ralfne

• Dahal-Koirala, Shiva; Balaban, Gabriel; Neumann, Ralf Stefan; Scheffer, Lonneke; Lundin, Knut & Greiff, Victor [Vis alle 9 forfattere av denne artikkelen] (2022). TCRpower: quantifying the detection power of T-cell receptor sequencing with a novel computational pipeline calibrated by spike-in sequences. Briefings in Bioinformatics. ISSN 1467-5463. 23(2), s. 1–14. doi: 10.1093/bib/bbab566. Fulltekst i vitenarkiv Vis sammendrag

  T-cell receptor (TCR) sequencing has enabled the development of innovative diagnostic tests for cancers, autoimmune diseases and other applications. However, the rarity of many T-cell clonotypes presents a detection challenge, which may lead to misdiagnosis if diagnostically relevant TCRs remain undetected. To address this issue, we developed TCRpower, a novel computational pipeline for quantifying the statistical detection power of TCR sequencing methods. TCRpower calculates the probability of detecting a TCR sequence as a function of several key parameters: in-vivo TCR frequency, T-cell sample count, read sequencing depth and read cutoff. To calibrate TCRpower, we selected unique TCRs of 45 T-cell clones (TCCs) as spike-in TCRs. We sequenced the spike-in TCRs from TCCs, together with TCRs from peripheral blood, using a 5′ RACE protocol. The 45 spike-in TCRs covered a wide range of sample frequencies, ranging from 5 per 100 to 1 per 1 million. The resulting spike-in TCR read counts and ground truth frequencies allowed us to calibrate TCRpower. In our TCR sequencing data, we observed a consistent linear relationship between sample and sequencing read frequencies. We were also able to reliably detect spike-in TCRs with frequencies as low as one per million. By implementing an optimized read cutoff, we eliminated most of the falsely detected sequences in our data (TCR α-chain 99.0% and TCR β-chain 92.4%), thereby improving diagnostic specificity. TCRpower is publicly available and can be used to optimize future TCR sequencing experiments, and thereby enable reliable detection of disease-relevant TCRs for diagnostic applications.

• Yao, Ying; Zia, Asima; Neumann, Ralf Stefan; Pavlović, Milena; Balaban, Gabriel & Lundin, Knut Erik Aslaksen [Vis alle 8 forfattere av denne artikkelen] (2021). T cell receptor repertoire as a potential diagnostic marker for celiac disease. Clinical Immunology. ISSN 1521-6616. 222:108621, s. 1–7. doi: 10.1016/j.clim.2020.108621. Fulltekst i vitenarkiv Vis sammendrag

  An individual's T cell repertoire is skewed towards some specificities as a result of past antigen exposure and subsequent clonal expansion. Identifying T cell receptor signatures associated with a disease is challenging due to the overall complexity of antigens and polymorphic HLA allotypes. In celiac disease, the antigen epitopes are well characterised and the specific HLA-DQ2-restricted T-cell repertoire associated with the disease has been explored in depth. By investigating T cell receptor repertoires of unsorted lamina propria T cells from 15 individuals, we provide the first proof-of-concept study showing that it could be possible to infer disease state by matching against a priori known disease-associated T cell receptor sequences.

• Risnes, Louise Fremgaard; Eggesbø, Linn Margrethe; Zühlke, Stephanie; Dahal-Koirala, Shiva; Neumann, Ralf Stefan & Lundin, Knut Erik Aslaksen [Vis alle 8 forfattere av denne artikkelen] (2021). Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease. Mucosal Immunology. ISSN 1933-0219. 14(4), s. 842–851. doi: 10.1038/s41385-021-00385-8. Fulltekst i vitenarkiv Vis sammendrag

  Gut intraepithelial γδ and CD8+ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αβ T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αβ T-cell clonotypes that may not be specific for the gluten antigen.

