Association of Protective HLA-A With HLA-B∗27 Positive Ankylosing Spondylitis.

OBJECTIVES: To further elucidate the role of the MHC in ankylosing spondylitis by typing 17 genes, searching for HLA-B∗27 independent associations and assessing the impact of sex on this male biased disease. METHODS: High-confidence two-field resolution genotyping was performed on 310 cases and 2196 controls using an n-1 concordance method. Protein-coding variants were called from next-generation sequencing reads using up to four software programs and the consensus result recorded. Logistic regression tests were applied to the dataset as a whole, and also in stratified sets based on sex or HLA-B∗27 status. The amino acids driving association were also examined. RESULTS: Twenty-five HLA protein-coding variants were significantly associated to disease in the population. Three novel protective associations were found in a HLA-B∗27 positive population, HLA-A∗24:02 (OR = 0.4, CI = 0.2-0.7), and HLA-A amino acids Leu95 and Gln156. We identified a key set of seven loci that were common to both sexes, and robust to change in sample size. Stratifying by sex uncovered three novel risk variants restricted to the female population (HLA-DQA1∗04.01, -DQB1∗04:02, -DRB1∗08:01; OR = 2.4-3.1). We also uncovered a set of neutral variants in the female population, which in turn conferred strong effects in the male set, highlighting how population composition can lead to the masking of true associations. CONCLUSION: Population stratification allowed for a nuanced investigation into the tightly linked MHC region, revealing novel HLA-B∗27 signals as well as replicating previous HLA-B∗27 dependent results. This dissection of signals may help to elucidate sex biased disease predisposition and clinical progression.

Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus.

OBJECTIVES: Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis. METHODS: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926. RESULTS: Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes. CONCLUSIONS: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation.

IL-22 Binding Protein Promotes the Disease Process in Multiple Sclerosis.

Genome-wide association studies have mapped the specific sequence variants that predispose for multiple sclerosis (MS). The pathogenic mechanisms that underlie these associations could be leveraged to develop safer and more effective MS treatments but are still poorly understood. In this article, we study the genetic risk variant rs17066096 and the candidate gene that encodes IL-22 binding protein (IL-22BP), an antagonist molecule of the cytokine IL-22. We show that monocytes from carriers of the risk genotype of rs17066096 express more IL-22BP in vitro and cerebrospinal fluid levels of IL-22BP correlate with MS lesion load on magnetic resonance imaging. We confirm the pathogenicity of IL-22BP in both rat and mouse models of MS and go on to suggest a pathogenic mechanism involving lack of IL-22-mediated inhibition of T cell-derived IFN-γ expression. Our results demonstrate a pathogenic role of IL-22BP in three species with a potential mechanism of action involving T cell polarization, suggesting a therapeutic potential of IL-22 in the context of MS.

A rare regulatory variant in the MEF2D gene affects gene regulation and splicing and is associated with a SLE sub-phenotype in Swedish cohorts.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with heterogeneous clinical presentation and complex etiology involving the interplay between genetic, epigenetic, environmental and hormonal factors. Many common SNPs identified by genome wide-association studies (GWAS) explain only a small part of the disease heritability suggesting the contribution from rare genetic variants, undetectable in GWAS, and complex epistatic interactions. Using targeted re-sequencing of coding and conserved regulatory regions within and around 215 candidate genes selected on the basis of their known role in autoimmunity and genes associated with canine immune-mediated diseases, we identified a rare regulatory variant rs200395694:G > T located in intron 4 of the MEF2D gene encoding the myocyte-specific enhancer factor 2D transcription factor and associated with SLE in Swedish cohorts (504 SLE patients and 839 healthy controls, p = 0.014, CI = 1.1-10). Fisher's exact test revealed an association between the genetic variant and a triad of disease manifestations including Raynaud, anti-U1-ribonucleoprotein (anti-RNP), and anti-Smith (anti-Sm) antibodies (p = 0.00037) among the patients. The DNA-binding activity of the allele was further studied by EMSA, reporter assays, and minigenes. The region has properties of an active cell-specific enhancer, differentially affected by the alleles of rs200395694:G > T. In addition, the risk allele exerts an inhibitory effect on the splicing of the alternative tissue-specific isoform, and thus may modify the target gene set regulated by this isoform. These findings emphasize the potential of dissecting traits of complex diseases and correlating them with rare risk alleles with strong biological effects.

