[Music] okay well thank you very much the organizers for inviting me to present you to you guys today so I'm a comparative immunologist we're not just interested in birds we're interested in immunology across the species and try and use comparisons to actually understand what the fundamental aspects of immunity are so today I am gonna deal very much with immunity and perhaps the title I have on my talk gives you a basis for what I was asked to do and actually I was asked to consider whether we should use nutritional interventions that are either pro-inflammatory or anti-inflammatory and really my bottom line on this is actually we should be looking for balance and it's been mentioned one or two times before balance in terms of microbiota and things like their immunities just the same there is no one easy way of describing what we should be doing so what I hope to do over the next 40 minutes or so is that is to literally take you on a journey through different types of areas of immunology how it relates to microbiota and I'm going to give you an example of what happens in terms of microbiome immune cell interactions both from the perhaps of the perspective of the immune system and also from the microbiota so I hope you find it interesting so the first thing that I did was I surveyed a whole bunch of the literature in terms of what it was that we were trying to answer the question that I was set and really the question very much is what are we steering what an earth is it that we're trying to steer in terms of bringing birds from the very young age to birds that have finished their production cycle and the bottom line is it's an absolutely moving target because what we're dealing with is we do it dealing with birds that are really changing immensely over that period of time and if we think about all the possible influences on gut health during that time on these are just some of them really one of the central players is actually the microbiota of many much of the time when we change the food or change the or add components to our food what we end up doing is modifying the microbiota the microbiota interacts with host genetics physiology immunity interacts with other aspects of the food and of course interacts with pathogens at the same time so what we're dealing with is actually a really complex scenario and we mustn't forget that it's very very easy to measure one or two things and think that we've got the answer I'm going to end up encouraging people to measure things much more broadly in terms of trying to understand the interactions and hopefully that will allow us to understand the mechanisms that are operating so one of the other classic scenarios if we were to think of the evolution of of the matter of of the interaction between the gut immune system in them and the microbiome and the food and all those sorts of events is that we must recognize that what we're doing in them in the poultry industry is we are we have changed the environment and the bird immensely that they're completely fine-tuned for production not necessarily forget health and now what we're trying to do is to reintroduce some sort of natural scenario where the bird wherever you look if you look in evolutionary time birds and their microbes or all animals and their microbes have actually come to a position of balance so what are we done we've changed the genetics we massively change the numbers that are there the production systems are speed at which these birds are growing is unbelievable and certainly not a situation that would have been present in the times when the whole interaction was evolving one of the other big effects that we do is we cigarette segregate the chicks away from the maternal environment we don't just segregate them physically by taking the eggs away and hatching them what we do is we disinfect the eggs we disinfect the environment we try and keep them as clean as possible this is completely unnatural and what it's hardly surprising that we end up with issues with gut health when we're trying to do so many things and I think what we've got to try and do is to bring the controlled aspects of creating a natural environment as we can and that will start helping us understand our helpers control various gut problems so I'm going to do with a whole raft of different things today and I just thought I'd sort of introduce some of the scenarios some of my pains of reading was I try to look at all sorts of different foods and additives and what what did we know about the mechanisms and one of the things that I'm most interested in is not just describing what happens but actually understanding mechanistically what's happening and I really quite struggled in the sense that a lot of different tests are done but they're they're rarely sort of comparable comparable between different types of studies and we've got all sorts of different ways that things can be modified and at the end of the day one of the conclusions that I really came to was that the central component as it was in that previous slide is that it's the microbiome that seems to be influenced really quite dramatically during that early stage of life and that can alter the gut immune system immensely which then in return alters the microbiome and then what you end up with is this cascade of events both the host and the microbe microbiome we shouldn't be thinking of them as separate entities we should be thinking of them as a single whole of ions and actually a changing circumstance over time one example I've got here from some of Ivan right-clicks work some of his published work is actually looking at some of the changes in the microbiome over time and I'm not going to go into any details here but to say except to say in the very very young animals this is changing incredibly rapidly these happen to be layers out in a commercial farm that he was looking at the the microbiota over a long period of time so there's a big change in the first few days of life those changes continue for men weeks and actually in the commercial layers you know you're out there at about 20-25 weeks before you get what we might consider a stable circumstance but actually the whole thing is stable because it's quite reproducible those changes are