• Dahal-Koirala, Shiva; Risnes, Louise Fremgaard; Neumann, Ralf Stefan; Christophersen, Asbjørn; Lundin, Knut E. A. & Sandve, Geir Kjetil [Vis alle 8 forfattere av denne artikkelen] (2021). Comprehensive Analysis of CDR3 Sequences in Gluten-Specific T-Cell Receptors Reveals a Dominant R-Motif and Several New Minor Motifs. Frontiers in Immunology. 12:639672, s. 1–13. doi: 10.3389/fimmu.2021.639672. Fulltekst i vitenarkiv Vis sammendrag

  Gluten-specific CD4+ T cells are drivers of celiac disease (CeD). Previous studies of gluten-specific T-cell receptor (TCR) repertoires have found public TCRs shared across multiple individuals, biased usage of particular V-genes and conserved CDR3 motifs. The CDR3 motifs within the gluten-specific TCR repertoire, however, have not been systematically investigated. In the current study, we analyzed the largest TCR database of gluten-specific CD4+ T cells studied so far consisting of TCRs of 3122 clonotypes from 63 CeD patients. We established a TCR database from CD4+ T cells isolated with a mix of HLA-DQ2.5:gluten tetramers representing four immunodominant gluten epitopes. In an unbiased fashion we searched by hierarchical clustering for common CDR3 motifs among 2764 clonotypes. We identified multiple CDR3α, CDR3β, and paired CDR3α:CDR3β motif candidates. Among these, a previously known conserved CDR3β R-motif used by TRAV26-1/TRBV7-2 TCRs specific for the DQ2.5-glia-α2 epitope was the most prominent motif. Furthermore, we identified the epitope specificity of altogether 16 new CDR3α:CDR3β motifs by comparing with TCR sequences of 231 T-cell clones with known specificity and TCR sequences of cells sorted with single HLA-DQ2.5:gluten tetramers. We identified 325 public TCRα and TCRβ sequences of which 145, 102 and 78 belonged to TCRα, TCRβ and paired TCRαβ sequences, respectively. While the number of public sequences was depended on the number of clonotypes in each patient, we found that the proportion of public clonotypes from the gluten-specific TCR repertoire of given CeD patients appeared to be stable (median 37%). Taken together, we here demonstrate that the TCR repertoire of CD4+ T cells specific to immunodominant gluten epitopes in CeD is diverse, yet there is clearly biased V-gene usage, presence of public TCRs and existence of conserved motifs of which R-motif is the most prominent.

• Ting, Yi Tian; Dahal-Koirala, Shiva; Kim, Hui Shi Keshia; Qiao, Shuo Wang; Neumann, Ralf Stefan & Lundin, Knut Erik Aslaksen [Vis alle 10 forfattere av denne artikkelen] (2020). A molecular basis for the T cell response in HLA-DQ2.2 mediated celiac disease. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 117(6), s. 3063–3073. doi: 10.1073/pnas.1914308117. Fulltekst i vitenarkiv Vis sammendrag

  The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4 + T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5–mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.

• Iversen, Rasmus; Roy, Bishnudeo; Stamnæs, Jorunn; Høydahl, Lene Støkken; Hnida, Kathrin & Neumann, Ralf Stefan [Vis alle 9 forfattere av denne artikkelen] (2019). Efficient T cell–B cell collaboration guides autoantibody epitope bias and onset of celiac disease. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 116(30), s. 15134–15139. doi: 10.1073/pnas.1901561116. Fulltekst i vitenarkiv Vis sammendrag

  B cells play important roles in autoimmune diseases through autoantibody production, cytokine secretion, or antigen presentation to T cells. In most cases, the contribution of B cells as antigen-presenting cells is not well understood. We have studied the autoantibody response against the enzyme transglutaminase 2 (TG2) in celiac disease patients by generating recombinant antibodies from single gut plasma cells reactive with discrete antigen domains and by undertaking proteomic analysis of anti-TG2 serum antibodies. The majority of the cells recognized epitopes in the N-terminal domain of TG2. Antibodies recognizing C-terminal epitopes interfered with TG2 cross-linking activity, and B cells specific for C-terminal epitopes were inefficient at taking up TG2-gluten complexes for presentation to gluten-specific T cells. The bias toward N-terminal epitopes hence reflects efficient T-B collaboration. Production of antibodies against N-terminal epitopes coincided with clinical onset of disease, suggesting that TG2-reactive B cells with certain epitope specificities could be the main antigen-presenting cells for pathogenic, gluten-specific T cells. The link between B cell epitopes, antigen presentation, and disease onset provides insight into the pathogenic mechanisms of a T cell-mediated autoimmune condition.