Common genetic variation in the autoimmune regulator (AIRE) locus is associated with autoimmune Addison's disease in Sweden.

Autoimmune Addison's disease (AAD) is the predominating cause of primary adrenal failure. Despite its high heritability, the rarity of disease has long made candidate-gene studies the only feasible methodology for genetic studies. Here we conducted a comprehensive reinvestigation of suggested AAD risk loci and more than 1800 candidate genes with associated regulatory elements in 479 patients with AAD and 2394 controls. Our analysis enabled us to replicate many risk variants, but several other previously suggested risk variants failed confirmation. By exploring the full set of 1800 candidate genes, we further identified common variation in the autoimmune regulator (AIRE) as a novel risk locus associated to sporadic AAD in our study. Our findings not only confirm that multiple loci are associated with disease risk, but also show to what extent the multiple risk loci jointly associate to AAD. In total, risk loci discovered to date only explain about 7% of variance in liability to AAD in our study population.

Memory T cells specific to citrullinated α-enolase are enriched in the rheumatic joint.

ACPA-positive rheumatoid arthritis (RA) is associated with distinct HLA-DR alleles and immune responses to many citrullinated self-antigens. Herein we investigated the T cell epitope confined within α-enolase326-340 in the context of HLA-DRB1*04:01 and assessed the corresponding CD4+ T cells in both the circulation and in the rheumatic joint. Comparative crystallographic analyses were performed for the native and citrullinated α-enolase326-340 peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for ex vivo and in vitro assessment of α-enolase-specific T cells in peripheral blood, synovial fluid and synovial tissue by flow cytometry. The native and modified peptides take a completely conserved structural conformation within the peptide-binding cleft of HLA-DRB1*04:01. The citrulline residue-327 was located N-terminally, protruding towards TCRs. The frequencies of T cells recognizing native eno326-340 were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno326-340 was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, p = 0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, p = 0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within α-enolase326-340 does not appear to lead to complete negative selection of cognate CD4+ T cells. In RA patient samples, only T cells recognizing the citrullinated version of α-enolase326-340 were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance.

EOMES-positive CD4+ T cells are increased in PTPN22 (1858T) risk allele carriers.

The presence of the PTPN22 risk allele (1858T) is associated with several autoimmune diseases including rheumatoid arthritis (RA). Despite a number of studies exploring the function of PTPN22 in T cells, the exact impact of the PTPN22 risk allele on T-cell function in humans is still unclear. In this study, using RNA sequencing, we show that, upon TCR-activation, naïve human CD4+ T cells homozygous for the PTPN22 risk allele overexpress a set of genes including CFLAR and 4-1BB, which are important for cytotoxic T-cell differentiation. Moreover, the protein expression of the T-box transcription factor Eomesodermin (EOMES) was increased in T cells from healthy donors homozygous for the PTPN22 risk allele and correlated with a decreased number of naïve CD4+ T cells. There was no difference in the frequency of other CD4+ T-cell subsets (Th1, Th17, Tfh, Treg). Finally, an accumulation of EOMES+ CD4+ T cells was observed in synovial fluid of RA patients with a more pronounced production of Perforin-1 in PTPN22 risk allele carriers. Altogether, we propose a novel mechanism of action of PTPN22 risk allele through the generation of cytotoxic CD4+ T cells and identify EOMES+ CD4+ T cells as a relevant T-cell subset in RA pathogenesis.

T cells are influenced by a long non-coding RNA in the autoimmune associated PTPN2 locus.