actually normal but we have to be thinking of each during the pros very rapid changes that we have a small influence can lead to really really big downstream effects I'm good I was asked to talk about immunity now I'm acutely aware there's a whole bunch of people in the audience who will now basically be going I'm going to switch off this immunity stuff is too complex I'll trying keep some fairly simple language trying to explain the concepts that we're dealing with the gut is the most complex and the largest immune organ in the body of any animal species it's bigger than if you put all the other immune compartments together it's got loads and loads of different populations of cells and these populations are cells do different things but the other important thing is these populations of cells interact with each other and are interacting with the external environment that we that is the lumen of the gut live cells like macrophages I'm sure most people have heard of those they're like the dustbin men the dustbin lorry of the immune system you've got other cells like dendritic cells which drive this group of cells called t-cells into dividing and that's going to become quite important as we move through the talk we've got B cells they're the cells that make antibodies and we've got the natural killer cells and many of those components you'd find all over the body and they link between other between other parts of the body and the gut but when you look in the gut you've got over representation of some components and I've just kept this quite simple you've got lots of B cells that are producing IgA the secretory antibody you've got rather special dendritic cells and I'm going to deal with that in a general situation in a few minutes and in birds in general we've got a high number of these rather unusual T cells called gamma delta T cells now to be honest I'm fascinated by them because they break all the rules we don't know much about their biology in any and system and their most studied in mice for example but mice don't have very many circulating gamma delta T cells so they're most studied in a system that hasn't got very many birds and many other animals have very very large numbers of gamma delta T cells this is where we're going to work out how they've early function the other thing that point that I wanted to make is that the epithelial cells themselves are part of the immune system they ourselves that are able to produce effector molecules like defensins and they can also be really intrinsically involved in activating and calling in cells of the immune system into the gut so they're often neglected and they shouldn't be neglected in terms of the interaction between the microbiota because of course they are indeed the interface between the internal and external world now this is a really sort of simple slide but I'm just going to point out for those and not immunologists the sort of main events that happen so really what we've got is we have detection events and there's a bunch of receptors called pattern recognition receptors and for some of you these include things like the toll-like receptors I'm not really going to talk about those today and but they're about recognizing what's good or what's bad essentially and they initiate the whole response initially the innate immune response that then moves on to the adaptive immune response that takes time to develop and the outcome hopefully in terms of pathogens might be pathogen removal or killing or control of a pathogen at least problem is is quite often the immune system is responsible for nearly all the pathologies that we actually see associated with pathogens relatively small amounts of it is directly driven by the pathogen the vast majority is driven by the by the immune system so if we have a sort of modify sort of gut epithelia here this is one of any night cells of the dendritic cell here's a macrophage these will engage with any microbes or pathogens particularly if they try and cross the epithelia and get into the deeper tissues the one difference is is that the macrophage will try and kill in Cicci whereas the dendritic so it's a bit of a scaredy-cat at the moment is TL ours are tickled it runs away and it runs away and goes and talks to t-cells makes them divide they then go back have a chat with B cells make antibodies like IgA or super activate the macrophage that is the basic sort of type mechanisms that operate in immunity and we're going to deal with some of those events in turn so let's start off with the dendritic cells and I said there's quite a lot of immune cells in the gut these particular images happen to be of mat of a mouse where all of the dendritic cells have been lit up with GFP okay so all of the green in this in this gut is actually dendritic cells and that shows you the intensity of the network of these first level immune surveillance cells as they're sitting there in the villi is a huge number of these cells waiting for action to happen some of them don't even wait for the action to happen they stick their dendrites across the epithelial barrier and wave them around out there where all those microbes are out in the lumen of the gut functionally what happens is that there are two real scenarios in the gut one of which is a steady state so they're always entering and leaving the intestine at a pretty steady state and if they do that then what happens is they have a prolonged period in the intestine and though dendritic cells that leave here are generally involved in regulation and introduction of oral tolerance if something goes wrong in the intestine then the dendritic cells spend less time essentially in that environment and when they exit they're much more likely to be inflammatory and drive classical inflammatory immune responses now those sort of effects a generic effects that occur in the gut but there is one other thing and I was asked to talk about diet in terms of immunity that I find it really quite neat and this is literally the story of the carrots so what happens is these dendritic cells that are conditioned out there in the gut are exposed to the environment in the gut associated with the gut and that includes a molecule called vitamin A that might be quite fretted quite