Biology subjects

Bentzen, A. K.

Publications and source records attributed to Bentzen, A. K..

7 recordsLinked to original sources

bioRxiv2025

ITRAP2, a flexible and robust strategy to assign antigen recognition of T-cells in a coupled single-cell TCR-pMHC assay

Determining T-cell specificity forms a crucial step toward understanding T-cell involvement in health and disease. Single-cell sequencing technologies allow for co-capture of TCR alpha and beta chains, and their antigen specificity can be determined through peptide-MHC (pMHC) multimer binding and capture of a co-attached barcode oligo. However, SC sequencing often includes a high level of dropouts and risk of cross-contamination. Similarly, barcoded pMHC readouts often suffer from significant background noise. These issues complicate the automatic assignment of pMHC recognition to TCR clonotypes. To overcome these challenges, we developed a method for data denoising - Improved T-cell Receptor Antigen Paring 2 (ITRAP2). This approach significantly reduces noise in single-cell pMHC readouts and allows for accurate identification of TCR specificity. ITRAP2 incorporates statistical tests and confidence metrics for each TCR-pMHC pairing, offering user flexibility in pairing rigor, and allows multiple pMHC assignments to the same T-cell clone in the event of cross-binding within the pMHC multimer library. We tested this method on an in-house generated dataset of 8141 single cells, screened for CD8 T-cell binding using a panel of 100 different barcode-labelled pMHC multimers holding virus-derived peptides, and on a larger public dataset from 10x Genomics with 208,589 T-cells evaluated for recognition using a panel of 50 different pMHCs. In both datasets, ITRAP2 was able to recover TCR-pMHC hits that were missed either when investigating the raw data or analyzing the data using alternative tools. Importantly, we demonstrate that the size of the pMHC multimer library is crucial for accurate pMHC-TCR pairing and that a minimum of 25 pMHC multimer should be included to optimally determine background characteristic, and assigning true positive events.

bioinformatics↗

bioRxiv2025

Mapping of CD8 T-cell recognition to latent EBV infection and neuroantigens reveals HLA-specific depletion of T-cell responses in multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disease with an unclear contribution from antigen-specific CD8 T cells. To enumerate adaptive immune responses associated with MS and to identify MS-related epitopes recognized by CD8 T cells, we evaluated T-cell recognition of 1,158 predicted major histocompatibility complex (MHC)-binding peptides derived from latent EBV antigens (laEBVs) and selected neuroantigens (NAgs) using DNA barcode-labelled pMHC multimers. HLA-B*07:02+ individuals living with relapsing-remitting MS (RRMS) and progressive MS (PMS) were characterized by a relative reduction in EBNA6-specific and MAG-specific CD8 T cells compared to healthy donors. Furthermore, both RRMS and PMS patients exhibited a delayed expansion of CMV- and laEBV-specific CD8 T cells compared to healthy donors indicating an age-specific interaction with disease. Female RRMS patients were moreover characterized by a specific reduction of laEBV-specific CD8 T cells compared to male RRMS patients. NAg-specific CD8 T cells recognized heterogeneous peptides, were of low cellular frequency in peripheral blood, and exhibited a circulating, naive-like, anergic phenotype. In contrast, virus-specific CD8 T cells were characterized by high-frequency cellular responses with an effector-like, tissue-homing phenotype. These immunophenotypes were largely shared between MS and healthy donors. Collectively, our data provide novel insights into CD8 T-cell recognition of both NAg and laEBV antigens in MS and constitute a key reference map of epitopes for further immunomonitoring of EBV- and self-specific CD8 T cells in health and disease.

immunology↗

bioRxiv2025

Antigen-scaffolds loaded with hyper-stable Neoleukin-2/15 expand antigen-specific T cells with a favorable phenotype for adoptive cell therapy

Adoptive cell therapy (ACT) has shown promising results in cancer treatment, however, achieving effective ex vivo expansion of potent, functionally active, and cytotoxic T cells remains challenging. To overcome this, we loaded the engineered cytokine Neoleukin-2/15 (Neo2/15) on our recently established artificial antigen-presenting scaffolds (Ag-scaffolds) to expand antigen-specific T cells. Neo2/15 selectively binds to IL-2R{beta}/{gamma} receptors, enhancing CD8+ T cell proliferation while limiting regulatory T cell expansion. Our study assessed the efficacy of Neo2/15-loaded Ag-scaffolds (Ag-Neo2/15 scaffolds) in expanding antigen-specific T cells from peripheral blood mononuclear cells (PBMCs) of healthy donors. We optimized Ag-scaffold configurations by varying the number of Neo2/15 molecules loaded on Ag-scaffolds and evaluated their impact on T-cell expansion and functionality. We showed that Ag-Neo2/15 scaffolds promoted significant T-cell expansion, with a comparable frequency of antigen-specific CD8+ T cells compared to IL-2/IL-21-loaded Ag-scaffolds (Ag-IL2/21 scaffolds). The CD8+ T cells expanded with Ag-Neo2/15 scaffolds exhibited potent TNF and IFN{gamma} production and expressed high levels of 4{beta}7 integrin, a homing molecule which is important for directing T cells to specific tissues, potentially enhancing their therapeutic potential. T cells expanded with Ag-Neo2/15 scaffolds had superior and durable cytotoxicity against tumor target cells compared to T cells expanded with Ag-IL2/21 scaffolds. These findings were further supported by our single-cell analysis revealing that T cells expanded with Ag-Neo2/15 scaffolds had higher cytotoxic scores and lower dysfunctionality scores compared to T cells expanded with Ag-IL2/21 scaffolds. The single-cell analysis also indicated increased expression of genes linked to cell division and enhanced proliferative capacity in Ag-Neo2/15 expanded T cells. Furthermore, TCR clonality analysis demonstrated that Ag-Neo2/15 scaffolds promoted the expansion of functionally superior T-cell clones. The top clones of CD8+ T cells expanded with Ag-Neo2/15 scaffolds exhibited a favorable phenotype, essential for effective antigen recognition and sustained T-cell mediated cytotoxicity. Our findings suggest that Ag-Neo2/15 scaffolds represent an advancement in ACT by producing high-quality, functional antigen-specific T cells. This method has the potential to improve clinical outcomes in cancer therapy by generating large numbers of highly functional T cells, thereby optimizing the balance between cytotoxicity and proliferation capacity with less exhausted T-cells in expansion protocols.