Non-coding SNPs in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) locus have been linked with several autoimmune diseases, including rheumatoid arthritis, type I diabetes, and inflammatory bowel disease. However, the functional consequences of these SNPs are poorly characterized. Herein, we show in blood cells that SNPs in the PTPN2 locus are highly correlated with DNA methylation levels at four CpG sites downstream of PTPN2 and expression levels of the long non-coding RNA (lncRNA) LINC01882 downstream of these CpG sites. We observed that LINC01882 is mainly expressed in T cells and that anti-CD3/CD28 activated naïve CD4+ T cells downregulate the expression of LINC01882. RNA sequencing analysis of LINC01882 knockdown in Jurkat T cells, using a combination of antisense oligonucleotides and RNA interference, revealed the upregulation of the transcription factor ZEB1 and kinase MAP2K4, both involved in IL-2 regulation. Overall, our data suggests the involvement of LINC01882 in T cell activation and hints towards an auxiliary role of these non-coding SNPs in autoimmunity associated with the PTPN2 locus.

Corrigendum: Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis.

[This corrects the article on p. 494 in vol. 7, PMID: 27895642.].

Novel risk genes for systemic lupus erythematosus predicted by random forest classification.

Genome-wide association studies have identified risk loci for SLE, but a large proportion of the genetic contribution to SLE still remains unexplained. To detect novel risk genes, and to predict an individual's SLE risk we designed a random forest classifier using SNP genotype data generated on the "Immunochip" from 1,160 patients with SLE and 2,711 controls. Using gene importance scores defined by the random forest classifier, we identified 15 potential novel risk genes for SLE. Of them 12 are associated with other autoimmune diseases than SLE, whereas three genes (ZNF804A, CDK1, and MANF) have not previously been associated with autoimmunity. Random forest classification also allowed prediction of patients at risk for lupus nephritis with an area under the curve of 0.94. By allele-specific gene expression analysis we detected cis-regulatory SNPs that affect the expression levels of six of the top 40 genes designed by the random forest analysis, indicating a regulatory role for the identified risk variants. The 40 top genes from the prediction were overrepresented for differential expression in B and T cells according to RNA-sequencing of samples from five healthy donors, with more frequent over-expression in B cells compared to T cells.

Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis.

Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA) patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from α-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified α-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS), the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS) elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue.

Immunoseq: the identification of functionally relevant variants through targeted capture and sequencing of active regulatory regions in human immune cells.

BACKGROUND: The observation that the genetic variants identified in genome-wide association studies (GWAS) frequently lie in non-coding regions of the genome that contain cis-regulatory elements suggests that altered gene expression underlies the development of many complex traits. In order to efficiently make a comprehensive assessment of the impact of non-coding genetic variation in immune related diseases we emulated the whole-exome sequencing paradigm and developed a custom capture panel for the known DNase I hypersensitive site (DHS) in immune cells - "Immunoseq". RESULTS: We performed Immunoseq in 30 healthy individuals where we had existing transcriptome data from T cells. We identified a large number of novel non-coding variants in these samples. Relying on allele specific expression measurements, we also showed that our selected capture regions are enriched for functional variants that have an impact on differential allelic gene expression. The results from a replication set with 180 samples confirmed our observations. CONCLUSIONS: We show that Immunoseq is a powerful approach to detect novel rare variants in regulatory regions. We also demonstrate that these novel variants have a potential functional role in immune cells.

DNA methylome analysis of acute lymphoblastic leukemia cells reveals stochastic de novo DNA methylation in CpG islands.

AIM: To identify regions of aberrant DNA methylation in acute lymphoblastic leukemia (ALL) cells of different subtypes on a genome-wide scale. MATERIALS & METHODS: Whole-genome bisulfite sequencing (WGBS) was used to determine the DNA methylation levels in cells from four pediatric ALL patients of different subtypes. The findings were confirmed by 450k DNA methylation arrays in a large patient set. RESULTS: Compared with mature B or T cells WGBS detected on average 82,000 differentially methylated regions per patient. Differentially methylated regions are enriched to CpG poor regions, active enhancers and transcriptional start sites. We also identified approximately 8000 CpG islands with variable intermediate DNA methylation that seems to occur as a result of stochastic de novo methylation. CONCLUSION: WGBS provides an unbiased view and novel insights into the DNA methylome of ALL cells.

