Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation.

T helper type 2 (Th2) cells are important regulators of mammalian adaptive immunity and have relevance for infection, autoimmunity, and tumor immunology. Using a newly developed, genome-wide retroviral CRISPR knockout (KO) library, combined with RNA-seq, ATAC-seq, and ChIP-seq, we have dissected the regulatory circuitry governing activation and differentiation of these cells. Our experiments distinguish cell activation versus differentiation in a quantitative framework. We demonstrate that these two processes are tightly coupled and are jointly controlled by many transcription factors, metabolic genes, and cytokine/receptor pairs. There are only a small number of genes regulating differentiation without any role in activation. By combining biochemical and genetic data, we provide an atlas for Th2 differentiation, validating known regulators and identifying factors, such as Pparg and Bhlhe40, as part of the core regulatory network governing Th2 helper cell fates.

Long noncoding RNA LIRIL2R modulates FOXP3 levels and suppressive function of human CD4+ regulatory T cells by regulating IL2RA.

Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the IL2RA locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the IL2RA locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., FOXP3, CTLA4, and PDCD1), upregulation of genes associated with effector T cells (e.g., SATB1 and GATA3), and loss of Treg-mediated suppression.

Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub.

T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

A proximal enhancer regulates RORA expression during early human Th17 cell differentiation.

Gene regulatory elements, such as enhancers, greatly influence cell identity by tuning the transcriptional activity of specific cell types. Dynamics of enhancer landscape during early human Th17 cell differentiation remains incompletely understood. Leveraging ATAC-seq-based profiling of chromatin accessibility and comprehensive analysis of key histone marks, we identified a repertoire of enhancers that potentially exert control over the fate specification of Th17 cells. We found 23 SNPs associated with autoimmune diseases within Th17-enhancers that precisely overlapped with the binding sites of transcription factors actively engaged in T-cell functions. Among the Th17-specific enhancers, we identified an enhancer in the intron of RORA and demonstrated that this enhancer positively regulates RORA transcription. Moreover, CRISPR-Cas9-mediated deletion of a transcription factor binding site-rich region within the identified RORA enhancer confirmed its role in regulating RORA transcription. These findings provide insights into the potential mechanism by which the RORA enhancer orchestrates Th17 differentiation.

Multi-omics analysis reveals drivers of loss of ß-cell function after newly diagnosed autoimmune type 1 diabetes: An INNODIA multicenter study.

AIMS: Heterogeneity in the rate of ß-cell loss in newly diagnosed type 1 diabetes patients is poorly understood and creates a barrier to designing and interpreting disease-modifying clinical trials. Integrative analyses of baseline multi-omics data obtained after the diagnosis of type 1 diabetes may provide mechanistic insight into the diverse rates of disease progression after type 1 diabetes diagnosis. METHODS: We collected samples in a pan-European consortium that enabled the concerted analysis of five different omics modalities in data from 97 newly diagnosed patients. In this study, we used Multi-Omics Factor Analysis to identify molecular signatures correlating with post-diagnosis decline in ß-cell mass measured as fasting C-peptide. RESULTS: Two molecular signatures were significantly correlated with fasting C-peptide levels. One signature showed a correlation to neutrophil degranulation, cytokine signalling, lymphoid and non-lymphoid cell interactions and G-protein coupled receptor signalling events that were inversely associated with a rapid decline in ß-cell function. The second signature was related to translation and viral infection was inversely associated with change in ß-cell function. In addition, the immunomics data revealed a Natural Killer cell signature associated with rapid ß-cell decline. CONCLUSIONS: Features that differ between individuals with slow and rapid decline in ß-cell mass could be valuable in staging and prediction of the rate of disease progression and thus enable smarter (shorter and smaller) trial designs for disease modifying therapies as well as offering biomarkers of therapeutic effect.

A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation.

Th17 cells are essential for protection against extracellular pathogens, but their aberrant activity can cause autoimmunity. Molecular mechanisms that dictate Th17 cell-differentiation have been extensively studied using mouse models. However, species-specific differences underscore the need to validate these findings in human. Here, we characterized the human-specific roles of three AP-1 transcription factors, FOSL1, FOSL2 and BATF, during early stages of Th17 differentiation. Our results demonstrate that FOSL1 and FOSL2 co-repress Th17 fate-specification, whereas BATF promotes the Th17 lineage. Strikingly, FOSL1 was found to play different roles in human and mouse. Genome-wide binding analysis indicated that FOSL1, FOSL2 and BATF share occupancy over regulatory regions of genes involved in Th17 lineage commitment. These AP-1 factors also share their protein interacting partners, which suggests mechanisms for their functional interplay. Our study further reveals that the genomic binding sites of FOSL1, FOSL2 and BATF harbour hundreds of autoimmune disease-linked SNPs. We show that many of these SNPs alter the ability of these transcription factors to bind DNA. Our findings thus provide critical insights into AP-1-mediated regulation of human Th17-fate and associated pathologies.

