High-throughput analysis of T cell-monocyte interaction in human tuberculosis.

The lack of efficient tools for identifying immunological correlates of tuberculosis (TB) protection or risk of disease progression impedes the development of improved control strategies. To more clearly understand the host response in TB, we recently established an imaging flow cytometer-based in-vitro assay, which assesses multiple aspects of T cell-monocyte interaction. Here, we extended our previous work and characterized communication between T cells and monocytes using clinical samples from individuals with different TB infection status and healthy controls from a TB endemic setting. To identify T cell-monocyte conjugates, peripheral blood mononuclear cells (PBMC) were stimulated with ds-Red-expressing Mycobacterium bovis bacille Calmette-Guérin or 6-kDa early secreted antigenic target (ESAT 6) peptides for 6 h, and analyzed by imaging flow cytometer (IFC). We then enumerated T cell-monocyte conjugates using polarization of T cell receptor (TCR) and F-actin as markers for synapse formation, and nuclear factor kappa B (NF-κB) nuclear translocation in the T cells. We observed a reduced frequency of T cell-monocyte conjugates in cells from patients with active pulmonary tuberculosis (pTB) compared to latent TB-infected (LTBI) and healthy controls. When we monitored NF-κB nuclear translocation in T cells interacting with monocytes, the proportion of responding cells was significantly higher in active pTB compared with LTBI and controls. Overall, these data underscore the need to consider multiple immunological parameters against TB, where IFC could be a valuable tool.

CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell-Specific Adaptor Protein.

The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr)629 and linker of activated T cells (LAT) pTyr171 , pTyr191 and pTyr226 .

The multiple sclerosis susceptibility genes TAGAP and IL2RA are regulated by vitamin D in CD4+ T cells.

Multiple sclerosis (MS) is an inflammatory, demyelinating disorder of the central nervous system that develops in genetically susceptible individuals. The majority of the MS-associated gene variants are located in genetic regions with importance for T-cell differentiation. Vitamin D is a potent immunomodulator, and vitamin D deficiency has been suggested to be associated with increased MS disease susceptibility and activity. In CD4+ T cells, we have analyzed in vitro vitamin D responsiveness of genes that contain an MS-associated single-nucleotide polymorphism (SNP) and with one or more vitamin D response elements in their regulatory regions. We identify IL2RA and TAGAP as novel vitamin D target genes. The vitamin D response is observed in samples from both MS patients and controls, and is not dependent on the genotype of MS-associated SNPs in the respective genes.

Reduced MCMV Δm157 viral clearance in the absence of TSAd.

The T cell specific adapter protein (TSAd) is expressed in activated T cells and NK cells. While TSAd is beginning to emerge as a critical regulator of Lck and Itk activity in T cells, its role in NK cells has not yet been explored. Here we have examined susceptibility to virus infections in a murine model using various viral infection models. We report that TSAd-deficient mice display reduced clearance of murine cytomegalovirus (MCMV) that lack the viral MHC class I homologue m157, which is critical for Ly49H-mediated NK cell recognition of infected cells. In this infection model, NK cells contribute in the early stages of the disease, whereas CD8+ T cells are critical for viral clearance. We found that mice infected with MCMV Δm157 displayed reduced viral clearance in the spleen as well as reduced proliferation in spleen NK cells and CD8+ T cells in the absence of TSAd. Though no other immunophenotype was detected in the infection models tested, these data suggests that in the absence of the Ly49H ligand activation, NK cell and CD8+ T cell responses may be compromised in TSAd-deficient mice.

Expression of the T cell-specific adapter protein in human tissues.

T cell-specific adapter protein (TSAd) encoded by the SH2D2A gene is expressed in activated T cells, NK cells and endothelial cells, but its tissue expression has not yet been mapped. Here, we have defined the specificity of two commercially available anti-TSAd monoclonal reagents using peptide arrays. We found them to bind separate epitopes in the C-terminal part of TSAd. We then used immunohistochemistry to examine TSAd expression in various human lymphoid and non-lymphoid tissues. Immunostaining of adjacent tissue sections revealed that a substantial fraction of CD3-positive cells in normal lymphoid and non-lymphoid tissues expressed TSAd. In particular, essentially all intra-epithelial T cells appeared to coexpress TSAd. In addition, TSAd expression was observed in endothelial cells of dermal microvessels, while it was not detected in endothelial cells of the other tested tissues. This work provides insight into the expression pattern of TSAd in various healthy human tissues.

