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High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics
Annabelle Gérard, Adam Woolfe, Guillaume Mottet, Marcel Reichen, Carlos Castrillon, Vera Menrath, Sami Ellouze, Adeline Poitou, Raphaël Doineau, Luis Briseno-Roa, Pablo Canales-Herrerias, Pascaline Mary, Gregory Rose, Charina Ortega, Matthieu Delincé, So
Nature Biotechnology - 38 715–721 - - 2020
Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. 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 bioassays2, 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.
The generality of transient compartmentalization and its associated error thresholds
Alex Blokhuis, Philippe Nghe , Luca Peliti , David Lacoste
J Theor Biol . - 487 110110 - doi: 10.1016/j.jtbi.2019.110110 - 2020
Can prelife proceed without cell division? A recently proposed mechanism suggests that transient compartmentalization could have preceded cell division in prebiotic scenarios. Here, we study transient compartmentalization dynamics in the presence of mutations and noise in replication, as both can be detrimental the survival of compartments. Our study comprises situations where compartments contain uncoupled autocatalytic reactions feeding on a common resource, and systems based on RNA molecules copied by replicases, following a recent experimental study. Using the theory of branching processes, we show analytically that two regimes are possible. In the diffusion-limited regime, replication is asynchronous which leads to a large variability in the composition of compartments. In contrast, in a replication-limited regime, the growth is synchronous and thus the compositional variability is low. Typically, simple autocatalysts are in the former regime, while polymeric replicators can access the latter. For deterministic growth dynamics, we introduce mutations that turn functional replicators into parasites. We derive the phase boundary separating coexistence or parasite dominance as a function of relative growth, inoculation size and mutation rate. We show that transient compartmentalization allows coexistence beyond the classical error threshold, above which the parasite dominates. Our findings invite to revisit major prebiotic transitions, notably the transitions towards cooperation, complex polymers and cell division.
Predicting Evolution Using Regulatory Architecture
Philippe Nghe , Marjon G J de Vos , Enzo Kingma , Manjunatha Kogenaru , Frank J Poelwijk , Liedewij Laan , Sander J Tans
Annu Rev Biophys - 6(49) 181-197 - doi: 10.1146/annurev-biophys-070317-032939 - 2020
The limits of evolution have long fascinated biologists. However, the causes of evolutionary constraint have remained elusive due to a poor mechanistic understanding of studied phenotypes. Recently, a range of innovative approaches have leveraged mechanistic information on regulatory networks and cellular biology. These methods combine systems biology models with population and single-cell quantification and with new genetic tools, and they have been applied to a range of complex cellular functions and engineered networks. In this article, we review these developments, which are revealing the mechanistic causes of epistasis at different levels of biological organization-in molecular recognition, within a single regulatory network, and between different networks-providing first indications of predictable features of evolutionary constraint.

Metabolic cost of rapid adaptation of single yeast cells
Gabrielle Woronoff, Philippe Nghe, Jean Baudry, Laurent Boitard, Erez Bra
PNAS - 117 (20) 10660-10666 - - 2020
We establish, using single-cell analysis of metabolism and division in a droplet microfluidic system, that yeast can adapt, resuming division, extremely rapidly to an unforeseen environmental challenge, and that adaptation is an active process, requiring the consumption of a characteristic amount energy. The adapted state is stable over at least several days, showing that this is a genuine adaptation process. The adaptation rate (10−3 cells per hour) is orders of magnitude higher than expected based on known mutation rates, suggesting an epigenetic origin, and the tight energetic coupling implies that there is active exploration of different states, and fixation of the solution(s) that allow adaptation.

Flux, toxicity and protein expression costs shape genetic interaction in a metabolic pathways
Gabrielle Woronoff, Philippe Nghe, Jean Baudry, Laurent Boitard, Erez Bra
Science Advances - 6 23 - DOI: 10.1126/sciadv.abb2236 - 2020
Our ability to predict the impact of mutations on traits relevant for disease and evolution remains severely limited by the dependence of their effects on the genetic background and environment. Even when molecular interactions between genes are known, it is unclear how these translate to organism-level interactions between alleles. We therefore characterized the interplay of genetic and environmental dependencies in determining fitness by quantifying ~4000 fitness interactions between expression variants of two metabolic genes, starting from various environmentally modulated expression levels. We detect a remarkable variety of interactions dependent on initial expression levels and demonstrate that they can be quantitatively explained by a mechanistic model accounting for catabolic flux, metabolite toxicity, and expression costs. Complex fitness interactions between mutations can therefore be predicted simply from their simultaneous impact on a few connected molecular phenotypes.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Yacine Bounab, Klaus Eyer, Sophie Dixneuf, Magda Rybczynska, Cécile Chauvel, Maxime Mistretta, Trang Tran, Nathan Aymerich, Guilhem Chenon, Jean-François Llitjos, Fabienne Venet, Guillaume Monneret, Iain A. Gillespie, Pierre Cortez, Virginie Moucadel, Al
Protocol - 15 2920–2955 - - 2020
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.

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
J Immunol July - 206 10 - DOI: - 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.

Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassa
M. Heo, G. Chenon, C. Castrillon, J. Bibette, P. Bruhns, A.D. Griffiths, J. Baudry and K. Eyer
Communications Biology - 3 614 - - 2020
Thresholds in origin of life scenarios
Jeancolas, C., Malaterre, C., & Nghe, P.
Iscience - 23(11) 101756 - - 2020
Predicting Evolutionary Constraints by Identifying Conflicting Demands in Regulatory Networks
M., Nghe, P., Poelwijk, F.J. and Tans, S.J.
Cell systems - 10(6) 526-534 - - 2020


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579 publications.