The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

Bidirectional epigenetic editing reveals hierarchies in gene regulation.

CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.

Organ- and Cell-Selective Delivery of mRNA In Vivo Using Guanidinylated Serinol Charge-Altering Releasable Transporters.

Selective RNA delivery is required for the broad implementation of RNA clinical applications, including prophylactic and therapeutic vaccinations, immunotherapies for cancer, and genome editing. Current polyanion delivery relies heavily on cationic amines, while cationic guanidinium systems have received limited attention due in part to their strong polyanion association, which impedes intracellular polyanion release. Here, we disclose a general solution to this problem in which cationic guanidinium groups are used to form stable RNA complexes upon formulation but at physiological pH undergo a novel charge-neutralization process, resulting in RNA release. This new delivery system consists of guanidinylated serinol moieties incorporated into a charge-altering releasable transporter (GSer-CARTs). Significantly, systematic variations in structure and formulation resulted in GSer-CARTs that exhibit highly selective mRNA delivery to the lung (∼97%) and spleen (∼98%) without targeting ligands. Illustrative of their breadth and translational potential, GSer-CARTs deliver circRNA, providing the basis for a cancer vaccination strategy, which in a murine model resulted in antigen-specific immune responses and effective suppression of established tumors.

Pathways for macrophage uptake of cell-free circular RNAs.

Circular RNAs (circRNAs) are stable RNAs present in cell-free RNA, which may comprise cellular debris and pathogen genomes. Here, we investigate the phenomenon and mechanism of cellular uptake and intracellular fate of exogenous circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid, energy dependent, and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake, with distinct gene expression programs depending on the route of RNA delivery. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1, Toll-like receptors, and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs, a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an "eat me" signal and danger-associated molecular pattern, indicating orderly pathways of recognition and disposal.

Epigenetic signature and key transcriptional regulators of human antigen-specific type 1 regulatory T cells.

Human adaptive immunity is orchestrated by effector and regulatory T (Treg) cells. Natural Tregs arise in the thymus where they are shaped to recognize self-antigens, while type 1 Tregs or Tr1 cells are induced from conventional peripheral CD4 + T cells in response to peripheral antigens, such as alloantigens and allergens. Tr1 cells have been developed as a potential therapy for inducing antigen-specific tolerance, because they can be rapidly differentiated in vitro in response to a target antigen. However, the epigenetic landscape and the identity of transcription factors (TFs) that regulate differentiation, phenotype, and functions of human antigen-specific Tr1 cells is largely unknown, hindering Tr1 research and broader clinical development. Here, we reveal the unique epigenetic signature of antigen-specific Tr1 cells, and TFs that regulate their differentiation, phenotype and function. We showed that in vitro induced antigen-specific Tr1 cells are distinct both clonally and transcriptionally from natural Tregs and other conventional CD4 + T cells on a single-cell level. An integrative analysis of Tr1 cell epigenome and transcriptome identified a TF signature unique to antigen-specific Tr1 cells, and predicted that IRF4, BATF, and MAF act as their transcriptional regulators. Using functional genomics, we showed that each of these TFs play a non-redundant role in regulating Tr1 cell differentiation, suppressive function, and expression of co-inhibitory and cytotoxic proteins. By using the Tr1-specific TF signature as a molecular fingerprint, we tracked Tr1 cells in peripheral blood of recipients of allogeneic hematopoietic stem cell transplantation treated with adoptive Tr1 cell therapy. Furthermore, the same signature identified Tr1 cells in resident CD4 + T cells in solid tumors. Altogether, these results reveal the epigenetic signature and the key transcriptional regulators of human Tr1 cells. These data will guide mechanistic studies of human Tr1 cell biology and the development and optimization of adoptive Tr1 cell therapies.

CoRAL accurately resolves extrachromosomal DNA genome structures with long-read sequencing.

Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in approximately 15% of early stage cancers and 30% of late-stage cancers. EcDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy-number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies. Paired-end short-read (Illumina) sequencing and mapping have been utilized to represent ecDNA amplifications using a breakpoint graph, where the inferred architecture of ecDNA is encoded as a cycle in the graph. Traversals of breakpoint graph have been used to successfully predict ecDNA presence in cancer samples. However, short-read technologies are intrinsically limited in the identification of breakpoints, phasing together of complex rearrangements and internal duplications, and deconvolution of cell-to-cell heterogeneity of ecDNA structures. Long-read technologies, such as from Oxford Nanopore Technologies, have the potential to improve inference as the longer reads are better at mapping structural variants and are more likely to span rearranged or duplicated regions. Here, we propose CoRAL (Complete Reconstruction of Amplifications with Long reads), for reconstructing ecDNA architectures using long-read data. CoRAL reconstructs likely cyclic architectures using quadratic programming that simultaneously optimizes parsimony of reconstruction, explained copy number, and consistency of long-read mapping. CoRAL substantially improves reconstructions in extensive simulations and 9 datasets from previously-characterized cell-lines as compared to previous short-read-based tools. As long-read usage becomes wide-spread, we anticipate that CoRAL will be a valuable tool for profiling the landscape and evolution of focal amplifications in tumors.

Approaches to probe and perturb long noncoding RNA functions in diseases.

