Antigen dominance hierarchies shape TCF1+ progenitor CD8 T cell phenotypes in tumors.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

CRISPR-Cas9 screens reveal regulators of ageing in neural stem cells.

Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing1-4. Several genetic interventions have been found to ameliorate old brain function5-8, but systematic functional testing of genes in old NSCs-and more generally in old cells-has not been done. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR-Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.

The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.

Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:

Driving Rel-iant Tregs toward an Identity Crisis.

Inhibiting Treg cell function in tumors is an attractive strategy to improve anti-cancer immunity. In a pair of papers in Immunity and Cell, Ghosh and colleagues show that the canonical NF-κB subunits p65 and c-Rel have non-redundant, critical roles in promoting Treg cell development and function (Oh et al., 2017). Targeting c-Rel blunts Treg cell immunosuppressive activity in the tumor microenvironment and enhances anti-tumor T cell responses (Grinberg-Bleyer et al., 2017).

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

State- and Circuit-Dependent Opponent Processing of Fear.

The presence of valence coding neurons in the basolateral amygdala (BLA) that form distinct projections to other brain regions implies functional opposition between aversion and reward during learning. However, evidence for opponent interactions in fear learning is sparse and may only be apparent under certain conditions. Here we test this possibility by studying the roles of the BLA→central amygdala (CeA) and BLA→nucleus accumbens (Acb) pathways in fear learning in male rats. First, we assessed the organization of these pathways in the rat brain. BLA→CeA and BLA→Acb pathways were largely segregated in the BLA but shared overlapping molecular profiles. Then we assessed activity of the BLA→CeA and BLA→Acb pathways during two different forms of fear learning-fear learning in a neutral context and fear learning in a reward context. BLA→CeA neurons were robustly recruited by footshock regardless of where fear learning occurred, whereas recruitment of BLA→Acb neurons was state-dependent because footshock only recruited this pathway in a reward context. Finally, we assessed the causal roles of activity in these pathways in fear learning. Photoinhibition of the BLA→CeA pathway during the footshock US impaired fear learning, regardless of where fear learning occurred. In contrast, photoinhibition of the BLA→Acb pathway augmented fear learning, but only in the reward context. Taken together, our findings show circuit- and state-dependent opponent processing of fear. Footshock activity in the BLA→Acb pathway limits how much fear is learned.

Acetyl-CoA carboxylase inhibition increases retinal pigment epithelial cell fatty acid flux and restricts apolipoprotein efflux.

Lipid-rich deposits called drusen accumulate under the retinal pigment epithelium (RPE) in the eyes of patients with age-related macular degeneration and Sorsby's fundus dystrophy (SFD). Drusen may contribute to photoreceptor degeneration in these blinding diseases. Stimulating ß-oxidation of fatty acids could decrease the availability of lipid with which RPE cells generate drusen. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate ß-oxidation and diminish lipid accumulation in fatty liver disease. In this report, we test the hypothesis that an ACC inhibitor, Firsocostat, can diminish lipid deposition by RPE cells. We probed metabolism and cellular function in mouse RPE-choroid tissue and human RPE cells. We used 13C6-glucose, 13C16-palmitate, and gas chromatography-linked mass spectrometry to monitor effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. We quantified lipid abundance, apolipoprotein E levels, and vascular endothelial growth factor release using liquid chromatography-mass spectrometry, ELISAs, and immunostaining. RPE barrier function was assessed by trans-epithelial electrical resistance (TEER). Firsocostat-mediated ACC inhibition increases ß-oxidation, decreases intracellular lipid levels, diminishes lipoprotein release, and increases TEER. When human serum or outer segments are used to stimulate lipoprotein release, fewer lipoproteins are released in the presence of Firsocostat. In a culture model of SFD, Firsocostat stimulates fatty acid oxidation, increases TEER, and decreases apolipoprotein E release. We conclude that Firsocostat remodels RPE metabolism and can limit lipid deposition. This suggests that ACC inhibition could be an effective strategy for diminishing pathologic drusen in the eyes of patients with age-related macular degeneration or SFD.

Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios.

Researchers choose different methods of making giant unilamellar vesicles to satisfy different constraints of their experimental designs. A challenge that arises when researchers use a variety of methods is that each method may produce vesicles with a different average lipid ratio, even if all experiments use lipids from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicle solutions made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either five-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts in lipid ratios (≤5 mol %) relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of saturated and unsaturated PC-lipids and cholesterol largely support results from the five-component mixture. In general, our results imply that experiments that increment lipid ratios in small steps will produce data that are highly sensitive to the technique used and to sample-to-sample variations. For example, sample-to-sample variations are ∼±2 mol % for five-component vesicles produced by a single technique. In contrast, experiments that explore larger increments in lipid ratio or that seek to explain general trends and new phenomena will be less sensitive to sample-to-sample variation and the method used.

Regulatory T Cells in Tumor-Associated Tertiary Lymphoid Structures Suppress Anti-tumor T Cell Responses.

Infiltration of regulatory T (Treg) cells into many tumor types correlates with poor patient prognoses. However, mechanisms of intratumoral Treg cell function remain to be elucidated. We investigated Treg cell function in a genetically engineered mouse model of lung adenocarcinoma and found that Treg cells suppressed anti-tumor responses in tumor-associated tertiary lymphoid structures (TA-TLSs). TA-TLSs have been described in human lung cancers, but their function remains to be determined. TLSs in this model were spatially associated with >90% of tumors and facilitated interactions between T cells and tumor-antigen-presenting dendritic cells (DCs). Costimulatory ligand expression by DCs and T cell proliferation rates increased in TA-TLSs upon Treg cell depletion, leading to tumor destruction. Thus, we propose that Treg cells in TA-TLSs can inhibit endogenous immune responses against tumors, and targeting these cells might provide therapeutic benefit for cancer patients.

The effect of noninstrumental information on reward learning.

Investigations of information-seeking often highlight people's tendency to forgo financial reward in return for advance information about future outcomes. Most of these experiments use tasks in which reward contingencies are described to participants. The use of such descriptions leaves open the question of whether the opportunity to obtain such noninstrumental information influences people's ability to learn and represent the underlying reward structure of an experimental environment. In two experiments, participants completed a two-armed bandit task with monetary incentives where reward contingencies were learned via trial-by-trial experience. We find, akin to description-based tasks, that participants are willing to forgo financial reward to receive information about a delayed, unchangeable outcome. Crucially, however, there is little evidence this willingness to pay for information is driven by an inaccurate representation of the reward structure: participants' representations approximated the underlying reward structure regardless of the presence of advance noninstrumental information. The results extend previous conclusions regarding the intrinsic value of information to an experience-based domain and highlight challenges of probing participants' memories for experienced rewards.

Revascularization techniques for complete portomesenteric venous occlusion in patients undergoing pancreatic resection.

INTRODUCTION: Pancreatic pathologies causing portomesenteric occlusion complicate extirpative pancreatic resection due to portomesenteric hypertension and collateral venous drainage. METHODS: Patients with portomesenteric occlusion undergoing pancreatectomy were identified between 2007 and 2020 at Stanford University Hospital. Demographic and clinical data, technique and perioperative factors, and post-operative outcomes were analyzed. RESULTS: Of twenty-seven (27) patients undergoing venous revascularization during pancreatectomy, most (15) were for pancreatic neuroendocrine tumor. Occlusions occurred mostly at the portosplenic confluence (15). Median occlusion length was 4.0 cm [3.1-5.8]. Regarding revascularization strategy, mesocaval shunting was used in 11 patients, in-line venous revascularization with internal jugular conduit in three patients, traditional venous resection and reconstruction in 9 patients, and thrombectomy in two patients. Median cohort operative time and estimated blood loss were 522 min [433-638] and 1000 mL [700-2500], respectively. Median length of stay was 10 days [8-14.5] with overall readmission rate of 37%. Significant complications occurred in 44% of patients despite only one (4%) perioperative mortality. DISCUSSION: Despite the technical complexity for managing portomesenteric occlusions, early revascularization strategies including mesocaval shunting or in-line venous revascularization are feasible and facilitate a safe pancreatic resection for surgically fit patients.

