Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4+ T Cell Immunity.

Differentiation of proinflammatory CD4+ conventional T cells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS+ PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.

Protracted neuronal recruitment in the temporal lobes of young children.

The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs1-4. The human EC continues to develop during childhood5, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5+RELN+ inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimer's disease, and previous work has linked cognitive decline to the loss of LAMP5+RELN+ cells6,7. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood.

A tissue checkpoint regulates type 2 immunity.

Group 2 innate lymphoid cells (ILC2s) and CD4+ type 2 helper T cells (TH2 cells) are defined by their similar effector cytokines, which together mediate the features of allergic immunity. We found that tissue ILC2s and TH2 cells differentiated independently but shared overlapping effector function programs that were mediated by exposure to the tissue-derived cytokines interleukin 25 (IL-25), IL-33 and thymic stromal lymphopoietin (TSLP). Loss of these three tissue signals did not affect lymph node priming, but abrogated the terminal differentiation of effector TH2 cells and adaptive lung inflammation in a T cell-intrinsic manner. Our findings suggest a mechanism by which diverse perturbations can activate type 2 immunity and reveal a shared local-tissue-elicited checkpoint that can be exploited to control both innate and adaptive allergic inflammation.

Global absence and targeting of protective immune states in severe COVID-19.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

Author Correction: Discovery of stimulation-responsive immune enhancers with CRISPR activation.

In this Letter, analysis of steady-state regulatory T (Treg) cell percentages from Il2ra enhancer deletion (EDEL) and wild-type (WT) mice revealed no differences between them (Extended Data Fig. 9d). This analysis included two mice whose genotypes were incorrectly assigned. Even after correction of the genotypes, no significant differences in Treg cell percentages were seen when data across experimental cohorts were averaged (as was done in Extended Data Fig. 9d). However, if we normalize the corrected data to account for variation among experimental cohorts, a subtle decrease in EDEL Treg cell percentages is revealed and, using the corrected and normalized data, we have redrawn Extended Data Fig. 9d in Supplementary Fig. 1. The Supplementary Information to this Amendment contains the corrected and reanalysed Extended Data Fig. 9d. The sentence "This enhancer deletion (EDEL) strain also had no obvious T cell phenotypes at steady state (Extended Data Fig. 9)." should read: "This enhancer deletion (EDEL) strain had a small decrease in the percentage of Treg cells (Extended Data Fig. 9).". This error does not affect any of the main figures in the Letter or the data from mice with the human autoimmune-associated single nucleotide polymorphism (SNP) knocked in or with a 12-base-pair deletion at the site (12DEL). In addition, we stated in the Methods that we observed consistent immunophenotypes of EDEL mice across three founders, but in fact, we observed consistent phenotypes in mice from two founders. This does not change any of our conclusions and the original Letter has not been corrected.

Discovery of stimulation-responsive immune enhancers with CRISPR activation.

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures. RESULTS: Here, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets. CONCLUSIONS: We demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

An ancestry-based approach for detecting interactions.

BACKGROUND: Epistasis and gene-environment interactions are known to contribute significantly to variation of complex phenotypes in model organisms. However, their identification in human association studies remains challenging for myriad reasons. In the case of epistatic interactions, the large number of potential interacting sets of genes presents computational, multiple hypothesis correction, and other statistical power issues. In the case of gene-environment interactions, the lack of consistently measured environmental covariates in most disease studies precludes searching for interactions and creates difficulties for replicating studies. RESULTS: In this work, we develop a new statistical approach to address these issues that leverages genetic ancestry, defined as the proportion of ancestry derived from each ancestral population (e.g., the fraction of European/African ancestry in African Americans), in admixed populations. We applied our method to gene expression and methylation data from African American and Latino admixed individuals, respectively, identifying nine interactions that were significant at P<5×10-8. We show that two of the interactions in methylation data replicate, and the remaining six are significantly enriched for low P-values (P<1.8×10-6). CONCLUSION: We show that genetic ancestry can be a useful proxy for unknown and unmeasured covariates in the search for interaction effects. These results have important implications for our understanding of the genetic architecture of complex traits.

Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

CD4+ CAR T-cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR T-cell therapy.

ABSTRACT: Multiple myeloma is characterized by frequent clinical relapses after conventional therapy. Recently, chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, although >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune-intrinsic mechanisms may contribute to this resistance. Although there were no preexisting T-cell features associated with clinical outcomes, we found that patients with a durable response to CAR T-cell treatment had greater persistence of their CAR T cells than patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T-effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR T cells. These cells expand in vivo early after infusion but express exhaustion markers (hepatitis A virus cellular receptor 2 [HAVCR2] and T-cell immunoglobulin and mucin domain-containing-3 [TIGIT]) and remain polyclonal. Finally, we demonstrate that nonclassical monocytes are enriched in the myeloma niche and may induce CAR T-cell dysfunction through mechanisms that include transforming growth factor ß. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR T-cell therapy.

Single-cell RNA-sequencing of peripheral blood mononuclear cells reveals widespread, context-specific gene expression regulation upon pathogenic exposure.

