Immune cells communicate via surface receptors controlling cell adhesion and signalling. Thus, immune receptors have to discriminate exquisitely molecules from self and non-self. Specific, reversible and force-sensitive ligand-receptor bonds are formed between the T cell receptor and MHC peptide, antigen and antibody as well as between adhesive receptor like integrins and their various ligands. We are deciphering these complex kinetics at the single molecule level with the aim of integrating them to understand collective effects at the cellular scale. For this, we develop and exploit different single molecule techniques, with a special expertise for laminar flow chamber, which, besides being a massively parallel technique, uniquely provide a control of interaction starting time. Our efforts contribute to unveil fundamental properties of these interactions as well as to exploit these concepts to design new therapeutic molecules.

 

T lymphocyte recognition (P. Bongrand, A. Brodovitch, L. Limozin, A. Pierres, PH. Puech, P. Robert; Collab. C. Boyer (CIML), A Van der Merwe (Oxford)). The detection of foreign material by T lymphocytes is both a key step of immune responses and a model of cell decision triggering. We addressed this model by several parallel approaches based on quantitative advanced biophysical methods. We measured the properties of single TCR binding on the cell surfaces with atomic force microscopy (AFM, PLoS ONE 2011) and laminar flow chamber (Biophys. J. 2012). T cell activation appears as dependent of TCR bonding lifetime under low force.

 

 

We measured the dynamics of T cell membrane interactions with planar surface with nearly nanometric and subsecond resolution, using reflection interference contrast microscopy (RICM) and evanescent wave illumination (TIRF). We found that T cells could detect the presence of foreign material within less than 10 seconds with highly motile microvilli and pulling motion (J. Immunol. 2013), starting ultimately active spreading, the earliest reporter of bona fide cell activation (J. Immunol. Methods 2011). T cells could quantitatively discriminate between specific antigens of varying potency within 45 seconds after initial contact (Eur. J. Immunol 2015). Quantitative analysis of TIRF images revealed that T cell plasma membranes displayed transverse undulations of several tens of nanometer amplitude at one hertz frequency (Cell Mol Bioengineering, 2015), an efficient way of mechanosensing the nearby surface through the TCR. The importance and significance of addressed problems was discussed in two review papers (Frontiers Immunol 2012; Annual Rev. Immunol 2015).

 

2D binding properties of antigen-antibody interactions (P. Bongrand, L. Limozin, V. Lo Schiavo, A. Pierres, PH. Puech, P. Robert; Collab. A. Nicolas (CNRS Grenoble), P. Decuzzi (Texas Univ.)). The laminar flow chamber is a potent tool to quantify 2D bond formation and rupture at the single molecular level. Functionalized microbeads are convected above a surface coated with complementary ligands at low density. Videorecording and analyses of bead trajectories provide the frequency of bond formation as well as bond duration. The laminar flow is controlling the duration or molecular encounter leading to bond formation, as well as the force applied to the bond once it is formed.

 

 

This technique was used on an antibody-antigen model, demonstrating a possible complex energy landscape characterized by a rough part (Biophys. J. 2011) and influenced by molecular environment at nanometer scale (Int. J. Nanotech. 2013). Adding systematic temperature variation to the method allowed us to measure this roughness (Sci. reports, 2016). A major point to understand receptor clustering is to quantify multiple bond breaking. We compared single to double antigen–antibody binding to dissect avidity mechanisms (PLoS ONE 2012). Through further improvements in the method, we measured the effect of much larger forces, both as ramps or steady force, necessary for multiple bond maturation and breaking studies (in preparation).