IL-6 mediates the hepatic acute phase response after prerenal azotemia in a clinically defined murine model.

Prerenal azotemia (PRA) is a major cause of acute kidney injury and uncommonly studied in preclinical models. We sought to develop and characterize a novel model of PRA that meets the clinical definition: acute loss of glomerular filtration rate (GFR) that returns to baseline with resuscitation. Adult male C57BL/6J wild-type (WT) and IL-6-/- mice were studied. Intraperitoneal furosemide (4 mg) or vehicle was administered at time = 0 and 3 h to induce PRA from volume loss. Resuscitation began at 6 h with 1 mL intraperitoneal saline for four times for 36 h. Six hours after furosemide administration, measured glomerular filtration rate was 25% of baseline and returned to baseline after saline resuscitation at 48 h. After 6 h of PRA, plasma interleukin (IL)-6 was significantly increased, kidney and liver histology were normal, kidney and liver lactate were normal, and kidney injury molecule-1 immunofluorescence was negative. There were 327 differentially regulated genes upregulated in the liver, and the acute phase response was the most significantly upregulated pathway; 84 of the upregulated genes (25%) were suppressed in IL-6-/- mice, and the acute phase response was the most significantly suppressed pathway. Significantly upregulated genes and their proteins were also investigated and included serum amyloid A2, serum amyloid A1, lipocalin 2, chemokine (C-X-C motif) ligand 1, and haptoglobin; hepatic gene expression and plasma protein levels were all increased in wild-type PRA and were all reduced in IL-6-/- PRA. This work demonstrates previously unknown systemic effects of PRA that includes IL-6-mediated upregulation of the hepatic acute phase response.NEW & NOTEWORTHY Prerenal azotemia (PRA) accounts for a third of acute kidney injury (AKI) cases yet is rarely studied in preclinical models. We developed a clinically defined murine model of prerenal azotemia characterized by a 75% decrease in measured glomerular filtration rate (GFR), return of measured glomerular filtration rate to baseline with resuscitation, and absent tubular injury. Numerous systemic effects were observed, such as increased plasma interleukin-6 (IL-6) and upregulation of the hepatic acute phase response.

Prolonged Impairment of Short-Chain Fatty Acid and L-Isoleucine Biosynthesis in Gut Microbiome in Patients With COVID-19.

BACKGROUND AND AIMS: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with altered gut microbiota composition. Phylogenetic groups of gut bacteria involved in the metabolism of short chain fatty acids (SCFAs) were depleted in SARS-CoV-2-infected patients. We aimed to characterize a functional profile of the gut microbiome in patients with COVID-19 before and after disease resolution. METHODS: We performed shotgun metagenomic sequencing on fecal samples from 66 antibiotics-naïve patients with COVID-19 and 70 non-COVID-19 controls. Serial fecal samples were collected (at up to 6 times points) during hospitalization and beyond 1 month after discharge. We assessed gut microbial pathways in association with disease severity and blood inflammatory markers. We also determined changes of microbial functions in fecal samples before and after disease resolution and validated these functions using targeted analysis of fecal metabolites. RESULTS: Compared with non-COVID-19 controls, patients with COVID-19 with severe/critical illness showed significant alterations in gut microbiome functionality (P < .001), characterized by impaired capacity of gut microbiome for SCFA and L-isoleucine biosynthesis and enhanced capacity for urea production. Impaired SCFA and L-isoleucine biosynthesis in gut microbiome persisted beyond 30 days after recovery in patients with COVID-19. Targeted analysis of fecal metabolites showed significantly lower fecal concentrations of SCFAs and L-isoleucine in patients with COVID-19 before and after disease resolution. Lack of SCFA and L-isoleucine biosynthesis significantly correlated with disease severity and increased plasma concentrations of CXCL-10, NT- proB-type natriuretic peptide, and C-reactive protein (all P < .05). CONCLUSIONS: Gut microbiome of patients with COVID-19 displayed impaired capacity for SCFA and L-isoleucine biosynthesis that persisted even after disease resolution. These 2 microbial functions correlated with host immune response underscoring the importance of gut microbial functions in SARS-CoV-2 infection pathogenesis and outcome.

