STAT4-associated natural killer cell tolerance following liver transplantation.

OBJECTIVE: Natural killer (NK) cells are important mediators of liver inflammation in chronic liver disease. The aim of this study was to investigate why liver transplants (LTs) are not rejected by NK cells in the absence of human leukocyte antigen (HLA) matching, and to identify a tolerogenic NK cell phenotype. DESIGN: Phenotypic and functional analyses on NK cells from 54 LT recipients were performed, and comparisons made with healthy controls. Further investigation was performed using gene expression analysis and donor:recipient HLA typing. RESULTS: NK cells from non-HCV LT recipients were hypofunctional, with reduced expression of NKp46 (p<0.05) and NKp30 (p<0.001), reduced cytotoxicity (p<0.001) and interferon (IFN)-γ secretion (p<0.025). There was no segregation of this effect with HLA-C, and these functional changes were not observed in individuals with HCV. Microarray and RT-qPCR analysis demonstrated downregulation of STAT4 in NK cells from LT recipients (p<0.0001). Changes in the expression levels of the transcription factors Helios (p=0.06) and Hobit (p=0.07), which control NKp46 and IFNγ expression, respectively, were also detected. Hypofunctionality of NK cells was associated with impaired STAT4 phosphorylation and downregulation of the STAT4 target microRNA-155. Conversely in HCV-LT NK cell tolerance was reversed, consistent with the more aggressive outcome of LT for HCV. CONCLUSIONS: LT is associated with transcriptional and functional changes in NK cells, resulting in reduced activation. NK cell tolerance occurs upstream of major histocompatibility complex (MHC) class I mediated education, and is associated with deficient STAT4 phosphorylation. STAT4 therefore represents a potential therapeutic target to induce NK cell tolerance in liver disease.

qKAT: a high-throughput qPCR method for KIR gene copy number and haplotype determination.

Killer cell immunoglobulin-like receptors (KIRs), expressed on natural killer cells and T cells, have considerable biomedical relevance playing significant roles in immunity, pregnancy and transplantation. The KIR locus is one of the most complex and polymorphic regions of the human genome. Extensive sequence homology and copy number variation makes KIRs technically laborious and expensive to type. To aid the investigation of KIRs in human disease we developed a high-throughput, multiplex real-time polymerase chain reaction method to determine gene copy number for each KIR locus. We used reference DNA samples to validate the accuracy and a cohort of 1698 individuals to evaluate capability for precise copy number discrimination. The method provides improved information and identifies KIR haplotype alterations that were not previously visible using other approaches.

LILRA6 copy number variation correlates with susceptibility to atopic dermatitis.

Leukocyte immunoglobulin-like receptors (LILR) are expressed mostly on myelomonocytic cells where they are mediators of immunological tolerance. Two LILR genes, LILRA3 and LILRA6, exhibit marked copy number variation. We assessed the contribution of these genes to atopic dermatitis (AD) by analysing transmission in 378 AD families. The data indicated that copies of LILRA6 were over-transmitted to affected patients. They are consistent with a contribution of LILR genes to AD. They could affect the equilibrium between activating and inhibitory signals in the immune response.

KIR haplotypes are associated with late-onset type 1 diabetes in European-American families.

Classical human leukocyte antigens (HLA) genes confer the strongest, but not the only, genetic susceptibility to type 1 diabetes. Killer cell immunoglobulin-like receptors (KIR), on natural killer (NK) cells, bind ligands including class I HLA. We examined presence or absence, with copy number, of KIR loci in 1698 individuals, from 339 multiplex type 1 diabetes families, from the Human Biological Data Interchange, previously genotyped for HLA. Combining family data with KIR copy number information allowed assignment of haplotypes using identity by descent. This is the first disease study to use KIR copy number typing and unambiguously define haplotypes by gene transmission. KIR A1 haplotypes were positively associated with T1D in the subset of patients without the high T1D risk HLA genotype, DR3/DR4 (odds ratio=1.29, P=0.0096). The data point to a role for KIR in type 1 diabetes risk in late-onset patients. In the top quartile (age of onset>14), KIR A2 haplotype was overtransmitted (63.4%, odds ratio=1.73, P=0.024) and KIR B haplotypes were undertransmitted (41.1%, odds ratio=0.70, P=0.0052) to patients. The data suggest that inhibitory 'A' haplotypes are predisposing and stimulatory 'B' haplotypes confer protection in both DR3/DR4-negative and late-onset patient groups.

The interaction of genetic determinants in the outcome of HCV infection: evidence for discrete immunological pathways.

