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What do you do if you want to see how an immune T-cell responds to meeting the antigen molecules that itís programmed to detect?
Geoff - My name is Geoff O'donoghue. I'm a post doc at UC Berkeley in the lab of Jay Groves. The general question we were interested by was how the immune system is able to detect form pathogens, things like virus and bacteria, and how this occurs at the molecular level. So, we were looking at T-cells, CD4 or helper T-cells and we were looking at the T-cell receptor and the T-cell receptor binds a pathogenic molecule on the surface of another cell. Itís just like a lock and a key basically at the receptor on the T-cell surface is a receptor and it has a ligand that itís looking for. Once it finds that, it binds and initiate an immune response specifically only for that one pathogen.
Chris - So, you look at these T-cells, CD4 cells specifically. How do you study that interaction?
Geoff - We study the interaction using optical imaging. Ideally, we would look at the interaction at the interphase between a living T-cell and what's called an antigen presenting cell. But thatís a very difficult thing to image because you have two curved surfaces trying to image the interphase between those two curved surfaces with high temporal and spatial precision is very difficult. The trick that we use is to replace the antigen presenting cell with, basically, a biomimetic surface that is flat. We put on the surface the support lipid bilayer. So, this is a very good mimic of a cell. Attached to this support lipid bilayer, we attached the pathogenic molecules. In those molecules are free to diffuse laterally in two dimensions and then interact the T-cells once we flowed those on top of this surface.
Chris - And you can see where these pathogenic molecules are going, can you?
Geoff - Yes. What we did was label each molecule on the surface with a tracer molecule where we can relatively easily observe. We basically observed a field of these individual molecules, diffusing around in two dimensions. Once we flowed the T-cells in, the T-cells bind some of those pathogenic molecules on a surface. Once that occurs, we observed that instead of diffusing around, the tracer molecules on the surface flow way down. And then we can relatively easily observe. The way that technique that we use was basically to use a very long camera exposure time, the images are similar to the long time lapse images you see of a city where the cars on a road are blurred out because they're moving so quickly, but the buildings in the background are very stationary and very clearly resolved. We took the same approach to distinguish between single molecules that are bound to their ligands in the living T-cell and once they were unbound, instantly diffusing on the surface.
Chris - And so, this enables you to tell when a T-cell has interacted with one of those ligand molecules on the biomimetic surface. Are you then able to tell how the T-cell responds to that interaction?
Geoff - Yes. So, the first thing we do is measure the number of seconds that we observe this interaction happening. That tells us something about the time component that the T-cell is detecting. And then weíre looking at a molecule inside the cell which is an enzyme and induces a cascade of chemical reactions inside the T-cell. And so, what we did was two-colour single molecule imaging. We looked at the pathogen on the surface and the catalyst inside the cell, and we measured the number of molecules that are recruited to the reaction site involved.
Chris - So, you can very neatly here, identify when the T-cell docks or binds onto the thing on the cell surface, or the pretend cell surface. You can see exactly when the T-cell begins to respond and how vigorously a T-cell responds. Thatís amazing! So, what did you find?
Geoff - What emerged was that interactions between the pathogenic material and its receptor were surprisingly long lived. It wasnít clear from the previous literature whether or not these would be short much, much less than a second or seconds to minutes long. We discovered that these interactions occurred for a surprisingly long time. what we also observed is that one molecule could induce a chemical cascade inside the T-cell. The T-cell is very sensitive and can detect single molecules or a handful of molecules that will induce a chemical reaction inside the cell. The next question is to look at not just one pathogen but to see how T-cells respond to a panel of these pathogens and then kind of get at that question, is a large number of short interactions equivalent to a small number of very long lived interactions. Thatís I think the next step and that would go some way towards kind of really getting at the question of how T-cells distinguish between self and non-self.