Personal neoantigen cancer vaccines: The momentum builds

Neoantigen-based cancer vaccines designed to target the unique immunogenic mutations arising in each patient’s tumor are breathing new life into a struggling approach. Data continue to demonstrate the importance of neoantigens in immune control of cancer. Despite manufacturing complexity, outstanding questions and desired further improvements, neoantigen vaccines are currently undergoing clinical evaluation

Requirement for the NF-kappa B family member Re1A in the development of secondary lymphoid organs

The transcription factor nuclear factor (NF)-kappaB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-kappaB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-kappaB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the trice that are born lack lymph nodes, foyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell-dependent antigen responses. Analyses of TNFR1/1RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs

Targeting individual cells by barcode in pooled sequence libraries

Transcriptional profiling of thousands of single cells in parallel by RNA-seq is now routine. However, due to reliance on pooled library preparation, targeting analysis to particular cells of interest is difficult. Here, we present a multiplexed PCR method for targeted sequencing of select cells from pooled single-cell sequence libraries. We demonstrated this molecular enrichment method on multiple cell types within pooled single-cell RNA-seq libraries produced from primary human blood cells. We show how molecular enrichment can be combined with FACS to efficiently target ultra-rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC) subset, in order to reduce the required sequencing effort to profile single cells by 100-fold. Our results demonstrate that DNA barcodes identifying cells within pooled sequencing libraries can be used as targets to enrich for specific molecules of interest, for example reads from a set of target cells.National Institute of Allergy and Infectious Diseases (U.S.) (U24AI11866803)National Human Genome Research Institute (U.S.) (RM1HG00619307)Broad Institute of MIT and HarvardBurroughs Wellcome Fund (Career Award at the Scientific Interface)National Science Foundation (U.S.). Graduate Research FellowshipNational Human Genome Research Institute (U.S.). Centers of Excellence in Genomic Science (RM1HG00619307)Massachusetts Institute of Technolog

Positional specificity of different transcription factor classes within enhancers

Gene expression is controlled by sequence-specific transcription factors (TFs), which bind to regulatory sequences in DNA. TF binding occurs in nucleosome-depleted regions of DNA (NDRs), which generally encompass regions with lengths similar to those protected by nucleosomes. However, less is known about where within these regions specific TFs tend to be found. Here, we characterize the positional bias of inferred binding sites for 103 TFs within ∼500,000 NDRs across 47 cell types. We find that distinct classes of TFs display different binding preferences: Some tend to have binding sites toward the edges, some toward the center, and some at other positions within the NDR. These patterns are highly consistent across cell types, suggesting that they may reflect TF-specific intrinsic structural or functional characteristics. In particular, TF classes with binding sites at NDR edges are enriched for those known to interact with histones and chromatin remodelers, whereas TFs with central enrichment interact with other TFs and cofactors such as p300. Our results suggest distinct regiospecific binding patterns and functions of TF classes within enhancers. Keywords: transcription factor binding; gene regulation; genomics; chromatin structureNational Human Genome Research Institute (U.S.) (Grant 2U54HG003067-10)National Institute of General Medical Sciences (U.S.) (Grant T32GM007753

Requirement for the NF-κB Family Member RelA in the Development of Secondary Lymphoid Organs

The transcription factor nuclear factor (NF)-κB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-κB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-κB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the mice that are born lack lymph nodes, Peyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell–dependent antigen responses. Analyses of TNFR1/RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs

TGF-β Suppresses β-Catenin-Dependent Tolerogenic Activation Program in Dendritic Cells

The mechanisms that underlie the critical dendritic cell (DC) function in maintainance of peripheral immune tolerance are incompletely understood, although the β-catenin signaling pathway is critical for this role. The molecular details by which β-catenin signaling is regulated in DCs are unknown. Mechanical disruption of murine bone marrow-derived DC (BMDC) clusters activates DCs while maintaining their tolerogenic potential and this activation is associated with β-catenin signaling, providing a useful model with which to explore tolerance-associated β-catenin signaling in DCs. In this report, we demonstrate novel molecular features of the signaling events that control DC activation in response to mechanical stimulation. Non-canonical β-catenin signaling is an essential component of this tolerogenic activation and is modulated by adhesion molecules, including integrins. This unique β-catenin-dependent signaling pathway is constitutively active at low levels, suggesting that mechanical stimulation is not necessarily required for induction of this unique activation program. We additionally find that the immunomodulatory cytokine TGF-β antagonizes β-catenin in DCs, thereby selectively suppressing signaling associated with tolerogenic DC activation while having no impact on LPS-induced, β-catenin-independent immunogenic activation. These findings provide new molecular insight into the regulation of a critical signaling pathway for DC function in peripheral immune tolerance

High-Resolution Sequencing and Modeling Identifies Distinct Dynamic RNA Regulatory Strategies

Cells control dynamic transitions in transcript levels by regulating transcription, processing, and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level; editing sites; and transcription, processing, and degradation rates of each transcript at a splice junction resolution during the LPS response of mouse dendritic cells. Four key regulatory strategies, dominated by RNA transcription changes, generate most temporal gene expression patterns. Noncanonical strategies that also employ dynamic posttranscriptional regulation control only a minority of genes, but provide unique signal processing features. We validate Tristetraprolin (TTP) as a major regulator of RNA degradation in one noncanonical strategy. Applying DRiLL to the regulation of noncoding RNAs and to zebrafish embryogenesis demonstrates its broad utility. Our study provides a new quantitative approach to discover transcriptional and posttranscriptional events that control dynamic changes in transcript levels using RNA sequencing data.National Human Genome Research Institute (U.S.) (Centers for Excellence in Genomics Science 1P50HG006193-01)Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Pioneer Award)Massachusetts Institute of Technology. William Asbjornsen Albert Memorial FellowshipXerox Fellowship Progra

Deciphering molecular circuits from genetic variation underlying transcriptional responsiveness to stimuli

Individual genetic variation affects gene expression in response to stimuli, often by influencing complex molecular circuits. Here we combine genomic and intermediate-scale transcriptional profiling with computational methods to identify variants that affect the responsiveness of genes to stimuli (responsiveness QTLs; reQTLs) and to position these variants in molecular circuit diagrams. We apply this approach to study variation in transcriptional responsiveness to pathogen components in dendritic cells from recombinant inbred mouse strains. We identify reQTLs that correlate with particular stimuli and position them in known pathways. For example, in response to a virus-like stimulus, a trans-acting variant acts as an activator of the antiviral response; using RNAi, we identify Rgs16 as the likely causal gene. Our approach charts an experimental and analytic path to decipher the mechanisms underlying genetic variation in circuits that control responses to stimuli