immunology↗

bioRxiv2024

Identification and characterization of neoantigen-reactive CD8+ T cells following checkpoint blockade therapy in a pan-cancer setting

BackgroundImmune checkpoint blockade (ICB) has been approved as first-line or second-line therapies for an expanding list of malignancies. T cells recognizing mutation-derived neoantigens are hypothesized to play a major role in tumor elimination. However, the dynamics and characteristics of such neoantigen-reactive T cells (NARTs) in the context of ICB are still limitedly understood. MethodsTo explore this, tumor biopsies and peripheral blood were obtained pre- and post-treatment from 20 patients with solid metastatic tumors, in a Phase I basket trial. From whole-exome sequencing and RNA-seq data, patient-specific libraries of neopeptides were predicted and screened with DNA barcode-labeled MHC multimers for CD8+ T cell reactivity, in conjunction with the evaluation of T cell phenotype. ResultsWe were able to detect NARTs in the peripheral blood and tumor biopsies for the majority of the patients; however, we did not observe any significant difference between the disease control and progressive disease patient groups, in terms of the breadth and magnitude of the detected NARTs. We also observed that the hydrophobicity of the peptide played a role in defining neopeptides resulting in NARTs response. A trend towards a treatment-induced phenotype signature was observed in the NARTs post-treatment, with the appearance of Ki67+ CD27+ PD-1+ subsets in the PBMCs and CD39+ Ki67+ TCF-1+ subsets in the TILs. Finally, the estimation of T cells from RNAseq was increasing post versus pre-treatment for disease control patients. ConclusionOur data demonstrates the possibility of monitoring the characteristics of NARTs from tumor biopsies and peripheral blood, and that such characteristics could potentially be incorporated with other immune predictors to understand further the complexity governing clinical success for ICB therapy.

immunology↗

bioRxiv2023

Simultaneous analysis of pMHC binding and reactivity unveils virus-specific CD8 T cell immunity to a concise epitope set

Knowledge of widely recognized T-cell epitopes against common virus infections are vital for immune monitoring and characterization of relevant antigen-specific CD8 T cells and their antigen receptors. We therefore aimed to establish a concise and validated epitope panel for monitoring human virus-specific immunity complete with data on both prevalence of recognition and reactivity in humans. To achieve this, we first establish TCR downregulation, and loss of peptide major histocompatibility (pMHC) multimer-binding, as an early and sensitive marker of T cell reactivity after peptide stimulation. We next applied TCR downregulation in a high-throughput assay by monitoring binding, and loss of binding (i.e. reactivity), to libraries of DNA-barcode labelled pMHC multimers in paired unstimulated/stimulated samples. This novel method allowed us to access T-cell responses in 48 donors towards 929 epitopes recorded in the Immune Epitope Database (IEDB) encompassing 29 virus common infections and 25 different HLA alleles. This yielded a concise panel of 137 virus epitopes, many of which were underrepresented in the public domain, recognized by T cells in peripheral blood. 84% of these epitopes exhibited prevalent reactivity to peptide stimulation, which was associated with effector and long-term memory phenotypes. Conversely, non-reactive responses correlated with naive and immunosenescence phenotypes. This study represents the largest effort to unbiasedly assess T-cell recognition and reactivity to common virus infections in healthy individuals providing a minimal epitope panel for monitoring adaptive immune responses in humans. Significance StatementCD8 T-cell epitopes are widely available in public databases yet many are not recognized in the general population. Here we undertook an exhaustive screening process using "state-of-the-art" methods to assess both T-cell recognition and reactivity against common virus infections, which holds significant implications for shaping T-cell immunity and disease protection. We identify 137 commonly recognized epitopes from common virus infections to which T cell responses are expected to occur in human donors. Importantly, several of the verified epitopes were underreported in public databases compared to their observed prevalence of recognition and high cellular frequency making this an important reference dataset and resource for immunologists studying antigen-specific T cells across different immunopathologies and contexts including autoimmunity, infectious disease and cancer immunotherapy.

immunology↗

bioRxiv2022

ATRAP - Accurate 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 is 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 ATRAP (Accurate 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.

immunology↗

bioRxiv2018

NetTCR: sequence-based prediction of TCR binding to peptide-MHC complexes using convolutional neural networks

Predicting epitopes recognized by cytotoxic T cells has been a long standing challenge within the field of immuno- and bioinformatics. While reliable predictions of peptide binding are available for most Major Histocompatibility Complex class I (MHCI) alleles, prediction models of T cell receptor (TCR) interactions with MHC class I-peptide complexes remain poor due to the limited amount of available training data. Recent next generation sequencing projects have however generated a considerable amount of data relating TCR sequences with their cognate HLA-peptide complex target. Here, we utilize such data to train a sequence-based predictor of the interaction between TCRs and peptides presented by the most common human MHCI allele, HLA-A*02:01. Our model is based on convolutional neural networks, which are especially designed to meet the challenges posed by the large length variations of TCRs. We show that such a sequence-based model allows for the identification of TCRs binding a given cognate peptide-MHC target out of a large pool of non-binding TCRs.

bioinformatics↗