familiar to us and the vitamin A turns out to be really important in the immune system because what happens is those dendritic cells are and the GAR pick up vitamin A and also some metabolic derivatives of vitamin A could retinoic acid and when they run off and this is be a mammal into a lymph node but if it in a bird we actually don't know where they're running off to when they run off they then go and talk to naive t-cells the moment our naive T cell recognizes an antigen or molecule on the surface of those dendritic cells and starts to divide it actually puts on a bunch of receptors on its surface one of those receptors is called the retinoic acid receptor and if that cell while in its first point of division experiences retinoic acid derived from from vitamin A then that t-cell will then change its surface and and put onto its surface homing receptors that will send it back to the gut so here the dendritic cell that's exited the gut is carrying with it an address and it hands that address across to the T cell and makes the T cell go back to the gut and that is the right but really based upon the environment and in this case a food Associated molecule Federman a i'm now going to move on to some of the T cells so these are some of the T cells I was asked to think consider are inflammatory versus regulatory signals now these T cells many of them are what we would call inflammatory or actively engage and try and kill pathogens but there are some subsets of T cells that go calm down guys they're the regulatory T cells and actually if we look at normal components of the the microbiota we already know that they drive forever entually different types of cells so the s FB is a segmented filamentous bacteria a really strongly drive a pro-inflammatory cell type some of the strains of Clostridium really strongly drive regulation and others really strongly drive both under different circumstances and it's probably about the environment they find themselves in as but as bugs as well so what we've got the most important thing here is actually balance between pro-inflammatory and regulatory if you've got too much inflammation you'll get pathology you'll lose those villi if you've got too much regulation then you won't be able to actively respond to any incoming pathogens what I'm going to do now is to move on to some of our data and actually talk about some of the processes that are involved and the first thing I'm going to do is I'm going to take things right to the extreme so what happens if we massively restrict a microbiota and look at germ-free or nota biotic birds and then ask questions about how is the immune system developing what are the influences as a microbiota in terms of this what we know is the gut structure is affected particularly the layer that lies just underneath the epithelia called the lamina propria it's full of immune cells that is massively restricted in terms of the number of immune cells that are entering the tissues the numbers of classical T cells and B cells are actually reduced and I'm going to show you some data and that and act and then I'm going to move on about the diversity of the receptors that are actually being induced in these animals so if we look at the macrophages in an innate cell type in germ-free individuals versus conventional versus individuals that are that have just received a single mono colonization with E coli or four different strains of bacteria there ain't much difference at all they've all got macrophages in their intestine they're driven they're in a microbiota independent fashion in contrast if you look at b-cells I think you'll see a difference so these are this is immune against the chemistry for b-cells so this is a conventional and I'm a lot of B cells in the intestine this is a germ-free well they're few and far between in the intestine and if you put a single eco Lian you get if you get a few spots a few more spots if you count them but if you put four bugs in then you start to see recon root reconstitution of the phenotype but this is just a number of B cells that are there the question is is what are they recognizing so in this situation in this situation these are using an antigen that's based upon an it the e.coli which was the single colonizer germ-free animals don't have any IgA against the e coli the monarch on Elysee with E coli have strong responses against the e coli if you put that eco lie in the context of three other bugs those responses in C coli are still detectable but they're actually reduced in terms of overall magnitude and if you look at a conventional flora they're really very very low indeed if we take another bacteria this fic cesium these are the same groups of birds so we can tell that this antibody is absolutely specific because the mono E coli exposed birds are not reacting to this entry caucus the Tetra birds are reacting to them but actually a much lower level than we see against the e coli so there are differences between the different microbiota components and the conventional birds are really quite variable in terms of their recognition so we've got recognition how can we follow some of the events that are going on and one of the things things that we've been doing quite a lot of that's a little bit unusual is actually trying to understand the repertoire of these T cells and B cells that act actively respondent and have the effect of function and we use two different methods we use one method which essentially is gives you a profile so the normal profile would look like this lots of different peaks in terms of the receptor profile if so this would represent a a normal population of cells where every cell has got a different receptor on so this is what happens with adaptive immunity in this room I haven't counted up how many people are here but each and every one of you is carrying about a hundred million different adaptive immune receptors so we don't like sequencing them all but that's what the population looks like if you get a pathogen come in or an antigenic stimulus the most important thing that happens to these small rather insignificant cells they've really are not very not very flashy in terms of looking at their structure the most important thing that happens is that