Crohn’s disease is an inflammatory bowel disease (IBD) commonly treated through anti-TNF blockade. However, most patients still relapse and inevitably progress. Comprehensive single-cell RNA-sequencing (scRNA-seq) atlases have largely sampled patients with established treatment-refractory IBD, limiting our understanding of which cell types, subsets, and states at diagnosis anticipate disease severity and response to treatment. Here, through combining clinical, flow cytometry, histology, and scRNA-seq methods, we profile diagnostic human biopsies from the terminal ileum of treatment-naïve pediatric patients with Crohn’s disease (pediCD; n=14), matched repeat biopsies (pediCD-treated; n=8) and from non-inflamed pediatric controls with functional gastrointestinal disorders (FGID; n=13). To resolve and annotate epithelial, stromal, and immune cell states among the 201,883 baseline single-cell transcriptomes, we develop a principled and unbiased tiered clustering approach, ARBOL. Through flow cytometry and scRNA-seq, we observe that treatment-naïve pediCD and FGID have similar broad cell type composition. However, through high-resolution scRNA-seq analysis and microscopy, we identify significant differences in cell subsets and states that arise during pediCD relative to FGID. By closely linking our scRNA-seq analysis with clinical meta-data, we resolve a vector of T cell, innate lymphocyte, myeloid, and epithelial cell states in treatment-naïve pediCD (pediCD-TIME) samples which can distinguish patients along the trajectory of disease severity and anti-TNF response. By using ARBOL with integration, we position repeat on-treatment biopsies from our patients between treatment-naïve pediCD and on-treatment adult CD. We identify that anti-TNF treatment pushes the pediatric cellular ecosystem towards an adult, more treatment-refractory state. Our study jointly leverages a treatment-naïve cohort, high-resolution principled scRNA-seq data analysis, and clinical outcomes to understand which baseline cell states may predict Crohn’s disease trajectory.

High-throughput phenotypic screens using biochemical perturbations and high-content readouts are constrained by limitations of scale. To address this, we establish a method of pooling exogenous perturbations followed by computational deconvolution to reduce required sample size, labor and cost. We demonstrate the increased efficiency of compressed experimental designs compared to conventional approaches through benchmarking with a bioactive small-molecule library and a high-content imaging readout. We then apply compressed screening in two biological discovery campaigns. In the first, we use early-passage pancreatic cancer organoids to map transcriptional responses to a library of recombinant tumor microenvironment protein ligands, uncovering reproducible phenotypic shifts induced by specific ligands distinct from canonical reference signatures and correlated with clinical outcome. In the second, we identify the pleotropic modulatory effects of a chemical compound library with known mechanisms of action on primary human peripheral blood mononuclear cell immune responses. In sum, our approach empowers phenotypic screens with information-rich readouts to advance drug discovery efforts and basic biological inquiry. Phenotypic screens in organoids and primary cells are scaled up using perturbation pooling.

High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.

The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in wild-type mice. Physiological organoid models of the differentiation of intestinal stem cells could find broader utility for the screening of biological targets and small molecules that can modulate the composition and function of other barrier epithelia.

