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Peptides Derived From Insulin Granule Proteins Are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice
Marie Eliane Azoury, Mahmoud Tarayrah, Georgia Afonso, Aurore Pais, Maikel L. Colli, Claire Maillard, Cassandra Lavaud, Laure Alexandre-Heymann, Sergio Gonzalez-Duque, Yann Verdier4, Joelle Vinh, Sheena Pinto, Soren Buus, Danièle Dubois-L
Diabetes - 69(12) 2678-2690 - https://doi.org/10.2337/db20-0013 - 2020
The antigenic peptides processed by β-cells and presented through surface HLA class I molecules are poorly characterized. Each HLA variant (e.g., the most common being HLA-A2 and HLA-A3) carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring β-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of β-cells, we analyzed the HLA-A3–restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1–50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neoepitopes, generated either via peptide cis-splicing or mRNA splicing (e.g., secretogranin-5 [SCG5]–009). As reported for HLA-A2–restricted peptides, several epitopes originated from β-cell granule proteins (e.g., SCG3, SCG5, and urocortin-3). Similarly, H-2Kd–restricted CD8+ T cells recognizing the murine orthologs of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes
Deciphering shell proteome within different Baltic populations of mytilid mussels illustrates important local variability and potential consequences in the context of changing marine conditions
JaisonArivalagan, Benjamin Marie, Giovanni Chiappetta, Joëlle Vinh, Xavier Gallet, Matthieu Lebon Saloua M'Zoudi Philippe Dubois Sophie Berland Arul Marie
ELSEVIER - 745 140878 - doi.org/10.1016/j.scitotenv.2020.140878 - 2020
Molluscs defend themselves against predation and environmental stressors through the possession of mineralized shells. Mussels are widely used to predict the effects of abiotic factors such as salinity and pH on marine calcifiers in the context of changing ocean conditions. Shell matrix proteins are part of the molecular control regulating the biomineralization processes underpinning shell production. Under changing environmental conditions, differential expression of these proteins leads to the phenotypic plasticity of shells seen in many mollusc species. Low salinity decreases the availability of calcium and inorganic carbon in seawater and consequently energetic constraints often lead to thin, small and fragile shells in Mytilid mussels inhabiting Baltic Sea. To understand how the modulation of shell matrix proteins alters biomineralization, we compared the shell proteomes of mussels living under full marine conditions in the North Sea to those living in the low saline Baltic Sea. Modulation of proteins comprising the Mytilus biomineralization tool kit is observed. These data showed a relative increase in chitin related proteins, decrease in SD-rich, GA-rich shell matrix proteins indicating that altered protein scaffolding and mineral nucleation lead to impaired shell microstructures influencing shell resistance in Baltic Mytilid mussels. Interestingly, proteins with immunity domains in the shell matrix are also found to be modulated. Shell traits such as periostracum thickness, organic content and fracture resistance qualitatively correlates with the modulation of SMPs in Mytilid mussels providing key insights into control of biomineralization at molecular level in the context of changing marine conditions.
Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein
Stan Perchepied, Nicolas Eskenazi, Chiara Giangrande, Julien Camperi, Thierry Fournier, Joëlle Vinh, Nathalie Delaunay, Valérie Pichon
Talanta - 209 120568 - doi.org/10.1016/j.talanta.2019.120171 - 2020
The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl
The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire
Magda Rybczynska, Jean Baudrya, Eyer Klaus
Vaccine - 38(33) 5337-5342 - doi.org/10.1016/j.vaccine.2020.05.066 - 2020
Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.

