Search

E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comβVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.

Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer mortality in the United States by 2030. Given that resistance to cytotoxic therapy is pervasive, there is a critical need to elucidate salient gene expression programs and spatial relationships among malignant and stromal cells in the tumor microenvironment (TME), particularly in residual disease. We developed and applied a single-nucleus RNA-seq (snRNA-seq) technique to 43 banked frozen primary PDAC specimens that either received neoadjuvant therapy (n=25) or were treatment-naïve (n=18). We discovered expression programs across malignant cell and fibroblast profiles that formed the basis for a refined molecular taxonomy, including a novel neural-like progenitor (NRP) malignant program enriched with neoadjuvant treatment in tumors and organoids, and associated with the worst prognosis in bulk profiles from independent cohorts. To elucidate how neoadjuvant treatment and cancer cell- and fibroblast-intrinsic programs modulate the composition of multicellular neighborhoods, we performed spatial profiling with the GeoMx[1] platform (NanoString) on 21 formalin-fixed paraffin-embedded sections using the human whole transcriptome atlas (WTA). Each tumor showed intra-tumoral heterogeneity in tissue architecture and regions of interest (ROIs) with diverse patterns of neoplastic cells, cancer-associated fibroblasts (CAFs), and immune cells were selected for profiling. We deconvolved the WTA data with our snRNA-seq cell type signatures and mapped expression programs onto the tumor architecture to reveal three distinct multicellular neighborhoods, which we annotated as classical, squamoid-basaloid, and treatment-enriched. The observed enrichment in post-treatment residual disease of multiple spatially-defined receptor-ligand interactions and a neighborhood featuring the NRP program, neurotropic CAF program, and CD8+ T cells may open new therapeutic opportunities. Next, we mapped malignant/CAF programs and immune cell subsets at single-cell spatial resolution by performing spatial molecular imaging (SMI[2]; NanoString CosMx) using a panel of 960 RNA targets on a subset of seven tumors (2 untreated, 5 treated) and captured over 200,000 cells with an average of more than 450 transcripts detected per cell. Correlating ROIs from whole-transcriptome DSP to matched fields of view in kiloplex SMI enabled further dissection of PDAC architecture and treatment-associated remodeling of cell type distributions and receptor-ligand interactions. Ongoing functional studies have begun to elucidate the key regulatory elements underlying the distinct treatment-associated NRP malignant program and its interactions with the TME. Overall, the complementary combination of snRNA-seq, whole-transcriptome DSP, and kiloplex SMI provides a high-resolution molecular framework that can be harnessed to augment precision oncology efforts in pancreatic cancer. [1] GeoMx DSP is for Research Use Only and not for use in diagnostic procedures. [2] CosMx SMI is for Research Use Only and not for use in diagnostic procedures. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Carina Shiau, Jennifer Su, Payman Yadollahpour, Jason W. Reeves, Youngmi Kim, Sean Kim, Mark Gregory, Prajan Divakar, Eric Miller, Michael Rhodes, Sarah Warren, Erroll Rueckert, Kit Fuhrman, Daniel R. Zollinger, Robin Fropf, Joseph M. Beechem, Arnav Mehta, Toni Delorey, Cristin McCabe, Jaimie L. Barth, Piotr Zelga, Cristina R. Ferrone, Motaz Qadan, Keith D. Lillemoe, Rakesh K. Jain, Jennifer Y. Wo, Theodore S. Hong, Ramnik Xavier, Orit Rozenblatt-Rosen, Andrew J. Aguirre, Carlos Fernandez-Del Castillo, Andrew S. Liss, Mari Mino-Kenudson, David T. Ting, Tyler Jacks, Aviv Regev. Multicellular spatial community featuring a novel neuronal-like malignant phenotype is enriched in pancreatic cancer after neoadjuvant chemotherapy and radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY12-04.

While advances in single cell genomics have helped to chart the cellular components of tumor ecosystems, it has been more challenging to characterize their specific spatial organization and functional interactions. Here, we combine single cell RNA-seq and spatial transcriptomics by Slide-seq, to create a detailed spatial map of healthy and dysplastic colon cellular ecosystems and their association with disease progression. We profiled an inducible genetic CRC mouse model that recapitulates key features of human CRC, assigned cell types and epithelial expression programs to spatial tissue locations in tumors, and computationally used them to identify the regional features spanning different cells in the same spatial niche. We find that tumors were organized in cellular neighborhoods, each with a distinct composition of cell subtypes, expression programs, and local cellular interactions. Three cellular neighborhood archetypes were associated with tumor progression, were active at the same time in different spatial parts of the same tumor, involved dysplasia-specific cellular layouts, and relied on distinct mechanisms: (1) inflammatory epithelial regions with endothelial cells and monocytes expressing angiogenesis, inflammation and invasion programs; (2) epithelial stem-like regions, associated with plasma and B cell activity; and (3) epithelial-to-mesenchymal transition (EMT) regions with dysplastic cells expressing a Wnt signaling program. Comparing to scRNA-seq and Slide-seq data from human CRC, we find that both cell composition and layout features were conserved in both species, with mouse archetypal neighborhoods correlated with malignancy and clinical outcome in human patient tumors, highlighting the relevance of our findings to human disease.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.

