Hello. I'm Michael Dustin from the University of Oxford and New York University School of Medicine. Today, I'm going to talk to you about the immunological synapse, Part 1 – antigen recognition. So, I'll follow this outline. I'll start with a general discussion of immune systems and their basic purpose; and the idea that there's innate immunity, which recognizes evolutionarily conserved patterns and sets up barriers to protect the host, and adaptive immunity, which can recognize any type of threat without any kind of prior experience of it, and how these systems work together; the physical challenges of antigen recognition for T-lymphocytes, which I'll introduce in a moment; and adhesion molecules that meet some of these challenges; and how these are coordinated in an immunological synapse. So, immunity is critical for essentially all forms of life. Once you start concentrating a lot of energy in a small package, there are always going to be other organisms, typically smaller, microbes, that will basically try to invade or attach to surfaces and steal that energy, effectively.

So, one needs to develop mechanisms to defend yourself. So, for example, even in organisms as simple as bacteria, they're developed this CRIPSR/CAS9 system to protect themselves against bacteriophage. In a classical example from innate immunity in invertebrates, in Drosophila they're essentially… the pattern recognition mechanisms were first discovered in the context of antifungal and antibacterial defense in Drosophila. You also have a bacterial infection in vertebrates and things like parasites, like malaria, that afflict millions worldwide. So, these are very significant threats. Innate immunity deals with the evolutionarily conserved components and adaptive immunity is something that was added in the vertebrates to basically defend against, essentially, more advanced and highly evolved pathogens that could evade innate immunity. So, why is it important to study immunity? Well, I guess from a human perspective, things like vaccination, which was the first effort of ourselves to manipulate our immune responses, you know, is essentially one of the greatest advances in protecting human health, where entire pathogenic species have been essentially eradicated by this kind of effective process where you can expose an individual to some form of a pathogen, even related or attenuated, or even components from these pathogens, and generate life-long protection.

So, we'll talk a little about how that works. Anti-cytokines therapies for rheumatoid arthritis are a type of immunotherapy that have greatly improved the lives of many people afflicted with these devastating diseases, autoimmune diseases in the context of rheumatoid arthritis, also other types of inflammatory diseases are addressed by a variety of these so-called biologic therapies. It's had a huge impact on human health. And the revolution in cancer immunotherapy, recently, based on checkpoint blockade and adoptive immunotherapy, and we'll touch more on that in Part 2, have provided new hope for people with previously incurable or very, you know, rarely curable diseases.

So, these are important contributions that studying the immune system has made to human health. So, in terms of introducing innate and adaptive immunity, we can think of these two components as kind of being somewhat of a pyramid, where the base is innate immunity. And, essentially, innate immunity is based on setting up barriers, which can be physical, chemical, or mechanical to pathogen attachment or invasion. When these are breached, there are a variety of induced so-called pattern recognition responses, like that picture of the Drosophila before, that was where this was first genetically defined. So, basically, innate immunity is effective against many organisms that would attempt to attack a larger animal. Innate immunity, I guess, is prevalent in bacteria, single cell organisms, plants, invertebrates, and vertebrates like us, of course.

So, if innate immunity is breached, basically, you have adaptive immunity. So, adaptive immunity is a system that's built on a set of receptors which are generated in the individual by somatic recombination… you could spend a whole talk just on these mechanisms, so I'm not going to say much more about this, but suffice it to say that they give you the ability to essentially recognize any molecular pattern that you would encounter, that you would be likely to encounter, and certainly across a population we really seem to have that capacity, although individuals may have holes, the whole population will basically cover a vast array of different types of potential molecular patterns that could be associated with pathogens, but are also associated with our own proteins, our own macromolecules, and lots of harmless environmental macromolecules. So, the rub with adaptive immunity and this pan-recognition is that it doesn't really know right from wrong, it doesn't know good from bad, and that's the job of innate immunity. So, these two systems need to…

