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John Trowsdale

Publications and source records attributed to John Trowsdale.

At least 19 recordsLinked to original sources
PubMed2007

Reciprocal regulation of human natural killer cells and macrophages associated with distinct immune synapses.

Natural killer (NK) cells directly lyse tumor or viral-infected cells but also an important role for NK cell cytotoxicity in regulating the extent of immune responses is emerging. Here, we show that autologous human macrophages activated NK cell proliferation and cytokine secretion, increased expression of activating receptors, and primed NK cell cytotoxicity against susceptible target cells. Ligation of NK cell 2B4, and not NKp30 (known to be important for DC-mediated NK cell activation), is critical for this macrophage-mediated NK cell activation. Reciprocally, however, NK cells regulated macrophage activity by directly killing macrophages stimulated by high doses of LPS. Cytolysis was triggered by NKG2D recognition of stress-inducible class I major histocompatibility complex (MHC)-like ligands on macrophages: high doses of LPS induced transcription and surface expression of ULBP1, ULBP2, and ULBP3 and surface expression of constitutively transcribed MICA. Thus, these data suggest a new function for NK cell cytotoxicity in eliminating overstimulated macrophages. Additionally, these interactions define, for the first time, 2 distinct activating NK cell synapses: lytic and nonlytic. Triggering NK cell proliferation and cytokine secretion, but not cytolysis, specifically associated with synaptic accumulation of macrophage F-actin and NK cell 2B4, while macrophages were killed when NK cell F-actin and macrophage ICAM-1 accumulated around a central cluster of NK cell NKG2D/DAP10.

Actins↗
PubMed2006

TRIM21 is a trimeric protein that binds IgG Fc via the B30.2 domain.

TRIMs comprise a large protein family that include anti-retroviral restriction factors such as TRIM5alpha. Auto-antibodies to TRIM21 (Ro52) are a common serological feature of patients with Sjogren's syndrome and systemic lupus erythematosus (SLE). We show that, in addition to this autoantibody response, TRIM21 binds specifically to the Fc region of human IgG isotypes 1, 2 and 4, via a conformation dependent interaction. The minimal binding epitope was identified as the C-terminal B30.2 domain. The interaction was independent of N-linked glycosylation of the IgG CH2 domain. TRIM21 formed a trimer that competed with protein A for binding to IgG Fc. We conclude that TRIM21 binds to the consensus CH2/CH3 domain interface in the Fc region, overlapping the binding site of several other proteins, including Staphylococcus aureus protein A and Streptococcus spp. protein G. The data suggest that the normal function of TRIM21 involves regulation of IgG functions and that TRIM/B30.2 molecules may have broader and unsuspected roles in innate immunity, beyond that of retroviral restriction.

Animals↗
PubMed2006

A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC.

The proteins encoded by the classical HLA class I and class II genes in the major histocompatibility complex (MHC) are highly polymorphic and are essential in self versus non-self immune recognition. HLA variation is a crucial determinant of transplant rejection and susceptibility to a large number of infectious and autoimmune diseases. Yet identification of causal variants is problematic owing to linkage disequilibrium that extends across multiple HLA and non-HLA genes in the MHC. We therefore set out to characterize the linkage disequilibrium patterns between the highly polymorphic HLA genes and background variation by typing the classical HLA genes and >7,500 common SNPs and deletion-insertion polymorphisms across four population samples. The analysis provides informative tag SNPs that capture much of the common variation in the MHC region and that could be used in disease association studies, and it provides new insight into the evolutionary dynamics and ancestral origins of the HLA loci and their haplotypes.

Genetic Predisposition to Disease↗
PubMed2006

Nature of allelic sequence polymorphism at the KIR3DL3 locus.

KIR3DL3 is a framework gene of the Leukocyte Receptor Complex, present in all individuals and haplotypes analysed to date. We describe 17 novel KIR3DL3 alleles, including seven single nucleotide polymorphic (SNP) positions within the coding region. Sequence variation within introns included a VNTR within intron 1. As KIR3DL3 mRNA is known to be expressed in decidual NK cells, we investigated the impact of KIR3DL3 allelic variation on pre-eclampsia. No statistical difference in allele frequency or polymorphism was observed between pre-eclampsia patient and control cohorts. Linkage disequilibrium (LD) analysis of exonic SNPs suggested that recombination may be a mechanism of generating sequence diversity within KIR3DL3. A potential recombination hotspot was located within intron 5. A strong LD was detected between polymorphism in exon 6 of KIR3DL3 and the KIR gene -2DL3 or -2DS2 loci, which define the centromeric end of two main haplotypes (A and B) of the KIR cluster. Comparison of primate KIR sequences indicated that the Ig domains of KIR3DL3 are highly conserved between chimpanzee, gorilla and humans. Investigation of KIR3DL3 dN/dS ratios indicated a greater level of synonymous mutations consistent with purifying selection, although positive selection was detected acting on two sites within the stem region.

Alleles↗
PubMed2006

Selective export of HLA-F by its cytoplasmic tail.

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.

14-3-3 Proteins↗
PubMed2006

Regulation of NKG2D ligand gene expression.

The activating immunoreceptor NKG2D has seven known host ligands encoded by the MHC class I chain-related MIC and ULBP/RAET genes. Why there is such diversity of NKG2D ligands is not known but one hypothesis is that they are differentially expressed in different tissues in response to different stresses. To explore this, we compared expression patterns and promoters of NKG2D ligand genes. ULBP/RAET genes were transcribed independent of each other in a panel of cell lines. ULBP/RAET gene expression was upregulated on infection with human cytomegalovirus; however, a clinical strain, Toledo, induced expression more slowly than did a laboratory strain, AD169. ULBP4/RAET1E was not induced by infection with either strain. To investigate the mechanisms behind the similarities and differences in NKG2D ligand gene expression a comparative sequence analysis of NKG2D ligand gene putative promoter regions was conducted. Sequence alignments demonstrated that there was significant sequence diversity; however, one region of high similarity between most of the genes is evident. This region contains a number of potential transcription factor binding sites, including those involved in shock responses and sites for retinoic acid-induced factors. Promoters of some NKG2D ligand genes are polymorphic and several sequence alterations in these alleles abolished putative transcription factor binding.

Alleles↗
PubMed2006

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history.

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.

Chromosome Mapping↗
PubMed2006

Molecular characterization of KIR3DL3.

Killer-cell immunoglobulin-like receptors (KIRs) are a structurally and functionally diverse family of molecules expressed by natural killer (NK) cells and T-cell subsets. The most centromeric gene in the human KIR cluster is KIR3DL3, a framework gene that is present in all haplotypes. KIR3DL3 has only one immunoreceptor tyrosine-based inhibitory motif and lacks the exon encoding the stem between the Immunoglobulin domains and the transmembrane region. We have investigated expression of KIR3DL3 in blood and decidual NK cells by reverse transcriptase polymerase chain reaction (RT-PCR) and protein analysis using a KIR3DL3-specific monoclonal antibody, CH21. KIR3DL3 mRNA was only detected in the CD56(bright) subset in cells from peripheral blood and in CD56(bright) decidual NK cells. The CD56(bright) NK92 cell line was also positive. Quantitative RT-PCR indicated a trend for higher expression of KIR3DL3 in female peripheral blood mononuclear cells compared to that in male. Using a bisulphite conversion method, we found that the promoter of KIR3DL3 was strongly methylated. Surface protein expression was detectable after demethylation. Like other KIRs, KIR3DL3 is highly polymorphic, and we detected 14 variants in 25 unrelated individuals. Nucleotide substitutions were scattered throughout the sequence, with a cluster of alleles at the start of the transmembrane region at the site where the remnant of the linking stem present in other KIR is found. We conclude that the KIR3DL3 gene is not a pseudogene but encodes a protein that is not expressed in healthy individuals. Protein expression might be induced under certain developmental or pathological situations.

Adult↗
PubMed2006

You say ITAM and I say ITIM, let's call the whole thing off: the ambiguity of immunoreceptor signalling.

Paradigms of protein tyrosine kinase (PTK)-mediated immunoreceptor signalling have developed largely from studies of molecules containing immunoreceptor tyrosine-based activation motifs (ITAM) and immunoreceptor tyrosine-based inhibition motifs (ITIM). In some circumstances, ITAM can mediate inhibition and ITIM can propagate activation signals. In addition to classical immune cells, some ITAM- and ITIM-encoding proteins are more widely expressed and are crucial to the development of haemopoietic cells, like osteoclasts and natural killer cells, as well as tissues such as bone and brain. Closer analysis of activating proteins reveals that some potentially encode ITIM within an ITAM. These 'closet' ITIM could, under conditions of partial ITAM phosphorylation, mediate inhibitory signalling.

Amino Acid Motifs↗
PubMed2006

Mother's little helpers: mechanisms of maternal-fetal tolerance.

The evolutionary adaptation in mammals that allows implantation of their embryos in the mother's womb creates an immunological problem. Although it ensures optimal nourishment and protection of the fetus throughout its early development, intimate contact with the mother's uterine tissue makes the fetus a potential target for her immune system. As half the fetal genes are derived from the father, the developing embryo and placenta must be considered a 'semi-allograft'. Such a mismatched organ transplant would be readily rejected without powerful immune suppression. During pregnancy, however, the semi-allogeneic fetus is protected from assault by the maternal immune system over an extended period of time. The mother's immune system seems to recognize the fetus as 'temporary self'. How this feat is managed is key to understanding immunological tolerance and intervention in treating disease.