The host's gene expression and gene regulatory response to pathogen exposure can be influenced by a combination of the host's genetic background, the type of and exposure time to pathogens. Here we provide a detailed dissection of this using single-cell RNA-sequencing of 1.3M peripheral blood mononuclear cells from 120 individuals, longitudinally exposed to three different pathogens. These analyses indicate that cell-type-specificity is a more prominent factor than pathogen-specificity regarding contexts that affect how genetics influences gene expression (i.e., eQTL) and co-expression (i.e., co-expression QTL). In monocytes, the strongest responder to pathogen stimulations, 71.4% of the genetic variants whose effect on gene expression is influenced by pathogen exposure (i.e., response QTL) also affect the co-expression between genes. This indicates widespread, context-specific changes in gene expression level and its regulation that are driven by genetics. Pathway analysis on the CLEC12A gene that exemplifies cell-type-, exposure-time- and genetic-background-dependent co-expression interactions, shows enrichment of the interferon (IFN) pathway specifically at 3-h post-exposure in monocytes. Similar genetic background-dependent association between IFN activity and CLEC12A co-expression patterns is confirmed in systemic lupus erythematosus by in silico analysis, which implies that CLEC12A might be an IFN-regulated gene. Altogether, this study highlights the importance of context for gaining a better understanding of the mechanisms of gene regulation in health and disease.

Factors associated with chemical burns in Zhejiang province, China: an epidemiological study.

BACKGROUND: Work-related burns are common among occupational injuries. Zhejiang Province is an industrial area with a high incidence of chemical burns. We aimed to survey epidemiological features of chemical burns in Zhejiang province to determine associated factors and acquire data for developing a strategy to prevent and treat chemical burns. METHODS: Questionnaires were developed, reviewed and validated by experts, and sent to 25 hospitals in Zhejiang province to prospectively collect data of 492 chemical burn patients admitted during one year from Sept. 1, 2008 to Aug. 31, 2009. Questions included victims' characteristics and general condition, injury location, causes of accident, causative chemicals, total body surface area burn, concomitant injuries, employee safety training, and awareness level of protective measures. Surveys were completed for each of burn patients by burn department personnel who interviewed the hospitalized patients. RESULTS: In this study, 417 victims (87.61%) got chemical burn at work, of which 355 victims (74.58%) worked in private or individual enterprises. Most frequent chemicals involved were hydrofluoric acid and sulfuric acid. Main causes of chemical injury accidents were inappropriate operation of equipment or handling of chemicals and absence of or failure to use effective individual protection. CONCLUSIONS: Most chemical burns are preventable occupational injuries that can be attributed to inappropriate operation of equipment or handling of chemicals, lack of employee awareness about appropriate action and lack of effective protective equipment and training. Emphasis on safety education and protection for workers may help protect workers and prevent chemical burns.

Single-cell transcriptional profiling of human thymic stroma uncovers novel cellular heterogeneity in the thymic medulla.

The thymus' key function in the immune system is to provide the necessary environment for the development of diverse and self-tolerant T lymphocytes. While recent evidence suggests that the thymic stroma is comprised of more functionally distinct subpopulations than previously appreciated, the extent of this cellular heterogeneity in the human thymus is not well understood. Here we use single-cell RNA sequencing to comprehensively profile the human thymic stroma across multiple stages of life. Mesenchyme, pericytes and endothelial cells are identified as potential key regulators of thymic epithelial cell differentiation and thymocyte migration. In-depth analyses of epithelial cells reveal the presence of ionocytes as a medullary population, while the expression of tissue-specific antigens is mapped to different subsets of epithelial cells. This work thus provides important insight on how the diversity of thymic cells is established, and how this heterogeneity contributes to the induction of immune tolerance in humans.

Inhibition of MET Signaling with Ficlatuzumab in Combination with Chemotherapy in Refractory AML: Clinical Outcomes and High-Dimensional Analysis.

Acute myeloid leukemia patients refractory to induction therapy or relapsed within one year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in pre-clinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single cell RNA sequencing using prospectively acquired patient specimens identified interferon response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well-tolerated with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies.

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a wholeblood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferonstimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and autodirected antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense. ONE SENTENCE SUMMARY: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.

Functional interpretation of single cell similarity maps.

We present Vision, a tool for annotating the sources of variation in single cell RNA-seq data in an automated and scalable manner. Vision operates directly on the manifold of cell-cell similarity and employs a flexible annotation approach that can operate either with or without preconceived stratification of the cells into groups or along a continuum. We demonstrate the utility of Vision in several case studies and show that it can derive important sources of cellular variation and link them to experimental meta-data even with relatively homogeneous sets of cells. Vision produces an interactive, low latency and feature rich web-based report that can be easily shared among researchers, thus facilitating data dissemination and collaboration.

Lineage dynamics of murine pancreatic development at single-cell resolution.

Organogenesis requires the complex interactions of multiple cell lineages that coordinate their expansion, differentiation, and maturation over time. Here, we profile the cell types within the epithelial and mesenchymal compartments of the murine pancreas across developmental time using a combination of single-cell RNA sequencing, immunofluorescence, in situ hybridization, and genetic lineage tracing. We identify previously underappreciated cellular heterogeneity of the developing mesenchyme and reconstruct potential lineage relationships among the pancreatic mesothelium and mesenchymal cell types. Within the epithelium, we find a previously undescribed endocrine progenitor population, as well as an analogous population in both human fetal tissue and human embryonic stem cells differentiating toward a pancreatic beta cell fate. Further, we identify candidate transcriptional regulators along the differentiation trajectory of this population toward the alpha or beta cell lineages. This work establishes a roadmap of pancreatic development and demonstrates the broad utility of this approach for understanding lineage dynamics in developing organs.

Genetic determinants of co-accessible chromatin regions in activated T cells across humans.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4+ T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression.