All Publications

  • Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut. Molecular systems biology Triana, S., Metz-Zumaran, C., Ramirez, C., Kee, C., Doldan, P., Shahraz, M., Schraivogel, D., Gschwind, A. R., Sharma, A. K., Steinmetz, L. M., Herrmann, C., Alexandrov, T., Boulant, S., Stanifer, M. L. 2021; 17 (4): e10232


    Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. However, despite the mounting evidence about SARS-CoV-2 infecting the human gut, little is known about the antiviral programs triggered in this organ. To address this gap, we performed single-cell transcriptomics of SARS-CoV-2-infected intestinal organoids. We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2 and, interestingly, found the lack of positive correlation between susceptibility to infection and the expression of ACE2. Infected cells activated strong pro-inflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon. These findings reveal that SARS-CoV-2 curtails the immune response and highlights the gut as a pro-inflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis.

    View details for DOI 10.15252/msb.202110232

    View details for PubMedID 33904651

  • Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells. eLife Wells, K. L., Miller, C. N., Gschwind, A. R., Wei, W., Phipps, J. D., Anderson, M. S., Steinmetz, L. M. 2020; 9


    Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

    View details for DOI 10.7554/eLife.60188

    View details for PubMedID 33226342

  • Targeted Perturb-seq enables genome-scale genetic screens in single cells. Nature methods Schraivogel, D., Gschwind, A. R., Milbank, J. H., Leonce, D. R., Jakob, P., Mathur, L., Korbel, J. O., Merten, C. A., Velten, L., Steinmetz, L. M. 2020


    The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer-target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer-target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.

    View details for DOI 10.1038/s41592-020-0837-5

    View details for PubMedID 32483332