Super-resolution imaging of early signaling in T cells
Supervisor: Prof Simon Davis
The dark secret at the heart of Immunology is that we don’t understand the very first step in the adaptive immune response, i.e. how the T-cell receptor (TCR) is “triggered” by binding to peptide/MHC. The TCR is composed of separate ligand binding and signal transducing subunits, and has no intrinsic enzymatic activity (1). Instead it is phosphorylated by the Src tyrosine kinase, Lck, attached to the inner leaflet of the membrane. There are three categories of explanations for how net phosphorylation is achieved (2). It is suggested that ligand-induced TCR dimerization or aggregation enhances receptor phosphorylation by increasing the proximity of weakly associated Lck molecules. Alternatively, ligand binding-induced conformational changes are thought to lead in some way to increased receptor phosphorylation. A third mechanism that we have proposed (2) postulates that the state of TCR phosphorylation is maintained by an equilibrium between kinases and large phosphatases, such as CD45, that is disturbed locally in favour of kinases when the TCR engages its ligand, owing to the exclusion of the phosphatases from regions of contact.
Supported membranes, partitioned with barriers to lateral mobility, have been used previously to probe the effects of spatial localization, receptor cluster size and ligand number on TCR triggering, in an approach called “spatial mutation” (3). Using fluorescence and super-resolution microscopy we have discovered that, during encounters of T cells with artificial and model cell membranes lacking TCR ligands, sub-μm “close-contacts” form that exclude CD45 and trap smaller molecules including the TCR (Fig. 1). We have also found that physical segregation of the TCR from the phosphatase under these conditions drives TCR-dependent signalling in T-cells, arguing against a strict requirement for ligand-induced effects in order for TCR signalling to be initiated in T cells.
What we now want to understand is what happens inside these close-contacts. In particular, we want to know if or how the activities of signaling molecules, in particular kinases, changes locally when the phosphatases are excluded.
To do this we will determine whether the signaling proteins change their conformations inside the close contact zones, and what types of new associations they form. In these experiments we will use Förster resonance energy transfer, and fluorescence tracking approaches, all at the single molecule and super-resolution level, in collaboration with Prof David Klenerman (Cambridge University) and Dr Christian Eggeling here at the Weatherall Institute. These techniques lie at the cutting edge of modern imaging techniques. We expect this work to offer up unprecedented insights into the mechanisms of signaling in T cells.
Single molecule tracks (coloured), are identified using super-resolution methods for labeled CD45 phosphatase (left) and TCR (right) molecules, against a background of bulk-labeled CD45 molecules (white) excluded from contacts that a T cell is making with a glass surface. The CD45, but not the TCR molecules, are excluded from the centre of the contact. Diameters of the contacts are 1-2μM.
- Smith-Garvin JE, Koretzky GA, Jordan MS. (2009) T cell activation. Annu Rev Immunol. 27, 591-619.
- Davis SJ, van der Merwe PA. (2006) The kinetic-segregation model: TCR triggering and beyond. Nat Immunol. 7, 803-9.
- Dustin ML, Groves JT. (2012) Receptor signaling clusters in the immune synapse. Annu Rev Biophys. 41, 543-56.