And then, basically, adaptive immunity evolved in vertebrates, it's important to say… and these two systems, innate immunity and adaptive immunity, communicate with each other through a process referred to, generally, as inflammation. So, this communication is critical, and this is one of the key things that's happening in this immunological synapse that I'm introducing here, so this is why I'm going through this, because this communication axis is critically transmitted through this, basically, cell-cell interface that we'll be describing. So, just to say a little bit about inflammation, so… this phenomena in, kind of, human health and, kind of, philosophy was recognized in the time of the ancient Greeks as having a number of attributes. Essentially, the meaning of the word is to set on fire, and the hallmarks are pain, redness, swelling, and heat, and this image, this movie that's playing in the background here, is essentially a picture of white blood cells, which are part of…

A type of white blood cell that's part of the innate immune system, lining up along a blood vessel, which is the structure, here, kind of highlighted in red because the plasma has a red fluorescent quantum dot, effectively, in it. So, we're imaging this in a live, anesthetized animal during an inflammatory reaction, and the release of these green fluorescent white blood cells from the vessel, and this leakage — the leakage of the red fluorescence signal from the vessel — is basically what's driving these responses in large part.

That's basically the classical signature of inflammation. So now, there's also, however… this is an infection driven inflammation… there's also something called sterile inflammation and there are a lot of nuances to the way the innate immune system would communicate to the adaptive immune system in the context of, you know, infection-driven versus sterile inflammation. So, if you look at an example of sterile inflammation, here, you have, basically, within the central nervous system… these are the phagocytes in the central nervous system called microglial cells, a certain type of cell that is part of the innate immune system. When there's this laser lesion that was created in the center of the image, and this will loop again, you see these cells… the neighboring cells respond to the death of their friend by walling off that site and essentially protecting the central nervous system from further damage from that insult, but there's no infection in this case, and there's no breach of any barrier, it's basically like, for example, like in a stroke, you see responses just like this, say, a blood clot.

There's no infection. There is a repair process that the immune system may participate in, but it's very different than infection, and the innate immune system will communicate to the adaptive immune system the nuance that there's injury that basically is not an infection, and then, in many cases, drive the appropriate response. Rarely, there are mistakes made, and you may end up with an autoimmune disease from a phenomenon like this, and this is something that we need to understand better. So, you can break down this kind of platform of innate immunity, you can break down further in to components — barriers; various cellular constituents like phagocytes, that's I've mentioned, in the context of those microglial cells in the brain; chemical defenses; various types of lymphoid cells; all your tissue cells can be recruited into this at some level during responses — and these cells would form a foundation for these various types of lymphocytes which engage in…

Which are the components of adaptive immunity, the cellular components. So, B cells — and the B, basically, in this context stands for bursa, which is the organ in birds in which they were first discovered — or T cells, two different major types of T cells, which are — T is for thymus, in this case, which is the organ that they develop in in both birds, where this was maybe initially studied developmentally, and in humans. So, basically, if you also… and then a way to remember B for B cell has also been in vertebrates… in other… well, in mammals, they develop in the bone marrow. Birds don't have bone marrow, so they have to have a different organ, but basically other types of vertebrates use the bone marrow for this.

So, B and bone marrow also works. So, these cells now have to talk to these cells and in order to to do it seems that we had to evolve a different cell type, and this is the dendritic cell, that basically sits in this kind of intermediate position, here. It's kind of a bridge between the two systems. And particularly the T cells have a critical communication with this dendritic cell. Finally, there are a couple things I want to mention about this. So, there are several types of Helper T cells that can essentially develop in response to signals from the dendritic cell that deal with different types of pathogens, so, say, viruses, extracellular bacteria, fungi, parasites, all have different modes of Helper T cells to deal with those, and that's a very important thing.