they divide so I used the analogy that the macrophage was the sort of dustbin man of the of the immune system clearing up all the mess and being in it being in Rome I think I should use a slightly different analogy for the lymphocyte this is essentially the Ferrari of the immune system if you wants a car to go really really fast you don't put all the extra bits on you take it all off and a lymphocyte which is the key cell in adaptive immunity it's basically stripped bare of anything that it doesn't need so the one thing you can do is replicate really fast and that's absolutely key in terms of adaptive immunity so we can follow those dos receptor changes and they tell us about the populations of cells so in a normal unselected population we see a diverse repertoire of cells in a selected population where one clone has grown out we see a much more focused population of cells and if we look in different situations and look at the the repertoire this in this case of alpha beta T cells what we can see is if we look in conventional animals at the repertoire of alpha beta T cells big normal distribution in the spleen but wherever we look in the gut what we get are actually biases in that in that repertoire and that's because there are local T cell clones that have grown up that are bigger than they were in the their fundamental initial repertoire so we see that complexity in terms of the repertoire in the gut and in different regions sometimes you've got shared clones sometimes you've got distinct clones but if you look in a germ-free then basically what you get is a normal distributed repertoire all over the place you've got no selection that's are happening what's interesting is when you get you culture in this case I'm on a floor with bacillus is what we ended up seeing was a single clone of cells that was grow out growing in all portions of the gun and we were able to show that this was actually actively responding to the bacillus so that's what happens if you have different types of gut microbiota and T cells ah in terms of alpha beta T cells that I'm going to move on to this other group of T cells which is the gamma gamma delta T cells and these are as I said a rather odd they don't follow the same rules there's lots of questions about whether they're part of the innate or adaptive immune response but what I think they are is probably part of both and I'm going to show you about microbiota dependent and independent biology of these cells I think these cells are quite fascinating and under-exploited in terms of being able to improve gut health so if you look at the simple gamma tau a repertoire in the spleen there are three large populations of gamma delta T cells in the chicken what you see what you see in colonized in during normal individuals and in germ-free individuals it's pretty diverse repertoires in all of the three different V gamma populations and we got really sort of trying to worry about subtle differences between the two and then we looked in the intestine and in colonized individuals you see some chrono biases but a pretty diverse population but when you go into germ-free individuals in V gamma one it's polyclonal in V gamma three it's polyclonal we got a little bit of a surprise because in one of those in the V gamma twos we basically saw a single peak on on this particular graph and when we sequence those those T Saurus up sorry when we sequence those T cell receptors they're all exactly the same clone so there's this large mono receptor crime that's in the gut of germ freeze at very very high levels when we look at a lot of individuals in both the small intestine and in the large intestine what we see is this large clone is present in the small intestine of all of our germ freeze and in the large intestine of all of our Jefferies and also present in the large intestine of actor actually our colonized individuals at really high frequencies I said that when we sequenced it this is essentially the same receptor everywhere this is the receptor sequence of the germ free ileum and the germ-free colon and actually in conventional colon we were also able to see this particular canonically rearranged sequence by canonical rearrangement it's essentially taking the segments that make up the t-cell receptor and sticking them together in the germ line configuration now there are other canonical gamma delta T cells that have been studied in the mouse and these gamma delta T cells include the V gamma v s and the V gamma sixes which are the very very first waves of gamma delta T cells ever made in the mouse and the V gamma v s all go to the skin and the V gamma Sixers all go to the uterus and to the tongue the third wave of gamma delta T cells are V gamma seven in the mouse they're not canonically rearranged but quite restricted and they all go to the gut so that you've got these waves of gamma delta T cells that are protecting the epithelia and we wanted to ask about the biology of this rather unusual canonically rearranged gamma delta T cell in the chicken and what we know is that gamma delta T cells are found in the earliest waves of thymocyte development and we but instead unlike what we see in the mouse what we could detect was all of the different TCR Gamma chains being rearranged at the same time but what we when we looked at the ontogeny of this particular V gamete v gamma to population of cells during embryonic development an early period post hatch what we see is we see this is the peak that we're interested in is there are 19 days of AM Bionic development you've got this dominance of that chrome which isn't so isn't so strong at 16 days of embryonic development these gamma delta T cells are exiting the beginning to exit the thymus at about 15 days of embryonic development and then it remain Taine's its dominance in this early period post hatch looking at it is slightly more fine scale so if we look at 15 days diverse repertoire 16 days diverse repertoire 17 days diverse repertoire 18 days diverse repertoire bang at 19 days it's all changed and now we have this very focused repertoire there's something rather odd going on here this is just before hatch this is a gamma dirty cell and what we know about gamma delta T cells as they interact a lot with