• Dahal-Koirala, Shiva; Neumann, Ralf Stefan; Jahnsen, Jørgen; Lundin, Knut Erik Aslaksen & Sollid, Ludvig Magne (2019). On the immune response to barley in celiac disease: Biased and public T-cell receptor usage to a barley unique and immunodominant gluten epitope. European Journal of Immunology. ISSN 0014-2980. s. 1–14. doi: 10.1002/eji.201948253. Fulltekst i vitenarkiv Vis sammendrag

  Celiac disease (CeD) is driven by CD4+ T‐cell responses to dietary gluten proteins of wheat, barley, and rye when deamidated gluten epitopes are presented by certain disease‐associated HLA‐DQ allotypes. About 90% of the CeD patients express HLA‐DQ2.5. In such patients, five gluten epitopes dominate the anti‐gluten T‐cell response; two epitopes unique to wheat, two epitopes present in wheat, barley, and rye and one epitope unique to barley. Despite presence of barley in commonly consumed food and beverages and hence being a prominent source of gluten, knowledge about T‐cell responses elicited by barley in CeD is scarce. Therefore, in this study, we explored T‐cell response toward the barley unique epitope DQ2.5‐hor‐3 (PIPEQPQPY) by undertaking HLA‐DQ:gluten peptide tetramer staining, single‐cell T‐cell receptor (TCR) αβ sequencing, T‐cell cloning, and T‐cell proliferation studies. We demonstrate that majority of the CeD patients generate T‐cell response to DQ2.5‐hor‐3, and this response is characterized by clonal expansion, preferential TCR V‐gene usage and public TCR features thus echoing findings previously made for wheat gluten epitopes. The knowledge that biased and public TCRs underpin the T‐cell response to all the immunodominant gluten epitopes in CeD suggests that such T cells are promising diagnostic and therapeutic targets.

• Eggesbø, Linn Margrethe; Risnes, Louise Fremgaard; Neumann, Ralf Stefan; Lundin, Knut Erik Aslaksen; Christophersen, Asbjørn & Sollid, Ludvig Magne (2019). Single-cell TCR sequencing of gut intraepithelial γδ T cells reveals a vast and diverse repertoire in celiac disease. Mucosal Immunology. ISSN 1933-0219. s. 1–9. doi: 10.1038/s41385-019-0222-9. Fulltekst i vitenarkiv Vis sammendrag

  A hallmark of celiac disease (CeD), a chronic condition driven by cereal gluten exposure, is increase of gut intraepithelial γδ T cells. This may indicate pathogenic involvement of γδ T cells and existence of disease-specific γδ T-cell receptors (TCRs) recognizing defined antigen(s). We performed high-throughput and paired γδ TCR sequencing of single intraepithelial γδ T cells of untreated CeD patients (n = 8; 1821 cells), CeD patients treated with a gluten-free diet (n = 5; 436 cells) and controls (n = 7; 1068 cells). We found that CeD patients, both untreated and treated, had larger and more diverse γδ TCR repertoires, more frequent usage of TRDV1 and TRDV3 and different patterns of TCRγ/TCRδ-pairing compared with controls. Although we observed no public CDR3δ sequences, there were several public CDR3γ sequences—many of which were shared by not only the CeD patients, but also by the controls. These public CDR3s were characterized by few N/P nucleotide insertions with germline and near-germline configuration, hence being easy to generate. Previous findings of CeD-specific CDR3 motifs were not replicated. Thus, being unable to raise evidence for CeD-specific γδ TCRs in this first large, paired γδ TCR single-cell sequencing study, we project challenges for identification of CeD-relevant γδ TCR ligands.

