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I also have to thank Dawn and and because they did a lot of the introduction. So they also allow me to be a bit quicker in this eight minutes. Um So, as I said today, I would like to talk to you about the role of T cells and in particularly regulatory T cells, or T rex inr imagination and how that changes with aging. So just basically, to do a brief introduction so that we are all in the same page. Um, Ms or multiple sclerosis, as you know, is that primary demyelinating disease in which usually T cells are considered the bad guys. They are the ones that target myelin, destroy myelin and lift the action vulnerable to regenerate to degeneration. But fortunately, um, inside our central nervous system, we have a population of adult brain stem cells. No, no solid underside, progenitor cells or obese is which basically have this remarkable capacity to restore myelin once is damage. And this, um, balance between inflammation and regeneration is key at the early stages of the disease. But unfortunately, as this progresses, and especially with aging, this balance is tilted inflammation going to the battle against regeneration and that leads to axonal loss. And today I would like to change the gear and basically, instead of thinking about this, tells about the bad. Guys were going to go back to the sales, having also a role in this repair process. Um, basically, for the sake of time, I have to cut down the introduction. But there was some lovely work done by the group here in Belfast before I joined by the Bone and the knees and other members of the group where they saw that regulatory. He's also have a role in driving OPC differentiation, understory smiling once it's lost. So but together today I want to go back to the story and look at it from the prisoner of aging. So why aging? Well, we know that agent affects all parts of the body, and remyelination is no exception. We are really good at restoring myelin once it's lost when we are younger, but this gets remarkably less efficient as we age here. You just have, um, to transversally sections of a damaged area in a rodent. Uh, CNS. What? You see that in the young one, we have a wrist or myelin, which you can see here with with electron dance, um, drinks. But this at exactly the same time. Point is pretty much absent in the age, um CNs, suggesting that remyelination is remarkably delay as we age. So since, uh, as I mentioned, theory has been identified as key factors that Dr Remyelination so you can see well, one way we could maybe restore this is we can basically boost the numbers, that of theories that we have and that might improve your imagination when we age. Well, unfortunately, the body itself has tried to do that. When we age, we already have more theory is that when we are young, this is just data from our our own hands. But it has been shown by multiple groups, and unfortunately, remyelination is still important. Therefore, there has to be something that impairs, um, that contributes to this important remyelination. Other groups in the US have shown, for example, that in skeletal muscle there's an impairment in the recruitment of T. Rex to the damage area. As we age, let's get to the in your muscle, and that contributes to an impaired repair. And if we basically managed to increase the number of cigarettes in the H muscle that could go to the lesion area. That helps repairing better. So we wondered whether that was also the case in our demyelinated lesions, and we went back to our animal models. And thanks to the health of a very talented PhD student, Jessica White, who basically master this very tricky protocol we were. We were able to stand for regulatory T cells in the lesion, and we look at whether we have more details or less details as we ate in the demyelinated area. Um, it's it has to be highlighted that we have very few. We can find very few directing the CNS even when it's damaged. But we didn't see any differences between young and age. Therefore, there has to be something else that is altered with aging that contributes to this impaired remyelination. So we wonder whether, as we age, correct themselves might have lost their capacity to drive remyelination. So to address this question, um, we went back to and in vitro model here. You just have a quick summary of how we did this in vitro model. We basically isolate neonatal oh, pieces which are fantastic at doing their job they can response because obviously, um, there's another question I'm going to I'm not going to touch upon today, which is whether the H o. P. C's are able to listen to the wrecks. But we will leave that question for another day. Um, so basically what neonatal pieces which are remarkably capable of, uh, differentiated and forming myelin. And then we isolated both young and H T. Rex and put entirely in contact with the pieces and see if both uh, T Rex both like Young and H. T. Rex, were equally able to drive focusing differentiation. And we did that by looking at the expression of to myelin protein CMP, a stingray and MBP in my agenda here in the pictures. And as you can see from the pictures and the quantification John correct were absolutely able to drive OPC differentiation. Um, and they especially did that by enhancing the expression of MPP, which is a later stage marker. So they were pushing opposites towards the later stage differentiation. But age correct. We're not able to do so, suggesting that they might have an impaired preventative capacity themselves, and this is quite important if we are thinking of exploding, uh, correction. Kind of in a therapeutic form because there's some clinical trials, uh, basically looking at expanding the theory of patients not only because of their anti inflammatory