epithelial cells that is homing to the intestine just before hatch so what do we think is happening this gamma delta T cells just like the other epithelial gamma delta T cells we know of in the mouse he's appears very early in the embryonic thymus but it seems to migrate out of the thymus and we can find it if we sequence a very deep sequencing in other places like the spleen we can find it in the gut at very low frequency but what happens between eighteen and E 19 is essentially all of these cells appear to migrate to the gun and we think there's something changing in the intestine and it's calling in these cells and then these cells are actually involved in some of the late stage embryonic development changes to the intestinal epithelium to prepare the bird for that horrible exhausting environment of hatching out into the microbio microbiologically richworld once the microbiota comes in these microbes actually then drive the diversification of all the other gamma delta T cells and of and the V gamma T's in to form a diverse repertoire and that's what we think that these cells are doing now I'm going to just change tack for the next few minutes and deal with microbiota immune interactions and and what I'm going to try and convince you is that the immune system is about stabilizing the complexity of the microbiota in all animals I don't think this is a very special circumstance in birds and what it what I'm gonna try and sort of describe to you is based upon a conversation I have with some theoretical ecologists quite a long time ago they can be very taxing conversations at times but what one one trap just turned around to me and said this microbiome stuff it's too complex it can't be stable it doesn't fit with a theory well I I I replied to him saying well unfortunately we know the microbiome can be stable so maybe the theory is not quite accurate but that's that's like a red rag to a bull in terms of some of the opinions that we have to deal with and then I wondered what would happen if I could create lots and lots and lots of slightly distinct niches in the gut honey is response was Oh that'd be that'd be okay so the analogy I'm going to use over the next a few minutes when I'm going to just deal with this particular area is it'd be like trying to understand finches from the Galapagos Islands if somebody come back with a ship ship load of finches and gave them to you and said oh look at these there's lots of different ones isn't that they would really make no sense but what was so crucial to Darwin is that actually he knew where they came from the finches were different on different islands and I'm going to try and convince you that the gut microbiome and the immune influence of the gut microbiome is based upon Island ecology so that's told you that outcome of it so if we think of the microbiota and we've heard a lot about it so I haven't really got to introduce it too much lots of people examine the poop a few people will examine the intestine this that I'm sorry this is prepared for a mammalian audience but examine the intestine and one of the big issues is wherever you examine what scaler we examine it you take a poop you've got an intestinal sort of structure this poop is bouncing off one wall onto the other wall and we got mixed up a bit people assume that this is ultimately mixed population it's representative of what's happening in the gut that's not true it's some sort of random representation of what might be happening in the gut clearly the bugs are there and sampling is really really important the scale at which you're suffering and the type of samples are really important and I hope to convince you of this and this was driven a little bit by some theoretical work done in Kevin father in in collaboration Kevin Foster by one of our students Kirsty McLaughlin who essentially looked at a bunch of mathematical models and I'm not going to go into the detail but I'll give you the bottom line is these models were very very unstable in terms of which microbes won and which microbes didn't so there was a stochastically in the model and there is somewhat familiar in some ways to some of the things that we're looking at but if you actually could provide some sort of spatial structure to an adhesion molecule that the bugs could bind to then this spatial structuring allowed spatial structuring of the microbial communities and then and then that became much much more stable something we know actually happens so one of the things that that requires is some sort of contemporary spatial structuring of a host provided adhesive molecule but we need massive diversity so we've got to explore what it is though that adhesive molecule might be and a strategy and this sort of holds true in all sorts of different animals as strategy was slightly crazy and I don't think Kirstie's ever forgiven me for this was to take a piece of intestine and chop it up and chop it up into lots of tiny bits so we have all the islands you know carrying on with the analogy of the Galactus we have all the islands so we chop it up into lots of different bits and then what we're going to do is we're going to analyze the microbiome and the immune repertoire in every piece so typically the size of the pieces have been one one by one millimeter squared or two by two millimeters squared we get quite similar results at those two scales yeah so what their first prediction from the model was we needed uneven distribution across microbes over small scales here's the grid the intensity of the color these are different microbes microbe a microbe B micro FC doesn't matter what their identity is the bottom line is if we look across the grid we see different patterns of different microbes they're proportional diversity is very different across the grid the second thing we needed to get some form of spatial structure in the microbial communities and we used a method which just allows us to look at the similarity or dissimilarity between any two frits chunks on the entire grid so we compare this particular chunk with every other piece on the grid and then we ask the question is it close is that is the comparator chunk close to the original one or far away and what we've done here is is simply plotted the distance