• Casado, Raquel Bartolomé; Landsverk, Ole J. B.; Chauhan, Sudhir Kumar; Richter, Lisa Maria; Phung, Danh & Greiff, Victor [Vis alle 18 forfattere av denne artikkelen] (2019). Resident memory CD8 T cells persist for years in human small intestine. Journal of Experimental Medicine (JEM). ISSN 0022-1007. 216(10), s. 2412–2426. doi: 10.1084/jem.20190414. Fulltekst i vitenarkiv Vis sammendrag

  Resident memory CD8 T (Trm) cells have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI, we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for >1 yr in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine producers, exhibiting a polyfunctional (IFN-γ+ IL-2+ TNF-α+) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders.

• Dahal-Koirala, Shiva; Ciacchi, Laura; Petersen, Jan; Risnes, Louise Fremgaard; Neumann, Ralf Stefan & Christophersen, Asbjørn [Vis alle 11 forfattere av denne artikkelen] (2018). Discriminative T-cell receptor recognition of highly homologous HLA-DQ2-bound gluten epitopes. Journal of Biological Chemistry. ISSN 0021-9258. 294(3), s. 941–952. doi: 10.1074/jbc.RA118.005736. Fulltekst i vitenarkiv Vis sammendrag

  Celiac disease (CeD) provides an opportunity to study the specificity underlying human T-cell responses to an array of similar epitopes presented by the same human leukocyte antigen II (HLA-II) molecule. Here, we investigated T-cell responses to the two immunodominant and highly homologous HLA-DQ2.5–restricted gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). Using HLA-DQ2.5–DQ2.5-glia-α1a and HLA-DQ2.5–DQ2.5-glia-ω1 tetramers and single-cell αβ T-cell receptor (TCR) sequencing, we observed that despite similarity in biased variable-gene usage in the TCR repertoire responding to these nearly identical peptide–HLA-II complexes, most of the T cells are specific for either of the two epitopes. To understand the molecular basis of this exquisite fine specificity, we undertook Ala substitution assays revealing that the p7 residue (Leu/Gln) is critical for specific epitope recognition by both DQ2.5-glia-α1a– and DQ2.5-glia-ω1–reactive T-cell clones. We determined high-resolution binary crystal structures of HLA-DQ2.5 bound to DQ2.5-glia-α1a (2.0 Å) and DQ2.5-glia-ω1 (2.6 Å). These structures disclosed that differences around the p7 residue subtly alter the neighboring substructure and electrostatic properties of the HLA-DQ2.5–peptide complex, providing the fine specificity underlying the responses against these two highly homologous gluten epitopes. This study underscores the ability of TCRs to recognize subtle differences in the peptide–HLA-II landscape in a human disease setting.

• Risnes, Louise Fremgaard; Christophersen, Asbjørn; Dahal-Koirala, Shiva; Neumann, Ralf Stefan; Sandve, Geir Kjetil & Sarna, Vikas Kumar [Vis alle 9 forfattere av denne artikkelen] (2018). Disease-driving CD4+ T cell clonotypes persist for decades in celiac disease. Journal of Clinical Investigation. ISSN 0021-9738. 128(6), s. 2642–2650. doi: 10.1172/JCI98819. Fulltekst i vitenarkiv Vis sammendrag