Giant mitochondria in cardiomyocytes: cellular architecture in health and disease.

Giant mitochondria are frequently observed in different disease models within the brain, kidney, and liver. In cardiac muscle, these enlarged organelles are present across diverse physiological and pathophysiological conditions including in ageing and exercise, and clinically in alcohol-induced heart disease and various cardiomyopathies. This mitochondrial aberration is widely considered an early structural hallmark of disease leading to adverse organ function. In this thematic paper, we discuss the current state-of-knowledge on the presence, structure and functional implications of giant mitochondria in heart muscle. Despite its demonstrated reoccurrence in different heart diseases, the literature on this pathophysiological phenomenon remains relatively sparse since its initial observations in the early 60s. We review historical and contemporary investigations from cultured cardiomyocytes to human tissue samples to address the role of giant mitochondria in cardiac health and disease. Finally, we discuss their significance for the future development of novel mitochondria-targeted therapies to improve cardiac metabolism and functionality.

Giant mitochondria in human liver disease.

This thematic review aims to provide an overview of the current state of knowledge about the occurrence of giant mitochondria or megamitochondria in liver parenchymal cells. Their presence and accumulation are considered to be a major pathological hallmark of the health and fate of liver parenchymal cells that leads to overall tissue deterioration and eventually results in organ failure. The first description on giant mitochondria dates back to the 1960s, coinciding with the availability of the first generation of electron microscopes in clinical diagnostic laboratories. Detailed accounts on their ultrastructure have mostly been described in patients suffering from alcoholic liver disease, chronic hepatitis, hepatocellular carcinoma and non-alcoholic fatty liver disease. Interestingly, from this extensive literature survey, it became apparent that giant mitochondria or megamitochondria present themselves with or without highly organised crystal-like intramitochondrial inclusions. The origin, formation and potential role of giant mitochondria remain to-date largely unanswered. Likewise, the biochemical composition of the well-organised crystal-like inclusions and their possible impact on mitochondrial function is unclear. Herein, concepts about the possible mechanism of their formation and three-dimensional architecture will be approached. We will furthermore discuss their importance in diagnostics, including future research outlooks and potential therapeutic interventions to cure liver disease where giant mitochondria are implemented.

Implementation of a Breast Intraoperative Oncoplastic Form to Aid Management of Oncoplastic Surgery.

INTRODUCTION: Oncoplastic breast conservation surgery (BCS) uses concurrent reduction and/or mastopexy with lumpectomy to improve aesthetic outcomes. However, tissue rearrangement can shift the original tumor location site in relation to external breast landmarks, resulting in difficulties during re-excision for a positive margin and accurate radiation targeting. We developed the Breast Intraoperative Oncoplastic (BIO) form to help depict the location of the tumor and breast reduction specimen. This study seeks to assess physician perspectives of the implementation outcomes. METHODS: From February 2021 to April 2021, the BIO form was used in 11 oncoplastic BCS cases at a single institution. With institutional review board approval, surgical oncologists (SOs), plastic surgeons (PSs), and radiation oncologists (ROs) were administered a 12-question validated survey on Acceptability of Intervention Measure (AIM), Intervention Appropriateness Measure (IAM), and Feasibility of Intervention Measure (FIM), using a 5-point Likert scale during initial implementation and at 6-month reassessment. RESULTS: Twelve physicians completed the survey initially (4 SOs, 4 PSs, and 4 ROs). The mean scores for Acceptability of Intervention Measure, Intervention Appropriateness Measure, and Feasibility of Intervention Measure were high (4.44, 4.56, and 4.56, respectively). Twelve completed the second survey (5 SOs, 3 PSs, and 4 ROs). The mean scores were marginally lower (4.06, 4.21, and 4.25). There were no significant differences when stratified by number of years in practice or specialty. Free text comments showed that 75% of physicians found the form helpful in oncoplastic BCS. CONCLUSIONS: The data indicate high feasibility, acceptability, and appropriateness of the BIO form. Results of this study suggest multidisciplinary benefits of implementing the BIO form in oncoplastic BCS.

