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Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation ofKlf4expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants ofKLF4revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmicKlf4expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.

Although 3D genome architecture is essential for long-range gene regulation, the significance of distal regulatory chromatin contacts is challenged by recent findings of low correlation between contact propensity and gene expression. To better understand the role of long-range interactions between distal regulatory elements during the early transformation from healthy colon to colorectal cancer, here we performed high resolution chromatin conformation capture for 33 samples including non-neoplastic mucosa, adenomatous polyps and adenocarcinomas, mostly from Familial Adenomatous Polyposis (FAP) patients. We identified hundreds of thousands of chromatin micro-structures, such as architectural stripes and loops, which originated from active cis-regulatory elements. Surprisingly, these structures progressively decayed throughout cancer progression, particularly at promoters. Meta-analyses revealed that this decay was independent of alterations in DNA methylation and chromatin accessibility. Interestingly, the degree of interaction loss was poorly correlated with gene expression changes. Instead, genes whose expression were disproportionately lower and higher than their relative promoter interaction in mucosa shifted their expression in polyps and adenocarcinomas to yield a more direct relationship between strength of interaction and gene expression. Our work provides the first high resolution 3D conformation maps during early cancer formation and progression, and provides novel insights into transcriptional readouts associated with fine-scale chromatin conformation alterations.

Aberrant shifts in DNA methylation have long been regarded as an early marker for cancer onset and progression. To chart DNA methylation changes that occur during the transformation from normal healthy colon tissue to malignant colorectal cancer (CRC), we collected over 50 samples from 15 familial adenomatous polyposis (FAP) and non-FAP colorectal cancer patients, and generated 30-70x whole-genome methylation sequencing (WGMS) runs via the novel Ultima Genomics ultra high-throughput sequencing platform. We observed changes in DNA methylation that occur early in the malignant transformation process, in gene promoters and in distal regulatory elements. Among these changes are events of hyper-methylation which are associated with a bivalent “poised” chromatin state at promoters and are CRC-specific. Distal enhancers show nonlinear dynamics, lose methylation in the progression from normal mucosa to dysplastic polyps but regain methylation in the adenocarcinoma state. Enhancers that gain chromatin accessibility in the adenocarcinoma state and are enriched with HOX transcription factor binding sites, a marker of developmental genes. This work demonstrates the feasibility of generating large high quality WGMS data using the Ultima Genomics platform and provides the first detailed view of methylation dynamics during CRC formation and progression in a model case.

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq, NOMe-seq and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m(6)A-SMAC-seq and m(6)A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, BCL6) and transcription factors regulating B cell differentiation (POU2AF1, HHEX). Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.

Physical access to DNA is a highly dynamic property of chromatin that plays an essential role in establishing and maintaining cellular identity. The organization of accessible chromatin across the genome reflects a network of permissible physical interactions through which enhancers, promoters, insulators and chromatin-binding factors cooperatively regulate gene expression. This landscape of accessibility changes dynamically in response to both external stimuli and developmental cues, and emerging evidence suggests that homeostatic maintenance of accessibility is itself dynamically regulated through a competitive interplay between chromatin-binding factors and nucleosomes. In this Review, we examine how the accessible genome is measured and explore the role of transcription factors in initiating accessibility remodelling; our goal is to illustrate how chromatin accessibility defines regulatory elements within the genome and how these epigenetic features are dynamically established to control gene expression.

SummaryMammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit, ACTL6A, is amplified at an early stage in the development of squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives its interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was sub-stoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near-saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition over the genome activating SCC genes and also enhanced the recruitment of transcription factor TEAD with its co-activator YAP, promoting their chromatin binding and enhancer accessibility. Both of these mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.HighlightsACTL6A occupancy within BAF complexes is sub-stoichiometric in normal epithelial cells.SCC cells upregulate ACTL6A thus increasing ACTL6A assembly with BAF complexes.Genome-wide chromatin profiling identifies ACTL6A-dependent regulatory regions.Increasing ACTL6A incorporation enhances TEAD-YAP binding to BAF complexes.ACTL6A overexpression counteracts polycomb-mediated repression at SCC signature genes.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting auto-reactive B cells. Dysfunction in these processes can lead to defects in immune response to pathogens or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC cell populations. Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.One sentence summarySingle-cell chromatin accessibility landscapes of immune cell subsets reveal regulatory potential of autoimmune-associated genetic variants during the germinal center response.

Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.

