To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4(+) T cells respond to intermittent TCR stimulation more efficiently than their CD8(+) T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.

Both classical and quantum electrodynamics predict the existence of dipole-dipole long-range electrodynamic intermolecular forces; however, these have never been hitherto experimentally observed. The discovery of completely new and unanticipated forces acting between biomolecules could have considerable impact on our understanding of the dynamics and functioning of the molecular machines at work in living organisms. Here, using two independent experiments, on the basis of different physical effects detected by fluorescence correlation spectroscopy and terahertz spectroscopy, respectively, we demonstrate experimentally the activation of resonant electrodynamic intermolecular forces. This is an unprecedented experimental proof of principle of a physical phenomenon that, having been observed for biomacromolecules and with long-range action (up to 1000 Å), could be of importance for biology. In addition to thermal fluctuations that drive molecular motion randomly, these resonant (and thus selective) electrodynamic forces may contribute to molecular encounters in the crowded cellular space.

Understanding the forces that enable protein interactions is a major scientific challenge. Indeed, Brownian diffusion alone, which is usually considered as the main engine of protein dynamics, does not explain the rapidity and efficiency of the biomolecular reactions at works in cells. In this work, we will present observations of the collective oscillations of a model protein driven out-of-equilibrium by means of optical excitation in aqueous medium using terahertz spectroscopy.

We describe the activation of out-of-equilibrium collective oscillations of a macromolecule as a classical phonon condensation phenomenon. If a macromolecule is modeled as an open system-that is, it is subjected to an external energy supply and is in contact with a thermal bath to dissipate the excess energy-the internal nonlinear couplings among the normal modes make the system undergo a nonequilibrium phase transition when the energy input rate exceeds a threshold value. This transition takes place between a state where the energy is incoherently distributed among the normal modes and a state where the input energy is channeled into the lowest-frequency mode entailing a coherent oscillation of the entire molecule. The model put forward in the present work is derived as the classical counterpart of a quantum model proposed a long time ago by Frohlich in an attempt to explain the huge speed of enzymatic reactions. We show that such a phenomenon is actually possible. Two different and complementary THz near-field spectroscopic techniques-a plasmonic rectenna and a microwire near-field probe-have been used in two different labs to eliminate artifacts. By considering an aqueous solution of a model protein, the bovine serum albumin, we find that this protein displays a remarkable absorption feature around 0.314 THz, when driven in a stationary out-of-thermal equilibrium state by means of optical pumping. The experimental outcomes are in very good qualitative agreement with the theory developed in the first part of the paper and in excellent quantitative agreement with the theoretical result, allowing us to identify the observed spectral feature with a collective oscillation of the entire molecule.

Fluorescence Correlation Spectroscopy (FCS) is widely used to detect and quantify diffusion processes at the molecular level. The molecules of which diffusion is studied are marked with fluorescent dyes. It is commonly maintained that this technique only applies to systems where the concentration of fluorescent molecules is low. Even if this is the optimal operational condition, we show that FCS can be used also at high concentrations (up to 50$\mu$M) of fluorescent molecules: the detector blinding due to highly fluorescent solutions of concentrated dyes can be avoided by using neutral optic density (OD) filters, and the initial condition of very bad signal to noise ratio (SNR) can be hampered by suitable statistical averaging, as usual in other contexts of signal analysis.

In the present paper, an experimental feasibility study on the detection of long-range intermolecular interactions through three-dimensional molecular diffusion in solution is performed. This follows recent theoretical and numerical analyses reporting that long-range electrodynamic forces between biomolecules could be identified through deviations from Brownian diffusion. The suggested experimental technique was fluorescence correlation spectroscopy (FCS). By considering two oppositely charged molecular species in aqueous solution, namely, lysozymes and fluorescent dye molecules (Alexa488), the diffusion coefficient of the dyes has been measured for different values of the concentration of lysozyme, that is, for different average distances between the oppositely charged molecules. For our model, long-range interactions are of electrostatic origin, suggesting that their action radius can be varied by changing the ionic strength of the solution. The experimental outcomes clearly prove the detectability of long-range intermolecular interactions by means of the FCS technique. Molecular dynamics simulations provide a clear and unambiguous interpretation of the experimental results.

Interactions électrodynamiques longues distances entre biomolécules L’étude des organismes vivants, la biologie, s’étend sur de nombreux domaines et notamment s’applique à comprendre le fonctionnement des êtres vivants. Les organismes les plus complexes comme les êtres Humains possèdent plusieurs niveaux d’organisation : ils sont constitués successivement d’organes, de tissus, de cellules, de biomolécules. On trouve plusieurs types de biomolécules dont les protéines, qui sont comme des minuscules outils qui permettent aux cellules de vivre et d’interagir avec leur environnement. Pour cela, les protéines doivent entrer en contact les unes avec les autres de manière très précise et déterminée. Cette thèse teste l’existence de forces électrodynamiques de longue portée qui leur permettraient d’interagir de manière rapide et guidée, via l’étude de l’absorption ou l’émission de ce type d’onde par des protéines, puis la diffusion de ces protéines en solution pour observer leur comportement.