properties, but also because of their protective properties. But if it happens that they are only able to drive a mile in preparing a certain window, we might have some issues with those approaches. So it's it's important to understand how h t Rex lose their capacity and whether we actually can restore it somehow. So to try to address whether we can restore the regenerative capacity, we went back to a proof of concept experiment. Um, I'm not going to go through all the details, but basically here we use the model that we have in the lab in which we can delete indigenous T Rex from mice using a diphtheria toxin receptor model. And then we can transfer the wrecks from other mice that are either young or age. And we can basically address the question whether a young micro environment can reverse this age environment and therefore that will actually let us know whether once cigarettes are aged, there's no way back or whether there's a way of reversing them back to be efficient and driving. Uh, regeneration. Um, so basically, the first thing we did is just to prove that our model was working. That means to make sure that we have depleted endogenous T rex, which we can do looking at a GFP tack that this transgenic might have. And you can see here in the first graph that basically will have a depletion of our endogenous T rex. And then it's looking at whether we have put back our theory X adequately, or at least to a certain extent, which we do by looking at Fox 33, which is a master transcription factor for identifying regulatory T cells. And as you can see here, we are able to identify back the populations of theories that we have injected. Do I have to highlight that we don't manage to go back to in the initial control levels? However, it's important to notice that there's no difference in the Constitution between young of an age, um, the wrecks. So then, once we have our system working as well as as we possibly can, um, ideally, we would like to go back to control levels. But that hasn't happened. So we went back to the spinal cord where we do our religions. We basically inject a toxin and the stress, smiling in the basically in the basal part of the spinal cord. And then we look at whether that opposites differentiate at the same rate in in normal groups. So basically what we have here is we have our DTR with PBS which had our control mice, they should we marinate normally and opposite to differentiate at the normal rate. Then we have the DT group in which we have depleted endogenous direct. But we haven't given any other t rex back. And this is our control group and very nicely recapitulate what it was shown in the previous paper that when you don't have your x o P C's differentiate less, which is what we measure here looking at both CC one and aspirin, which are both markers of differentiated little lymphocytes. And then we'll have our John T. Rex. So basically, this is the group that has depleted endogenous T rex and have gotten back young cataracts from another mask that doesn't have the the production receptor, and therefore it will not be depleted during experiment and we restore the numbers of differentiated like on the sides. And surprisingly, if we look at the dark Blue column, which is our age group, this one got the endogenous direct depleted and got age correct Back in, uh, this one also restores the ability of oh, pieces to differentiate, suggesting that basically the age of the theory is not irreversible. And if we put them in the right environment, they can restore, at least partially, the capacity to drive OPC differentiation. Well, this is a nice proof of concept of study, but I don't think patients will be very happy with this approach. So we need to find another way in which we can potentially revert, uh, direct agent to try to see if we can rescue this regenerative capacity. So basically, uh, you got a long story short, Um, because I only have eight minutes. I'm not going to go through all the painful analysis of to transcript comic databases, but basically, we were lucky to, uh, in the lab, have two different script comic databases. One was comparing young age theories, and the other one was comparing. Um was basically looking at the changes that office is undergo once they are exposed to the air condition media. So by combining those two datasets, um, doing some sort of protein protein interaction analysis, um, using different sources, I came across a list of about 10 candidates that basically seem to be down, regulated with aging. Incorrect. And also their partners could be upregulated once offices are exposed to the rec. I mean, there's several hypotheses that is, we decided first to go with the concept of the simplest one, which is that correct. Lose something with agent that is needed for opposite differentiation. That doesn't mean that there might not be. Other possibility is like something that negatively affect offices that increases in h T. Rex, but we have to start from somewhere. So by doing that, we went back to our in vitro cultures that I saw you initially and out of these candidates, we decided to start with the ones that were expressing the surface of the T. Rex because they were easier to tackle. So we did an antibody blocking approaching which isolated young cataracts in this case, and we block the candidates. Some of the candidates that we have identified in the screening with an antibody for half an hour. And once those it was blocked. We went back to the opposite and put them together and basically see if those theories were able to drive for differentiation at the same rate as you can see here. Quite a few of them did, like 30. 