versus the dissimilarity between the different chunks and as you move further away on the grid then they get on average more dissimilar than neighboring regions so we've got some sort of spatial structuring what's quite cute is that you can then take this analysis and serially take one microbe or one sequence out of that microbiota analysis and that's essentially bootstrap the scenario and then ask for what is the patterns of microbes that we see that when we remove them we get an increased dissimilarity space association those are microbes that we tend to find everywhere or a big decrease in the dissimilarity space relationship and these are microbes that are very spatially structured within the very small segment a whole centimeter of interesting we then asked what happens in the same way to the adaptive immune system IgA repertoire shows exactly the same pattern as you move away in distance we see all the fragments are different but as you move away and distance they're spatially structured TCR beta the alpha beta T cells are doing exactly the same we see spatial structure and association with the microbes TCR gamma very different in every segment lots and lots of difference but they don't structure in terms of our organization so they're a very different relationship that's going on here so really I'm going to summarize this stuff before moving on I'm going to skip the last section and move on to the end and really what we've got here I think is immune generated adhesion as a mechanism for spatial structuring of the microbiome we've got an uneven distribution of microbes across small scales we've got spatial structuring of microbial communities across incredibly small scales we've also got contemporary space restructuring for host product provided adhesive molecules IgA IgA we all think of as being an anti pathogen molecule how about IgA actually being a community structuring molecule its primary role rather than antigen rather than anti pathogen and we're trying to use this spatial structure to link community members microbial community members and IgA specificities and then we ask want to ask questions and this is where I appeal to you with all the different infection models because I think that different infection model models would it will disrupt this very delicate balance of the microbiome in different ways some by promoting high levels of IgA against the pathogen others by doing different things and what we were really interested in doing is actually exploring that so how do we think this might work we think that microbiota immune interactions are quite important it's become quite topical in the literature I think what we're putting on it is a spatial context that's a little bit different so if we've got the Mountains and the valley valleys that are the that are represented by the villi and you've got particular b-cells producing antibody of particular specificities inside the each fillers the antibody that's produced the iga that's produced is going to be translocated primarily across the epithelia of that villus it's not really going to diffuse over and get across into the neighboring villas so what we think is you've got communities of b-cells whether it's one Villa stem than our 50 villa I don't know but communities of b-cells inhabiting space and that in their neighboring space you've got another community of b-cells and a neighboring space you've got another and maybe it's repeated depending upon how far away you're looking these IgA molecules then essentially stick together Fred and his mates here George and his mates there and Carl and his mates over there over here I'm basically establishing founder communities that the the differentially organized across space in the gut if we disturbed that with a pathogen what happens is that this all gets into a big mix mash and these bugs start fighting with each other and that's where we get unusual dysbiosis despotic effects but it recovers remarkably well and the reason we think it recovers well is that actually these b-cells are still there they're just waiting for the epithelium to repair and then they can re-establish their local communities so that's what that's a sort of research part now I'm going to skip through a couple of slides I was going to talk a little bit about campy I thought this was too much but let me just skip through these and that the paper hopefully will be out next week and just really come to the last piece that I want to talk talk through with you how do we assess the effect of manipulations that was the task I was set I've just picked a couple of little bits and pieces it's a math wreck let us recognize as a complex moving target massively complex massively different so what do I think that we should be doing yeah we can all measure lots of parameters and we should carry on doing so actually we should measure as many and things as possible we should apply multiple methods but respect their both their benefits in terms of interpretation and the difficulties in interpreting we've got to be very very careful in the way that we anyone can apply a method it's about interpreting it and really the bottom line is we've got to try and consider the biology and I really am absolutely fanatical about the fact that we should be moving all the way backwards and forwards seamlessly between the field and experimental systems all experiments are a form of model all field situations are hyper complex but we've got to be moving backwards and forwards between these to try and collectively come up with similar solutions and that means using all of the tools that are available and generating some more but really thinking very carefully about the function of the immune system it's not about measuring stuff it's about how it works and that's an area that I think in the next few years we're gonna see some big changes let me just thank you for listening and the key players in my lab are listed here on that with their different areas and this work would not have been possible without many of you that are in the audience how many people that you know that haven't made it here to this meeting those collaborations are absolutely key so it's being able to do this work thank you very much [Applause] [Music]