PH Puech, A Sadoun
Collaboration Y Hamon (CIML), P Pierobon (Institut Curie), J Husson (LaDHyX)
We are tackling the APC vs. T cell situation by having a model APC adhered onto an AFM lever, contacting a (Ca2+reporter loaded) T cell of known shape. This method, called Single Cell Force Spectroscopy (SCFS), allows to evaluate the contact mechanics between cells, the evolution of the contact and the forces needed for separation, from the single molecule to the entire cell scales. It has been recently applied to T cell / APC contact for long contact times, after formation of the immune synapse but (a) only adhesion at cell scale was recorded and (b) no simultaneous observation of the early activation of the cells was ever proposed, to our knowledge.
We then added to SCFS our technique for combining simultaneously AFM/fluorescence to record the activation pattern before, during and after contact. We quantified APC / T cell adhesion for short contact times mimicking early T/APC interactions, the APC bearing only MHC molecules and varying the presented peptide, and measuring the cell/cell contact mechanics and adhesion from single molecule to groups of molecules, by varying the contact conditions (force, duration, frequency).
We plan to increase the complexity of the system by adding coactivation and adhesion molecules at APC surface, using primary T cells, and systems inspired by the interaction of T and B cells in the germinal centers. We expect by such to dissect the early physical determinants of T cell activation, such as which forces are required or created by T cells to integrate a biochemical signal / biomechanical signal.
Formation of the invariant NKT/ APC immune synapse and Subversion by parasite
We are interested in understanding how invariant NKT cells recognize glycolipid antigens upon infection by pathogens and how they interact with antigen presenting cells following this recognition.
NKT cells are glycolipid-reactive T cells with powerful immunoproperties. However, despite their critical role in infectious diseases, how pathogens subvert their functions and achieve immune evasion remains largely unknown.
To tackle this question, we will investigate the role of membrane remodeling upon NKT cell engagement with antigen presenting cells (APC)- defined as immunological synapse (IS) – in prompting NKT cell activation and analyze the structural and functional impact of pathogens on this IS. The parasite Leishmania will be used as an ideal infectious model owing to its capacity to exploit APCs, as hosts, a feature with major effects on APC/T interactions and immune response initiation.