Diversity within the innate and adaptive immune response to hepatitis C is important in determining spontaneous resolution (SR) and treatment response. The aim of this study was to analyze how these variables interact in combination; furthering our understanding of the mechanisms that drive successful immunological clearance. Multivariate analysis was performed on retrospectively collected data for 357 patients previously genotyped for interferon (IFN)-λ3/4, killer cell immunoglobulin (KIR), human leukocyte antigen (HLA) class I and II and tapasin. High resolution KIR genotyping was performed for individuals with chronic infection and haplotypes determined. Outcomes for SR, IFN response and cirrhosis were examined. Statistical analysis included univariate methods, χ(2) test for trend, multivariate logistic regression, synergy and principal component analysis (PCA). Although KIR2DL3:HLA-C1C1 (P = 0.027), IFN-λ3/4 rs12979860 CC (P = 0.027), tapasin G in individuals with aspartate at residue 114 of HLA-B (TapG:HLA-B(114D) ) (P = 0.007) and HLA-DRB1*04:01 (P = 0.014) were associated with SR with a strong additive influence (χ(2) test for trend P < 0.0001); favorable polymorphisms did not interact synergistically, nor did patients cluster by outcome. In the treatment cohort, IFN-λ3/4 rs12979860 CC was protective in hepatitis C virus (HCV) G1 infection and KIR2DL3:HLA-C1 in HCV G2/3. In common with SR, variables did not interact synergistically. Polymorphisms predictive of viral clearance did not predict disease progression. In summary, different individuals resolve HCV infection using discrete and non-interacting immunological pathways. These pathways are influenced by viral genotype. This work provides novel insights into the complexity of the interaction between host and viral factors in determining the outcome of HCV infection.

2DL1, 2DL2 and 2DL3 all contribute to KIR phenotype variability on human NK cells.

Natural killer (NK) cells are lymphocytes that function as part of the innate immune system. Their activity is controlled by a range of inhibitory and activating receptors, including the important killer-cell immunoglobulin-like receptors (KIR). The KIR are a multi-gene family of receptors that interact with the human leukocyte antigen (HLA) class I family of molecules and are characterised by extensive allelic polymorphism. Their expression on the cell surface of NK cells is highly variable, but the factors responsible for this variability are not yet clearly understood. In the current study, we investigated KIR expression in a healthy human cohort that we had previously characterised in depth at a genetic level, with KIR allele typing and HLA class I ligand genotypes available for all donors (n=198). Allelic polymorphism significantly affected the phenotypic expression of all KIR analysed, whereas HLA ligand background influenced the expression levels of 2DL1 and 2DL3. In particular, we found that although 2DL2 may influence 2DL1 expression, this appears to be owing to variation in 2DL1 copy number. Finally, the inhibitory receptor LILRB1 had higher expression levels in individuals with B/B KIR genotypes, suggesting a possible relationship between KIR and non-KIR receptors, which serves to balance NK cell activation potential.

Killer immunoglobulin-like receptor gene repertoire influences viral load of primary human cytomegalovirus infection in renal transplant patients.

Killer cell immunoglobulin-like receptors (KIR) are highly polymorphic members of the immunoglobulin superfamily, which influence the response of natural killer cells and some T-lymphocyte subsets. Analysis of a cohort of previously human cytomegalovirus (HCMV)-negative patients, who developed primary HCMV infection following HCMV-positive renal transplant (n=76), revealed an increase in the frequency of KIR genes located on the telomeric region of B haplotypes (Tel B). The presence of Tel B in combination with the KIR ligand HLA-C2 was significantly more frequent in this subgroup. These genetic factors were associated with resistance to HCMV infection in a second cohort (n=65), where the Tel B genes KIR2DL5, -2DS1, 2DS5 and -3DS1 were all significantly associated with high viral loads. Furthermore, the KIR haplotype Tel A when in combination with the KIR ligand HLA-C1 was significantly protective against the development of severe infection. Our results suggest that KIR are a significant factor in the control of primary HCMV infection, and that determination of KIR gene repertoire may help in detection of renal transplant patients who were most at risk.

Human MHC architecture and evolution: implications for disease association studies.