Targeted serum proteomics of longitudinal samples from newly diagnosed youth with type 1 diabetes distinguishes markers of disease and C-peptide trajectory.

AIMS/HYPOTHESIS: There is a growing need for markers that could help indicate the decline in beta cell function and recognise the need and efficacy of intervention in type 1 diabetes. Measurements of suitably selected serum markers could potentially provide a non-invasive and easily applicable solution to this challenge. Accordingly, we evaluated a broad panel of proteins previously associated with type 1 diabetes in serum from newly diagnosed individuals during the first year from diagnosis. To uncover associations with beta cell function, comparisons were made between these targeted proteomics measurements and changes in fasting C-peptide levels. To further distinguish proteins linked with the disease status, comparisons were made with measurements of the protein targets in age- and sex-matched autoantibody-negative unaffected family members (UFMs). METHODS: Selected reaction monitoring (SRM) mass spectrometry analyses of serum, targeting 85 type 1 diabetes-associated proteins, were made. Sera from individuals diagnosed under 18 years (n=86) were drawn within 6 weeks of diagnosis and at 3, 6 and 12 months afterwards (288 samples in total). The SRM data were compared with fasting C-peptide/glucose data, which was interpreted as a measure of beta cell function. The protein data were further compared with cross-sectional SRM measurements from UFMs (n=194). RESULTS: Eleven proteins had statistically significant associations with fasting C-peptide/glucose. Of these, apolipoprotein L1 and glutathione peroxidase 3 (GPX3) displayed the strongest positive and inverse associations, respectively. Changes in GPX3 levels during the first year after diagnosis indicated future fasting C-peptide/glucose levels. In addition, differences in the levels of 13 proteins were observed between the individuals with type 1 diabetes and the matched UFMs. These included GPX3, transthyretin, prothrombin, apolipoprotein C1 and members of the IGF family. CONCLUSIONS/INTERPRETATION: The association of several targeted proteins with fasting C-peptide/glucose levels in the first year after diagnosis suggests their connection with the underlying changes accompanying alterations in beta cell function in type 1 diabetes. Moreover, the direction of change in GPX3 during the first year was indicative of subsequent fasting C-peptide/glucose levels, and supports further investigation of this and other serum protein measurements in future studies of beta cell function in type 1 diabetes.

Early DNA methylation changes in children developing beta cell autoimmunity at a young age.

AIMS/HYPOTHESIS: Type 1 diabetes is a chronic autoimmune disease of complex aetiology, including a potential role for epigenetic regulation. Previous epigenomic studies focused mainly on clinically diagnosed individuals. The aim of the study was to assess early DNA methylation changes associated with type 1 diabetes already before the diagnosis or even before the appearance of autoantibodies. METHODS: Reduced representation bisulphite sequencing (RRBS) was applied to study DNA methylation in purified CD4+ T cell, CD8+ T cell and CD4-CD8- cell fractions of 226 peripheral blood mononuclear cell samples longitudinally collected from seven type 1 diabetes-specific autoantibody-positive individuals and control individuals matched for age, sex, HLA risk and place of birth. We also explored correlations between DNA methylation and gene expression using RNA sequencing data from the same samples. Technical validation of RRBS results was performed using pyrosequencing. RESULTS: We identified 79, 56 and 45 differentially methylated regions in CD4+ T cells, CD8+ T cells and CD4-CD8- cell fractions, respectively, between type 1 diabetes-specific autoantibody-positive individuals and control participants. The analysis of pre-seroconversion samples identified DNA methylation signatures at the very early stage of disease, including differential methylation at the promoter of IRF5 in CD4+ T cells. Further, we validated RRBS results using pyrosequencing at the following CpG sites: chr19:18118304 in the promoter of ARRDC2; chr21:47307815 in the intron of PCBP3; and chr14:81128398 in the intergenic region near TRAF3 in CD4+ T cells. CONCLUSIONS/INTERPRETATION: These preliminary results provide novel insights into cell type-specific differential epigenetic regulation of genes, which may contribute to type 1 diabetes pathogenesis at the very early stage of disease development. Should these findings be validated, they may serve as a potential signature useful for disease prediction and management.

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes.

AIMS/HYPOTHESIS: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. METHODS: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. RESULTS: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate <0.05. CONCLUSIONS/INTERPRETATION: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.

Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.

CRISPR/Cas9 guided genome and epigenome engineering and its therapeutic applications in immune mediated diseases.