A Novel HLA-DRB1*10:01-Restricted T Cell Epitope From Citrullinated Type II Collagen Relevant to Rheumatoid Arthritis.

OBJECTIVE: Antibodies against citrullinated type II collagen (Cit-CII) are common in the sera and synovial fluid of patients with rheumatoid arthritis (RA); however, the known T cell epitope of CII is not dependent on citrullination. The aim of this study was to identify and functionally characterize the Cit-CII-restricted T cell epitopes that are relevant to RA. METHODS: Peripheral blood mononuclear cells (PBMCs) from HLA-DRB1*10:01-positive patients with RA and healthy donors were stimulated in vitro with candidate CII peptides. CD154 up-regulation was measured as a marker of antigen-specific activation, and anti-HLA-DR-blocking experiments confirmed HLA restriction. Cytokine production was measured using a Luminex technique. Direct peptide-binding assays using HLA-DRB1*10:01 and HLA-DRB1*04:01 monomeric proteins were performed. The T cell receptor (TCR) ß-chain of CD154-enriched antigen-specific T cells was analyzed using high-throughput sequencing. RESULTS: A novel Cit-CII peptide was identified based on its ability to activate CD4+ T cells from HLA-DRB1*10:01-positive individuals. When stimulated in vitro, Cit-CII autoreactive T cells produced proinflammatory cytokines. Cit-CII(311-325) bound (with low affinity) to HLA-DRB1*10:01 but not to HLA-DRB1*04:01, while the native form was unable to bind either protein. In addition, highly expanded clones were identified in the TCRß repertoire of Cit-CII(311-325) -stimulated PBMCs. CONCLUSION: These results illustrate the ability of the citrullination process to create T cell epitopes from CII, a cartilage-restricted protein that is relevant to RA pathogenesis. The exclusive binding of Cit-CII(311-325) to HLA-DRB1*10:01 suggests that recognition of citrullinated epitopes might vary between individuals carrying different RA-associated HLA-DR molecules.

Activated T cells enhance interferon-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes.

OBJECTIVES: Patients with systemic lupus erythematosus (SLE) have an ongoing interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs). We investigated whether T cells can promote IFN-α production by pDCs. METHODS: Human pDCs were stimulated with immune complexes (ICs) containing U1 small nuclear ribonucleic proteins particles and SLE-IgG (RNA-IC) in the presence of T cells or T cell supernatants. T cells were activated by anti-CD3/CD28 antibodies or in a mixed leucocyte reaction. IFN-α and other cytokines were determined in culture supernatants or patient sera with immunoassays. The effect of interleukin (IL) 3 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) on pDCs was examined by the use of antibodies, and the expression of CD80/CD86 was determined using flow cytometry. RESULTS: Activated T cells and supernatants from activated T cells increased IFN-α production by >20-fold. The stimulatory effect of T cell supernatants was reduced after depletion of GM-CSF (81%) or by blocking the GM-CSF receptor (55%-81%). Supernatant from activated T cells, furthermore, increased the frequency of CD80 and CD86 expressing pDCs stimulated with RNA-IC from 6% to 35% (p<0.05) and from 10% to 26% (p<0.01), respectively. Activated SLE T cells enhanced IFN-α production to the same extent as T cells from healthy individuals and a subset of patients with SLE had increased serum levels of GM-CSF. CONCLUSIONS: Activated T cells enhance IFN-α production by RNA-IC stimulated pDCs via GM-CSF and induce pDC maturation. Given the increased serum levels of GM-CSF in a subset of patients with SLE, these findings suggest that activated T cells may upregulate type I IFN production in SLE.

Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.

IFN-α production by plasmacytoid dendritic cell associations with polymorphisms in gene loci related to autoimmune and inflammatory diseases.