Multiple sclerosis-associated single-nucleotide polymorphisms in CLEC16A correlate with reduced SOCS1 and DEXI expression in the thymus.

Genome-wide association studies have revealed that the 16p13 chromosomal region, including CLEC16A, DEXI, CIITA and SOCS1, is associated with susceptibility to autoimmune diseases. As non-coding single-nucleotide polymorphisms (SNPs) may confer susceptibility to disease by affecting expression of nearby genes, we examined whether autoimmune-associated intronic CLEC16A SNPs (rs12708716, rs6498169 and rs7206912) correlate with the expression of CLEC16A itself as well as neighboring genes in whole-blood and thymic samples. Real-time quantitative PCR analyses show that SOCS1 and DEXI expression was lower in thymic samples carrying at least one of the CLEC16A risk alleles compared with non-carriers of the risk allele. Linear regression analysis revealed a significant correlation between the expression level of CLEC16A and that of SOCS1 and DEXI in thymic samples. These data indicate a possible regulatory role for multiple sclerosis-associated non-coding CLEC16A SNPs and a common control mechanism for the expression of CLEC16A, SOCS1 and DEXI.

Exploring the CLEC16A gene reveals a MS-associated variant with correlation to the relative expression of CLEC16A isoforms in thymus.

Genomewide association studies have implicated the CLEC16A gene in several autoimmune diseases, including multiple sclerosis (MS) and type 1 diabetes. However, the most associated single-nucleotide polymorphism (SNP) varies, and causal variants are still to be defined. In MS, two SNPs in partial linkage disequilibrium with each other, rs6498169 and rs12708716, have been validated at genomewide significance level. To explore the CLEC16A association in MS in more detail, we genotyped 57 SNPs in 807 Norwegian MS patients and 1027 Norwegian controls. Six highly associated SNPs emerged and were then replicated in two large independent sample sets (Norwegian and British), together including 1153 MS trios, 2308 MS patients and 4044 healthy controls. In combined analyses, SNP rs12708716 gave the strongest association signal in MS (P=5.3 x 10⁻8, odds ratio 1.18, 95% confidence interval=1.11-1.25), and was found to be superior to the other SNP associations in conditional logistic regression analyses. Expression analysis revealed that rs12708716 genotype was significantly associated with the relative expression levels of two different CLEC16A transcripts in thymus (P=0.004), but not in blood, possibly implying a thymus- or cell-specific splice regulation.

X chromosome inactivation in females with multiple sclerosis.

The aetiology of multiple sclerosis (MS) is unknown. Autoimmune mechanisms are most probably involved. Loss of immunological tolerance to self-antigens is a common feature of autoimmune disorders. Response to X-linked self-antigens could be influenced by X-chromosome inactivation, and contribute to the gender bias observed in autoimmune disorders. Previous studies have indicated an association between skewed X inactivation and autoimmune thyroid disease and scleroderma. To investigate a potential role of X inactivation in MS, we compared the X-inactivation pattern in 568 female MS patients with controls. We found no difference in degree of skewing between patients (median 64%) and controls (median 65%) (P = 0.474). The X-inactivation pattern did thus not explain the female predominance of MS patients in general. As the aetiology of different subgroups of MS may differ, patients were grouped according to disease course: relapsing-remitting (RR-MS), secondary progressive (SP-MS) and primary progressive (PP-MS). A comparison of the X-inactivation pattern between subgroups indicated a possible difference in degree of skewing between patients with a progressive versus a relapsing course (P = 0.05).

The impact of HLA-A and -DRB1 on age at onset, disease course and severity in Scandinavian multiple sclerosis patients.

The human leucocyte antigen (HLA) class II haplotype DRB1*15-DQB1*06 (DR15-DQ6) is associated with susceptibility to multiple sclerosis (MS), and HLA class I associations in MS have also been reported. However, the influence of HLA class I and II alleles on clinical phenotypes in MS has not yet been completely studied. This study aimed at evaluating the impact of HLA-A and -DRB1 alleles on clinical variables in Scandinavian MS patients. The correlation between HLA-A or -DRB1 alleles and age at onset, disease course and Multiple Sclerosis Severity Score (MSSS) were studied in 1457 Norwegian and Swedish MS patients by regression analyses and Kruskal-Wallis rank sum test. Presence of HLA-DRB1*15 was correlated with younger age at onset of disease (corrected P = 0.009). No correlation was found between HLA-A and the variables studied. This study analysed the effect of HLA-A on clinical variables in a large Scandinavian sample set, but could not identify any significant contribution from HLA-A on the clinical phenotype in MS. However, associations between HLA-DRB1*15 and age at onset of MS were reproduced in this extended Scandinavian MS cohort.