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth stunting, and diminished oral vaccine responses. Although EE has been shown to be the by-product of a recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE by performing high-throughput, single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE in Lusaka, Zambia (eight HIV-negative and three HIV-positive), six adults without EE in Boston, United States, and two adults in Durban, South Africa, which we complemented with published data from three additional individuals from the same clinical site. We analyzed previously defined bulk-transcriptomic signatures of reduced villus height and decreased microbial translocation in EE and showed that these signatures may be driven by an increased abundance of surface mucosal cells-a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we determined cell subsets whose fractional abundances associate with EE severity, small intestinal region, and HIV infection. Furthermore, by comparing duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and increased proinflammatory cytokine gene expression in a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells in the EE cohort. Together, our work elucidates epithelial and immune correlates of EE and nominates cellular and molecular targets for intervention.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in lowand middle-income countries. It is thought to be a primary cause of most global growth-stunting cases and a key contributing factor to childhood malnutrition and diminished oral vaccine responses. While EE has been shown to be the by-product of recurrent enteric infection, to date, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE severity by performing high-throughput single-cell RNAsequencing on 33 small intestinal biopsies from 11 adults with EE from Lusaka, Zambia (8 HIVnegative, 3 HIV-positive) and 6 adults without EE in Boston, USA. Using the resulting cellular atlas, we scored existing bulk-transcriptomic signatures of reduced villus height and decreased plasma LPS levels in EE, finding that these signatures may be driven by an increased abundance of surface mucosal cells–a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we identified several cell subsets whose fractional abundances associated with histological determined EE severity, small intestinal region, and HIV infection. Furthermore, by comparing distal duodenal EE samples with those from two U.S. control cohorts, we identified broadly decreased epithelial proliferative signaling, lower fractional abundances of goblet cells, and a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells but with increased pro-inflammatory cytokine expression in EE. Altogether, our work illuminates the epithelial and immune correlates of EE severity and provides new molecular targets for intervention. Main Text: INTRODUCTION Environmental enteropathy (EE) is a subclinical condition of the small intestine that is driven by enteropathogen exposure through environmental contamination (1, 2). Also referred to as Environmental Enteric Dysfunction (EED), EE impacts millions of children and adults around the world. It is associated with stunted growth, neurocognitive impairment, reduced oral vaccine efficacy, and life-long increased risk of metabolic syndrome (1, 3, 4).Water, sanitation, and hygiene (WASH) interventions for preventing EE have proven ineffective, and ongoing work is assessing alternative therapeutic interventions such as antibiotics, anti-inflammatory therapeutics, and dietary supplementation (5). However, development of effective treatments has been hindered by a limited understanding of the underlying mechanisms of EE. Studies of the tissue biology of EE have been practically limited by operational constraints. Obtaining small intestinal biopsies from patients with EE requires esophago-gastroduodenoscopy (EGD)—a procedure that, while generally safe, carries increased risk to perform in undernourished pediatric populations. Accordingly, novel approaches and initial mechanistic studies using invasive techniques should ethically be completed in affected adult populations before profiling pediatric patients. While the largest global health consequences of EE are seen in children, this condition also negatively impacts adult quality of life via its associated increased intestinal permeability, which can lead to systemic inflammation, increased risk of metabolic diseases, and reduced intestinal absorption (1, 6). Given the environmental nature of enteric infections, finding unaffected controls for studies of EE in affected populations is similarly challenging. Thus, EE is often contextualized to health by either comparing intermediate EE with severe EE (7) or by comparing EE patients to control cohorts in the United States or the United Kingdom (8). The validity of these international comparisons is supported by the environmental nature of EE and the resolution of EE in Peace Corps volunteers upon repatriation to the United States (9). Pathologically, EE in the proximal small intestine is continuous (not patchy), does not extend beyond the mucosa, and is characterized by reduced villus height, increased villus fusion, greater crypt depth, and increased microbial translocation (10). However, in a study of Zambian children with EE and non-responsive growth stunting over time, reduced villus height was associated with decreased circulating levels of LPS (a measure of microbial translocation) (11). In agreement, a bulk transcriptomic study of Zambia children with enteropathy due to severe acute malnutrition (SAM) showed that many genes differentially upregulated in biopsies with reduced villus height were downregulated in biopsies from participants with high circulating levels of LPS (7). These studies raise the intriguing possibility that EE is an adaptive response to potentially lethal enteropathogen exposure that comes at the cost of reduced absorptive capacity and thus impaired growth. However, a detailed picture of the cellular changes underpinning these processes is currently missing. Histological analysis of EE has revealed increased abundance of lymphocytes (rather than granulocytes), abnormalities of secretory cells, reduced goblet cell numbers, and abnormal Paneth cell morphology (8, 12). Low plasma levels of tryptophan in children with growth stunting (13) and the amelioration of villus blunting in Zambian adults given tryptophan, glutamine, and leucine supplementation suggest that amino acid deficiency may play a role in epithelial remodeling in EE (14). Bulk transcriptomic studies of EE duodenal biopsies, meanwhile, have revealed increased expression of NADPH oxidases, CXC chemokines, mucin genes, matrix metalloprotease genes, interferon stimulated genes, and antimicrobial genes including LCN2, DUOX2, and DUOXA2 (15, 16). In addition, immunohistochemistry staining of the DUOX2 protein has been validated as a marker distinguishing tissues from Bangladeshi children with EE from control samples obtained from North American children with healthy tissue or with celiac disease (16). Furthermore, transcriptomic analysis of feces from Malawian children has illustrated increased innate immune activity and interferon signaling, as well as reduced expression of mucins and pro-proliferative genes (including EGFR and MAPK7), in severe EE (17). However, previous work has lacked the single-cell resolution required to localize these changes to specific epithelial and immune cell subsets in the small intestine. High-throughput single-cell genomic profiling holds transformative promise for understanding the cellular populations, phenotypic states, and signaling changes that underlie EE (18). In the small intestine, it is challenging to use bulk level measurements to resolve the states of rarer cell types (such as intestinal stem cells, enteroendocrine cells, or tissue resident lymphocytes) that play crucial roles in tissue maintenance (19). In contrast, single-cell RNA-sequencing (scRNA-seq) can be used to comprehensively profile the distinct cellular subsets that compose complex tissues, thereby enabling analysis of rare yet influential cell subsets, and discovery of altered cell states and signaling. Illustratively, the unprecedented cellular resolution provided by scRNA-seq has been leveraged to identify novel features of human epithelial inflammation in the skin(20), the nasal passages (21), and the large intestine (22). While scRNA-seq has also been used to study the healthy human small intestine (23), to our knowledge, it has yet to be leveraged to understand inflammation in this organ. Here, we applied the Seq-Well platform for massively-parallel scRNA-seq (24) to profile small intestinal biopsies from 11 adults from a community in Zambia where EE is known to be ubiquitous (25). Across these individuals, we profiled 27 biopsies spanning 3 small intestinal regions, HIV-positive and HIV-negative patients, and a range of histological EE severity scores. This provided capacity to define the cellular subsets associated with these covariates. In addition, by comparing EE biopsies with those from two control groups in the USA, we found broadly decreased epithelial proliferative signaling, and lower abundances of goblet cells and a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells as well as proinflammatory cytokines in EE. Altogether, our data provide new insight into epithelial remodeling and immune signaling in EE, suggesting several novel therapeutic targets for further investigation. RESULTS scRNA-seq atlas of the proximal small intestine with and without EE We collected 27 small intestinal biopsies from 11 Zambian volunteers with EE (8 HIV-negative, 3 HIV-positive). For all 11, we profiled duodenal bulb and distal duodenum; for a subset, we also collected jejunal samples (Table S1, S2). Biopsies displayed varying levels of EE severity, with villus height:crypt depth ratios ranging from 0.64:1 to 2.38:1, compared to a normal ratio of 3:1 or greater (Fig. 1A, Table S3) (8). Across these samples, we sought to identify the cellular correlates of intestinal region, HIV infection, and EE histological severity. However, due to both the widespread prevalence of EE in Zambia and a lack of existing screening methods to identify patients with EE, we could not obtain control biopsies from patients without EE in Zambia. Thus, we chose to distinguish the cellular and molecular features of EE biology by comparing to samples from patients in Boston, USA where EE can safely be assumed not to occur, and to mitigate the possibility of unexpected disease effects we included two control groups. We profiled distal duodenum samples from 3 patients undergoing screening for eosinophilic esophagitis (EoE) and 3 patients undergoing duodenal resection for pancreatic malignancy (Table S1, S2). Among the former, pathology did not report eosinophilic gastroenteritis for any patients screened for EoE, and revealed that one patient did not have EoE; duodenal resections from pancreatic cancer patients

In humans and nonhuman primates, Mycobacterium tuberculosis lung infection yields a complex multicellular structure—the tuberculosis granuloma. All granulomas are not equivalent, however, even within the same host: in some, local immune activity promotes bacterial clearance, while in others, it allows persistence or outgrowth. Here, we used single-cell RNA-sequencing to define holistically cellular responses associated with control in cynomolgus macaques. Granulomas that facilitated bacterial killing contained significantly higher proportions of CD4+ and CD8+ T cells expressing hybrid Type1-Type17 immune responses or stem-like features and CD8-enriched T cells with specific cytotoxic functions; failure to control correlated with mast cell, plasma cell and fibroblast abundance. Co-registering these data with serial PET-CT imaging suggests that a degree of early immune control can be achieved through cytotoxic activity, but that more robust restriction only arises after the priming of specific adaptive immune responses, defining new targets for vaccination and treatment.