Adoption of cytoreductive surgery in the management of peritoneal malignancies-Global trends.

Cytoreductive surgery (CRS) has now been accepted as an integral component in the management of gastrointestinal and gynecological cancers with peritoneal metastases. Since the adoption of CRS is influenced by access to advanced medical facilities, trained multidisciplinary teams, and funding, there is wide variability in incorporation of CRS into routine clinical practice between high- versus low- and middle-income countries. This review highlights the global trends in the adoption of CRS for peritoneal malignancies with a specific focus on the establishment of CRS programs and barriers to incorporate CRS into routine clinical care in low- and middle-income countries.

High-throughput analysis of lipidomic phenotypes of methicillin-resistant Staphylococcus aureus by coupling in situ 96-well cultivation and HILIC-ion mobility-mass spectrometry.

Antimicrobial resistance is a major threat to human health as resistant pathogens spread globally, and the development of new antimicrobials is slow. Since many antimicrobials function by targeting cell wall and membrane components, high-throughput lipidomics for bacterial phenotyping is of high interest for researchers to unveil lipid-mediated pathways when dealing with a large number of lab-selected or clinical strains. However, current practice for lipidomic analysis requires the cultivation of bacteria on a large scale, which does not replicate the growth conditions for high-throughput bioassays that are normally carried out in 96-well plates, such as susceptibility tests, growth curve measurements, and biofilm quantitation. Analysis of bacteria grown under the same condition as other bioassays would better inform the differences in susceptibility and other biological metrics. In this work, a high-throughput method for cultivation and lipidomic analysis of antimicrobial-resistant bacteria was developed for standard 96-well plates exemplified by methicillin-resistant Staphylococcus aureus (MRSA). By combining a 30-mm liquid chromatography (LC) column with ion mobility (IM) separation, elution time could be dramatically shortened to 3.6 min for a single LC run without losing major lipid features. Peak capacity was largely rescued by the addition of the IM dimension. Through multi-linear calibration, the deviation of retention time can be limited to within 5%, making database-based automatic lipid identification feasible. This high-throughput method was further validated by characterizing the lipidomic phenotypes of antimicrobial-resistant mutants derived from the MRSA strain, W308, grown in a 96-well plate.

The impact of rare variation on gene expression across tissues.

Rare genetic variants are abundant in humans and are expected to contribute to individual disease risk. While genetic association studies have successfully identified common genetic variants associated with susceptibility, these studies are not practical for identifying rare variants. Efforts to distinguish pathogenic variants from benign rare variants have leveraged the genetic code to identify deleterious protein-coding alleles, but no analogous code exists for non-coding variants. Therefore, ascertaining which rare variants have phenotypic effects remains a major challenge. Rare non-coding variants have been associated with extreme gene expression in studies using single tissues, but their effects across tissues are unknown. Here we identify gene expression outliers, or individuals showing extreme expression levels for a particular gene, across 44 human tissues by using combined analyses of whole genomes and multi-tissue RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project v6p release. We find that 58% of underexpression and 28% of overexpression outliers have nearby conserved rare variants compared to 8% of non-outliers. Additionally, we developed RIVER (RNA-informed variant effect on regulation), a Bayesian statistical model that incorporates expression data to predict a regulatory effect for rare variants with higher accuracy than models using genomic annotations alone. Overall, we demonstrate that rare variants contribute to large gene expression changes across tissues and provide an integrative method for interpretation of rare variants in individual genomes.