Publications

Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the antibacterial 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.

https://doi.org/10.1038/s42003-020-01296-3

The removal of ultrathin amorphous polymer films in contact with an aqueous gelled solution containing small amounts of good solvent is addressed by means of specular and off-specular neutron reflectometry. The distribution of heavy water and benzyl alcohol is revealed inside Laropal A81, often employed as a protective varnish layer for Culture Heritage in the restoration of easel paintings. The swelling kinetics, interface roughness, and film morphologies were recorded as a function of temperature and increasing benzyl alcohol concentration in the dispersion of Pemulen TR-2, a hydrophobically modified acrylic acid copolymer. The addition of small amounts of good solvent results in the appearance of water-filled cavities inside the varnish, which grow with time. It is shown that while increasing the solvent concentration greatly enhances the hole growth kinetics, an increase in temperature above the glass transition temperature does not have such a big effect on the kinetics.

We have discovered the existence of extremely polydisperse High Internal-Phase-Ratio Emulsions (HIPEs) in which the internal-phase droplets, present at 95% volume fraction, remain spherical and organize themselves in the available space according to Apollonian packing rules. Such Apollonian emulsions are obtained from dispersing oil dropwise in water in the presence of very little surfactant, and allowing them to evolve at rest for at least a week. The packing structure of the droplets was confirmed through size distribution measurements that evolved spontaneously towards power laws with the known Apollonian exponents, as well as comparison of the structure factors of aged HIPEs measured by Small-Angle X-ray Scattering with that of a numerically simulated Random Apollonian Packing. Thanks to the perfect sphericity of the droplets, Apollonian emulsions were found to display Newtonian flow even at such extremely high volume fraction. We argue that these fascinating space-filling assemblies of spherical droplets are a result of coalescence and fragmentation processes obeying simple geometrical rules of conserving total volume and sphericity, and minimizing the elastic energy associated with interactions of neighbouring droplets.

We characterize the motility of athermal swimming droplets within the framework of a persistent random walk. Just like active colloids, their trajectories can be modeled with a constant velocity $V$ and a slow angular diffusion, but the random changes in direction are not thermally driven. Instead, $V$ is determined by the interfacial tension gradient along the droplet surface, while reorientation of the surfactant gradient leads to changes in direction with a persistence time $\tau$. We show that the origin of locomotion is the difference in the critical micellar concentration in the front and the back of the droplet, $\Delta \mathrm{CMC}$. Tuning this parameter by salt controls $V$ from 3 to 15 diameters $d/s$. Surfactant concentration has little effect on speed, but leads to a dramatic decrease in $\tau$ over 4 orders of magnitude. The corresponding range of the persistence length $â„“=V\tau$ extends beyond the realm of synthetic or living swimmers, in which $V$ is limited by fuel consumption and $\tau$ is set by thermal fluctuations or biological activity, respectively. Our tunable swimmers are ideal candidates for the study of the departure from equilibrium to high levels of activity. We show that their collective behavior exhibits the formation of active clusters of a well-defined size.

Measuring the structure factor, S(q), of a dispersion of particles by small-angle X-ray scattering provides a unique method to investigate the spatial arrangement of colloidal particles. However, it is impossible to find the exact location of the particles from S(q) because some information is inherently lacking in the measured signal. The two standard ways to analyse an experimental S(q) are then to compare it either with structure factors computed from simulated systems or with analytical ones calculated from approximated systems. However, such approaches may prove inadequate for dispersions of variously polydisperse particles. While Vrij, Bloom and Stell established a mean-field approach that could yield fairly accurate approximations for experimental S(q), this solution has remained underused because of its mathematical complexity. In the present work, the complete Percus–Yevick solution for general polydisperse hard-sphere systems is derived in a concise form that is straightforward to use. The form of the solution has been simplified enough to provide experimentalists with ready solutions of several commonly encountered particle-radius distributions in real systems (Schulz, truncated normal and inverse Gaussian). The approach is also illustrated with a case study of the exponential radius distribution. Finally, the application of the proposed solution to the power-law radius distribution is discussed in detail by comparing the calculations with experimentally measured S(q) for an Apollonian packing of spherical droplets recently reported in high-internal-phase-ratio emulsions.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.

https://www.nature.com/articles/s41596-020-0354-0

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.

DOI: https://doi.org/10.4049/jimmunol.2000112

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 modelling and statistical inference is a concrete way to investigate biological processes underlying affinity maturation (such as selection permissiveness), hardly accessible through measurements.

DOI: https://doi.org/10.7554/eLife.55678

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.

https://doi.org/10.1016/j.vaccine.2020.05.066

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

https://doi.org/10.1038/s41587-020-0466-7

Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation. This process allows S. cerevisiae to adapt on a physiological timescale, but related phenomena may also be important in other processes, such as cellular differentiation, cellular reprogramming, and the emergence of drug resistance in cancer.

https://doi.org/10.1073/pnas.1913767117

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.

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 (R) 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.

Monte Carlo simulations, fully constrained by experimental parameters, are found to agree well with a measured phase diagram of aqueous dispersions of nanoparticles with a moderate size polydispersity over a broad range of salt concentrations, c(s), and volume fractions, phi. Upon increasing phi, the colloids freeze first into coexisting compact solids then into a body centered cubic phase (bcc) before they melt into a glass forming liquid. The surprising stability of the bcc solid at high phi and c(s) is explained by the interaction (charge) polydispersity and vibrational entropy.