  Little is known about the repertoire dynamics and persistence of pathogenic T cells in HLA-associated disorders. In celiac disease, a disorder with a strong association with certain HLA-DQ allotypes, presumed pathogenic T cells can be visualized and isolated with HLA-DQ:gluten tetramers, thereby enabling further characterization. Single and bulk populations of HLA-DQ:gluten tetramer–sorted CD4+ T cells were analyzed by high-throughput DNA sequencing of rearranged TCR-α and -β genes. Blood and gut biopsy samples from 21 celiac disease patients, taken at various stages of disease and in intervals of weeks to decades apart, were examined. Persistence of the same clonotypes was seen in both compartments over decades, with up to 53% overlap between samples obtained 16 to 28 years apart. Further, we observed that the recall response following oral gluten challenge was dominated by preexisting CD4+ T cell clonotypes. Public features were frequent among gluten-specific T cells, as 10% of TCR-α, TCR-β, or paired TCR-αβ amino acid sequences of total 1813 TCRs generated from 17 patients were observed in 2 or more patients. In established celiac disease, the T cell clonotypes that recognize gluten are persistent for decades, making up fixed repertoires that prevalently exhibit public features. These T cells represent an attractive therapeutic target.

• Bråte, Jon; Neumann, Ralf Stefan; Fromm, Bastian; Haraldsen, Arthur Alexander Blørstad; Tarver, James E. & Suga, Hiroshi [Vis alle 11 forfattere av denne artikkelen] (2018). Unicellular origin of the animal microRNA machinery. Current Biology. ISSN 0960-9822. 28(20), s. 3288–3295.e5. doi: 10.1016/j.cub.2018.08.018. Fulltekst i vitenarkiv Vis sammendrag

  The emergence of multicellular animals was associated with an increase in phenotypic complexity and with the acquisition of spatial cell differentiation and embryonic development. Paradoxically, this phenotypic transition was not paralleled by major changes in the underlying developmental toolkit and regulatory networks. In fact, most of these systems are ancient, established already in the unicellular ancestors of animals [1, 2, 3, 4, 5]. In contrast, the Microprocessor protein machinery, which is essential for microRNA (miRNA) biogenesis in animals, as well as the miRNA genes themselves produced by this Microprocessor, have not been identified outside of the animal kingdom [6]. Hence, the Microprocessor, with the key proteins Pasha and Drosha, is regarded as an animal innovation [7, 8, 9]. Here, we challenge this evolutionary scenario by investigating unicellular sister lineages of animals through genomic and transcriptomic analyses. We identify in Ichthyosporea both Drosha and Pasha (DGCR8 in vertebrates), indicating that the Microprocessor complex evolved long before the last common ancestor of animals, consistent with a pre-metazoan origin of most of the animal developmental gene elements. Through small RNA sequencing, we also discovered expressed bona fide miRNA genes in several species of the ichthyosporeans harboring the Microprocessor. A deep, pre-metazoan origin of the Microprocessor and miRNAs comply with a view that the origin of multicellular animals was not directly linked to the innovation of these key regulatory components.

• Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Neumann, Ralf Stefan; Bjerregaard-Andersen, Kaare; Nilssen, Nicolay Rustad & Sollid, Ludvig Magne [Vis alle 8 forfattere av denne artikkelen] (2018). Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system. PLOS ONE. 13:e0195868(4), s. 1–15. doi: 10.1371/journal.pone.0195868. Fulltekst i vitenarkiv Vis sammendrag

  There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

• Roy, Bishnudeo; Neumann, Ralf Stefan; Snir, Omri; Iversen, Rasmus; Sandve, Geir Kjetil & Lundin, Knut Erik Aslaksen [Vis alle 7 forfattere av denne artikkelen] (2017). High-Throughput Single-Cell Analysis of B Cell Receptor Usage among Autoantigen-Specific Plasma Cells in Celiac Disease. Journal of Immunology. ISSN 0022-1767. 199((2)), s. 782–791. doi: 10.4049/jimmunol.1700169. Fulltekst i vitenarkiv Vis sammendrag

