Project description:Identification of neoepitopes that are effective in cancer therapy is a major challenge in creating cancer vaccines. Here, using an entirely unbiased approach, we have queried all possible neoepitopes in a mouse cancer model and asked which of those are effective in mediating tumor rejection, and independently, in eliciting a measurable CD8 response. This analysis uncovers a new universe of effective anticancer neoepitopes which have strikingly different properties from conventional epitopes and suggests a novel algorithm to predict them. It also reveals that our current methods of prediction discard the majority of true anticancer neoepitopes. These results from a single mouse model have been validated in another, independent mouse cancer model, and are consistent with data with human studies. T cells elicited by the active neoepitopes identified here demonstrate a stem-like early dysfunctional phenotype associated with effective responses against viruses and tumors of transgenic mice. These abundant and novel anticancer neoepitopes can be exploited for generation of personalized human cancer vaccines.
Project description:Identification of neoepitopes that are effective in cancer therapy is a major challenge in creating cancer vaccines. Here, using an entirely unbiased approach, we queried all possible neoepitopes in a mouse cancer model and asked which of those are effective in mediating tumor rejection and, independently, in eliciting a measurable CD8 response. This analysis uncovered a large trove of effective anticancer neoepitopes that have strikingly different properties from conventional epitopes and suggested an algorithm to predict them. It also revealed that our current methods of prediction discard the overwhelming majority of true anticancer neoepitopes. These results from a single mouse model were validated in another antigenically distinct mouse cancer model and are consistent with data reported in human studies. Structural modeling showed how the MHC I-presented neoepitopes had an altered conformation, higher stability, or increased exposure to T cell receptors as compared with the unmutated counterparts. T cells elicited by the active neoepitopes identified here demonstrated a stem-like early dysfunctional phenotype associated with effective responses against viruses and tumors of transgenic mice. These abundant anticancer neoepitopes, which have not been tested in human studies thus far, can be exploited for generation of personalized human cancer vaccines.
Project description:Microglia provide a front-line defense against neuroinvasive viral infection, however, determination of their bona fide transcriptional profiles under conditions of health and disease is challenging. Here, we used a combination of experimental tools to delineate the overall transcriptional landscape of microglia during viral infection. By exploiting the ribosomal tagging approach, we developed the concept of enrichment of relevant marker genes by comparing immunoprecipitated RNA with total RNA. Enriched transcripts corresponding to genes expressed in target cells were instrumental in defining bona fide signatures of microglia. With this approach, we generated a comprehensive and accurate transcriptome of microglia at the in situ environment under conditions of health and virus infection. These unified microglial signatures may serve as a benchmark to retrospectively assess ex vivo artefacts from available atlases. Leveraging the microglial translatome, we found enrichment of genes implicated in T-cell activation and cytokine production during the course of VSV infection. These data linked microglia with T-cell re-stimulation and further underscored that microglia shape anti-viral T-cell responses in the brain. Collectively, this study faithfully defines the transcriptional landscape of microglia in steady state and during viral encephalitis and highlights cellular interactions between microglia and T cells that contribute to the control of virus dissemination.
Project description:Accurate annotation of regulatory RNAs is a complex task but nevertheless essential as sRNA molecular and functional studies ensue from it. Several formerly considered small RNAs (sRNA) are now known to be parts of UTR transcripts. In light of experimental data, we review hundreds of Staphylococcus aureus putative regulatory RNAs. We pinpoint those that are likely acting in trans and are not expressed from the opposite strand of a coding gene. We conclude that HG003, a NCTC8325 derivative strain, has about 50 bona fide sRNAs, indicating that these RNAs are less numerous than commonly stated.
Project description:Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remains unclear. We develop a hypermutated model of non-small cell lung cancer, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L+CD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and decreased exhaustion in effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage.
Project description:The post-translational modification of proteins by ubiquitination is a highly regulated process that involves a dynamic, three-step enzymatic cascade, where more than 600 E3 ligases play a critical role in recognizing specific substrates for modification. Separating bona fide targets of E3s from E3-interacting proteins remains a major challenge in the field. In this study, we present BioE3, a novel approach for identifying substrates of ubiquitin-like (UbL) E3 ligases of interest. Using BirA-E3 ligase fusion proteins and bioUbLs, the method facilitates site-specific biotinylation of UbL-modified substrates for proteomic identification. We demonstrate that the BioE3 system can identify both known and novel targets of two RING-type ubiquitin E3 ligases: RNF4, known to be involved in DNA damage response and the regulation of PML nuclear bodies, and MIB1, implicated in endocytosis, autophagy, and centrosomal protein homeostasis. We further show the versatility of BioE3 by identifying targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, we show that BioE3 works with HECT-type E3 ligases and identify novel targets of NEDD4 involved in vesicular trafficking. BioE3 is a powerful tool that enables identification of bona fide substrates of UbL E3 ligases and how they change with chemical perturbations, which may be useful for the emerging applications in targeted protein degradation (TPD). BioE3 may also be applicable for UbLs beyond Ub and SUMO, as well as other E3 ligase classes. The resulting knowledge can shed light on the regulation of cellular processes by the complex UbL network, advancing our understanding of fundamental biological mechanisms.
Project description:Placentation disorders such as severe fetal growth restriction and preeclampsia have their origin in the first trimester of pregnancy, whereas symptoms typically present later on. To be able to study the pathogenesis of these diseases, there is a need for a reliable model system of early placenta development with known pregnancy outcomes. Therefore, we aimed to optimize the generation of human induced trophoblast stem cells (iTSCs) from term umbilical cord using a direct reprogramming approach. In this, we added three supplements—A-485 (CREBBP/EP300 inhibitor), BMP4 or EPZ-6438 (PRC2 inhibitor)—that could potentially boost iTSC induction. The generated iTSCs fulfilled the criteria for bona fide first-trimester trophoblasts and exhibited transcriptional similarity to TSCs derived from first-trimester placental tissue, while the supplements tested did not substantially enhance iTSC induction.