SARS-CoV-2 infection leads to a broad range of outcomes and immune responses, with the development of neutralizing antibodies generally correlated with protection against reinfection. Here, we have characterized both neutralizing activity and T cell responses in a cluster of subjects with mild disease linked to a single spreading event. Surprisingly, we observed sex-specific associations between spike- and particularly nucleoprotein-specific T cell responses and neutralization, with pro-inflammatory cytokines being linked to higher titers only in males. Using single cell immunoprofiling, which provided matched transcriptome and T-cell receptor (TCR) profiles in restimulated CD4 + and CD8 + cells from these subjects, we identified differences in type I IFN signaling that may underlie this difference in antibody generation. Finally, we also identified several TCRs associated with cytokine producing T cells. Altogether, our work maps the breadth of immunological outcomes of SARS-CoV2 infections and highlight the potential role of sex-specific feedback loops during the generation of neutralizing antibodies.

Summary The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.

Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract SARS-CoV-2 and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA-seq and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses., B cell genomics reveals transcriptionally distinct populations that modulate antibody responses to SARS-CoV-2, with the identification of a monoclonal antibody that locks the virus spike trimer to neutralize recent variants, SARS and heterologous RBDs.

This protocol describes the dissociation of primary human colorectal cancer (CRC) resection specimens into single cell suspensions for single cell RNA-seq applications. It was successfully used for droplet-based single cell RNA-seq of more than 60 patient specimens using the 10x Genomics platform, including 21 HTAPP CRC cohort samples. It can also be used upstream of other single cell RNA-seq approaches such as plate-based SS2 single cell RNA-seq protocols.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients’ demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease. Characterizing PDAC by mRNA profiling remains particularly challenging. Previously identified bulk expression subtypes were influenced by contaminating stroma and have not yet informed clinical management, whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for frozen archival PDAC specimens and used it to study both untreated tumors and those that received neoadjuvant chemotherapy and radiotherapy (CRT). Gene expression programs learned across untreated malignant cell and fibroblast profiles uncovered a clinically relevant molecular taxonomy with improved prognostic stratification compared to prior classifications. Moreover, in the increasingly-adopted neoadjuvant treatment context, there was a depletion of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states, which may be more resilient to cytotoxic treatment. Spatially-resolved transcriptomics revealed an association between malignant cells expressing these basal-like programs and higher immune infiltration with increased lymphocytic content, whereas those exhibiting classical-like programs were linked to sparser macrophage-predominant microniches, perhaps pointing to susceptibility to distinct therapeutic strategies. Our refined molecular taxonomy and spatial resolution can help advance precision oncology in PDAC through informative stratification in clinical trials and insights into differential therapeutic targeting leveraging the immune system.

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS. Cellular states capable of promoting tumor progression and resisting therapies exist in heterogeneous tumors. Marjanovic et al. discover that a high-plasticity cell state common to mouse and human lung tumors drives cellular heterogeneity, is highly tumorigenic and drug resistant, and associates with poor patient prognosis.

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using two novel preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, which are similar to tumor-infiltrating lymphocytes of human PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical to maintain immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.