A follow-up study of Nordic multiple sclerosis candidate gene regions.

In this study, the results from three Nordic linkage disequilibrium screens in multiple sclerosis (MS) were investigated, in a new sample set of 314 Nordic MS trios from Denmark, Norway, Sweden and Iceland. Among 30 non-HLA and two HLA microsatellite markers individually genotyped, eight markers displayed distorted transmission with uncorrected P-value<0.05, ranked in this order: D6S2443 (6p21.32, HLA class II) (P corrected=0.01), D2S2201 (2p24), D19S552 (19q13), D3S3584 (3q21), D17S975 (17q11), D1S2627 (1p22), D6S273 (6p21.33, HLA class III) and D12S1051 (12q23). These non-HLA regions need further investigation as possible MS candidate gene regions in our population.

Genetics in multiple sclerosis: past and future perspectives.

The enormous development in the field of molecular genetics during the last decades has lead to optimism concerning the possibilities for identifying the causes of multiple sclerosis (MS) through genetic studies. However, we have learned that dense mapping of large sample sets is needed, which only can be achieved through large collaborative studies. The contribution from each yet unidentified gene is probably weaker than that of the well established human leukocyte antigen association. The ultimate goal of the search for susceptibility genes in MS is to develop diagnostic tools and better treatments that can prevent or reduce the development of symptoms of this often devastating disease.

Molecular genetic studies of natives on Easter Island: evidence of an early European and Amerindian contribution to the Polynesian gene pool.

Most archaeological and linguistic evidence suggest a Polynesian origin of the population of Easter Island (Rapanui), and this view has been supported by the identification of Polynesian mitochondrial DNA (mtDNA) polymorphisms in prehistoric skeletal remains. However, some evidence of an early South American contact also exists (the sweet potato, bottle gourd etc.), but genetic studies have so far failed to show an early Amerindian contribution to the gene pool on Easter Island. To address this issue, we analyzed mtDNA and Y chromosome markers and performed high-resolution human leukocyte antigen (HLA) genotyping of DNA harvested from previously collected sera of 48 reputedly nonadmixed native Easter Islanders. All individuals carried mtDNA types and HLA alleles previously found in Polynesia, and most men carried Y chromosome markers of Polynesian origin, providing further evidence of a Polynesian origin of the population of Easter Island. A few individuals carried HLA alleles and/or Y chromosome markers of European origin. More interestingly, some individuals carried the HLA alleles A*0212 and B*3905, which are of typical Amerindian origin. The genealogy of some of the individuals carrying these non-Polynesian HLA alleles and their haplotypic backgrounds suggest an introduction into Easter Island in the early 1800s, or earlier. Thus, there may have been an early European and Amerindian contribution to the Polynesian gene pool of Easter Island.

Primary sclerosing cholangitis is associated with extended HLA-DR3 and HLA-DR6 haplotypes.

Primary sclerosing cholangitis (PSC) is associated with the human leukocyte antigen (HLA)-DRB1*0301-DQA1*0501-DQB1*0201 (DR3) and HLA-DRB1*1301-DQA1*0103-DQB1*0603 (DR6) haplotypes. Recently, the extended HLA class I region has been found to harbour genes that modulate or confer susceptibility independently of the HLA class II genes in several immune-mediated diseases. The aim of the present study was to evaluate the influence of genes in the extended HLA class I region on susceptibility to PSC. Seven microsatellite markers (MIB, D6S265, D6S2222, D6S464, D6S2223, D6S2225 and D6S2239) were analysed together with HLA class II alleles in 219 Norwegian patients with PSC and 282 random controls. To control for associations because of linkage disequilibrium (LD), 142 HLA-DR3 homozygous and 187 DR6-positive controls were included. The unstratified analysis showed significant associations with the alleles MIB*349 [odds ratio (OR) = 3.0, corrected P value (P(c)) = 3 x 10(-12)], D6S265*122 (OR = 1.7, P(c)= 0.004), D6S464*209 (OR = 1.8, P(c)= 0.03) and D6S2225*147 (OR = 2.7, P(c)= 4 x 10(-6)), which were mainly secondary to the DR3 association. When stratifying for DR6, an association with the D6S265*122 allele was still observed (OR = 3.7, P(c)= 0.0004). In the presence of the D6S265*122 allele, the risk to develop PSC conferred by DR6 was increased four times compared with the risk conferred by DR6 alone. In addition, a novel negative association of PSC with DR11 was observed (OR = 0.21, P(c)= 2 x 10(-4)). In conclusion, our study shows that a gene in LD with D6S265 contributes to susceptibility to develop PSC in individuals carrying DR6. Moreover, we found that the PSC-associated DR3 haplotype extends more telomeric than that previously reported. We also report a possible protective effect of DR11 on development of PSC.

Genetic association between juvenile rheumatoid arthritis and polymorphism in the SH2D2A gene.

T-cell-specific adapter protein (TSAd) involved in the negative control of T-cell activation is encoded by the SH2D2A gene. Our recent studies indicate that homozygosity for short (ie GA(13) and GA(16)) alleles of the SH2D2A gene promoter is associated with development of multiple sclerosis. To study whether the same SH2D2A promoter polymorphism also contributes to the genetic susceptibility to develop juvenile rheumatoid arthritis (JRA), we examined 210 JRA patients and 558 healthy unrelated controls from Norway. The frequency of the short allele GA(13) was increased among the JRA patients compared to control (0.098 vs 0.05; P(n=8)=0.042). There was a significant increased frequency of HLA-DRB1(*)08-positive patients carrying two copies of 'short' alleles GA(13) and/or GA(16) compared to healthy controls (16% vs 6%; P(n=4)=0.016). Our data indicate that the 'short' alleles of the SH2D2A promoter could contribute to the genetic susceptibility to JRA.

Tumour necrosis factor receptor superfamily member 6 gene mutation detection by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a tool for diagnostic screening of polymorphisms in the tumour necrosis factor receptor superfamily member 6 (TNFRSF6) also known as CD95, Apo-1 or Fas gene. Exons 1-9 of the TNFRSF6 gene were amplified from genomic DNA of 38 individuals, of which three were known to carry mutations in the TNFRSF6 gene. The TNFRSF6 gene amplicons were analysed for heterozygosity by DHPLC. Samples that displayed heterozygous variation by DHPLC were further analysed by sequencing. Comparison of DHPLC analysis with sequencing results showed an overall 100% concordance for samples in which heterozygosity was detected by DHPLC. Importantly, DHPLC was in all cases able to demonstrate the presence or absence of mutations in exon 9 encoding the death domain of the TNFRSF6 gene, which have been implied as the most frequent genetic cause of autoimmune lymphoproliferative syndrome. Comparison of DHPLC analysis with sequencing results showed an overall 100% concordance for samples in which heterozygosity was detected by DHPLC. In conclusion, DHPLC is a suitable method for the detection of genetic variation in the TNFRSF6 gene.

Genes in the HLA class I region may contribute to the HLA class II-associated genetic susceptibility to multiple sclerosis.

In order to analyze whether loci in the human leukocyte antigen (HLA) class I region may contribute to the HLA class II-associated genetic susceptibility to multiple sclerosis (MS), we examined selected microsatellite markers in 177 Nordic sib-pair families, 222 British sib-pair families, 323 sporadic Norwegian MS patients and 386 Norwegian controls. All samples were, in addition, genotyped for the HLA-DR DQ haplotype, and the Norwegian case-control samples were also typed for HLA-A and -B loci. In the Norwegian sporadic MS patients association was seen with HLA-A, HLA-B, and with the D6S265 marker, located 100 kb centromeric to HLA-A. Associations with HLA-A and D6S265 loci were also suggested when restricting the analysis to HLA-DR15 haplotypes. In the sib-pair data a similar trend was seen with marker D6S265. Higher genotypic relative risk (GRR) was found for individuals who carry both HLA-DR15 and -A3 (GRR = 15), compared to those who carry only HLA-DR15 (GRR = 7), only HLA-A3 (GRR = 3) or none of these alleles (GRR = 1). The highest risk was conferred by a combination of HLA-DR15 and -A3 (odds ratio (OR) = 5.2). These results suggest that HLA-A or a gene in linkage disequilibrium with it may contribute to the HLA class II-associated genetic susceptibility to MS.