If you make a mistake about that you can end up with the wrong response for the pathogen and that can lead to pathogen escape and disease in some situations. And the other thing that I want to point out is that there's another, kind of, a variation on a Helper T cell c alled a regulatory T cell, or Treg. These cells are very critically matched, in some respects, to dendritic cells, and they control the activity of the dendritic cells in an antigen-specific way… I'll get to the antigen in a moment, but they essentially… it's a type of cell, a similar type of adaptive receptor, this pan-recognition process. They tend to be actually self-reactive and they suppress responses in the context of self-recognition, so they actually are critical in protecting us from autoimmune disease. If you lose these cells, say, due to a primary genetic immunodeficiency, you don't have a lack of immunity, you have an excess of immunity, and that's actually almost worse, that can kill you faster than the lack of immunity in some contexts, and this is because it's your own immune system attacking your body, which, again, has devastating consequences.

Now, the other thing I wanted to point out was Killer T cells recognize components on host cells that we'll talk about in a moment. If these are subverted by, say, viral or bacterial immune evasion mechanisms, then you might think you would be vulnerable to attack by those pathogens, but in fact there's this Natural Killer cell type that steps in and recognizes the loss of those molecules that are involved in that communication and kills those cells.

So, tumor cells or virally infected cells that might lose molecules required for the communication with the T cells are basically attacked by Natural Killer cells, so you have this missing self-recognition which is also critical in protecting yourselves. So, that gives you kind of an overview of the cells of adaptive and innate immunity. So, a critical thing, I've used the term antigen a couple times and I think I need to define that at this point. So, antigen… the term comes from antibody generation, but it also applies to T cells, which don't use antibodies. So, B cells, again, make antibodies, which, again, start out as a receptor on the surface of the B cell and are then eventually secreted from a later developed form of B cell called a plasma cell. So, these antibodies recognize intact forms of the antigens. So essentially, this is… the image here is a viral coat protein called influenza hemagglutinin with three antibody fragments, here in purple, these three fragments here, basically in kind of a…

It's a trimeric structure, the hemagglutinin, so there are three copies of the antibody binding site in the intact protein, and that's the process you're seeing. This antibody binding would neutralize the function of that viral protein and prevent further cycles of infection, so this is a critical way the host defends itself against viruses and a critical… making these kinds of antibodies is a critical target of vaccination, so what you want to do when you're designing a vaccine is make these neutralizing antibodies, and for a highly mutable virus like influenza, you want to make antibodies that are broadly neutralizing.

That would be the holy grail at this point, so, this would allow us to say… now, we have these seasonal flu vaccines because the antibodies are very specific, are very strain specific. If you could make vaccines that generated these broadly neutralizing antibodies, you could have broader coverage and less need to vaccinate every year. T cells, on the other hand… so, the B cells see the intact proteins… the T cells cannot see the intact proteins at all, so they don't have any ability to recognize a structure like this on a virus or on any other type of pathogen. What happens is the dendritic cell that I mentioned before will internalize the antigen, often in viral particles or whole bacteria — they're a type of phagocyte, they can take in large structures that are almost as big as themselves in some contexts — they break them down, digest those complex macromolecules into peptides, and then bind these to histocompatibility proteins.

So, what you're seeing here in this structure is the surface, the upper surface, pretty much what the T cell would see, with this… the peptide binding groove, it's almost like a hot dog bun in some ways, holding this linear peptide, which is derived from proteins that are taken up by the dendritic cell. These proteins can be from pathogens, they can be from yourself, they can be from harmless things that you're breathing in or out, you know, allergens, things that aren't really going to hurt you but you might respond to.

So, all of these different degradation products of these proteins are binding to these MHC molecules. So, this term MHC is Major Histocompatibility Complex. That terms comes from the fact that these molecules also control transplantation. So, if you look at skin transplantation or organ transplantation, there are differences between us, in a population, that reflect different types of these peptide binding proteins.

It's important the population have that diversity because you could imagine with this peptide binding process, there's some specificity here. Some individuals may not be able to bind peptides from some pathogens, then they'd have a hole in their repertoire. So, this… individuals, then, may be susceptible to that particular pathogen, but in the population, because there's more diversity in the population of these molecules, it makes you able to defend yourself against a wide array of pathogens. However, it also prevents transplantation, or at least makes transplantation challenging and requires immunosuppression, sometimes for the life of the individual.