  Characterization of Ag-specific BCR repertoires is essential for understanding disease mechanisms involving humoral immunity. This is optimally done by interrogation of paired H chain V region (VH) and L chain V region (VL) sequences of individual and Ag-specific B cells. By applying single-cell high-throughput sequencing on gut lesion plasma cells (PCs), we have analyzed the transglutaminase 2 (TG2)-specific VH:VL autoantibody repertoire of celiac disease (CD) patients. Autoantibodies against TG2 are a hallmark of CD, and anti-TG2 IgA-producing gut PCs accumulate in patients upon gluten ingestion. Altogether, we analyzed paired VH and VL sequences of 1482 TG2-specific and 1421 non–TG2-specific gut PCs from 10 CD patients. Among TG2-specific PCs, we observed a striking bias in IGHV and IGKV/IGLV gene usage, as well as pairing preferences with a particular presence of the IGHV5-51:IGKV1-5 pair. Selective and biased VH:VL pairing was particularly evident among expanded clones. In general, TG2-specific PCs had lower numbers of mutations both in VH and VL genes than in non–TG2-specific PCs. TG2-specific PCs using IGHV5-51 had particularly few mutations. Importantly, VL segments paired with IGHV5-51 displayed proportionally low mutation numbers, suggesting that the low mutation rate among IGHV5-51 PCs is dictated by the BCR specificity. Finally, we observed selective amino acid changes in VH and VL and striking CDR3 length and J segment selection among TG2-specific IGHV5-51:IGKV1-5 pairs. Hence this study reveals features of a disease- and Ag-specific autoantibody repertoire with preferred VH:VL usage and pairings, limited mutations, clonal dominance, and selection of particular CDR3 sequences. © 2017 American Association of Immunologists

• Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva; Neumann, Ralf Stefan & Bergseng, Elin [Vis alle 15 forfattere av denne artikkelen] (2017). A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions. JCI Insight. 2(17). doi: 10.1172/jci.insight.95193. Fulltekst i vitenarkiv Vis sammendrag

  Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3β loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3β loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3β. This allowed prediction of expanded DQ2.5-glia-α2–reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues. This research was originally published in JCI Insight. © 2018 American Society for Clinical Investigation

• Kumar, Surendra; Krabberød, Anders Kristian; Neumann, Ralf Stefan; Michalickova, Katerina; Zhao, Sen & Zhang, Xiaoli [Vis alle 7 forfattere av denne artikkelen] (2015). BIR pipeline for preparation of phylogenomic data. Evolutionary Bioinformatics. 11, s. 79–83. doi: 10.4137/EBO.S10189. Fulltekst i vitenarkiv
• Bråte, Jon; Adamski, Marcin; Neumann, Ralf Stefan; Shalchian-Tabrizi, Kamran & Adamska, Maja (2015). Regulatory RNA at the root of animals: dynamic expression of developmental lincRNAs in the calcisponge Sycon ciliatum. Proceedings of the Royal Society of London. Biological Sciences. ISSN 0962-8452. 282(1821), s. 1–9. doi: 10.1098/rspb.2015.1746. Fulltekst i vitenarkiv Vis sammendrag

  Long non-coding RNAs (lncRNAs) play important regulatory roles during animal development, and it has been hypothesized that an RNA-based gene regulation was important for the evolution of developmental complexity in animals. However, most studies of lncRNA gene regulation have been performed using model animal species, and very little is known about this type of gene regulation in non-bilaterians. We have therefore analysed RNA-Seq data derived from a comprehensive set of embryogenesis stages in the calcareous sponge Sycon ciliatum and identified hundreds of developmentally expressed intergenic lncRNAs (lincRNAs) in this species. In situ hybridization of selected lincRNAs revealed dynamic spatial and temporal expression during embryonic development. More than 600 lincRNAs constitute integral parts of differentially expressed gene modules, which also contain known developmental regulatory genes, e.g. transcription factors and signalling molecules. This study provides insights into the non-coding gene repertoire of one of the earliest evolved animal lineages, and suggests that RNA-based gene regulation was likely present in the last common ancestor of animals.