60 12 didn't show any difference compared to normal. T. Rex, Um, so that L Y succeed. However, there's two candidates, which is, uh, the one for six, or Femcon or integrin Alpha two, which, when you block them with the antibody, have a decreased capacity to drive opposite differentiation, suggesting that at least in vitro, these two candidates might have a role. And it might be partially Why, um, when the expression decreases in h T Rex, they might be less able to drive opposite differentiation again, at least in vitro. Uh, in the well, there might be some other candidates, or quite likely, um, and with this, I just would like to go to my conclusions just to recapitulate. I have We have seen you that a gym person ability of t. Rex to drive OPC differentiation. However, that's not a set in stone thing. It can be reverted if we put a a direct in. A young medical environment agent significantly enters the direct transcript. And I haven't told you the data here, but just for information is about 1700 genes are altered with aging interests. Um, And then we have identified at least two candidates, which are integral, Alpha two, and and come back in. Vitro seems to be partially responsible for the ability of counteracts to drive opposite differentiation and also partially related to the age associated decline in TRX and remyelination. And with this, I would like to first thank all of you for your attention the organizers, my lab here in Belfast, my new my lab in a week and all the collaborators and the past and current funding. Uh, it turns out that there was a little bit of a problem in the program at the timing, but that's not a problem, because that gives us the opportunity to to ask any questions from the rectum. I want to shine very, very nice work. You said that you were surprised that result where the age to rags didn't when you took them. And, uh, you got as much degeneration as you had with the younger ones. And you said that they must have been rejuvenated or something. What are the markers of rejuvenation? I mean, they could, you know, you could look at whether so it's known, for example, like every other further increase in essence, and theories are particularly sensitive to San s times as the yet So we could probably look at, you know, CD two K. I always say from one of the contestants 27 and CT to K. You know what I wanted? I mean, I always get that name from, uh, we could probably look at them, but we haven't taken. So I say they must have been rejuvenated because we actually haven't taken the cells back out and look at whether they are actively rejuvenated in the prescription or we have a There's very few T rex in the lesions, so I can see one or two. But even if I stand for those sorts of markets, I couldn't be able to comfortably see that they are rejuvenated. I can definitely say that they have restored their function in terms of obesity differentiation and my hypothesis that they probably are rejuvenated as it happens. Like with parapertussis experiment on those, You mean? Uh, I actually have a look at it in the transcript. Uh, sorry, I haven't looked at it. Uh, I actually don't have a look at it, but since, uh, work has some that when you basically knock out that product in this remyelination still happens, I cannot ignore it. It's a target. Uh, so, yeah, we ideally, if we would have time and money, we should get the cells back out and see if you compare the transcript on back to the original one. Um, so that's my hypothesis. I cannot say for 30 high, great work, and it's not because I am in the middle of that, but, uh, you mentioned this recommendation of the T rex and they have the capability to revert their aging. What do you propose is the environmental factors that make aging or young a T Rex? Oh, well, that's probably a million dollar question. I don't think I have the answer. I guess it's a combination of generally my gut feeling. I put it in general proinflammatory environment that seems to have been irritating in every cell in the body So I guess they are exposed to tons of cytokines and and probably, um, less of there. So apparently there was one paper. I think it was an influence. Or or that they show that there's all these inflammatory cytokines coming up that they basically make t rex more like an effect or t sal as they age. So I guess it's the general inflammatory environment in which they are in. I don't really I wouldn't put my I wouldn't be able to say one factor that would rejuvenate that, uh, one thing I was wondering, um, following up a journalist and works, for example. We're thinking of interleukin two and how they surviving the tissue. So one thing I was asking him the other day in London was whether with aging, would have less intelligent to, for example, in the CNN, which has low so things like that. But I I wouldn't know which one could be a factor. Very quick. One. I'm in the answer. It depends on the question. Is a really, really nice story area. Just a very quick question. So when you adopted to transfer t rex back, have you look, do they reach the Penis or the demyelinated area or not. They do. Yeah. I mean, I cannot do it very well because ideally, you want 3. 45.1 point two, which we don't have, but I sustained for 30 for folks. Be three e f p. And I've seen folks be 3 34 positive cells that are GFP negative in the in the C n s back. Unfortunately, I don't know if they reach to the same extent because we're having some issues with the Fox Be three antibody, second batch, Um, that that we're trying to solve percentage Do you think is going to? So if my standing is completely reliable, which I need to double check, um, at the beginning, you get in. The PBS is about 85% are the ones that you find rgf be positive in the adoptive transfer, it reverses 85% of the ones you find that the f b negative, but same numbers, I would say, is what they said. Thank you very much. So