Assessing Protein Immunogenicity with a Dendritic Cell Line-Derived Endolysosomal Degradome

Background: The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently available, we developed an in vitro assay that exploits the link between a proteins immunogenicity and its susceptibility to endolysosomal proteolysis. Methodology: We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry. Results: Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays. Conclusions: Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress

International audience; High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14 days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15 µM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5 µM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress

Mass spectrometry based fingerprinting of DC-derived endolysosomal fractions.

<p>%, percentage to which identified peptides cover the full length protein sequence; AEP, Asparagine endopeptidase; ARF, ADP-ribosylation factor; LAMP, Lysosome-associated membrane glycoprotein; LMP, lysosome membrane protein; pep., identified peptides; Rab, Ras-like protein; VAMP, Vesicle-associated membrane protein.</p

Chronology of Bet v 1 peptide cluster formation during endolysosomal proteolysis.

<p>Peptides sequenced by mass spectrometry after 0.5, 1, 3, 5, 12, 24, 36, and 48 h of <i>in vitro</i> digestion with microsomal fractions from mDCs (blue), BMDCs (green) and JAWS II DCs (brown) are shown for Bet v 1.0101 (A) and Bet v 1.0401 (B), respectively. Regions of predominant peptide clusters (cl_x-x) are highlighted as bars colored in different shades of grey depending on their temporal occurrence (average appearance of early clusters: ≤1 h, intermediate clusters: >1 h and <5 h, and late clusters: ≥5 h. Bet v 1 secondary structures (α-helices and β-sheets) are indicated as framed boxes. Amino acids (n = 7) that differ between the 2 Bet v 1 isoforms are highlighted in orange.</p

Cathepsin S digest of Bet v 1.

<p>Cathepsin S cleavage sites are depicted in the bar diagram showing their location in the Bet v 1.0101 sequence (<i>x-axis</i>) and the number of different peptides generated per cleavage site (<i>y-axis</i>). According to their temporal accessibility, cathepsin S sites are highlighted in red (early), orange (intermediate), and yellow (late) in the bar diagram. Generated peptides are shown as purple lines. Bet v 1.0101 secondary structures (α-helices and β-sheets) are indicated as framed boxes. Regions of Bet v 1.0101 peptide clusters (cl_x-x) generated by endolysosomal proteases isolated from DCs are depicted as grey boxes.</p

Regions of peptide clusters generated by endolysosomal proteolysis.

<p>Peptides sequenced by mass spectrometry after 0.5, 1, 3, 5, 12, 24, 36, and 48 h of <i>in vitro</i> digestion with microsomal fractions from mDCs (blue), BMDCs (green) and JAWS II DCs (brown) clustered in distinct regions along the protein sequence of RNase (A) and peroxidase (B) model antigens. Secondary structures (α-helices and β-sheets) are indicated as framed boxes.</p

Kinetics of endolysosomal proteolysis.

<p>2.5 µg of protein samples were analyzed by SDS-PAGE and GelCode® Blue staining after 0, 0.5, 1, 3, 5, 12, 24, 36 and 48 h of <i>in vitro</i> degradation using endolysosomal fractions isolated from human mDCs, murine BMDCs, and the DC line JAWS II (A). For each protein, the half-life during endolysosomal proteolysis was calculated from scanned and densitometrically quantified protein bands (B).</p

Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in the complementary CNS in vitro models

The present study was performed in an attempt to develop an in vitro integrated testing strategy to evaluate neurotoxicity of drugs during development phase. A number of endpoints was analyzed using two complementary brain cell culture models, and an in vitro blood-brain barrier model after acute, sub-chronic, and repeated-dose treatments with a series of selected drugs. The developed in vitro BBB model allowed to detect toxic effects on the BBB and to evaluate drug transport through the BBB for prediction free brain concentrations of studied drugs. The electrical activity of cortical neuronal networks recorded with a micro-electrode array was found to be a good tool to predict the neuroactivity and neurotoxicity of drugs and it is suggested as a first-step high content screening test. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found well suitable for OMICs analyzes. The obtained data suggest that an in vitro integrated testing strategy (ITS), including toxicity to and transport through BBB, as well as metabolomics, proteomics and neuronal electrical activity, measured in stable rodent brain cell culture systems (in the future human stem cell-derived neuronal models), may considerably improve current drug-induced neurotoxicity evaluation. Robustness of this ITS has to be further evaluated with a larger number of studied drugs.JRC.I.5-Systems Toxicolog

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress.

High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the characterized nephrotoxin Cyclosporine A (CsA) at therapeutic and supra therapeutic concentrations for 14 days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15 µM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein- response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5 µM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress.JRC.I.1-Chemical Assessment and Testin