• Neumann, Ralf Stefan; Kumar, Surendra & Shalchian-Tabrizi, Kamran (2014). BLAST output visualization in the new sequencing era. Briefings in Bioinformatics. ISSN 1467-5463. 15(4), s. 484–503. doi: 10.1093/bib/bbt009. Fulltekst i vitenarkiv
• Neumann, Ralf Stefan; Kumar, Surendra; Haverkamp, Thomas Hendricus Augustus & Shalchian-Tabrizi, Kamran (2014). BLASTGrabber: A bioinformatic tool for visualization, analysis and sequence selection of massive BLAST data. BMC Bioinformatics. 15(128). doi: 10.1186/1471-2105-15-128. Fulltekst i vitenarkiv Vis sammendrag

  Background Advances in sequencing efficiency have vastly increased the sizes of biological sequence databases, including many thousands of genome-sequenced species. The BLAST algorithm remains the main search engine for retrieving sequence information, and must consequently handle data on an unprecedented scale. This has been possible due to high-performance computers and parallel processing. However, the raw BLAST output from contemporary searches involving thousands of queries becomes ill-suited for direct human processing. Few programs attempt to directly visualize and interpret BLAST output; those that do often provide a mere basic structuring of BLAST data. Results Here we present a bioinformatics application named BLASTGrabber suitable for high-throughput sequencing analysis. BLASTGrabber, being implemented as a Java application, is OS-independent and includes a user friendly graphical user interface. Text or XML-formatted BLAST output files can be directly imported, displayed and categorized based on BLAST statistics. Query names and FASTA headers can be analysed by text-mining. In addition to visualizing sequence alignments, BLAST data can be ordered as an interactive taxonomy tree. All modes of analysis support selection, export and storage of data. A Java interface-based plugin structure facilitates the addition of customized third party functionality. Conclusion The BLASTGrabber application introduces new ways of visualizing and analysing massive BLAST output data by integrating taxonomy identification, text mining capabilities and generic multi-dimensional rendering of BLAST hits. The program aims at a non-expert audience in terms of computer skills; the combination of new functionalities makes the program flexible and useful for a broad range of operations.

Se alle arbeider i NVA

• Eggesbø, Linn Margrethe; Risnes, Louise Fremgaard; Neumann, Ralf Stefan; Lundin, Knut E. A.; Christophersen, Asbjørn Otto & Sollid, Ludvig Magne (2021). Single-cell TCR repertoire analysis reveals highly polyclonal composition of human intraepithelial CD8⁺ αβ T lymphocytes in untreated celiac disease. European Journal of Immunology. ISSN 0014-2980. 51(6), s. 1542–1545. doi: 10.1002/eji.202048974. Fulltekst i vitenarkiv
• Haraldsen, Arthur Alexander Blørstad; Bråte, Jon; Neumann, Ralf Stefan; Fromm, Bastian; Grini, Paul Eivind & Shalchian-Tabrizi, Kamran (2016). Pre-metazoan origin of animal microRNAs. Fulltekst i vitenarkiv
• Bjorbækmo, Marit Markussen; Neumann, Ralf Stefan; Haraldsen, Arthur Alexander Blørstad & Shalchian-Tabrizi, Kamran (2015). HIDDEN PROTIST PARASITES: HOW CAN THEY BE DETECTED? Fulltekst i vitenarkiv
• Bjorbækmo, Marit Markussen; Neumann, Ralf Stefan; Haraldsen, Arthur Alexander Blørstad & Shalchian-Tabrizi, Kamran (2015). Expanding our knowledge about parasites using PATHOS-DB (The PArasiTe–HOSt-DataBase). Fulltekst i vitenarkiv
• Høydahl, Lene Støkken; Neumann, Ralf Stefan; Qiao, Shuo Wang; Sollid, Ludvig Magne; Sandlie, Inger & Løset, Geir Åge (2011). Development of a High Affinity TCR Reagent by Phage Display to Study Antigen Presentation in Celiac Disease. Scandinavian Journal of Immunology. ISSN 0300-9475. 73(4), s. 372–372. Fulltekst i vitenarkiv

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