Recent developments in the nucleic acid editing technologies have provided a powerful tool to precisely engineer the genome and epigenome for studying many aspects of immune cell differentiation and development as well as several immune mediated diseases (IMDs) including autoimmunity and cancer. Here, we discuss the recent technological achievements of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based RNA-guided genome and epigenome editing toolkit and provide an insight into how CRISPR/Cas9 (CRISPR Associated Protein 9) toolbox could be used to examine genetic and epigenetic mechanisms underlying IMDs. In addition, we will review the progress in CRISPR/Cas9-based genome-wide genome and epigenome screens in various cell types including immune cells. Finally, we will discuss the potential of CRISPR/Cas9 in defining the molecular function of disease associated SNPs overlapping gene regulatory elements.

The role of Interleukin-32 in autoimmunity.

Interleukin-32 (IL-32) is a pro-inflammatory cytokine that induces other cytokines involved in inflammation, including tumour necrosis factor (TNF)-α, IL-6 and IL-1ß. Recent evidence suggests that IL-32 has a crucial role in host defence against pathogens, as well as in the pathogenesis of chronic inflammation. Abnormal IL-32 expression has been linked to several autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel diseases, and a recent study suggested the importance of IL-32 in the pathogenesis of type 1 diabetes. However, despite accumulating evidence, many molecular characteristics of this cytokine, including the secretory route and the receptor for IL-32, remain largely unknown. In addition, the IL-32 gene is found in higher mammals but not in rodents. In this review, we outline the current knowledge of IL-32 biological functions, properties, and its role in autoimmune diseases. We particularly highlight the role of IL-32 in rheumatoid arthritis and type 1 diabetes.

snpEnrichR: analyzing co-localization of SNPs and their proxies in genomic regions.

Motivation: Co-localization of trait associated SNPs for specific transcription-factor binding sites or regulatory regions in the genome can yield profound insight into underlying causal mechanisms. Analysis is complicated because the truly causal SNPs are generally unknown and can be either SNPs reported in GWAS studies or other proxy SNPs in their linkage disequilibrium. Hence, a comprehensive pipeline for SNP co-localization analysis that utilizes all relevant information about both the genotyped SNPs and their proxies is needed. Results: We developed an R package snpEnrichR for SNP co-localization analysis. The software integrates different tools for random SNP generation and genome co-localization analysis to automatize and help users to create custom SNP co-localization analysis. We show via an example that including proxy SNPs in SNP co-localization analysis enhances the sensitivity of co-localization detection. Availability and implementation: The software is available at https://github.com/kartiek/snpEnrichR.

Analysis of the plasma proteome using iTRAQ and TMT-based Isobaric labeling.

Over the past decade, chemical labeling with isobaric tandem mass tags, such as isobaric tags for relative and absolute quantification reagents (iTRAQ) and tandem mass tag (TMT) reagents, has been employed in a wide range of different clinically orientated serum and plasma proteomics studies. In this review the scope of these works is presented with attention to the areas of research, methods employed and performance limitations. These applications have covered a wide range of diseases, disorders and infections, and have implemented a variety of different preparative and mass spectrometric approaches. In contrast to earlier works, which struggled to quantify more than a few hundred proteins, increasingly these studies have provided deeper insight into the plasma proteome extending the numbers of quantified proteins to over a thousand.

L1TD1 - a prognostic marker for colon cancer.

BACKGROUND: Prognostic markers specific to a particular cancer type can assist in the evaluation of survival probability of patients and help clinicians to assess the available treatment modalities. METHODS: Gene expression data was analyzed from three independent colon cancer microarray gene expression data sets (N = 1052). Survival analysis was performed for the three data sets, stratified by the expression level of the LINE-1 type transposase domain containing 1 (L1TD1). Correlation analysis was performed to investigate the role of the interactome of L1TD1 in colon cancer patients. RESULTS: We found L1TD1 as a novel positive prognostic marker for colon cancer. Increased expression of L1TD1 associated with longer disease-free survival in all the three data sets. Our results were in contrast to a previous study on medulloblastoma, where high expression of L1TD1 was linked with poor prognosis. Notably, in medulloblastoma L1TD1 was co-expressed with its interaction partners, whereas our analysis revealed lack of co-expression of L1TD1 with its interaction partners in colon cancer. CONCLUSIONS: Our results identify increased expression of L1TD1 as a prognostic marker predicting longer disease-free survival in colon cancer patients.

Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming.

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.

Hyper-phosphorylation of Sequestosome-1 Distinguishes Resistance to Cisplatin in Patient Derived High Grade Serous Ovarian Cancer Cells.

