examples.) In addition, we have seen one example of a blast-specific HLA-A null allele. One HLA-A allele (HLA-A*0264) and one HLA-B allele (HLA-B*400104) were subsequently identified in other laboratories and submitted to the international ImMunoGeneTics project (IMGT) database.

The identification and characterisation of a novel null HLA-DRB1 allele.

Since the introduction of DNA-based human leukocyte antigen (HLA) typing a number of discrepancies with serological typing have been documented. At the time of submission of this abstract (July 2005 ImMunoGeneTics project (IMGT) project database release) 42 HLA class I and II null alleles had been described characterised by a lack of expression of cell surface antigen. These null alleles can be accounted for by a number of demonstrated molecular mechanisms including insertion, deletion and point mutation and may lead to a nonsense codon, splicing defect or premature stop codon.

14th International HLA and Immunogenetics Workshop: report on KIR receptors and their applications.

During the last few years many laboratories have developed a keen interest in detecting killer immunoglobulin-like receptor (KIR) receptor genes in various populations, diseases and in stem cell transplantation. At the 14th International Histocompatibility Workshop held in Melbourne in December 2006, many of these laboratories presented their findings at a special session. To introduce this work, we provide an introduction to KIR receptors and an outline of previous applications of KIR receptor typing prior to the Workshop.

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.

Killer immunoglobulin-like receptors and HLA act both independently and synergistically to modify HIV disease progression.

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.

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.

Assign 2.0: software for the analysis of Phred quality values for quality control of HLA sequencing-based typing.

As improvements to DNA sequencing technology have resulted in increasing the throughput of DNA sequencing, the bottleneck for high throughput DNA sequencing-based typing (SBT) has shifted to sequence analysis, genotyping and quality control (QC). Consistent high-quality DNA sequence is required in order to reduce manual verification and editing of sequence electropherograms. However, identifying systematic changes in quality is difficult to achieve without the aid of sophisticated sequence analysis programs dedicated to this purpose. We describe a computer software program called Assign 2.0, which integrates sequence QC analysis and genotyping in order to facilitate high-throughput SBT. Assign 2.0 performs an analysis of Phred quality values in order to produce quality scores for a sample and a sequencing run. This enables sample-to-sample and run-to-run QC monitoring and provides a mechanism for the comparison of sequence quality between various genes, various reagents and various protocols with the aim of improving the overall quality of DNA sequence data. This, in turn, will result in reducing sequence analysis as a bottleneck for high-throughput SBT.

Maternal KIR repertoire is not associated with recurrent spontaneous abortion.

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.

A multicenter international evaluation of single-tube amplification protocols for sequencing-based typing of HLA-DRB1 and HLA-DRB3,4,5.

We have described previously a novel single-tube polymerase chain reaction (PCR) amplification (STAmp) protocol for the efficient sequencing-based typing (SBT) of human leukocyte antigen (HLA)-DRB1. The PCR amplification mix includes primers to each of seven allele group-sequence motifs. We have applied this principle to the simultaneous SBT of HLA-DRB3, -DRB4, and -DRB5 using locus specific primers. We report here a multicenter international evaluation of the STAmp protocols performed as a component of the 13th International Histocompatibility Workshop. Identical amplification primer mixes, sequencing primers, and DNA were sent to participating laboratories. The primer mixes contained the amplification primers and the PCR buffer. Each laboratory was requested to amplify the DNA with the primer mixes and perform SBT on the resulting PCR protocols, using their own protocols, and return the typing results for analysis. The reported results indicated that the expected sequence could be obtained with a variety of PCR amplification and sequencing platforms and protocols. There were difficulties but these seemed unrelated to STAmp reagents and suggest that optimal SBT results can be obtained if bi-directional sequencing is performed and software is used for sequence verification and editing. This indicates that SBT by STAmp can be applied in many laboratories for high-throughput HLA-DRB1 and HLA-DRB3,4,5 SBT.

The beneficial role of inhibitory KIR genes of HLA class I NK epitopes in haploidentically mismatched stem cell allografts may be masked by residual donor-alloreactive T cells causing GVHD.