The ATAC-seq assay has emerged as the most useful, versatile, and widely adaptable method for profiling accessible chromatin regions and tracking the activity of cis-regulatory elements (cREs) in eukaryotes. Thanks to its great utility, it is now being applied to map active chromatin in the context of a very wide diversity of biological systems and questions. In the course of these studies, considerable experience working with ATAC-seq data has accumulated and a standard set of computational tasks that need to be carried for most ATAC-seq analyses has emerged. Here, we review and provide examples of common such analytical procedures (including data processing, quality control, peak calling, identifying differentially accessible open chromatin regions, and variable transcription factor (TF) motif accessibility) and discuss recommended optimal practices.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.

The ATAC-seq assay has emerged as the most useful, versatile, and widely adaptable method for profiling accessible chromatin regions and tracking the activity of cis-regulatory elements (cREs) in eukaryotes. Thanks to its great utility, it is now being applied to map active chromatin in the context of a very wide diversity of biological systems and questions. In the course of these studies, considerable experience working with ATAC-seq data has accumulated and a standard set of computational tasks that need to be carried for most ATAC-seq analyses has emerged. Here, we review and provide examples of common such analytical procedures (including data processing, quality control, peak calling, identifying differentially accessible open chromatin regions, and variable transcription factor (TF) motif accessibility) and discuss recommended optimal practices.

Significance Autism is a complex neurodevelopmental disorder whose causative mechanisms are unclear. Taking advantage of a unique cohort with recessively inherited autism, we identified six families with biallelic mutation of the neuronal-specific subunit of the BAF complex, ACTL6B (also known as BAF53b). Relative to all other genes, ACTL6B was the most statistically significant mutated gene in the recessive autism cohort. We describe autism-relevant phenotypes in human brain organoids and in mouse and fly models. We foresee the outcomes from this study will be the following: 1) a link between neuronal activity-dependent transcriptional repression and autism; 2) a characterization of mouse and fly models to study ACTL6B mutant autism; and 3) an understanding the role of ACTL6B and nBAF complexes in neuronal transcriptional regulation., Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such “early activation” genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.

Summary ATAC-seq is a versatile, adaptable, and widely adopted technique for mapping open chromatin regions. However, some biological systems, such as primary neurons, present unique challenges to its application. Conventional ATAC-seq would require the dissociation of the primary neurons after plating but dissociating them leads to rapid cell death and major changes in cell state, affecting ATAC-seq results. We have developed this modified ATAC-seq protocol to address this challenge for primary neurons, providing a high-quality and high-resolution accessible chromatin profile. For complete details on the use and execution of this protocol, please refer to Maor-Nof et al. (2021)., Graphical abstract, Highlights • Neuronal ATAC maps chromatin accessibility in primary neurons. • Neuronal ATAC avoids damage to neurons during cell handling and nuclei preparation. • Neuronal ATAC provides high-quality and high resolution accessible chromatin profiles., ATAC-seq is a versatile, adaptable, and widely adopted technique for mapping open chromatin regions. However, some biological systems, such as primary neurons, present unique challenges to its application. Conventional ATAC-seq would require the dissociation of the primary neurons after plating but dissociating them leads to rapid cell death and major changes in cell state, affecting ATAC-seq results. We have developed this modified ATAC-seq protocol to address this challenge for primary neurons, providing a high-quality and high-resolution accessible chromatin profile.

DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53−/−) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53−/− mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53−/− mESCs display increased cellular heterogeneity both in the “naïve” state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity.

Active regulatory elements in eukaryotes are typically characterized by an open, nucleosome-depleted chromatin structure; mapping areas of open chromatin has accordingly emerged as a widely used tool in the arsenal of modern functional genomics. However, existing approaches for profiling chromatin accessibility are limited by their reliance on DNA fragmentation and short read sequencing, which leaves them unable to provide information about the state of chromatin on larger scales or reveal coordination between the chromatin state of individual distal regulatory elements. To address these limitations, we have developed a method for profiling accessibility of individual chromatin fibers at multi-kilobase length scale (SMAC-seq, or Single-Molecule long-read Accessible Chromatin mapping sequencing assay), enabling the simultaneous, high-resolution, single-molecule assessment of the chromatin state of distal genomic elements. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases (CpG and GpC 5-methylcytosine (5mC) and N6-methyladenosine (m6A) enzymes) and the ability of long-read single-molecule nanopore sequencing to directly read out the methylation state of individual DNA bases. Applying SMAC-seq to the budding yeast Saccharomyces cerevisiae, we demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule protection footprints of nucleosomes and transcription factors, and quantify the correlation between the chromatin states of distal genomic elements.

Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells. In contrast, somatic cells transmit considerable epigenetic information to progenies. Paradoxically, the persistence of the somatic epigenome makes it more vulnerable to noise, since random epimutations can accumulate to massively perturb the epigenomic ground state. The rate of epigenetic perturbation depends on the genomic context, and, in particular, DNA methylation loss is coupled to late DNA replication dynamics. Epigenetic perturbation is not observed in the pluripotent state, because the rapid turnover-based equilibrium continuously reinforces the canonical state. This dynamic epigenetic equilibrium also explains how the epigenome can be reprogrammed quickly and to near perfection after induced pluripotency.

Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ∼10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded.

Dermal DC mobilization requires docking to CCL21 on lymphatic endothelium, Dendritic cells (DCs) must travel through lymphatics to carry skin antigens into lymph nodes. The processes controlling their mobilization and migration have not been completely delineated. We studied how DCs in live mice respond to skin inflammation, transmigrate through lymphatic endothelium, and propagate in initial lymphatics. At steady state, dermal DCs remain sessile along blood vessels. Inflammation mobilizes them, accelerating their interstitial motility 2.5-fold. CCR7-deficient BMDCs crawl as fast as wild-type DCs but less persistently. We observed discrete depositions of CCL21 complexed with collagen-IV on the basement membrane of initial lymphatics. Activated DCs move directionally toward lymphatics, contact CCL21 puncta, and migrate through portals into the lumen. CCR7-deficient DCs arrive at lymphatics through random migration but fail to dock and transmigrate. Once inside vessels, wild-type DCs use lamellipodia to crawl along lymphatic endothelium and, sensing lymph flow, proceed downstream. DCs start drifting freely only in collecting lymphatics. These results demonstrate in vivo that the CCL21–CCR7 axis plays a dual role in DC mobilization: promoting both chemotaxis and arrest of DCs on lymphatic endothelium. Intralymphatic crawling, in which DCs combine active adhesion-based migration and directional cues from lymph flow, represents a new step in DC mobilization which may be amenable to regulation.

DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Poor tumor vascularization is an obstacle to immunotherapy by CTLs. It impairs tumor infiltration but also introduces hypoxia, known to interfere with T cell migration. It is yet unknown how suboptimal vascularization affects CTL migration and function within tumors. To study this question, we combined immunohistochemistry of human melanoma samples with two-photon imaging in live mice. Orthotopically implanted B16-OVA tumors were studied after adoptive transfer of in-vitro matured antigen-specific OT-I CTLs. In patients, CD8 T cells concentrated around peripheral vessels in the tumor and sparsely infiltrated avascular areas. This pattern was clearest in poorly-infiltrated tumors. In mice, OT-I CTLs, as well as polyclonal CTLs, entered tumors through inflamed intratumoral vessels, and crawled only in oxygenated areas within 50 µm of flowing blood vessels. Gradually, CTLs formed dense perivascular swarms, which eliminated nearby target cells leading to patchy tumor clearance around blood vessels. Occluding intratumoral blood vessels triggered immediate arrest of CTL motility, which was quickly reversed when flow was resumed. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Live CTL imaging in vitro showed deceleration under hypoxic conditions and when oxidative phosphorylation was blocked. To circumvent intratumoral CTL dysfunction we attempted to increase vascular density by implanting tumors in matrices containing bFGF. bFGF-laced tumors were more easily rejected after transfer of CTLs and displayed delayed growth in untreated mice but were not affected in mice deficient in CD8 T-cells. CTLs infiltrated such tumors in normal numbers, but displayed enhanced motility in highly-vascular tumors, suggesting that enhanced rejection resulted from improved intratumoral CTL migration. Taken together, the results suggest that hypoxia limits CTL function away from blood vessels, and that alleviating it may synergize with immunotherapy. Citation Format: Tali Feferman, Yoav Manaster, Yael Gruper, Zohar Shipony, Guy Shakhar. Releasing oxygen-restricted CTL function: Insights from live intratumoral imaging. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B102.

Nature 504, 282–286 (2013); doi:10.1038/nature12745 The reduced representation bisulphite sequencing (RRBS) data generated and analysed in this Letter were not originally uploaded to the Gene Expression Omnibus (GEO), but can now be found under accession number GSE52617 (within the GSE52824 super series).

Nature 502, 65–70 (2013); doi:10.1038/nature12587 In this Article, the reduced representation bisulphite sequencing (RRBS) data obtained and analysed were not initially uploaded to the Gene Expression Omnibus (GEO) record, but can now be found under accession number GSE64115, within the GSE49767 super series.