610 Background: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistance to chemotherapy and radiotherapy is a major obstacle to improving clinical outcomes. Hence, there is an urgent need to elucidate the gene expression programs, spatial context, and interactions among different cell types in residual disease after neoadjuvant treatment. Methods: We optimized and applied single-nucleus RNA-seq (snRNA-seq) to 43 frozen primary PDAC tumors. Eighteen were treatment-naïve, 14 received FOLFIRINOX followed by radiotherapy with 5-FU or capecitabine (CRT), and 5 were subjected to CRT combined with losartan on protocol (CRTL). We performed unsupervised clustering of single nucleus profiles and then annotated and quantified cell subsets. Malignant and fibroblast gene expression programs were identified by consensus non-negative matrix factorization (cNMF). We mapped our cell type signatures and expression programs onto the tumor architecture using whole-transcriptome digital spatial profiling (DSP) to uncover distinct multicellular spatial neighborhoods and intercellular interactions that compose PDAC and are remodeled by neoadjuvant treatment. Results: Consistent with treatment effect, the proportion of malignant cells was significantly lower in tumors treated with neoadjuvant therapy. Within the immune compartment, CRTL was associated with a higher fraction of CD8+ T cells and Tregs compared to untreated and CRT tumors. Differential expression analysis of CD8+ T cells revealed greater effector function (e.g., IL2, CCL4, CCL5) and reduced quiescence/dysfunction markers (e.g., TIGIT, TCF7, KLF2, LEF1) associated with CRTL. We discovered expression programs across malignant and fibroblast profiles that formed a refined molecular taxonomy, including a novel neuronal-like malignant program enriched in the neoadjuvant groups and associated with the worst prognosis in independent cohorts. Ex vivo treatment of organoids derived from an untreated PDAC with FOLFIRINOX chemotherapy and radiotherapy recapitulated enrichment of the neuronal-like program. Whole-transcriptome DSP revealed three distinct multicellular neighborhoods: classical, squamoid-basaloid, and treatment-enriched. The observed enrichment in post-treatment residual disease of multiple spatially-defined receptor-ligand interactions and a neighborhood featuring colocalization of the neuronal-like malignant program, neurotropic CAF program, and CD8+ T cells may open new opportunities for therapeutic targeting in PDAC. Conclusions: Our work provides a high-resolution molecular framework for understanding the inter- and intra-tumoral heterogeneity of pancreatic cancer, spatial organization into discrete multicellular communities, and treatment-associated reprogramming as a blueprint for exploring novel therapeutic strategies tailored to residual disease.

This protocol describes the dissociation of primary human colorectal cancer (CRC) resection specimens into single cell suspensions for single cell RNA-seq applications. It was successfully used for droplet-based single cell RNA-seq of more than 60 patient specimens using the 10x Genomics platform, including 20 HTAPP CRC cohort samples. It can also be used upstream of other single cell RNA-seq approaches such as plate-based SS2 single cell RNA-seq protocols.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

A molecular classification of pancreatic ductal adenocarcinoma (PDAC) that informs clinical management remains elusive. Previously identified bulk expression subtypes in the untreated setting were influenced by contaminating stroma whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Two consensus subtypes have arisen from these prior efforts: (1) classical-like, and (2) basal-like. Basal-like tumors were associated with worse survival in the metastatic setting but attempts to refine this binary classification have failed to further stratify patient survival. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for banked frozen PDAC specimens and studied a cohort of untreated resected primary tumors (n ~ 20). Gene expression programs learned across malignant cell and cancer-associated fibroblast (CAF) profiles uncovered a clinically-relevant molecular taxonomy with improved prognostic stratification compared to prior classifications. Digital spatial profiling revealed an association between malignant cells expressing basal-like programs and greater immune infiltration with relatively fewer macrophages, whereas those exhibiting classical-like programs were linked to inflammatory CAFs and macrophage-predominant microniches. Recent clinical trials have supported the increasing adoption of neoadjuvant therapy to aggressively address the risk of micro-metastatic spread and to circumvent concerns of treatment tolerance in the postoperative setting. There is an urgent need to understand how preoperative treatment impacts residual tumor cells and their interactions with other cell types in the tumor microenvironment to identify additional therapeutic vulnerabilities that can be exploited. Towards this end, we performed snRNA-seq on an unmatched cohort of neoadjuvant-treated resected primary tumors (n ~ 25) with most cases involving FOLFIRINOX chemotherapy followed by chemoradiation. Remarkably, the quality of single-nucleus mRNA profiles was comparable between heavily pre-treated and untreated specimens. We identified differentially expressed genes between treated and untreated samples to infer cell-type specific reprogramming in the residual tumor. This analysis revealed that in the neoadjuvant treatment context, there was lower expression of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states. Our refined molecular taxonomy and spatial resolution may help advance precision oncology in PDAC through informative stratification in clinical trials and insights into compartment-specific therapies. Citation Format: William L. Hwang, Karthik A. Jagadeesh, Jimmy A. Guo, Hannah I. Hoffman, Payman Yadollahpour, Jason Reeves, Eugene Drokhlyansky, Nicholas Van Wittenberghe, Samouil Farhi, Denis Schapiro, George Eng, Jason M. Schenkel, William A. Freed-Pastor, Orr Ashenberg, Clifton Rodrigues, Domenic Abbondanza, Toni Delorey, Devan Phillips, Jorge Roldan, Debora Ciprani, Marina Kern, Jaimie L. Barth, Daniel R. Zollinger, Kit Fuhrman, Robin Fropf, Joseph Beechem, Colin Weekes, Cristina R. Ferrone, Jennifer Y. Wo, Theodore S. Hong, Orit Rozenblatt-Rosen, Andrew J. Aguirre, Mari Mino-Kenudson, Carlos Fernandez-del- Castillo, Andrew S. Liss, David T. Ting, Tyler Jacks, Aviv Regev. Multi-compartment reprogramming and spatially-resolved interactions in frozen pancreatic cancer with and without neoadjuvant chemotherapy and radiotherapy at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 94.

Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.

Almost all humans are colonized with Candida albicans. However, in immunocompromised individuals, this benign commensal organism becomes a serious, life-threatening pathogen. Here, we describe and analyze the regulatory networks that modulate innate responses in the host niches. We identified Zcf15 and Zcf29, two Zinc Cluster transcription Factors (ZCF) that are required for C. albicans virulence. Previous sequence analysis of clinical C. albicans isolates from immunocompromised patients indicates that both ZCF genes diverged during clonal evolution. Using in vivo animal models, ex vivo cell culture methods, and in vitro sensitivity assays, we demonstrate that knockout mutants of both ZCF15 and ZCF29 are hypersensitive to reactive oxygen species (ROS), suggesting they help neutralize the host-derived ROS produced by phagocytes, as well as establish a sustained infection in vivo. Transcriptomic analysis of mutants under resting conditions where cells were not experiencing oxidative stress revealed a large network that control macro and micronutrient homeostasis, which likely contributes to overall pathogen fitness in host niches. Under oxidative stress, both transcription factors regulate a separate set of genes involved in detoxification of ROS and down-regulating ribosome biogenesis. ChIP-seq analysis, which reveals vastly different binding partners for each transcription factor (TF) before and after oxidative stress, further confirms these results. Furthermore, the absence of a dominant binding motif likely facilitates their mobility, and supports the notion that they represent a recent expansion of the ZCF family in the pathogenic Candida species. Our analyses provide a framework for understanding new aspects of the interface between C. albicans and host defense response, and extends our understanding of how complex cell behaviors are linked to the evolution of TFs.

The outcome of fungal infections depends on interactions with innate immune cells. Within a population of macrophages encountering Candida albicans, there are distinct host-pathogen trajectories; however, little is known about the molecular heterogeneity that governs these fates. Here we developed an experimental system to separate interaction stages and single macrophage cells infected with C. albicans from uninfected cells and assessed transcriptional variability in the host and fungus. Macrophages displayed an initial up-regulation of pathways involved in phagocytosis and proinflammatory response after C. albicans exposure that declined during later time points. Phagocytosed C. albicans shifted expression programs to survive the nutrient poor phagosome and remodeled the cell wall. The transcriptomes of single infected macrophages and phagocytosed C. albicans displayed a tightly coordinated shift in gene expression co-stages and revealed expression bimodality and differential splicing that may drive infection outcome. This work establishes an approach for studying host-pathogen trajectories to resolve heterogeneity in dynamic populations., The outcomes of the interactions between individual host cells and pathogens are heterogeneous. Here, the authors assess transcriptional variability in both host and pathogen during infection of macrophages with the fungus Candida albicans, using sorted subpopulations and single macrophages.

The transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of C. gattii in rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccase LAC1 to expression of LAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response to C. gattii. This work highlights the dynamic roles of key C. gattii virulence genes in response to macrophages., Cryptococcus gattii is a pathogenic yeast of humans and other animals which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual-transcriptome sequencing (RNA-seq) experiment for four lineages of C. gattii (lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons of in vitro to ex vivo gene expression levels indicated that lineage VGII is transcriptionally divergent from non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment, and ergosterol production. Several paralogous genes demonstrated subfunctionalization between lineages, including upregulation of capsule biosynthesis-related gene CAP2 and downregulation of CAP1 in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule gene CAS3 in VGIV, which have lost the CAS31 gene. Differential expression of one in five C. gattii genes was detected following coincubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and downregulation of capsule attachment genes. We also found that VGII switches expression of two laccase paralogs (from LAC1 to LAC2) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of C. gattii by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages of C. gattii and the diversity of transcriptional responses at this host-pathogen interface. IMPORTANCE The transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of C. gattii in rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccase LAC1 to expression of LAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response to C. gattii. This work highlights the dynamic roles of key C. gattii virulence genes in response to macrophages.

