scholarly journals Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models

2021 ◽  
Author(s):  
Tyler Jacks ◽  
Alex Jaeger ◽  
Lauren Stopfer ◽  
Emma Sanders ◽  
Demi Sandel ◽  
...  
Keyword(s):  
Transcript Abundance ◽  
Genetically Engineered ◽  
Ductal Adenocarcinoma ◽  
Affinity Tag ◽  
Class I Mhc ◽  
Mhc I ◽  
Novel Approach ◽  
In Vitro Investigation

Abstract Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the “immunopeptidome”. Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of tumor-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the KrasLSL-G12D/+; p53fl/fl (KP) mouse model (KP; KbStrep). This novel approach has allowed us to isolate tumor-specific MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome through tumor progression and show that in vivo MHC-I presentation is shaped by post-translational mechanisms. We also uncovered novel, putative LUAD tumor associated antigens (TAAs). Many peptides that were recurrently presented in vivo exhibited very low expression of the cognate mRNA, provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the KbStrep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.

scholarly journals Coronaviruses induce the expression of the peptide antigen transporter TAP1.

2020 ◽  
Author(s):  
Shahan Mamoor
Keyword(s):  
Host Cell ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
The United States ◽  
Class I ◽  
Class I Mhc ◽  
Mhc I

Coronavirus infection is an emerging public health threat in the United States and worldwide (1). We mined published microarray data to perform systems-level analysis of host cell transcription following infection with multiple coronavirus types in order to identify therapeutic targets and host cell vulnerabilities (2, 3). We identified the antigen peptide transported TAP1 as differentially expressed in vitro in a human cell line following infection with human coronavirus 229E and in vivo in the lungs of mice following infection with the severe acute respiratory distress syndrome SARS coronavirus. While TAP1 functions to enhance presentation of class I-associated peptides to CD8 cytotoxic T-cells, TAP1 also enhances presentation of major histocompatibility class I (MHC-I) receptors (4). One criteria natural killer cells use to lyse cells is the absence of surface MHC-I (5), and hepatitis C virus up-regulates TAP1 to increase surface expression of MHC-I and inhibit NK-cell cytotoxicity and promote infection (6). We propose coronaviruses similarly up-regulate TAP1 to impair natural killer cell-mediated cytotoxicity responses and avoid recognition by NK cells.

scholarly journals eIF4A supports an oncogenic translation program in pancreatic ductal adenocarcinoma

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Karina Chan ◽  
Francis Robert ◽  
Christian Oertlin ◽  
Dana Kapeller-Libermann ◽  
Daina Avizonis ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Translational Control ◽  
Treatment Options ◽  
Central Carbon Metabolism ◽  
Genetically Engineered ◽  
Metabolic Reprogramming ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Tumour

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with limited treatment options. Although metabolic reprogramming is a hallmark of many cancers, including PDA, previous attempts to target metabolic changes therapeutically have been stymied by drug toxicity and tumour cell plasticity. Here, we show that PDA cells engage an eIF4F-dependent translation program that supports redox and central carbon metabolism. Inhibition of the eIF4F subunit, eIF4A, using the synthetic rocaglate CR-1-31-B (CR-31) reduced the viability of PDA organoids relative to their normal counterparts. In vivo, CR-31 suppresses tumour growth and extends survival of genetically-engineered murine models of PDA. Surprisingly, inhibition of eIF4A also induces glutamine reductive carboxylation. As a consequence, combined targeting of eIF4A and glutaminase activity more effectively inhibits PDA cell growth both in vitro and in vivo. Overall, our work demonstrates the importance of eIF4A in translational control of pancreatic tumour metabolism and as a therapeutic target against PDA.

scholarly journals Abrogation of IFN-γ Signaling May not Worsen Sensitivity to PD-1/PD-L1 Blockade

2020 ◽  
Vol 21 (5) ◽  
pp. 1806
Author(s):  
Julie Vackova ◽  
Adrianna Piatakova ◽  
Ingrid Polakova ◽  
Michal Smahel
Keyword(s):  
Tumor Cells ◽  
Predictive Biomarkers ◽  
Predictive Marker ◽  
Loss Of Function ◽  
Class I Mhc ◽  
Mhc I ◽  
Ifn Γ ◽  
Β Receptor

Programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockade is a promising therapy for various cancer types, but most patients are still resistant. Therefore, a larger number of predictive biomarkers is necessary. In this study, we assessed whether a loss-of-function mutation of the interferon (IFN)-γ receptor 1 (IFNGR1) in tumor cells can interfere with anti-PD-L1 therapy. For this purpose, we used the mouse oncogenic TC-1 cell line expressing PD-L1 and major histocompatibility complex class I (MHC-I) molecules and its TC-1/A9 clone with reversibly downregulated PD-L1 and MHC-I expression. Using the CRISPR/Cas9 system, we generated cells with deactivated IFNGR1 (TC-1/dIfngr1 and TC-1/A9/dIfngr1). In tumors, IFNGR1 deactivation did not lead to PD-L1 or MHC-I reduction on tumor cells. From potential inducers, mainly IFN-α and IFN-β enhanced PD-L1 and MHC-I expression on TC-1/dIfngr1 and TC-1/A9/dIfngr1 cells in vitro. Neutralization of the IFN-α/IFN-β receptor confirmed the effect of these cytokines in vivo. Combined immunotherapy with PD-L1 blockade and DNA vaccination showed that IFNGR1 deactivation did not reduce tumor sensitivity to anti-PD-L1. Thus, the impairment of IFN-γ signaling may not be sufficient for PD-L1 and MHC-I reduction on tumor cells and resistance to PD-L1 blockade, and thus should not be used as a single predictive marker for anti-PD-1/PD-L1 cancer therapy.

scholarly journals Cholera Toxin Impairs the Differentiation of Monocytes into Dendritic Cells, Inducing Professional Antigen-Presenting Myeloid Cells

2010 ◽  
Vol 79 (3) ◽  
pp. 1300-1310 ◽  
Author(s):  
Filippo Veglia ◽  
Ester Sciaraffia ◽  
Antonella Riccomi ◽  
Dora Pinto ◽  
Donatella R. M. Negri ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cholera Toxin ◽  
Interleukin 4 ◽  
Dependent Manner ◽  
Necrosis Factor Alpha ◽  
Class I Mhc ◽  
Mhc I ◽  
Antigen Presenting

ABSTRACTCholera toxin (CT) is a potent adjuvant for mucosal vaccination; however, its mechanism of action has not been clarified completely. It is well established that peripheral monocytes differentiate into dendritic cells (DCs) bothin vitroandin vivoand that monocytes are thein vivoprecursors of mucosal CD103−proinflammatory DCs. In this study, we asked whether CT had any effects on the differentiation of monocytes into DCs. We found that CT-treated monocytes, in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), failed to differentiate into classical DCs (CD14lowCD1ahigh) and acquired a macrophage-like phenotype (CD14highCD1alow). Cells differentiated in the presence of CT expressed high levels of major histocompatibility complex class I (MHC-I) and MHC-II and CD80 and CD86 costimulatory molecules and produced larger amounts of IL-1β, IL-6, and IL-10 but smaller amounts of tumor necrosis factor alpha (TNF-α) and IL-12 than did monocytes differentiated into DCs in the absence of CT. The enzymatic activity of CT was found to be important for the skewing of monocytes toward a macrophage-like phenotype (Ma-DCs) with enhanced antigen-presenting functions. Indeed, treatment of monocytes with scalar doses of forskolin (FSK), an activator of adenylate cyclase, induced them to differentiate in a dose-dependent manner into a population with phenotype and functions similar to those found after CT treatment. Monocytes differentiated in the presence of CT induced the differentiation of naïve T lymphocytes toward a Th2 phenotype. Interestingly, we found that CT interferes with the differentiation of monocytes into DCsin vivoand promotes the induction of activated antigen-presenting cells (APCs) following systemic immunization.

scholarly journals “Induction of pancreatic tumor-selective ferroptosis through modulation of cystine import”