Long noncoding RNAs (lncRNAs) are a class of RNA molecules exceeding 200 nucleotides in length that lack long open-reading frames. Transcribed predominantly by RNA polymerase II (>500nt), lncRNAs can undergo splicing and are produced from various regions of the genome, including intergenic regions, introns, and in antisense orientation to protein-coding genes. Aberrations in lncRNA expression or function have been associated with a wide variety of diseases, including cancer, cardiovascular diseases, diabetes, and neurodegeneration. Despite the growing recognition of select lncRNAs as key players in cellular processes and diseases, several challenges obscure a comprehensive understanding of their functional landscape. Recent technological innovations, such as in sequencing, affinity-based techniques, imaging, and RNA perturbation, have advanced functional characterization and mechanistic understanding of disease-associated lncRNAs.

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for induction of ecDNA with extensive characterization of newly formed ecDNA to examine ecDNA biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of a 1 Mb and 1.8 Mb ecDNA both reaching 15%. We show non-homologous end joining and microhomology mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

Charge-altering releasable transporters enhance mRNA delivery in vitro and exhibit in vivo tropism.

The introduction of more effective and selective mRNA delivery systems is required for the advancement of many emerging biomedical technologies including the development of prophylactic and therapeutic vaccines, immunotherapies for cancer and strategies for genome editing. While polymers and oligomers have served as promising mRNA delivery systems, their efficacy in hard-to-transfect cells such as primary T lymphocytes is often limited as is their cell and organ tropism. To address these problems, considerable attention has been placed on structural screening of various lipid and cation components of mRNA delivery systems. Here, we disclose a class of charge-altering releasable transporters (CARTs) that differ from previous CARTs based on their beta-amido carbonate backbone (bAC) and side chain spacing. These bAC-CARTs exhibit enhanced mRNA transfection in primary T lymphocytes in vitro and enhanced protein expression in vivo with highly selective spleen tropism, supporting their broader therapeutic use as effective polyanionic delivery systems.

Coordinated inheritance of extrachromosomal DNA species in human cancer cells.

The chromosomal theory of inheritance has dominated human genetics, including cancer genetics. Genes on the same chromosome segregate together while genes on different chromosomes assort independently, providing a fundamental tenet of Mendelian inheritance. Extrachromosomal DNA (ecDNA) is a frequent event in cancer that drives oncogene amplification, dysregulated gene expression and intratumoral heterogeneity, including through random segregation during cell division. Distinct ecDNA sequences, herein termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells. However, how multiple ecDNA species within a tumor cell are assorted and maintained across somatic cell generations to drive cancer cell evolution is not known. Here we show that cooperative ecDNA species can be coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. EcDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy number gains in multiple ecDNA species prior to any selection. Computational modeling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Finally, we show that coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations.

Transcriptional immune suppression and upregulation of double stranded DNA damage and repair repertoires in ecDNA-containing tumors.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood. We examined gene expression patterns of 870 tumors of varied histological types, to identify transcriptional correlates of ecDNA. Here we show that ecDNA containing tumors impact four major biological processes. Specifically, ecDNA containing tumors upregulate DNA damage and repair, cell cycle control, and mitotic processes, but downregulate global immune regulation pathways. Taken together, these results suggest profound alterations in gene regulation in ecDNA containing tumors, shedding light on molecular processes that give rise to their development and progression.

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells.

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

Circular RNA vaccine induces potent T cell responses.

Circular RNAs (circRNAs) are a class of RNAs commonly found across eukaryotes and viruses, characterized by their resistance to exonuclease-mediated degradation. Their superior stability compared to linear RNAs, combined with previous work showing that engineered circRNAs serve as efficient protein translation templates, make circRNA a promising candidate for RNA medicine. Here, we systematically examine the adjuvant activity, route of administration, and antigen-specific immunity of circRNA vaccination in mice. Potent circRNA adjuvant activity is associated with RNA uptake and activation of myeloid cells in the draining lymph nodes and transient cytokine release. Immunization of mice with engineered circRNA encoding a protein antigen delivered by a charge-altering releasable transporter induced innate activation of dendritic cells, robust antigen-specific CD8 T cell responses in lymph nodes and tissues, and strong antitumor efficacy as a therapeutic cancer vaccine. These results highlight the potential utility of circRNA vaccines for stimulating potent innate and T cell responses in tissues.

The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner.

Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

LINE-associated cryptic splicing induces dsRNA-mediated interferon response and tumor immunity.

RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance.

Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state.

Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings in vivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.

Breakage fusion bridge cycles drive high oncogene copy number, but not intratumoral genetic heterogeneity or rapid cancer genome change.

Oncogene amplification is a major driver of cancer pathogenesis. Breakage fusion bridge (BFB) cycles, like extrachromosomal DNA (ecDNA), can lead to high copy numbers of oncogenes, but their impact on intratumoral heterogeneity, treatment response, and patient survival are not well understood due to difficulty in detecting them by DNA sequencing. We describe a novel algorithm that detects and reconstructs BFB amplifications using optical genome maps (OGMs), called OM2BFB. OM2BFB showed high precision (>93%) and recall (92%) in detecting BFB amplifications in cancer cell lines, PDX models and primary tumors. OM-based comparisons demonstrated that short-read BFB detection using our AmpliconSuite (AS) toolkit also achieved high precision, albeit with reduced sensitivity. We detected 371 BFB events using whole genome sequences from 2,557 primary tumors and cancer lines. BFB amplifications were preferentially found in cervical, head and neck, lung, and esophageal cancers, but rarely in brain cancers. BFB amplified genes show lower variance of gene expression, with fewer options for regulatory rewiring relative to ecDNA amplified genes. BFB positive (BFB (+)) tumors showed reduced heterogeneity of amplicon structures, and delayed onset of resistance, relative to ecDNA(+) tumors. EcDNA and BFB amplifications represent contrasting mechanisms to increase the copy numbers of oncogene with markedly different characteristics that suggest different routes for intervention.