Of course, inducing transplant tolerance, then, is sometimes experimentally or, you know, therapeutically, that we'd like to be able to achieve. Okay, so now what I want to describe is how immune cells come in contact with antigen in the body. So, if you imagine an infection in the skin, you have a break in the skin, some microbes have entered and started to replicate, innate immunity has tried to deal with this, but failed, the organism is increasing in numbers, so now you have an increasing amount of particulate material or small molecules, proteins and things, being released by the growing pathogens, and these are draining, now, through lymphatics to structures referred to as lymph nodes, which are basically filters which are packed with T and B lymphocytes, and also sites where dendritic cells congregate to show antigens to T cells, and B cells basically become exposed to materials that are draining to the lymph node from these tissue sites. So, this set of movies from Facundo Batista's lab basically show how the B cells, which are these antigen-specific B cells, which are these red cells, so they have a particular antibody on their surface that recognizes the antigen that they are using in these experiments, which is green.

So, what you see here is the filter capturing the… filter at the outside of the lymph nodes, which is cellular actually, it's phagocytes, capturing the antigen, and then the B cells, it's kind of looping between these three views — the large view and then two detailed views, one at the filter boundary and then one at the place where the T cells are — and what you can see basically is the B cells, at this edge where these phagocytic cells are capturing the antigen, displaying it in a way that the B cells can test if their antigen receptor has the right specificity to capture and concentrate that antigen, then they will process that, make the MHC-peptide complex as I described before, and then they very quickly go to this zone where the T cells are…

So basically there's a boundary between the place where most of the T cells stay and most of the B cells stay, they're usually segregated, kept apart, but under the conditions where antigen comes into the system they come together at that junction and have a chance to test… the T cells test their antigen receptor, to determine if it recognizes any of the MHC-peptide complexes being presented by those B cells and if they get a match, that is a situation where you start to get help for the B cell to make high-affinity antibodies against that pathogen, starting with the receptor that they used to capture the antigen and then trying to improve it by mutating it and reselecting it, again, with continual advice from the T cells. The T cells, in that situation, have already received instructions from the dendritic cells, which are also looking at the same pathogen and helping the T cell identify what kind of response is needed.

So, this is a highly coordinated process and I just wanted to point out the… use this movie to point out the dynamics of this process. So, this is another… a static electron micrograph of a T cell and a dendritic cell. Now, you know this is not the way things actually happen in vivo, the system would be much more dynamic than the still image conveys, but I just want to basically use this image to say a little bit about this interface, the immunological synapse between the T cell and the dendritic cell.

So, this is… again, T cells only see these MHC-associated peptide fragments, which are on the surface of the dendritic cell or the B cell, as I just mentioned. The dendritic cell and the B cell take them up differently, but they're basically… eventually the T cell would recognize the same structure on either cell type. The T cell receptor is also only on the surface, there's no soluble T cell receptor, so basically the T cell and the antigen presenting cell, whether it's a dendritic cell or a B cell, are always going to be dealing with this…

The dimensions of these molecules, which will only span about 13 nanometers (nm) between the two cells, and this is a structure, an X-ray crystallography-based structure, of a T cell receptor, kind of the specific part of the T cell receptor, then the MHC-peptide complex, there's the peptide, this is the histocompatibility antigen… you know, so essentially this is only 13 nm long and these cells are about 10 microns or so across, which is 10,000 nm. So, basically the gap between these cells is very small compared to the cells and the cells have to get very close to each other to achieve this recognition. I guess the other aspect that I've already touched on is that the dendritic cells are these very dynamic cells in the tissues, they're part of…

This motility is involved in essentially allowing them to drink up large amounts of fluid and engulf particles, which are basically… could be either derived from the host, other host cells, or from a pathogen… takes them into a lysosomal compartment, degrades them partially — not completely, not to amino acids, but to peptides. Those peptides come into contact with the MHC molecules that have been recently synthesized, those molecules become receptive to the peptide, bind the peptide, and then go to the surface as the dendritic cells move to a lymph node. Once these dendritic cells get to the lymph node, they basically distribute in the T cell zones and essentially take up a position in a network, and then continue to undergo a very high level of surface dynamics. They have a very large surface area, so they'll come in contact with about 1,000 T cells per hour, and in this movie there's some antigen-specific T cells, and control T cells that are light or dark blue.

