Consortium-based genome-wide meta-analysis for childhood dental caries traits.

Prior studies suggest dental caries traits in children and adolescents are partially heritable, but there has been no large-scale consortium genome-wide association study (GWAS) to date. We therefore performed GWAS for caries in participants aged 2.5-18.0 years from nine contributing centres. Phenotype definitions were created for the presence or absence of treated or untreated caries, stratified by primary and permanent dentition. All studies tested for association between caries and genotype dosage and the results were combined using fixed-effects meta-analysis. Analysis included up to 19 003 individuals (7530 affected) for primary teeth and 13 353 individuals (5875 affected) for permanent teeth. Evidence for association with caries status was observed at rs1594318-C for primary teeth [intronic within ALLC, odds ratio (OR) 0.85, effect allele frequency (EAF) 0.60, P 4.13e-8] and rs7738851-A (intronic within NEDD9, OR 1.28, EAF 0.85, P 1.63e-8) for permanent teeth. Consortium-wide estimated heritability of caries was low [h2 of 1% (95% CI: 0%: 7%) and 6% (95% CI 0%: 13%) for primary and permanent dentitions, respectively] compared with corresponding within-study estimates [h2 of 28% (95% CI: 9%: 48%) and 17% (95% CI: 2%: 31%)] or previously published estimates. This study was designed to identify common genetic variants with modest effects which are consistent across different populations. We found few single variants associated with caries status under these assumptions. Phenotypic heterogeneity between cohorts and limited statistical power will have contributed; these findings could also reflect complexity not captured by our study design, such as genetic effects which are conditional on environmental exposure.

SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments.

Identifying which mutation(s) within a given genotype is responsible for an observable phenotype is important in many aspects of molecular biology. Here, we present SigniSite, an online application for subgroup-free residue-level genotype-phenotype correlation. In contrast to similar methods, SigniSite does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set phenotype. As output, SigniSite displays a sequence logo, depicting the strength of the phenotype association of each residue and a heat-map identifying 'hot' or 'cold' regions. SigniSite was benchmarked against SPEER, a state-of-the-art method for the prediction of specificity determining positions (SDP) using a set of human immunodeficiency virus protease-inhibitor genotype-phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found to outperform SPEER. SigniSite is available at: http://www.cbs.dtu.dk/services/SigniSite/.

A systematic evaluation of state-of-the-art deconvolution methods in spatial transcriptomics: insights from cardiovascular disease and chronic kidney disease.

A major challenge in sequencing-based spatial transcriptomics (ST) is resolution limitations. Tissue sections are divided into hundreds of thousands of spots, where each spot invariably contains a mixture of cell types. Methods have been developed to deconvolute the mixed transcriptional signal into its constituents. Although ST is becoming essential for drug discovery, especially in cardiometabolic diseases, to date, no deconvolution benchmark has been performed on these types of tissues and diseases. However, the three methods, Cell2location, RCTD, and spatialDWLS, have previously been shown to perform well in brain tissue and simulated data. Here, we compare these methods to assess the best performance when using human data from cardiovascular disease (CVD) and chronic kidney disease (CKD) from patients in different pathological states, evaluated using expert annotation. In this study, we found that all three methods performed comparably well in deconvoluting verifiable cell types, including smooth muscle cells and macrophages in vascular samples and podocytes in kidney samples. RCTD shows the best performance accuracy scores in CVD samples, while Cell2location, on average, achieved the highest performance across all test experiments. Although all three methods had similar accuracies, Cell2location needed less reference data to converge at the expense of higher computational intensity. Finally, we also report that RCTD has the fastest computational time and the simplest workflow, requiring fewer computational dependencies. In conclusion, we find that each method has particular advantages, and the optimal choice depends on the use case.

Benchmarking data-driven filtering for denoising of TCRpMHC single-cell data.

Pairing of the T cell receptor (TCR) with its cognate peptide-MHC (pMHC) is a cornerstone in T cell-mediated immunity. Recently, single-cell sequencing coupled with DNA-barcoded MHC multimer staining has enabled high-throughput studies of T cell specificities. However, the immense variability of TCR-pMHC interactions combined with the relatively low signal-to-noise ratio in the data generated using current technologies are complicating these studies. Several approaches have been proposed for denoising single-cell TCR-pMHC specificity data. Here, we present a benchmark evaluating two such denoising methods, ICON and ITRAP. We applied and evaluated the methods on publicly available immune profiling data provided by 10x Genomics. We find that both methods identified approximately 75% of the raw data as noise. We analyzed both internal metrics developed for the purpose and performance on independent data using machine learning methods trained on the raw and denoised 10x data. We find an increased signal-to-noise ratio comparing the denoised to the raw data for both methods, and demonstrate an overall superior performance of the ITRAP method in terms of both data consistency and performance. In conclusion, this study demonstrates that Improving the data quality from high throughput studies of TCRpMHC-specificity by denoising is paramount in increasing our understanding of T cell-mediated immunity.

