 Good evening, ladies and gentlemen. I am James Hucklesby. I am in the first year of my PhD down the road at the School of Biological Sciences. I am supervised by E.J. Lowe, Scott Graham and Kate Angel. And I am here tonight to talk to you about how plasma and system proteins regulate endothelial presentation of C-cell 21 and subsequent T-cell electrification. Which is an absolute mouthful. But I hope by the end of this presentation you'll understand how this is keeping you alive. So the first thing we have to ask ourselves is what is one of these T-cell things. And T-cells are these little cells that patrol your body and there's 400 billion of them depending on how well or secure you are at any time. They constantly travel around your body and the blood flow. And they have to be able to access every tissue because they are the immune sentinels of the body. They are looking for anything foreign that might harm you. So they look for things like cancers and virally infected cells and if they find them they can directly kill them. They are also really important in producing antibodies that protect you against things like bacteria, viruses and fungi. However they also involve the production of cytokines which are little molecules that cause allergy and asthma. So the balance of T-cells in your body is really important. So to give you a slightly more graphic example this balance is really key to preventing autoimmune disease and cancer. So if we look at these images the one on the left up here is the inside of someone's colon and that big red spot is not normal. That person has bowel cancer so they don't have enough T-cells so the cancer is able to continue to proliferate or eventually leave the bowel and kill them. Conversely the person on the other side has Crohn's disease. They have too many T-cells which means they are in the process of attacking themselves so all that kind of whitish material that's all pus that's coming out of their intestine because they're having this immune response against themselves. So it's really important to keep you happy and healthy that you're sitting somewhere in the middle between those two. So it's really really important we find out how this balance occurs. So the main way that we can regulate this is by looking at how T-cells circulate in the blood and how they exit. So this is my schematic diagram of what the inside of a blood vessel looks like with the blood vessel at the bottom and the blood flowing over the top. And if there are no signals then the T-cell will just go past. There's nothing interesting there, it'll go past. However there are signals that can be placed on the surface that tell the T-cell to stop and exit. One of these is CCL 21 and this is the one that I'm going to talk about in this presentation. So having the CCL 21 up there, we can now make the T-cell come past, it sees the CCL 21, it stops, has a bit of a think about its environment and then exits into the tissue beneath. So now you've got a T-cell in the tissue, it can do whatever it's going to do, be it a tissue, a virus or a cancer. Now this process has been really well defined and we know that the cells can modulate how much CCL 21 they put onto the surface and they can regulate the protein that sticks to while it's there. But no one has yet demonstrated a good mechanism for getting rid of the stuff when you're done with it, which seems like a bit of a hole in the situation. So that's what we set out to solve. So we want to ask the question, how does the body then regulate the CCL 21 on the endothelium? So the hypothesis that the Birch lab has been working with is that this enzyme, Plasmin, is able to remove this from the surface. So Plasmin's been known about since the late 60s and it's really important for breaking down blood clots and it does that by cutting up the fibres that hold them together. It's what we call a protease, a protein that cuts other proteins. However, more recently, it's been shown to have a huge variety of other functions, including cleaning up various immune factors. So we thought, could it leave CCL 21 in the blood where it's normally found? This is where this process gets a smidge more complicated, because if you had active Plasmin in your blood, you would all be bleeding to death as we speak. So it's got to be regulated. And the way your body does that is it has this precursor called Plasminogen that's bound to the surface. And that is then activated by either TPA or UPA, which are two factors called tissue Plasminogen Activator or urokinase Plasminogen Activator. They're produced by cells only when required and they convert Plasminogen into active Plasmin. And this whole process so far is very well defined. However, we hypothesize that this Plasmin will then cut the top of the CCL 21 and cause the T cell to go past. So this is what I set out to investigate. Now, in order to do that, I did a series of experiments. I'm going to talk through those now and show you how we built up this diagram. So the first thing we needed to know was, can we have a blood vessel? It's really