The Quantitative Assessment of the Secreted IgG Repertoire after Recall to Evaluate the Quality of Immunizations
Klaus Eyer, Carlos Castrillon, Guilhem Chenon, Jérôme Bibette, Pierre Bruhns, Andrew D. Griffiths and Jean Baudry
The Journal of Immunology - 205 8 - DOI: https://doi.org/10.4049/jimmunol.2000112 - 2020
One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.
Quantitative modeling of the effect of antigen dosage on B-cell affinity distributions in maturating germinal centers
Marco Molari, Klaus Eyer, Jean Baudry, Simona Cocco, Rémi Monasson
Nature Protocols - 15 2920–2955 - DOI: 10.7554/eLife.55678 - 2020
Affinity maturation is a complex dynamical process allowing the immune system to generate antibodies capable of recognizing antigens. We introduce a model for the evolution of the distribution of affinities across the antibody population in germinal centers. The model is amenable to detailed mathematical analysis and gives insight on the mechanisms through which antigen availability controls the rate of maturation and the expansion of the antibody population. It is also capable, upon maximum-likelihood inference of the parameters, to reproduce accurately the distributions of affinities of IgG-secreting cells we measure in mice immunized against Tetanus Toxoid under largely varying conditions (antigen dosage, delay between injections). Both model and experiments show that the average population affinity depends non-monotonically on the antigen dosage. We show that combining quantitative modeling and statistical inference is a concrete way to investigate biological processes underlying affinity maturation (such as selection permissiveness), hardly accessible through measurements.
Motion of oil in water induced by osmosis in a confined system
Erwan Crestel, Anežka Kvasnicková, Enric Santanach-Carreras , Jérôme Bibette, and Nicolas Bremond
Phys. Rev. Fluids - 5 104003 - - 2020
Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassay
Millie Heo, Guilhem Chenon, Carlos Castrillon, Jérôme Bibette, Pierre Bruhns, Andrew D. Griffiths, Jean Baudry & Klaus Eyer
Communications Biology - 614 5337-5342 - https://doi.org/10.1038/s42003-020-01296-3 - 2020
Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the anti-bacterial antibody repertoire, we developed an in-droplet bioassay with single-antibody resolution. The assay not only allowed us to identify whether the secreted antibodies recognized a bacterial surface antigen, but also to estimate the apparent dissociation constant (KD app) of the interaction and the density of the recognized epitope on the bacteria. Herein, we found substantial differences within the KD app/epitope density profiles in mice immunized with various species of heat-killed bacteria. The experiments further revealed a high cross-reactivity of the secreted IgG repertoires, binding to even unrelated bacteria with high affinity. This application confirmed the ability to quantify the anti-bacterial antibody repertoire and the utility of the developed bioassay to study the interplay between bacteria and the humoral response.

Apollonian packing in polydisperse emulsions
Sylvie Kwok, Robert Botet, Lewis Sharpnack and Bernard Cabane
Nature Protocols - 16 2426-2430 - https://doi.org/10.1039/C9SM01772K - 2020
We have discovered the existence of polydisperse high internal-phase-ratio emulsions (HIPE) in which the internal-phase droplets, present at 95% volume fraction, remain spherical and organise themselves according to Apollonian packing rules. These polydisperse HIPEs are formed by emulsifying oil dropwise in a surfactant-poor aqueous continuous phase. After stirring has ceased, their droplet size distributions begin to evolve spontaneously and continuously through coalescence towards well-defined power laws with the Apollonian exponent. Small-angle X-ray Scattering performed on aged HIPEs demonstrate that the droplet packing structure agrees with that of a numerically simulated random Apollonian packing. We argue that when such concentrated emulsions are allowed to evolve, the coalescing droplets must obey volume and sphericity conservation. This leads to a mechanism that differs from typical coalescence in dilute emulsions.

Swelling, dewetting and breakup in thin polymer films for cultural heritage
Amélie Castel, Philipp Gutfreund, Bernard Cabaned and Yahya Rharbi
Soft Matter - 16 1485-1497 - https://doi.org/10.1039/C9SM01976F - 2020
The removal of ultrathin amorphous polymer films in contact with nonsolvent/solvent binary mixtures is addressed by means of neutron reflectometry and atomic force microscopy. The high resolution of neutron scattering makes it possible to resolve the distribution profiles of heavy water and benzyl alcohol inside Laropal®A81, often employed as a protective varnish layer for Culture Heritage in restoration of easel paintings. The swelling kinetics and distribution profiles were recorded as a function of time and increasing benzyl alcohol concentration in water. The varnish film swells by penetration of the good solvent. At higher concentrations water-filled cavities appear inside the varnish and grow with time. Contrary to homogeneous dissolution dewetting is observed at late stages of exposure to the liquid which leads to the Breakup of the film. The high resolution measurements are compared to bulk behaviour characterized by the ternary phase diagram and the Flory–Huggins interaction parameters are calculated and used to predict the swelling and solvent partition in the films. Distinct differences of the thin film to bulk behaviour are found. The expectations made previously for the behaviour of solvent/non-solvent mixtures on the removal of thin layers in the restoration of easel paintings should be revised in view of surface interactions.



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579 publications.