Session 4: Molecular View of Adaptive Immunity
Transcript of Part 1: The Immunological Synapse: Antigen Recognition
00:07.2 Hello. 00:09.3 I'm Michael Dustin from the University of Oxford 00:11.2 and New York University School of Medicine. 00:14.1 Today, I'm going to talk to you about 00:16.1 the immunological synapse, 00:17.2 Part 1 - antigen recognition. 00:21.2 So, I'll follow this outline. 00:24.1 I'll start with a general discussion 00:26.3 of immune systems 00:29.0 and their basic purpose; 00:31.3 and the idea that there's innate immunity, 00:36.1 which recognizes 00:38.3 evolutionarily conserved patterns 00:40.2 and sets up barriers to protect the host, 00:42.3 and adaptive immunity, 00:44.1 which can recognize any type of threat 00:47.1 without any kind of prior experience of it, 00:50.0 and how these systems work together; 00:53.0 the physical challenges 00:54.3 of antigen recognition for T-lymphocytes, 00:57.1 which I'll introduce in a moment; 00:58.3 and adhesion molecules 01:01.2 that meet some of these challenges; 01:03.2 and how these are coordinated 01:05.2 in an immunological synapse. 01:09.2 So, immunity is critical 01:11.3 for essentially all forms of life. 01:14.2 Once you start concentrating a lot of energy 01:17.0 in a small package, 01:18.2 there are always going to be other organisms, 01:20.1 typically smaller, microbes, 01:22.2 that will basically try to 01:25.1 invade or attach to surfaces 01:28.2 and steal that energy, effectively. 01:32.1 So, one needs to develop 01:36.1 mechanisms to defend yourself. 01:39.1 So, for example, 01:41.2 even in organisms as simple as bacteria, 01:43.2 they're developed this 01:45.2 CRIPSR/CAS9 system 01:47.2 to protect themselves against bacteriophage. 01:50.2 In a classical example 01:53.2 from innate immunity in invertebrates, 01:56.3 in Drosophila 01:58.2 they're essentially... 02:00.1 the pattern recognition mechanisms 02:02.0 were first discovered in the context 02:04.1 of antifungal and antibacterial defense in Drosophila. 02:08.1 You also have a bacterial infection 02:11.1 in vertebrates 02:14.1 and things like parasites, like malaria, 02:16.2 that afflict millions worldwide. 02:19.1 So, these are very significant threats. 02:23.0 Innate immunity deals with the evolutionarily conserved components 02:25.3 and adaptive immunity 02:27.3 is something that was added in the vertebrates 02:29.2 to basically defend against, 02:31.2 essentially, more advanced 02:33.2 and highly evolved pathogens 02:35.0 that could evade innate immunity. 02:37.3 So, why is it important to study immunity? 02:41.3 Well, I guess from a human perspective, 02:44.2 things like vaccination, 02:46.3 which was the first effort of ourselves 02:52.1 to manipulate our immune responses, 02:54.1 you know, is essentially one of the greatest advances 02:57.0 in protecting human health, 02:58.2 where entire pathogenic species 03:02.1 have been essentially eradicated 03:05.1 by this kind of effective process 03:07.3 where you can expose an individual 03:09.3 to some form of a pathogen, 03:12.0 even related or attenuated, 03:14.2 or even components from these pathogens, 03:17.2 and generate life-long protection. 03:20.0 So, we'll talk a little about how that works. 03:22.1 Anti-cytokines therapies 03:25.0 for rheumatoid arthritis 03:26.2 are a type of immunotherapy 03:28.2 that have greatly improved the lives of many people 03:30.2 afflicted with these devastating diseases, 03:34.1 autoimmune diseases in the context of rheumatoid arthritis, 03:36.2 also other types of inflammatory diseases 03:38.1 are addressed by a variety 03:41.1 of these so-called biologic therapies. 03:43.1 It's had a huge impact on human health. 03:46.1 And the revolution in cancer immunotherapy, 03:48.1 recently, 03:49.3 based on checkpoint blockade 03:51.2 and adoptive immunotherapy, 03:53.1 and we'll touch more on that in Part 2, 03:56.2 have provided new hope for people with previously 03:59.2 incurable or very, you know, rarely curable diseases. 04:02.2 So, these are important contributions 04:04.1 that studying the immune system 04:06.3 has made to human health. 04:09.1 So, in terms of introducing innate and adaptive immunity, 04:13.0 we can think of these two components 04:15.2 as kind of being somewhat of a pyramid, 04:17.2 where the base is innate immunity. 04:21.2 And, essentially, innate immunity 04:23.3 is based on setting up barriers, 04:25.2 which can be physical, chemical, or mechanical 04:29.0 to pathogen attachment or invasion. 04:33.1 When these are breached, 04:34.2 there are a variety of induced 04:36.2 so-called pattern recognition responses, 04:38.1 like that picture of the Drosophila before, 04:39.3 that was where this was first genetically defined. 04:44.1 So, basically, 04:45.3 innate immunity is effective 04:48.0 against many organisms that would attempt 04:50.0 to attack a larger animal. 04:53.1 Innate immunity, I guess, is prevalent in bacteria, 04:57.1 single cell organisms, 04:59.1 plants, invertebrates, 05:00.2 and vertebrates like us, of course. 05:02.3 So, if innate immunity is breached, 05:05.0 basically, you have adaptive immunity. 