HLA allele mismatches will provoke T-cell alloreactivity after allogeneic stem cell transplantation. As donors and recipients are usually HLA matched, the public HLA epitopes that are recognized by natural killer (NK) cells (NK epitopes) are rarely mismatched, and therefore there is rarely potential for NK alloreactivity arising from the absence of ligands for inhibitory killer immunoglobulin-like receptors (KIR). Transplants using related donors sharing only one haplotype (haploidentical donors) represent a setting in which NK epitopes are often mismatched, thus resulting in the potential for NK alloreactivity. We have analyzed engraftment, acute graft vs host disease (GVHD), leukemia relapse, and survival in 62 haploidentical transplants in relationship with potential NK alloreactivity, inhibitory, and activating KIR genes of class I HLA NK epitopes. Potential NK alloreactivity in the rejection direction was not associated with any outcome variable. Potential NK alloreactivity in the GVHD direction was associated with an increased incidence of severe GVHD and poorer patient survival but not with non-engraftment nor leukemia relapse. A higher number of activating KIR receptors in the genome of the donor was associated with a higher prevalence of GVHD. These results suggest that lack of extensive T-cell depletion in haploidentical transplantation is associated with high GVHD rates and diminishes the benefits of NK-cell alloreactivity.

Quality assessment program for genotypic antiretroviral testing improves detection of drug resistance mutations.

Genotypic antiretroviral testing is now widely used for the management of patients who are undergoing antiretroviral therapy for human immunodeficiency virus infection. The assays are complex, and there is considerable potential for variation between laboratories. Informative and ongoing quality assessment programs (QAPs) which address all aspects of testing are required. The panel distribution of clinical material is a critical component of QAPs. We report on the results and data from a recent panel. Four cryopreserved plasma samples from treated donors were distributed to nine laboratories. Three laboratories performed testing by commercial assays, and six laboratories used in-house assays, with one laboratory reporting results from two in-house assays. There was complete concordance between results for 95.9% of the nucleotide sequence and 94.5% of the amino acid sequence. Despite this overall high level of concordance, the degree of concordance at drug resistance mutation (DRM) sites when DRMs were present was considerably less (38% of DRM sites). Consequently, only 3 of the 10 methods reported 100% of DRMs as present. This elevated discrepancy rate is almost certainly a result of variability in the identification of mixtures of nucleotides (mixtures) at any site within the sequence. In addition, laboratories differed in the number of codons in the reverse transcriptase gene that were sequenced and their ability to amplify all samples. This panel distribution demonstrated a requirement for laboratory participation in ongoing QAPs and the optimization of assays with standards that contain mixtures.

Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir.

BACKGROUND: The use of abacavir--a potent HIV-1 nucleoside-analogue reverse-transcriptase inhibitor--is complicated by a potentially life-threatening hypersensitivity syndrome in about 5% of cases. Genetic factors influencing the immune response to abacavir might confer susceptibility. We aimed to find associations between MHC alleles and abacavir hypersensitivity in HIV-1-positive individuals treated with abacavir. METHODS: MHC region typing was done in the first 200 Western Australian HIV Cohort Study participants exposed to abacavir. Definite abacavir hypersensitivity was identified in 18 cases, and was excluded in 167 individuals with more than 6 weeks' exposure to the drug (abacavir tolerant). 15 individuals experienced some symptoms but did not meet criteria for abacavir hypersensitivity. p values were corrected for comparisons of multiple HLA alleles (p(c)) by multiplication of the raw p value by the estimated number of HLA alleles present within the loci examined. FINDINGS: HLA-B*5701 was present in 14 (78%) of the 18 patients with abacavir hypersensitivity, and in four (2%) of the 167 abacavir tolerant patients (odds ratio 117 [95% CI 29-481], p(c)<0.0001), and the HLA-DR7 and HLA-DQ3 combination was found in 13 (72%) of hypersensitive and five (3%) of tolerant patients (73 [20-268], p(c)<0.0001 ). HLA-B*5701, HLA-DR7, and HLA-DQ3 were present in combination in 13 (72%) hypersensitive patients and none of the tolerant patients (822 [43-15 675], p(c)<0.0001). Other MHC markers also present on the 57.1 ancestral haplotype to which the three markers above belong confirmed the presence of haplotype-specific linkage disequilibrium, and mapped potential susceptibility loci to a region bounded by C4A6 and HLA-C. Within the entire abacavir-exposed cohort (n=200), presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 had a positive predictive value for hypersensitivity of 100%, and a negative predictive value of 97%. INTERPRETATION: Genetic susceptibility to abacavir hypersensitivity is carried on the 57.1 ancestral haplotype. In our population, withholding abacavir in those with HLA-B*5701, HLA-DR7, and HLA-DQ3 should reduce the prevalence of hypersensitivity from 9% to 2.5% without inappropriately denying abacavir to any patient.