MBASED: allele-specific expression detection in cancer tissues and cell lines

Allele-specific gene expression, ASE, is an important aspect of gene regulation. We developed a novel method MBASED, meta-analysis based allele-specific expression detection for ASE detection using RNA-seq data that aggregates information across multiple single nucleotide variation loci to obtain a gene-level measure of ASE, even when prior phasing information is unavailable. MBASED is capable of one-sample and two-sample analyses and performs well in simulations. We applied MBASED to a panel of cancer cell lines and paired tumor-normal tissue samples, and observed extensive ASE in cancer, but not normal, samples, mainly driven by genomic copy number alterations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-014-0405-3) contains supplementary material, which is available to authorized users

Nuclear positioning rather than contraction controls ordered rearrangements of immunoglobulin loci

Progenitor-B cells recombine their immunoglobulin (Ig) loci to create unique antigen receptors. Despite a common recombination machinery, the Ig heavy and Ig light chain loci rearrange in a stepwise manner. We studied pre-pro-B cells and Rag-/- progenitor-B cells to determine whether Ig locus contraction or nuclear positioning is decisive for stepwise rearrangements. We found that both Ig loci were contracted in pro-B and pre-B cells. Igh relocated from the nuclear lamina to central domains only at the pro-B cell stage, whereas, Igê remained sequestered at the lamina, and only at the pre-B cell stage located to central nuclear domains. Finally, in vitro induced re-positioning of Ig alleles away from the nuclear periphery increased germline transcription of Ig loci in pre-pro-B cells. Thus, Ig locus contraction juxtaposes genomically distant elements to mediate efficient recombination, however, sequential positioning of Ig loci away from the nuclear periphery determines stage-specific accessibility of Ig loci

The 3-D structure of the immunoglobulin heavy chain locus : implications for long-range genomic interactions

The immunoglobulin heavy chain (Igh) locus is organized into distinct regions that encode multiple variable (Vh), diversity (Dh), joining (Jh) and constant (Ch) gene segments. DNA recombination takes place between the Vh, Dh and Jh segments at the Igh locus in developing B cells. The locus undergoes large-scale contraction to facilitate this recombination. However, its structural organization is unknown. It is likely that the structural organization plays a role in this process. By simultaneously visualizing three subregions of the Igh locus using 3D fluorescence in-situ hybridization, we show that looping of the distal Vh segments to the CH segments is observable in pro-B cells. This looping occurs at a significantly higher frequency in pro-B cells compared to CD8⁺ T cells. This indicated that there is a structural reorganization of the locus and not just a simple contraction of the chromatin fiber. To decipher the topology of the locus, 12 genomic markers were used that spanned the entire locus. Spatial distance distributions between different combinations of these markers were determined and compared to computer simulations of different models of chromatin structure. These comparisons revealed that the data agreed with a topology that predicted higher order organization of the chromatin fiber into multiple subcompartments connected by linkers (Multi-Loop Subcompartment model). Compartmentalization of the locus was visualized by labeling the entire locus with hybridization markers. Relative locations of the different Igh sub-regions in 3D space were determined using a trilateration technique. Striking conformational changes can be seen between pre- pro-B and pro-B cells, when the locus transitions from a de-contracted to a contracted state. The implications of the higher order organization of the locus on long-range genomic interactions are discussed. It is evident that the higher order organization is necessary for promoting long- range genomic interactions that would facilitate V(D)J recombination at the Igh locus. In absence of higher order organization, the expected frequency of interactions are much lesse

Cancer neoantigens and immunogenicity: mutation position matters

Cancer mutations can elicit protective immunity. Computational methods are critical for selecting these neoantigens for immunotherapy. While significant progress has been made in the field in predicting peptide presentation, our understanding of which mutated peptide is recognized as foreign by T cells remains limited. We used mouse vaccination studies to examine the features of immunogenic neoantigens and demonstrated that the mutation position is an important criterion for predicting neoantigens

Visualization of looping involving the immunoglobulin heavy-chain locus in developing B cells