Platinum-resistance is a major limitation to effective chemotherapy regimens in high-grade serous ovarian cancer (HGSOC). To better understand the mechanisms involved we characterized the proteome and phosphoproteome in cisplatin sensitive and resistant HGSOC primary cells using a mass spectrometry-based proteomic strategy. PCA analysis identified a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines. The most phosphorylated protein in cisplatin resistant cells was sequestosome-1 (p62/SQSTM1). Changes in expression of apoptosis and autophagy related proteins Caspase-3 and SQSTM1, respectively, were validated by Western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while SQSTM1 revealed increased expression in the resistant cell line relative to sensitive cell line. Furthermore, site-specific phosphorylation on 20 amino acid residues of SQSTM1 was detected indicating a hyper-phosphorylation phenotype. This elevated hyper-phosphorylation of SQSTM1 in resistant HGSOC cell lines was validated with Western blot analysis. Immunofluoresence staining of s28-pSQSTM1 showed inducible localization to autophagosomes upon cisplatin treatment in the sensitive cell line while being constitutively expressed to autophagosomes in the resistant cell. Furthermore, SQSTM1 expression was localized in cancer cells of clinical high-grade serous tumors. Here, we propose hyper-phosphorylation of SQSTM1 as a marker and a key proteomic change in cisplatin resistance development in ovarian cancers by activating the autophagy pathway and influencing down-regulation of apoptosis.

Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3.

BACKGROUND: Regulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood. RESULTS: To gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset. CONCLUSION: The data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.

Immunological biomarkers for the development and progression of type 1 diabetes.

Immune biomarkers of type 1 diabetes are many and diverse. Some of these, such as the autoantibodies, are well established but not discriminative enough to deal with the heterogeneity inherent to type 1 diabetes progression. As an alternative, high hopes are placed on T cell assays, which give insight into the cells that actually target the beta cell or play a crucial role in maintaining tolerance. These assays are approaching a level of robustness that may allow for solid conclusions on both disease progression and therapeutic efficacy of immune interventions. In addition, 'omics' approaches to biomarker discovery are rapidly progressing. The potential emergence of novel biomarkers creates a need for the introduction of bioinformatics and 'big data' analysis systems for the integration of the multitude of biomarker data that will be available, to translate these data into clinical tools. It is worth noting that it is unlikely that the same markers will apply to all individuals. Instead, individualised signatures of biomarkers, combining autoantibodies, T cell profiles and other biomarkers, will need to be used to classify at-risk patients into various categories, thus enabling personalised prediction, prevention and treatment approaches. To achieve this goal, the standardisation of assays for biomarker discovery, the integration of analyses and data from biomarker studies and, most importantly, the careful clinical characterisation of individuals providing samples for these studies are critical. Longitudinal sample-collection initiatives, like INNODIA, should lead to novel biomarker discovery, not only providing a better understanding of type 1 diabetes onset and progression, but also yielding biomarkers of therapeutic efficacy of interventions to prevent or arrest type 1 diabetes.

Enterovirus-associated changes in blood transcriptomic profiles of children with genetic susceptibility to type 1 diabetes.

AIMS/HYPOTHESIS: Enterovirus infections have been associated with the development of type 1 diabetes in multiple studies, but little is known about enterovirus-induced responses in children at risk for developing type 1 diabetes. Our aim was to use genome-wide transcriptomics data to characterise enterovirus-associated changes in whole-blood samples from children with genetic susceptibility to type 1 diabetes. METHODS: Longitudinal whole-blood samples (356 samples in total) collected from 28 pairs of children at increased risk for developing type 1 diabetes were screened for the presence of enterovirus RNA. Seven of these samples were detected as enterovirus-positive, each of them collected from a different child, and transcriptomics data from these children were analysed to understand the individual-level responses associated with enterovirus infections. Transcript clusters with peaking or dropping expression at the time of enterovirus positivity were selected as the enterovirus-associated signals. RESULTS: Strong signs of activation of an interferon response were detected in four children at enterovirus positivity, while transcriptomic changes in the other three children indicated activation of adaptive immune responses. Additionally, a large proportion of the enterovirus-associated changes were specific to individuals. An enterovirus-induced signature was built using 339 genes peaking at enterovirus positivity in four of the children, and 77 of these genes were also upregulated in human peripheral blood mononuclear cells infected in vitro with different enteroviruses. These genes separated the four enterovirus-positive samples clearly from the remaining 352 blood samples analysed. CONCLUSIONS/INTERPRETATION: We have, for the first time, identified enterovirus-associated transcriptomic profiles in whole-blood samples from children with genetic susceptibility to type 1 diabetes. Our results provide a starting point for understanding the individual responses to enterovirus infections in blood and their potential connection to the development of type 1 diabetes. DATA AVAILABILITY: The datasets analysed during the current study are included in this published article and its supplementary information files ( www.btk.fi/research/computational-biomedicine/1234-2 ) or are available from the Gene Expression Omnibus (GEO) repository (accession GSE30211).