Biology subjects

Gentili, M.

Publications and source records attributed to Gentili, M..

5 recordsLinked to original sources

bioRxiv2020

An efficient lentiviral CRISPRi approach to silence genes in primary human monocytes

Human primary monocytes are critical in controlling immune responses and tissue homeostasis. However, to identify and study molecular components that underlie the function of these cells, there remains a need to develop methods to perturb genes in these cells. Here we report lentiviral-based delivery of dCas9-KRAB and guide RNAs to efficiently silent target genes, such as CD45 and CD209. We further show that sgRNAs against TICAM1 dampen proinflammatory cytokine and interferon expression in response to lipopolysaccharide. When delivered by lentivirus, sgRNAs are incorporated into the genome, thus enabling pooled screening to perturb and identify coding and non-coding elements that contribute to the functions of primary human monocytes.

immunology↗

bioRxiv2020

Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients

COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.

systems biology↗

bioRxiv2020

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

bioengineering↗

bioRxiv2020

SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.

immunology↗

bioRxiv2020

Compromised nuclear envelope integrity drives tumor cell invasion

While mutations leading to a fragile envelope of the cell nucleus are well known to cause diseases such as muscular dystrophies or accelerated aging, the pathophysiological consequences of the recently discovered mechanically induced nuclear envelope ruptures in cells harboring no mutation are less known. Here we show that repeated loss of nuclear envelope integrity in nuclei experiencing mechanical constraints promotes senescence in nontransformed cells, and induces an invasive phenotype including increased collagen degradation in human breast cancer cells, both in vitro and in a mouse xenograft model of breast cancer progression. We show that these phenotypic changes are due to the presence of chronic DNA damage and activation of the ATM kinase. In addition, we found that depletion of the cytoplasmic exonuclease TREX1 is sufficient to abolish the DNA damage in mechanically challenged nuclei and to suppress the phenotypes associated with the loss of nuclear envelope integrity. Our results also show that TREX1-dependent DNA damage induced by physical confinement of tumor cells inside the mammary duct drives the progression of in situ breast carcinoma to the invasive stage. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

cell biology↗