This is an image in an experimental setting where we kind of knew the specificity of these cells and we were looking at their interactions with the dendritic cells, but if you looked at all the T cells in this tissue the image would just be packed with T cells. So they're, you know, really, this image would contain tens of thousands of T cells, and those T cells would be moving around, coming in contact with these dendritic cells, again, looking for a fit between the antigen receptor and the MHC-peptide complexes.

So, the initial encounter for any kind of antigen with a T cell would be on th dendritic cells, they are the best cell for initiating T cell responses. The activated T cells, which are relatively… the antigen-specific T cells, which are relatively rare, become activated and undergo a proliferative burst, which greatly increases their numbers, so a T cell can go from being, you know, 1 in 100,000 to being about 5 or 10% of your total number of T cells in about 5 days during an antiviral response, so this proliferative burst can be quite dramatic.

And then these effector T cells will then exit the lymph node or move to the B cell follicles, and once they exit the lymph node they'll go to sites of inflammation via the blood, and once they're entered those sites of inflammation they'll be prepared to kill virally infected cells, for the Killer T cells, or help other cells in the system basically coordinate their response to the pathogen, those are the Helper T cells, and, again, because the dendritic cells have instructed those two cells to take on certain attributes, they should be well-equipped to deal with the type of pathogen that they encounter once they get to the site of inflammation in the body. So, this is, again, a very well-coordinated system, but the recognition process underlying this then faces a lot of challenges related to working within this… working with these constraints in the system. So, again, just to summarize these challenges, the T cell receptor (TCR) and the MHC-peptide complex (pMHC) are small; the MHC-peptide complexes are rare because they're competing with all of these self proteins and other types of proteins that are essentially present in the tissues that are in addition to the proteins from the pathogens; the affinity of this interaction is low, I haven't really touched on that very much but this is…

Compared to antibodies, the affinity of the T cell receptor interaction with an MHC-peptide complex is about three orders of magnitude lower than what you typically see for antibodies binding to their intact antigens; and the T cell and the dendritic cell are moving, so you have this, you know, kind of search going on, so the cells really have relatively little time to decide whether they have a fit or not, they have to do that in a few minutes, basically, in a response that may go for u p to a couple of weeks overall. So, how do you deal with these challenges? So it turned out in maybe around the mid-1980s that we didn't know very much about this. We knew that there was this antigen recognition process, we were beginning to understand the T cell receptor in the late 80s, this picture of the MHC-peptide complex became more clear, and at the same time investigators started to explore this issue of how this recognition process works.

And basically one of the key things that this transmission electron micrograph shows is this very close interface between a target cell and a cytotoxic T cell. So, the cytotoxic T cell will kill the target cell, in this case based on allorecognition, which is the mode of recognition you have in transplantation, so basically seeing foreign MHC proteins. This is a very strong type of recognition, but it's clear that the antigen recognition process itself can't account for this very tight interface, this very extensive interface. It would seem like you'd need something else to do this, so investigators started immunizing mice with the T cells and then trying to screen for monoclonal antibodies, so basically individuals antibodies — so, using the immune system to study the immune system — that would essentially block this recognition process, and they found a number of antigens, essentially in this case, functional molecules of the T cell, that were involved in this process.

So, here we have a little schematic that introduces a few of these. So, the antigen receptor and the MHC complex provide the specificity, but a set of non-polymorphic molecules were defined in these studies for which antibodies binding to those proteins would inhibit the functional process, and these included LFA-1, or lymphocyte function-associated 1, which is a member of the integrin family; ICAM-1, which is actually a member of the immunoglobulin superfamily, so it's related to antibodies; and CD2 and LFA-3, also sometimes referred to as as CD58, which are also members of the immunoglobulin superfamily, which interact across these gaps. So, these molecules are all present in around something on the order of 50,000-100,000 copies per cell, but all of these molecules are capable of interaction, whereas maybe only a very small fraction of the MHC molecules have the appropriate peptide. So, these molecules effectively give the T cell the ability to make these short, these tight interfaces, but this then posed somewhat of a problem, which is that if the T cell is going to survey all these different cells and has this ability to stick to then, how is that regulated? And it turned out that you needed another layer of understanding in this to kind of start to understand the whole process, and actually this really comes into, what is the immunological synapse? How does it work? So, the T cell receptor itself is a signaling molecule.