Improved T cell receptor antigen pairing through data-driven filtering of sequencing information from single cells.

Novel single-cell-based technologies hold the promise of matching T cell receptor (TCR) sequences with their cognate peptide-MHC recognition motif in a high-throughput manner. Parallel capture of TCR transcripts and peptide-MHC is enabled through the use of reagents labeled with DNA barcodes. However, analysis and annotation of such single-cell sequencing (SCseq) data are challenged by dropout, random noise, and other technical artifacts that must be carefully handled in the downstream processing steps. We here propose a rational, data-driven method termed ITRAP (improved T cell Receptor Antigen Paring) to deal with these challenges, filtering away likely artifacts, and enable the generation of large sets of TCR-pMHC sequence data with a high degree of specificity and sensitivity, thus outputting the most likely pMHC target per T cell. We have validated this approach across 10 different virus-specific T cell responses in 16 healthy donors. Across these samples, we have identified up to 1494 high-confident TCR-pMHC pairs derived from 4135 single cells.

NetTCR-2.1: Lessons and guidance on how to develop models for TCR specificity predictions.

T cell receptors (TCR) define the specificity of T cells and are responsible for their interaction with peptide antigen targets presented in complex with major histocompatibility complex (MHC) molecules. Understanding the rules underlying this interaction hence forms the foundation for our understanding of basic adaptive immunology. Over the last decade, efforts have been dedicated to developing assays for high throughput identification of peptide-specific TCRs. Based on such data, several computational methods have been proposed for predicting the TCR-pMHC interaction. The general conclusion from these studies is that the prediction of TCR interactions with MHC-peptide complexes remains highly challenging. Several reasons form the basis for this including scarcity and quality of data, and ill-defined modeling objectives imposed by the high redundancy of the available data. In this work, we propose a framework for dealing with this redundancy, allowing us to address essential questions related to the modeling of TCR specificity including the use of peptide- versus pan-specific models, how to best define negative data, and the performance impact of integrating of CDR1 and 2 loops. Further, we illustrate how and why it is strongly recommended to include simple similarity-based modeling approaches when validating an improved predictive power of machine learning models, and that such validation should include a performance evaluation as a function of "distance" to the training data, to quantify the potential for generalization of the proposed model. The conclusion of the work is that, given current data, TCR specificity is best modeled using peptide-specific approaches, integrating information from all 6 CDR loops, and with negative data constructed from a combination of true and mislabeled negatives. Comparing such machine learning models to similarity-based approaches demonstrated an increased performance gain of the former as the "distance" to the training data was increased; thus demonstrating an improved generalization ability of the machine learning-based approaches. We believe these results demonstrate that the outlined modeling framework and proposed evaluation strategy form a solid basis for investigating the modeling of TCR specificities and that adhering to such a framework will allow for faster progress within the field. The final devolved model, NetTCR-2.1, is available at https://services.healthtech.dtu.dk/service.php?NetTCR-2.1.

Reduced expression of OXPHOS and DNA damage genes is linked to protection from microvascular complications in long-term type 1 diabetes: the PROLONG study.

Type 1 diabetes is a chronic autoimmune disease requiring insulin treatment for survival. Prolonged duration of type 1 diabetes is associated with increased risk of microvascular complications. Although chronic hyperglycemia and diabetes duration have been considered as the major risk factors for vascular complications, this is not universally seen among all patients. Persons with long-term type 1 diabetes who have remained largely free from vascular complications constitute an ideal group for investigation of natural defense mechanisms against prolonged exposure of diabetes. Transcriptomic signatures obtained from RNA sequencing of the peripheral blood cells were analyzed in non-progressors with more than 30 years of diabetes duration and compared to the patients who progressed to microvascular complications within a shorter duration of diabetes. Analyses revealed that non-progressors demonstrated a reduction in expression of the oxidative phosphorylation (OXPHOS) genes, which were positively correlated with the expression of DNA repair enzymes, namely genes involved in base excision repair (BER) machinery. Reduced expression of OXPHOS and BER genes was linked to decrease in expression of inflammation-related genes, higher glucose disposal rate and reduced measures of hepatic fatty liver. Results from the present study indicate that at transcriptomic level reduction in OXPHOS, DNA repair and inflammation-related genes is linked to better insulin sensitivity and protection against microvascular complications in persons with long-term type 1 diabetes.

NetTCR-2.0 enables accurate prediction of TCR-peptide binding by using paired TCRα and ß sequence data.