Physical separation of HLA-A alleles by denaturing high-performance liquid chromatography.

Genomic typing of polymorphic loci may be hampered by ambiguous typing results. Moreover, robust methods for simultaneous sequencing of two alleles present in a given sample may be difficult to establish. We used denaturing high-performance liquid chromatography (DHPLC) for physical separation of HLA-A alleles before sequence-based genomic typing (SBT). Physical separation was achieved by resolution of heteroduplexes between the sample alleles and a modified reference probe by DHPLC followed by selective reamplification of the sample alleles present in heteroduplexes. Complementary strands of the reference probe and sample alleles for heteroduplex induction were obtained by lambda-exonuclease digestion. HLA-A genotyping of 101 individuals using DHPLC-SBT yielded better typing resolution compared with serological typing and genotyping by the sequence-specific primer-polymerase chain reaction (SSP-PCR) method. Physical separation of alleles using a modified reference probe allows for development of fully automated methods for genomic typing of highly polymorphic loci such as HLA.

The use of modified primer competitors to enhance yields and specificity of HLA class I amplification by polymerase chain reaction (PCR).

Various PCR based techniques have been developed for genomic HLA typing. The fidelity of these techniques is highly dependent upon the specificity of the primers for the given HLA locus. Due to the high degree of homology between HLA class I loci, few primer sites that selectively amplify genes at a given HLA class I locus may be identified. To avoid coamplification of homologous loci, we designed and applied primer competitors for PCR amplification of HLA-A, -B and -C loci. Primer competitors identical to the 3' end of the specific primers and completely degenerate in the 5' end were designed and titrated into the respective HLA-locus PCR mixtures. We found that inclusion of primer competitors in the PCR reaction increased the specificity and yields of HLA class I amplifications, in particular when crude DNA preparation was used as template. This was particularly true for DNA preparations of low quality. The method described here may be useful for various protocols for downstream genomic typing of HLA-A, -B and -C alleles. In particular the method is useful when DNA is in scarce supply (i.e., for extensive PCR based allelic typing) or when high yields and locus specificity of amplicons are needed (i.e., sequencing-based typing).

A genome-wide screen for linkage in Nordic sib-pairs with multiple sclerosis.

Genetic factors influence susceptibility to multiple sclerosis but the responsible genes remain largely undefined, association with MHC class II alleles being the only established genetic feature of the disease. The Nordic countries have a high prevalence of multiple sclerosis, and to further explore the genetic background of the disease, we have carried out a genome-wide screen for linkage in 136 sibling-pairs with multiple sclerosis from Denmark, Finland, Norway and Sweden by typing 399 microsatellite markers. Seventeen regions where the lod score exceeds the nominal 5% significance threshold (0.7) were identified-1q11-24, 2q24-32, 3p26.3, 3q21.1, 4q12, 6p25.3, 6p21-22, 6q21, 9q34.3, 10p15, 10p12-13, 11p15.5, 12q21.3, 16p13.3, 17q25.3, 22q12-13 and Xp22.3. Although none of these regions reaches the level of genome-wide significance, the number observed exceeds the 10 that would be expected by chance alone. Our results significantly add to the growing body of linkage data relating to multiple sclerosis.

HLA profile of three ethnic groups living in the North-Western region of Russia.

HLA class II alleles were determined by PCR-SSO and PCR-SSP typing of DNA samples from 55 Nentsy, 81 Saami and 73 Pomor individuals from the North-European part of Russia. The results were compared with similar data from Russians. A high frequency of the DRB1*04-DQA1*0301-DQB1*0302 haplotype and a low frequency of the DRB1*11-DQA1*0501-DQB1*0301 haplotype, observed in all three ethnic groups, may indicate a common aboriginal component in their ancestry. Saami and Pomors displayed a similar pattern of allele and haplotype distribution, with the exception of the DRB1*04-DQA1*0304-DQB1*0301 haplotype, which was significantly higher among Saami compared Nentsy, Pomors and Russians. Nentsy individuals had a particularly high frequency of the DRB1*09-DQA1*0301-DQB1*0303 and the DRB1*12-DQA1*0501-DQB1*0301 haplotypes. Genetic distances and correspondence analysis show that Pomors have a close relationship with Norwegians and Finns, whereas Nentsy and Saami are more closely related to Oriental populations.