So, this is basically a schematic of the T cell receptor — these parts here are involved in antigen recognition, these parts here are involved in signal transduction, they're non-covalently associated with each other, so it's quite a complicated feat to basically build this complex, that was studied quite a bit — but the key to the signaling process is that these cytoplasmic motifs contain tyrosine residues and they're phosphorylated by kinases, and this is a kind of a schematic of this process from Art Weiss' lab.

So, Art Weiss described this ZAP-70 kinase, there's also this so-called Lck, or lymphocyte kinase, that's a Src family kinase, it's associated with a co-receptor, CD4, that also interacts with the MHC proteins that are involved in Helper T cell function. So, when you have recognition between the T cell receptor and the MHC-peptide complex and, again, in this 13 nanometer or so gap, you have CD4 that comes in, binding the MHC molecule, and this is a non-antigen-specific process, so the antigen specificity just comes from this interaction, and then you have the Lck that phosphorylates the cytoplasmic domains of the complex, and that recruits ZAP-70, then ZAP-70 starts hitting other substrates and this becomes an amplified phosphotyrosine cascade, leading to things like phospholipase C-gamma activation, which leads to calcium and Ras-MAP kinase activation, and basically a whole cascade controlling both immediate behavior of the cell and transcriptional effects, and proliferation — cell cycle control gives you that proliferative burst — cytokine production — diffusible molecules that allow the cells to communicate…

So this is basically the heart of the recognition process. So, this also talks to the adhesion systems, and this was discovered through experiments that actually I was involved in, so I'll describe them a little bit. So, basically, we radiolabeled T cells that were taken from peripheral blood of a human, and we had substrates that we could coat with adhesion molecules like ICAM-1, and then we would incubate these radiolabeled cells on the adhesion molecule-coated substrates, and what we found is that if you took cells right out of human peripheral blood they did not stick to ICAM-1, so the adhesion molecules were inactive, as kind of illustrated here in timelines, but if you engaged the T cell receptor with antibodies, and also this works with eventually the MHC-peptide complexes, you dramatically increase the level of adhesion, and then this is transient.

So, why is it transient? So, what we think is that you have the adhesion molecules, which we've kind of illustrated as these little closed hands at this point, because they're not functional, and then these receptors, which I just showed you the schematic of before, much more complicated, but just very simply schematized. So, the antibody that we're putting in is crosslinking the antigen receptors and triggering signals in the T cell that activate the adhesion molecules, and now the hands are opening, they're ready to grab the ICAM-1 on the substrate, and that's when you see this peak of adhesion.

And then once these T cell receptor complexes that are crosslinked get internalized and degraded, that terminates the signal and the adhesion molecules go back to being inactive. So, you have this kind of power steering for the immune system, that antigen recognition is linked to the adhesion molecule function that allows the T cells to tune their interaction with antigen presenting cells. If they see something that has a good antigen, they latch onto it. Otherwise, they could have very transient, casual interactions. So, if we look at this by time-lapse microscopy, we can basically see that the… using substrates that have two different components on them, one coated with the adhesion molecule and MHC-peptide complex, and then another, kind of a backfill, with just the adhesion molecule. If we then look at the T cells, so these are individual T cells in time-lapse imaging, the T cells on the adhesion molecule alone crawl very rapidly, because they have weak adhesion. Then, when you go across this line, now you're in an area with the appropriate MHC-peptide complex for these T cells, and the T cells, basically, that are crossing that line stop moving, accumulate along this edge, and the T cells that have basically fallen onto this part of the substrate show much less motility than the T cells out here.