The immunoglobulin heavy-chain (IgH) locus undergoes large-scale contraction in B cells poised to undergo IgH V(D)J recombination. We considered the possibility that looping of distinct IgH V regions plays a role in promoting long-range interactions. Here, we simultaneously visualize three subregions of the IgH locus, using three-dimensional fluorescence in situ hybridization. Looping within the IgH locus was observed in both B- and T-lineage cells. However, monoallelic looping of IgH V regions into close proximity of the IgH DJ cluster was detected in developing B cells with significantly higher frequency when compared with hematopoietic progenitor or CD8(+) T-lineage cells. Looping of a subset of IgH V regions, albeit at lower frequency, was also observed in RAG-deficient pro-B cells. Based on these observations, we propose that Ig loci are repositioned by a looping mechanism prior to IgH V(D)J rearrangement to facilitate the joining of Ig variable, diversity, and joining segments

Graph-pMHC: Graph Neural Network Approach to MHC Class II Peptide Presentation and Antibody Immunogenicity

<p>Antigen presentation on MHC Class II (pMHCII presentation) plays an essential role in the adaptive immune response to extracellular pathogens and cancerous cells. But it can also reduce the efficacy of large-molecule drugs by triggering an anti-drug response. Significant progress has been made in pMHCII presentation modeling due to the collection of large-scale pMHC mass spectrometry datasets (ligandomes) and advances in  machine learning. Here, we develop graph-pMHC, a graph neural network approach to predict pMHCII presentation. We derive adjacency matrices for pMHCII using Alphafold2-multimer, and address the peptide-MHC binding groove alignment problem with a simple graph enumeration strategy. We demonstrate that graph-pMHC dramatically outperforms methods with suboptimal inductive biases, such as the multilayer-perceptron-based NetMHCIIpan-4.0 (+20.17% absolute average precision). Finally, we create an antibody drug immunogenicity dataset from clinical trial data, and develop a method for measuring anti-antibody immunogenicity risk using pMHCII presentation models. Our model increases ROC AUC by 2.57% compared to just filtering peptides by hits in OASis alone for predicting antibody drug immunogenicity.</p&gt

The 3D-structure of the Immunoglobulin Heavy Chain Locus: implications for long-range genomic interactions [supplemental data]

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (VH), diversity (DH), joining (JH) and constant (CH) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is

A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate

It is now established that the transcription factors E2A, EBF1 and Foxo1 have critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1, as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate

Expanding cross-presenting dendritic cells enhances oncolytic virotherapy and is critical for long-term anti-tumor immunity

Immunotherapies directly enhancing anti-tumor CD8(+) T cell responses have yielded measurable but limited success, highlighting the need for alternatives. Anti-tumor T cell responses critically depend on antigen presenting dendritic cells (DC), and enhancing mobilization, antigen loading and activation of these cells represent an attractive possibility to potentiate T cell based therapies. Here we show that expansion of DCs by Flt3L administration impacts in situ vaccination with oncolytic Newcastle Disease Virus (NDV). Mechanistically, NDV activates DCs and sensitizes them to dying tumor cells through upregulation of dead-cell receptors and synergizes with Flt3L to promote anti-tumor CD8(+) T cell cross-priming. In vivo, Flt3L-NDV in situ vaccination induces parallel amplification of virus- and tumor-specific T cells, including CD8(+) T cells reactive to newly-described neoepitopes, promoting long-term tumor control. Cross-presenting conventional Type 1 DCs are indispensable for the anti-tumor, but not anti-viral, T cell response, and type I IFN-dependent CD4(+) Th1 effector cells contribute to optimal anti-tumor immunity. These data demonstrate that mobilizing DCs to increase tumor antigen cross-presentation improves oncolytic virotherapy and that neoepitope-specific T cells can be induced without individualized, ex vivo manufactured vaccines. Strategies to advance T cell based immune therapies are mostly focusing on the improvement of CD8 T cell effector functions, such as cytotoxicity or recruitment to the tumor. Here authors show that by combining in situ vaccination with oncolytic Newcastle Disease Virus and Flt3L-driven dendritic cell expansion, the anti-tumor T cell response is amplified via increased antigen cross-presentation.Funding Agencies|Swedish Research Council [2017-00565]; Swedish Society for Medical Research, SSMF [P16-0026]; National Institutes of Health (NIH) [R01CA229818]; NIH [R01CA229818, R01CA257195, R37 CA246239]; Alliance for Cancer Gene Therapy; Applebaum Foundation; Cancer Research Institute (CRI) Lloyd Old STAR Award; Damon Runyon Cancer Research Foundation Clinical Investigator Award</p