Major histocompatibility complex (MHC) variation is a key determinant of susceptibility and resistance to a large number of infectious, autoimmune and other diseases. Identification of the MHC variants conferring susceptibility to disease is problematic, due to high levels of variation and linkage disequilibrium. Recent cataloguing and analysis of variation over the complete MHC has facilitated localization of susceptibility loci for autoimmune diseases, and provided insight into the MHC's evolution. This review considers how the unusual genetic characteristics of the MHC impact on strategies to identify variants causing, or contributing to, disease phenotypes. It also considers the MHC in relation to novel mechanisms influencing gene function and regulation, such as epistasis, epigenetics and microRNAs. These developments, along with recent technological advances, shed light on genetic association in complex disease.

Analysis of the BTNL2 truncating splice site mutation in tuberculosis, leprosy and Crohn's disease.

The region on chromosome 6 encoding the major histocompatibility complex (MHC) is associated with a number of autoimmune and infectious diseases. Primary susceptibility to many of these has been localized to a region containing the human leukocyte antigen (HLA)-DR and -DQ genes. A recent study of sarcoidosis has provided evidence of an independent effect, associated with a truncating single nucleotide polymorphism (SNP) of a nearby gene, BTNL2. This gene may encode an immune receptor involved in costimulation. Sarcoidosis, tuberculoid leprosy, tuberculosis (TB) and Crohn's disease all have similar immunological features, including a Th1 response with granuloma formation. In addition mycobacteria have been identified or suggested to be causative pathogens in such conditions. We genotyped the truncating BTNL2 SNP in 92 TB and 72 leprosy families from Brazil and carried out family-based association studies. We could not find evidence of overtransmission of the truncating allele in TB. There was an association with susceptibility to leprosy (P=0.04), however, this is most likely due to linkage disequilibrium with HLA-DR. We also genotyped 476 UK Caucasian cases of Crohn's disease with 760 geographically matched controls and found no evidence of a disease association. We conclude that the truncating BTNL2 SNP is not important in this group of Th1 dominated granulomatous diseases.

The LRC haplotype project: a resource for killer immunoglobulin-like receptor-linked association studies.

There is increasing evidence for epistatic interactions between gene products (e.g. KIR) encoded within the Leukocyte Receptor Complex (LRC) with those (e.g. HLA) of the Major Histocompatibility Complex (MHC), resulting in susceptibility to disease. Identification of such associations at the DNA level requires comprehensive knowledge of the genetic variation and haplotype structure of the underlying loci. The LRC haplotype project aims to provide this knowledge by sequencing common LRC haplotypes.

Extent and distribution of linkage disequilibrium in three genomic regions.

The positional cloning of genes underlying common complex diseases relies on the identification of linkage disequilibrium (LD) between genetic markers and disease. We have examined 127 polymorphisms in three genomic regions in a sample of 575 chromosomes from unrelated individuals of British ancestry. To establish phase, 800 individuals were genotyped in 160 families. The fine structure of LD was found to be highly irregular. Forty-five percent of the variation in disequilibrium measures could be explained by physical distance. Additional factors, such as allele frequency, type of polymorphism, and genomic location, explained <5% of the variation. Nevertheless, disequilibrium was occasionally detectable at 500 kb and was present for over one-half of marker pairs separated by <50 kb. Although these findings are encouraging for the prospects of a genomewide LD map, they suggest caution in interpreting localization due to allelic association.

Single nucleotide polymorphism and linkage disequilibrium within the TCR alpha/delta locus.

Much attention is being given to the identification of common disease genes through whole-genome linkage disequilibrium (LD) screens with single nucleotide polymorphisms (SNPs). Simulation studies have suggested that useful LD is unlikely to extend beyond 3 kb, and that > 500,000 SNPs may be needed for comprehensive coverage of the genome. The TCR alpha/delta locus on chromosome 14q contains many V, J and D segments that combine with constant domains to produce either an alpha or a delta chain of the T cell receptor. Multiple SNPs have been recognized within the V segments, and it has been suggested that variation within the locus may modify the course of autoimmune and allergic diseases. We have examined LD within an 850 kb section of the TCR alpha/delta locus on chromosome 14q by typing 24 V gene segment SNPs and two microsatellites. One hundred and fifty-nine nuclear and extended families were genotyped in order to derive haplotypes, and the pair-wise LD between SNPs was investigated in 600 haplotypes from unrelated individuals (the parents). The mean extent of useful LD was much greater than suggested by simulations: significant LD was relatively common at 250 kb and was detectable beyond 500 kb. The mean extent of LD was twice as far between alleles of low frequency than between common alleles. The distribution of LD was highly irregular and concentrated in three distinct islands. The results differ from those obtained by simulation, and if they are typical of other genomic regions, suggest that the minimum number of markers necessary for comprehensive LD mapping may be reduced by at least an order of magnitude.