L. Limozin, PH. Puech

Collaboration: K. Sengupta (CINaM), R. Varma (NIH), Y. Hamon (CIML)

We interrogate T lymphocyte physiology by designing carefully controlled artificial surfaces to stimulate cell activation and spreading. Our original artificial surfaces exhibit fixed or mobile ligands, and arrays of submicrometric adhesive patches surrounded with repulsive zones (Nanoletters 2013, 2015). These surfaces are compatible with high resolution surface microscopy like TIRFM and RICM. With these tools, we have demonstrated that (1) ligand mobility controls lymphocyte spreading and we proposed a physical mechanism based on friction to explain these results (Biophys. J. 2014), (2) cell adhesion area is determined by global ligand density, but local cell-substrate interface and receptor clustering is tuned by underlying adhesive patch geometry (Integr. Biol. 2016). We have also found a non-monotonous response of spreading area to substrate rigidity, T-cell spreading mediated by anti-CD3 being maximal for a substrate rigidity of around 50 kPa.Examples of T-cell spreading on nanopatterned substrates (left; TCR labelled) and substrates of variable rigidity (right; adhesion via TCR only or TCR+Integrin)

We introduced the use of “shaped cells” since the spreading state of cells, on any adhesive molecule, is strongly inhomogeneous, hence giving rise to strong dispersion of the mechanical parameters of the population. The cells are adhered on micro-stamped adhesive / repulsive patterns that will impose a cell shape. Since the T cells are small and extremely activable, the pattern design require specific dimensions and passivation strategies that were developed in the frame of the PhD of A. Sadoun (Labex INFORM). We determined the best adhesive / repulsive molecular combination, the most favourable concentration conditions and cell densities, in order to achieve patterned T cells and model APCs (transfected COS-7 cells) with very little basal activation of T cells and a good reactivation pattern. The cell lines used, transfected or not, labelled or not with Ca2+ reporter molecules for the T cells, were characterized mechanically using AFM indentation (article in preparation).

Pi FW, P Dillard, L Limozin, A Charrier, K Sengupta (2013) Nano letters 13:3372-3378. Pubmed

Pi F, Dillard P, Alameddine R, Benard E, Wahl A, Ozerov I, Charrier A, Limozin L, Sengupta K (2015) NanoLetters, 15: 5178-5184. Pubmed

Dillard P, Varma R, Sengupta K, Limozin L (2014) Biophys. J. 107:2629-2638. Pubmed

Dillard P, Pi F, Lellouch AC, Limozin L, Sengupta K (2016) . Integrative Biology 8:287. Pubmed

Wahl A, Dinet C, Dillard P, Puech P-H, Limozin L, Sengupta S (2019) PNAS 116:5908-5913. Pubmed