Unagi: Deep Generative Model for Deciphering Cellular Dynamics and In-Silico Drug Discovery in Complex Diseases (preprint)

Human diseases are characterized by intricate cellular dynamics. Single-cell sequencing provides critical insights, yet a persistent gap remains in computational tools for detailed disease progression analysis and targeted in-silico drug interventions. We introduce UNAGI, a deep generative neural network tailored to analyze time-series single-cell transcriptomic data. This innovative tool captures the complex cellular dynamics underlying disease progression, enhancing drug perturbation modeling and discovery. When applied to a dataset from patients with Idiopathic Pulmonary Fibrosis (IPF), UNAGI adeptly learns disease-informed cell embeddings that sharpen our understanding of disease progression, leading to the identification of potential therapeutic drug candidates. Validation via proteomics reveals the accuracy of UNAGI’s cellular dynamics analyses, and the use of the Fibrotic Cocktail treated human Precision-cut Lung Slices confirms UNAGI’s predictions that Nifedipine, an antihypertensive drug, may have antifibrotic effects on human tissues. UNAGI's versatility extends to other diseases, including a COVID dataset, demonstrating adaptability and confirming its broader applicability in decoding complex cellular dynamics beyond IPF, amplifying its utility in the quest for therapeutic solutions across diverse pathological landscapes.

Type I Interferon Transcriptional Network Regulates Expression of Coinhibitory Receptors in Human T cells (preprint)

While inhibition of T cell co-inhibitory receptors has revolutionized cancer therapy, the mechanisms governing their expression on human T cells have not been elucidated. Type 1 interferon (IFN-I) modulates T cell immunity in viral infection, autoimmunity, and cancer, and may facilitate induction of T cell exhaustion in chronic viral infection. Here we show that IFN-I regulates co-inhibitory receptor expression on human T cells, inducing PD-1/TIM-3/LAG-3 while surprisingly inhibiting TIGIT expression. High-temporal-resolution mRNA profiling of IFN-I responses enabled the construction of dynamic transcriptional regulatory networks uncovering three temporal transcriptional waves. Perturbation of key transcription factors on human primary T cells revealed unique regulators that control expression of co-inhibitory receptors. We found that the dynamic IFN-I response in vitro closely mirrored T cell features with IFN-I linked acute SARS-CoV-2 infection in human, with high LAG3 and decreased TIGIT expression. Finally, our gene regulatory network identified SP140 as a key regulator for differential LAG3 and TIGIT expression, which were validated at the level of protein expression. The construction of IFN-I regulatory networks with identification of unique transcription factors controlling co-inhibitory receptor expression may provide targets for enhancement of immunotherapy in cancer, infectious diseases, and autoimmunity.

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity (preprint)

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyzed the transcriptomes of 605,904 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observed a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD epithelial cells expressed higher levels of genes linked directly to the efficiency of viral replication and innate immune response. Additionally, we identified basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Type I Interferon Transcriptional Network Regulates Expression of Coinhibitory Receptors in Human T cells (preprint)

While inhibition of T cell co-inhibitory receptors has revolutionized cancer therapy, the mechanisms governing their expression on human T cells have not been elucidated. Type 1 interferon (IFN-I) modulates T cell immunity in viral infection, autoimmunity, and cancer, and may facilitate induction of T cell exhaustion in chronic viral infection 1,2 . Here we show that IFN-I regulates co-inhibitory receptors expression on human T cells, inducing PD-1/TIM-3/LAG-3 while surprisingly inhibiting TIGIT expression. High-temporal-resolution mRNA profiling of IFN-I responses enabled the construction of dynamic transcriptional regulatory networks uncovering three temporal transcriptional waves. Perturbation of key transcription factors on human primary T cells revealed both canonical and non-canonical IFN-I transcriptional regulators, and identified unique regulators that control expression of co-inhibitory receptors. To provide direct in vivo evidence for the role of IFN-I on co-inhibitory receptors, we then performed single cell RNA-sequencing in subjects infected with SARS-CoV-2, where viral load was strongly associated with T cell IFN-I signatures. We found that the dynamic IFN-I response in vitro closely mirrored T cell features with acute IFN-I linked viral infection, with high LAG3 and decreased TIGIT expression. Finally, our gene regulatory network identified SP140 as a key regulator for differential LAG3 and TIGIT expression. The construction of co-inhibitory regulatory networks induced by IFN-I with identification of unique transcription factors controlling their expression may provide targets for enhancement of immunotherapy in cancer, infectious diseases, and autoimmunity.

Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells (preprint)

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.