difficult to use a blood vessel in a whole human because you can't see what's going on. So we had to grow our own. And the way we did this was we used the HMEC1 cell line. This is an immortalized cell, which means it was taken out of someone's skin, treated with a virus, and now we can grow it forever. Now, this is a little bit of a dodgy process, but it's been really well shown in the literature that this is still effectively a blood vessel. So we use this for our study. The first experiment we had to do was check that, in fact, Plasminogen could firstly stick to the surface because if it's not stuck to the surface, it's gone in the blood flow. And secondly, that it could be activated. So what we did was we bound Plasminogen to the surface, gave the cells a wash, and then it had to this activating TVA. And as you can see from the graph behind me, the cells that received nothing and the cells that received just Plasminogen, absolutely no activity at all. And then the cells that received TVA showed a significant increase. And by measuring this, using a special colored substrate over four hours, you can see that there is Plasminogen activity only when we added TVA. And this gave us confidence that we now have a blood vessel in which the Plasmin system is acting as it should. The big issue with this assays, to get these nice numbers, is that we look for a color change in a molecule that gets cut. But that's nothing like what CCL21 looks like. So we then ask the question, can we do the same thing with CCL21? And to do that, we developed an assay called an ELISA, which allows us to measure the amount of just the top of CCL21 in solution. So the higher this graph goes, the more CCL21 has been cut off the surface. And what you'll notice here is we've got a very similar result to our previous experiment. The cells that received just CCL21 in the blue and the ones that received CCL21 and Plasminogen in the red, no significant increase. However, once we add that activating factor, we immediately see a lot of CCL21 released within the first four hours. So this was the first evidence we had that the whole stack so far was working well. Now that's all very well, but this process only works if you can get a T-cell involved. So in order to do that, we had to create a slightly more sophisticated blood vessel. And the way I did that was I grew my endothelial cells again on the bottom of these special channels that are about 0.35 of a millimetre across. So they're really, really quite small. The advantage of growing them in this format is I can move very precise amounts of fluid over the surface, and I can put T-cells in that fluid. So I found a friendly postdoc and we borrowed some of his T-cells, and then I stained them bright green so you can see what's going on. And if I now play this video, you'll notice the T-cells are whipping past, I've speeded this video up 60 times so it's nice and quick, and you can see at the top little green dots starting to stick to that layer of cells, and none of the cells are sticking at the bottom. Now because that's hard to see, I've gone through and counted the cells and graphed them, and you'll notice that the cells that receive just active proteas on the surface have significantly more cell sticking than those cells that also had TPA. And this lines up with our model and tells us that our whole stack of proteins is working effectively. So that's really convincing. So now that we've got this system, where could we take it from here? Well, we know that this plasma energy system is highly regulated, and that this gives us multiple therapeutic options. So it's been shown previously that mice that lack this TPA, this activating factor, have more immune cells infiltrating during kidney and liver diseases. And no mechanisms have been shown for this, but our mechanism fits perfectly. So that's really convincing. Secondly, it's been shown that Pi-1 expression has increased in inflammatory diseases, and that knocks out the TPA. Again, this could be causing immune infiltration in diseases such as obesity, where you don't want it in what is effectively the process. So that's another really exciting finding. And finally, we've had drugs that modulate the plasma energy system for people with clotting diseases for many, many years. And there's a huge potential that these could be repurposed. So the smalling the Nobel Prize was one for modulating these cells with drugs that are hundreds of thousands of dollars. Potentially, we could use drugs that just sit on the shelf and are very cheap because they're used for people by years and years. And finally, some acknowledgments. This research was primarily presented supervised by Nigel Birch, who sadly passed away about a month ago after a battle with cancer. It was also supervised by E.J. Loaf, and I'd like to thank the rest of the Birch group for their huge support, as well as the Graham Group, the guys who work the BioFlux, because it's a bit of a thinkative system, and the Rod Dumber and the Marson Fund for their digital support. Thank you very much.