Cryptococcus gattiiis a pathogenic yeast of humans and other animals, which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual RNA-seq experiment for four lineages ofC. gattii(VGI-IV) interacting with mouse macrophages at 1hr, 3hr and 6hr post infection. Comparison ofin vitrotoex vivogene expression indicates lineage VGII is transcriptionally divergent to non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment and ergosterol production. Various paralogs demonstrate sub-functionalisation between lineages including an upregulation of capsule biosynthesis-related geneCAP2, and downregulation ofCAP1in VGIII. Isolates also compensate for lineage-specific gene-losses by over-expression of genetically similar paralogs, including an over-expression of capsule geneCAS3in VGIV having lostCAS31. Differential expression of one in fiveC. gattiigenes was detected following co-incubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and a downregulation of capsule attachment genes. We also show that VGII switches expression of two laccase paralogs (fromLAC1toLAC2) during co-incubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages ofC. gattiiby upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This study highlights the evolutionary breadth of expression profiles amongst the lineages ofC. gattiiand the diversity of transcriptional responses at this host-pathogen interface.ImportanceThe transcriptional profiles of related pathogens and their response to host induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages ofC. gattiiin rich media. Our analyses identified key processes including cell capsule, ergosterol production and melanin that are differentially expressed between lineages, and we find that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone sub-functionalisation for various paralogs including capsule biosynthesis and attachment genes. Most genes appeared down-regulated during co-incubation with macrophages, with the largest decrease observed for capsule attachment genes, which appears coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched expression of the laccaseLAC1toLAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow derived macrophages, marking a role in the host response toC. gattii. This work highlights the dynamic roles of keyC. gattiivirulence genes in response to macrophages.

How the immune system readies for battle Although gene expression is tightly controlled at both the RNA and protein levels, the quantitative contribution of each step, especially during dynamic responses, remains largely unknown. Indeed, there has been much debate whether changes in RNA level contribute substantially to protein-level regulation. Jovanovic et al. built a genome-scale model of the temporal dynamics of differential protein expression during the stimulation of immunological dendritic cells (see the Perspective by Li and Biggin). Newly stimulated functions involved the up-regulation of specific RNAs and concomitant increases in the levels of the proteins they encode, whereas housekeeping functions were regulated posttranscriptionally at the protein level. Science , this issue 10.1126/science.1259038 ; see also p. 1066

Candida albicans is both a member of the healthy human microbiome and a major pathogen in immunocompromised individuals. Infections are typically treated with azole inhibitors of ergosterol biosynthesis often leading to drug resistance. Studies in clinical isolates have implicated multiple mechanisms in resistance, but have focused on large-scale aberrations or candidate genes, and do not comprehensively chart the genetic basis of adaptation. Here, we leveraged next-generation sequencing to analyze 43 isolates from 11 oral candidiasis patients. We detected newly selected mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss-of-heterozygosity (LOH) events. LOH events were commonly associated with acquired resistance, and SNPs in 240 genes may be related to host adaptation. Conversely, most aneuploidies were transient and did not correlate with drug resistance. Our analysis also shows that isolates also varied in adherence, filamentation, and virulence. Our work reveals new molecular mechanisms underlying the evolution of drug resistance and host adaptation., National Science Foundation (U.S.). Graduate Research Fellowship Program, Howard Hughes Medical Institute, Helen Hay Whitney Foundation (Postdoctoral Fellowship), Alfred P. Sloan Foundation, National Institutes of Health (U.S.) (Grant 8DP1CA174427), National Institutes of Health (U.S.) (Grant 2R01CA119176-01)

Changes in transcriptional regulatory networks can significantly contribute to species evolution and adaptation. However, identification of genome-scale regulatory networks is an open challenge, especially in non-model organisms. Here, we introduce multi-species regulatory network learning (MRTLE), a computational approach that uses phylogenetic structure, sequence-specific motifs, and transcriptomic data, to infer the regulatory networks in different species. Using simulated data from known networks and transcriptomic data from six divergent yeasts, we demonstrate that MRTLE predicts networks with greater accuracy than existing methods because it incorporates phylogenetic information. We used MRTLE to infer the structure of the transcriptional networks that control the osmotic stress responses of divergent, non-model yeast species and then validated our predictions experimentally. Interrogating these networks reveals that gene duplication promotes network divergence across evolution. Taken together, our approach facilitates study of regulatory network evolutionary dynamics across multiple poorly studied species. Keywords: regulatory networks; network inference; evolution of gene regulatory networks; evolution of stress response; yeast; probabilistic graphical model; phylogeny; comparative functional genomics, National Science Foundation (U.S.) (Grant DBI-1350677), National Institutes of Health (U.S.) (Grant R01CA119176-01), National Institutes of Health (U.S.) (Grant DP1OD003958-01)

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.