2019 ◽  
Author(s):  
Michael A. Badgley ◽  
Daniel Kremer ◽  
H. Carlo Maurer ◽  
Kathleen E. DelGiorno ◽  
Ho-Joon Lee ◽  
...  
Keyword(s):  
Pancreatic Tumor ◽  
Genetically Engineered ◽  
Ductal Adenocarcinoma ◽  
Oxygen Species ◽  
The Third ◽  
The Us ◽  
Immune Therapies ◽  
Tumor Selective

AbstractPancreatic ductal adenocarcinoma (PDA) is the third-leading cause of cancer mortality in the US and is highly resistant to classical, targeted, and immune therapies. We show that human PDA cells are dependent on the provision of exogenous cystine to avert a catastrophic accumulation of lipid reactive oxygen species (ROS) that, left unchecked, leads to ferroptotic cell death, both in vitro and in vivo. Using a dual-recombinase genetically engineered model, we found that acute deletion of Slc7a11 led to tumor-selective ferroptosis, tumor stabilizations/regressions, and extended overall survival. The mechanism of ferroptosis induction in PDA cells required the concerted depletion of both glutathione and coenzyme A, highlighting a novel branch of ferroptosis-relevant metabolism. Finally, we found that cystine depletion in vivo using the pre-IND agent cyst(e)inase phenocopied Slc7a11 deletion, inducing tumor-selective ferroptosis and disease stabilizations/regressions in the well-validated KPC model of PDA.One Sentence SummaryGenetic and pharmacological targeting of cystine import induces pancreatic cancer-selective ferroptosis in vivo.

scholarly journals Ultra-strong bio-glue from genetically engineered polypeptides

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chao Ma ◽  
Jing Sun ◽  
Bo Li ◽  
Yang Feng ◽  
Yao Sun ◽  
...  
Keyword(s):  
Soft Tissues ◽  
Covalent Bond ◽  
Genetically Engineered ◽  
Supramolecular Interactions ◽  
Molar Ratio ◽  
High Adhesion ◽  
Strong Adhesion ◽  
Order Of Magnitude

AbstractThe development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.

scholarly journals Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 692
Author(s):  
Giulia Franzoni ◽  
Antonio Anfossi ◽  
Chiara Grazia De Ciucis ◽  
Samanta Mecocci ◽  
Tania Carta ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Macrophage Polarization ◽  
Surface Protein ◽  
Antimicrobial Activities ◽  
Toll Like Receptor ◽  
Activation Markers ◽  
Class I Mhc ◽  
Toll Like Receptor 2

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly activated innate immune cells, including porcine monocyte-derived macrophages (moMΦ). In this study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine moMΦ. We observed enhanced expression of activation markers (MHC class I, MHC class II DR, CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-Pam2Cys also upregulated the gene expression of several IFN-α subtypes, p65, NOS2, and molecules with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However, Mag-Pam2Cys downregulated the expression of IFN-α3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful consequences. We hope that our in vitro results will lay the foundation for the further evaluation of this diacylated lipopeptide as an immunopotentiator in vivo.

scholarly journals Heterodimer Formation of the Homodimeric ABC Transporter OpuA

2021 ◽  
Vol 22 (11) ◽  
pp. 5912
Author(s):  
Patricia Alvarez-Sieiro ◽  
Hendrik R. Sikkema ◽  
Bert Poolman
Keyword(s):  
Abc Transporter ◽  
Conformational Dynamics ◽  
Host Organism ◽  
Affinity Tag ◽  
Practical Applications ◽  
Fundamental Research ◽  
Protein Multimerization ◽  
Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

scholarly journals Designing P. aeruginosa synthetic phages with reduced genomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diana P. Pires ◽  
Rodrigo Monteiro ◽  
Dalila Mil-Homens ◽  
Arsénio Fialho ◽  
Timothy K. Lu ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Antibacterial Properties ◽  
Genetically Engineered ◽  
In Vivo Studies ◽  
Infection Model ◽  
Promising Therapeutic Approach ◽  
Genes Encoding ◽  
First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

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