The type I interferon (IFN) system is persistently activated in systemic lupus erythematosus (SLE) and many other systemic autoimmune diseases. Studies have shown an association between SLE and several gene variants within the type I IFN system. We investigated whether single-nucleotide polymorphisms (SNPs) associated with SLE and other autoimmune diseases affect the IFN-α production in healthy individuals. Plasmacytoid dendritic cells (pDCs), B cells and NK cells were isolated from peripheral blood of healthy individuals and stimulated with RNA-containing immune complexes (ICs), herpes simplex virus (HSV) or the oligonucleotide ODN2216. IFN-α production by pDCs alone or in cocultures with B or NK cells was measured by an immunoassay. All donors were genotyped with the 200K ImmunoChip, and a 5 bp CGGGG length polymorphism in the IFN regulatory factor 5 gene (IRF5) was genotyped by PCR. We found associations between IFN-α production and 18-86 SNPs (P ≤ 0.001), depending on the combination of the stimulated cell types. However, only three of these associated SNPs were shared between the cell-type combinations. Several SNPs showed novel associations to the type I IFN system among all the associated SNPs, whereas some loci have been described earlier for their association with SLE. Furthermore, we found that the SLE-risk variant of the IRF5 CGGGG-indel was associated with lower IFN-α production. We conclude that the genetic variants affecting the IFN-α production highlight the intricate regulation of the type I IFN system and the importance of understanding the mechanisms behind the dysregulated type I IFN system in SLE.

AGAMOUS subfamily MADS-box genes and the evolution of seed cone morphology in Cupressaceae and Taxodiaceae.

In this comparative developmental genetics study, we test hypotheses based on fossil and morphological data on reproductive organ morphology and evolution in conifers--specifically, the ovule-bearing organ in Cupressaceae and Taxodiaceae. Genes homologous to the Arabidopsis gene AGAMOUS are expressed in ovuliferous scales of spruces (Picea) throughout development. Previous studies have shown that the AGAMOUS subfamily of MADS-box genes predates the split between angiosperms and gymnosperms, and that these genes have in part conserved functions in reproductive development among seed plants, especially in the specification of identity of the ovule-bearing organs. These data indicate that their expression in conifer families other than Pinaceae might be used as markers for organs homologous to the Pinaceae ovuliferous scale. Here we have isolated putative AGAMOUS orthologs from Cupressaceae and Taxodiaceae and analyzed their expression pattern in seed cones to test for the presence of morphological homologs of ovuliferous scales. Our results were not congruent with the hypothesis that the tooth of the Cryptomeria seed cone is homologous to the Picea ovuliferous scale. Likewise, the hypothesis that the bracts of Thujopsis and Juniperus contain fused ovuliferous scales was not supported. However, we found expression of AGAMOUS homologs in the sterile bracts of Cupressaceae seed cones at late developmental stages. This expression probably represents a novel gene function in these conifer families, since no corresponding expression has been identified in Pinaceae. Our study suggests that the evolutionary history of modern conifer cones is more diverse than previously thought.

The MADS-box gene DAL1 is a potential mediator of the juvenile-to-adult transition in Norway spruce (Picea abies).

Progression through the plant life cycle involves change in many essential features, most notably in the capacity to reproduce. The transition from a juvenile vegetative and non-reproductive to an adult reproductive phase is gradual and can take many years; in the conifer Norway spruce, Picea abies, typically 20-25 years. We present a detailed analysis of the activities of three regulatory genes with potential roles in this transition in Norway spruce: DAL1, a MADS-box gene related to the AGL6 group of genes from angiosperms, and the two LEAFY-related genes PaLFY and PaNLY. DAL1 activity is initiated in the shoots of juvenile trees at an age of 3-5 years, and then increases with age, whereas both LFY genes are active throughout the juvenile phase. The activity of DAL1 further shows a spatial pattern along the stem of the tree that parallels a similar gradient in physiological and morphological features associated with maturation to the adult phase. Constitutive expression of DAL1 in transgenic Arabidopsis plants caused a dramatic attenuation of both juvenile and adult growth phases; flowers forming immediately after the embryonic phase of development in severely affected plants. Taken together, our results support the notion that DAL1 may have a regulatory role in the juvenile-to-adult transition in Norway spruce.