Prediction of T-cell receptor (TCR) interactions with MHC-peptide complexes remains highly challenging. This challenge is primarily due to three dominant factors: data accuracy, data scarceness, and problem complexity. Here, we showcase that "shallow" convolutional neural network (CNN) architectures are adequate to deal with the problem complexity imposed by the length variations of TCRs. We demonstrate that current public bulk CDR3ß-pMHC binding data overall is of low quality and that the development of accurate prediction models is contingent on paired α/ß TCR sequence data corresponding to at least 150 distinct pairs for each investigated pMHC. In comparison, models trained on CDR3α or CDR3ß data alone demonstrated a variable and pMHC specific relative performance drop. Together these findings support that T-cell specificity is predictable given the availability of accurate and sufficient paired TCR sequence data. NetTCR-2.0 is publicly available at https://services.healthtech.dtu.dk/service.php?NetTCR-2.0 .

Immunopeptidomic Data Integration to Artificial Neural Networks Enhances Protein-Drug Immunogenicity Prediction.

Recombinant DNA technology has, in the last decades, contributed to a vast expansion of the use of protein drugs as pharmaceutical agents. However, such biological drugs can lead to the formation of anti-drug antibodies (ADAs) that may result in adverse effects, including allergic reactions and compromised therapeutic efficacy. Production of ADAs is most often associated with activation of CD4 T cell responses resulting from proteolysis of the biotherapeutic and loading of drug-specific peptides into major histocompatibility complex (MHC) class II on professional antigen-presenting cells. Recently, readouts from MHC-associated peptide proteomics (MAPPs) assays have been shown to correlate with the presence of CD4 T cell epitopes. However, the limited sensitivity of MAPPs challenges its use as an immunogenicity biomarker. In this work, MAPPs data was used to construct an artificial neural network (ANN) model for MHC class II antigen presentation. Using Infliximab and Rituximab as showcase stories, the model demonstrated an unprecedented performance for predicting MAPPs and CD4 T cell epitopes in the context of protein-drug immunogenicity, complementing results from MAPPs assays and outperforming conventional prediction models trained on binding affinity data.

Liver nucleotide biosynthesis is linked to protection from vascular complications in individuals with long-term type 1 diabetes.

Identification of biomarkers associated with protection from developing diabetic complications is a prerequisite for an effective prevention and treatment. The aim of the present study was to identify clinical and plasma metabolite markers associated with freedom from vascular complications in people with very long duration of type 1 diabetes (T1D). Individuals with T1D, who despite having longer than 30 years of diabetes duration never developed major macro- or microvascular complications (non-progressors; NP) were compared with those who developed vascular complications within 25 years from diabetes onset (rapid progressors; RP) in the Scandinavian PROLONG (n = 385) and DIALONG (n = 71) cohorts. The DIALONG study also included 75 healthy controls. Plasma metabolites were measured using gas and/or liquid chromatography coupled to mass spectrometry. Lower hepatic fatty liver indices were significant common feature characterized NPs in both studies. Higher insulin sensitivity and residual ß-cell function (C-peptide) were also associated with NPs in PROLONG. Protection from diabetic complications was associated with lower levels of the glycolytic metabolite pyruvate and APOCIII in PROLONG, and with lower levels of thiamine monophosphate and erythritol, a cofactor and intermediate product in the pentose phosphate pathway as well as higher phenylalanine, glycine and serine in DIALONG. Furthermore, T1D individuals showed elevated levels of picolinic acid as compared to the healthy individuals. The present findings suggest a potential beneficial shunting of glycolytic substrates towards the pentose phosphate and one carbon metabolism pathways to promote nucleotide biosynthesis in the liver. These processes might be linked to higher insulin sensitivity and lower liver fat content, and might represent a mechanism for protection from vascular complications in individuals with long-term T1D.

TCRpMHCmodels: Structural modelling of TCR-pMHC class I complexes.

The interaction between the class I major histocompatibility complex (MHC), the peptide presented by the MHC and the T-cell receptor (TCR) is a key determinant of the cellular immune response. Here, we present TCRpMHCmodels, a method for accurate structural modelling of the TCR-peptide-MHC (TCR-pMHC) complex. This TCR-pMHC modelling pipeline takes as input the amino acid sequence and generates models of the TCR-pMHC complex, with a median Cα RMSD of 2.31 Å. TCRpMHCmodels significantly outperforms TCRFlexDock, a specialised method for docking pMHC and TCR structures. TCRpMHCmodels is simple to use and the modelling pipeline takes, on average, only two minutes. Thanks to its ease of use and high modelling accuracy, we expect TCRpMHCmodels to provide insights into the underlying mechanisms of TCR and pMHC interactions and aid in the development of advanced T-cell-based immunotherapies and rational design of vaccines. The TCRpMHCmodels tool is available at http://www.cbs.dtu.dk/services/TCRpMHCmodels/ .