Activation of T cells using nano-functionalized surfaces

The activation of T lymphocytes which ultimately leads to acquired immunity in vertebrates is the prevailing element during the recognition process of antigenic (i.e. foreign) substances derived from viral, bacterial, fungal or mutated endogenous proteins. Prior to recognition, proteins are converted to short peptide fragments p (10-15 aminoacids) via a process called antigen processing by antigen presenting cells (APC) such as B cells and dentritic cells. These fragments are presented by the major histocompatibility complex (MHC) on the APC membrane. Productive binding of a specific T cell receptor (TCR) to the MHC loaded with a peptide derived from an antigen (pMHC) triggers initially clustering of the TCR. The recognition process itself is exquisitely sensitive and specific. T lymphocytes sense the presence of even a single antigenic pMHC on the APC amongst tens of thousands endogenous ones which are very similar in structure. It is still controversially discussed how the intercellular binding of a few TCRs to its ligand is related to the intracellular signaling cascade yielding T lymphocyte activation. Is the lymphocyte-mediated TCR clustering a mechanism to boost receptor triggering or just a way to mark them for intracellular degradation? As a PhD student, I applied different single molecule fluorescence microscopy techniques to study the interaction of cell-bound TCR and its ligand pMHC in situ. As a surrogate APC membrane, I used a supported lipid bilayer consisting of the two lipids POPC and DOGS-Ni-NTA. The latter one is able to bind histidine-tagged proteins, here fluorescently labeled pMHC, the costimulatory molecule B7-1 and the integrin intercellular adhesion molecule 1. These proteins represent the minimum set required to achieve peptide-specific T lymphocyte activation. In the proposed project, I will use different patterning techniques to design nano-functionalized surfaces for analyzing the spatio-molecular requirements of the specific T lymphocyte activation. A technique developed by Prof. Dr. J. P. Spatz using nanolithography with self-assembly of diblock copolymer micelles containing HAuCl4 allows for a surface patterning with gold nanoparticles (diameter 1-15 nm) with a variable interparticle spacing. A subsequent functionalization of the gold nanoparticles with specific proteins at a ratio of one biomolecule per nanoparticle permits the examination of the interaction between single proteins and their counterpart on living cells as a function of their overall density and spatial arrangement. The proposed project aims at combining these surfaces with a super-resolution microscopy technique in order to visualize the effect of different spatial arrangements (clustered, peripheral or central, randomly distributed) of pMHCs bound to the gold nanoparticles on T lymphocyte activation using quantitative Ca2+-measurements. Furthermore, a combination of the nano-patterned surfaces with a lipid bilayer system studied during my thesis (Huppa et al., Nature 2010 (463) 963) will be tested for its ability to mimic the cell membrane of an APC more accurately. In preliminary experiments I have shown that a lipid bilayer is formed on a nano-patterned surface and functionalized proteins in the lipid bilayer are still mobile.