Animals↗
PubMed2006

KIR/HLA pleiotropism: protection against both HIV and opportunistic infections.

The compound genotype KIR3DS1/HLA-B Bw4-80I, which presumably favors natural killer cell activation, has been implicated in protection against HIV disease. We show that this genotype confers dual protection over the course of HIV disease; early direct containment of HIV viral load, and late specific defense against opportunistic infections, but not AIDS-related malignancies. The double protection of KIR3DS1/Bw4-80I in an etiologically complex disease such as AIDS, along with the disease specificity of its effects is conceptually novel and underscores the intricacy of host immunogenetics against HIV/AIDS.

AIDS-Related Opportunistic Infections↗
PubMed2005

Association of the truncating splice site mutation in BTNL2 with multiple sclerosis is secondary to HLA-DRB1*15.

The major histocompatibility complex human leukocyte antigen (HLA)-DRB1*15 (DR2) haplotype is strongly associated with risk of multiple sclerosis (MS). The primary susceptibility has been localized to only approximately 200 kb encompassing the HLA-DR and -DQ loci. Further dissection of disease association with this region is demanding because of the high levels of linkage disequilibrium (LD). Recently, evidence was obtained for the involvement of a gene, potentially encoding an immune co-receptor, in another DR2-associated inflammatory condition, sarcoidosis. The implicated gene, BTNL2, is adjacent to DR and is in strong LD with HLA-DRB1. This fact, combined with a sequence relationship between BTNL2 and myelin oligodendrocyte glycoprotein, an autoantigen associated with MS, makes the gene an attractive candidate. To determine whether BTNL2 contributes to MS, we genotyped 1136 well-characterized MS families from the UK and the USA, as well as an African-American case-control data set, making this among the largest genetic studies in MS. Family-based and case-control association studies were performed for the BTNL2 and HLA-DRB1 loci. In all family data sets, the protein-truncating allele of BTNL2, implicated in sarcoidosis, was significantly over-transmitted to cases (combined data sets: global P=2.4x10(-11)). Given that the protein-truncating allele of BTNL2 virtually always occurred with DRB1*15, an effect could only be tested in DRB1*15-negative individuals or pedigrees. However, despite adequate power to detect an independent association, no difference in transmission of BTNL2 alleles or genotypes was observed in DRB1*15-negative individuals with MS. Conditional logistic regression modeling also strongly supported the conclusion that BTNL2 does not confer additional disease risk. The association of BTNL2 with MS observed in the African-American data set was also secondary to the primary DRB1*15 association.

Black or African American↗
PubMed2005

Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb' region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.

Amino Acid Sequence↗
PubMed2005

HLA-C and killer cell immunoglobulin-like receptor genes in idiopathic bronchiectasis.

RATIONALE: In idiopathic bronchiectasis, lung inflammation and chronic bacterial infection lead to progressive lung damage. A possible role for natural killer (NK) cells is suggested by the observation that familial bronchiectasis occurs in a rare group of individuals with impaired HLA class I expression and consequent NK cell dysfunction. OBJECTIVE: Because the HLA-C locus and killer cell immunoglobulin-like receptors (KIRs) are of key importance for NK cell recognition, we analyzed HLA-C/KIR combinations by genotyping patients with idiopathic bronchiectasis. METHODS: Genomic DNA from 96 individuals with idiopathic bronchiectasis and 101 control subjects was analyzed by polymerase chain reaction with sequence-specific primers. High-resolution HLA-C genotyping was performed in addition to KIR analysis. RESULTS: HLA-Cw*03 alleles and, in particular, HLA-C group 1 homozygosity are associated with the presence of bronchiectasis. Analysis of the relationship between HLA-C and KIR genes suggests a shift to activatory NK cell function. CONCLUSION: This is the first demonstration of genetic susceptibility in idiopathic bronchiectasis. The association with HLA-C group 1 homozygosity, and the interplay between HLA-C and KIR genes, argue for a role for NK cells in the progressive lung damage seen in this disease. This will require further investigation using functional studies.

Bronchiectasis↗
PubMed2005

Features of MHC and NK gene clusters.

When comparing the immune genome to the genome in general, a higher prevalence for association with disease is the only genetic feature significant in immune genes as a group. However, some genetic features, such as marked levels of polymorphism and gene duplication, are present in subsets of immune genes, namely the Major Histocompatibility Complex (MHC) and Natural Killer (NK) cell receptor gene complexes. In this review, we discuss features of MHC and NK receptor gene clusters, their epistatic interactions, and the impact of both on association to disease.

Animals↗
PubMed2005

A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery.

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.

Acylation↗