So, this is basically the search strategy. Search and, then once it's found its cognate antigen presenting cell, it'll stop for a while, not forever, but just for a few hours, exchange information, initiate its proliferative burst or execute an effector function, and then eventually move on and go on to other… so this is, again, a highly motile system, so this would be a transient stopping effect, that then would be related to the antigen receptor signaling dynamics. So, this gets us to the immunological synapse. So, the coordination between the adhesion molecule and the antigen receptor. It's not just timing, as I just showed you, but also spatial, so this was kind of a breakthrough in the mid-1990s based on deconvolution microscopy, this technology that was developed by Agard and Sedat basically for looking at chromosomes, applied by Avi Kupfer, who was then at the University of Colorado, now he's at Johns Hopkins, to essentially look at the…

Used fixed conjugates between T cells and B cells that are antigen specific, and look at where the T cell receptor, and the adhesion molecules, and LFA-1 are sitting, and what you see from the side is that there's this cluster of T cell receptors in this optical section, there's a hole in the adhesion molecules, but now if you take this three-dimensional reconstruction of this conjugate and rotate it so that now you're looking at the… maybe the T cell's view of this process, you can now see this bullseye-like organization. So, this is what we refer to as a mature immunological synapse, so you have this segregation of the T cell receptor from the adhesion molecule, again suggesting another layer of organization, both of this interface as a communication medium for the T cell and, in this case, a B cell, but could also be applied to a dendritic cell, and essentially these images evoked many hypotheses about how this was working. So, one of our contributions to the study of the immunological synapse was to set up this reconstitution system where we have a supported lipid bilayer, this is a technology developed in Harden McConnnell's lab at Stanford, presenting purified ICAM-1 and MHC-peptide complexes in a laterally mobile form with a live T cell.

So, when the T cell comes in contact with the substrate, the T cell is activated by these molecules, and because these molecules are laterally mobile, the T cell is capable of reorganizing these purified proteins into the pattern of the immunological synapse described by Kupfer in the cell-cell junction model. So, basically this is a functional reconstitution of the synapse and the optics of this system allowed us to study the dynamics of the immunological synapse. So, this is one of the original movies of the initial engagement of the T cell receptors, which surprisingly was in the more periphery of the junction, and then it's centripetal movement into that central cluster. So, this illustrated for us the dynamics of the immunological synapse and the idea that the membrane cytoskeleton complex of the T cell was able to sort of cell-autonomously assemble this junction, as long as the molecules were presented by the antigen presenting cell in a laterally mobile form. So, this system also allowed us to determine that this T cell has single-molecule sensitivity for these MHC-peptide complexes, so it really started to allow us to solve many of the problems that we encountered in thinking about how the T cell would accomplish this, even without using the dendritic cells, by using these artificial systems and then taking these questions or hypotheses from this system back into the in vivo setting, with live cells.

So, we now know how we can use the immunological synapse to overcome many of these challenges, but there are still many questions about, say, how this, say, single-molecule sensitivity is achieved. One of these is basically, how do you coordinate this cytoskeletal machinery and the membrane of the T cell to accomplish this? How does the cytoskeleton of the antigen presenting cell modify this? Essentially, how do these different components, the different central clusters, the ring of adhesion molecules, smaller elements that are involved, how do they actually function? And what goes wrong when the system fails, like when you have pathogen or tumor escape, or autoimmunity? What's going wrong and can we fix it? So, these are all very important questions that we're trying to deal with, using both these artificial platforms, in vivo imaging approaches, and, you know, trying to develop new ways to study this process in vitro and in vivo.

So, I just want to acknowledge my colleagues who contributed to this work, starting at Washington University, Harvard Medical School, New York University, and now Oxford. And I think… obviously I've reviewed a lot of work from many other colleagues in the field, and basically there are citations in the talk that basically point those out, and lots of additional other reading that could be pursued. And I hope you'll rejoin me for Part 2 and Part 3 of this series.

So, thank you. Bye-bye..



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