05:07.0 So, adaptive immunity 05:09.0 is a system that's built on a set of receptors 05:13.2 which are generated in the individual 05:15.2 by somatic recombination... 05:17.2 you could spend a whole talk 05:20.0 just on these mechanisms, 05:21.2 so I'm not going to say much more about this, 05:23.1 but suffice it to say that 05:25.1 they give you the ability to essentially recognize 05:27.1 any molecular pattern 05:29.1 that you would encounter, 05:30.2 that you would be likely to encounter, 05:32.1 and certainly across a population 05:34.1 we really seem to have that capacity, 05:36.0 although individuals may have holes, 05:38.0 the whole population 05:40.1 will basically cover a vast array 05:42.2 of different types of 05:46.1 potential molecular patterns 05:48.0 that could be associated with pathogens, 05:49.2 but are also associated with our own proteins, 05:51.2 our own macromolecules, 05:53.3 and lots of harmless environmental macromolecules. 05:56.0 So, the rub with adaptive immunity 05:58.1 and this pan-recognition 05:59.3 is that it doesn't really know right from wrong, 06:02.3 it doesn't know good from bad, 06:04.2 and that's the job of innate immunity. 06:06.1 So, these two systems need to... 06:08.2 and then, basically, 06:10.2 adaptive immunity evolved in vertebrates, 06:12.0 it's important to say... 06:13.2 and these two systems, 06:15.1 innate immunity and adaptive immunity, 06:16.3 communicate with each other 06:18.2 through a process referred to, 06:20.1 generally, as inflammation. 06:21.3 So, this communication is critical, 06:24.2 and this is one of the key things 06:26.1 that's happening in this immunological synapse 06:27.3 that I'm introducing here, 06:30.1 so this is why I'm going through this, 06:31.3 because this communication axis 06:33.3 is critically transmitted through this, 06:36.1 basically, cell-cell interface 06:39.3 that we'll be describing. 06:41.1 So, just to say a little bit about inflammation, 06:43.0 so... this phenomena in, kind of, 06:47.3 human health and, kind of, philosophy 06:50.3 was recognized in the time of the ancient Greeks 06:53.2 as having a number of attributes. 06:56.1 Essentially, the meaning of the word 06:58.0 is to set on fire, 06:59.2 and the hallmarks are pain, redness, swelling, and heat, 07:04.3 and this image, this movie that's playing in the background here, 07:07.1 is essentially a picture of white blood cells, 07:12.1 which are part of... 07:14.0 a type of white blood cell that's part of the innate immune system, 07:16.2 lining up along a blood vessel, 07:19.2 which is the structure, here, 07:21.1 kind of highlighted in red because the plasma has a red fluorescent 07:25.1 quantum dot, effectively, in it. 07:27.1 So, we're imaging this in a live, anesthetized animal 07:30.2 during an inflammatory reaction, 07:32.3 and the release of these green fluorescent 07:35.3 white blood cells from the vessel, 07:38.2 and this leakage 07:40.1 #NAME? 07:42.3 from the vessel -- 07:44.2 is basically what's driving these responses in large part. 07:49.0 That's basically the classical signature of inflammation. 07:51.3 So now, there's also, however... 07:55.0 this is an infection driven inflammation... 07:56.1 there's also something called sterile inflammation 07:58.1 and there are a lot of nuances 08:00.2 to the way the innate immune system would communicate 08:02.1 to the adaptive immune system 08:04.2 in the context of, you know, 08:06.1 infection-driven versus sterile inflammation. 08:08.3 So, if you look at an example of sterile inflammation, here, 08:11.1 you have, basically, within the central nervous system... 08:14.1 these are the phagocytes in the central nervous system 08:18.0 called microglial cells, a certain type of cell 08:20.1 that is part of the innate immune system. 08:22.1 When there's this laser lesion that was created in the center of the image, 08:25.3 and this will loop again, 08:27.1 you see these cells... 08:28.2 the neighboring cells respond to the death of their friend 08:31.3 by walling off that site 08:34.2 and essentially protecting the central nervous system 08:38.1 from further damage from that insult, 08:40.2 but there's no infection in this case, 08:43.1 and there's no breach of any barrier, 08:45.0 it's basically like, for example, 08:47.0 like in a stroke, 08:48.3 you see responses just like this, say, a blood clot. 08:50.3 There's no infection. 08:52.1 There is a repair process 08:54.1 that the immune system may participate in, 08:55.3 but it's very different than infection, 08:57.2 and the innate immune system 08:59.2 will communicate to the adaptive immune system 09:01.1 the nuance that there's injury 09:03.1 that basically is not an infection, 09:05.1 and then, in many cases, 09:07.2 drive the appropriate response. 09:08.3 Rarely, there are mistakes made, 09:10.1 and you may end up with an autoimmune disease 09:11.3 from a phenomenon like this, 09:13.2 and this is something that we need to understand better. 09:16.1 So, you can break down this kind of platform of innate immunity, 09:20.2 you can break down further in to components 09:22.3 -- barriers; 09:24.2 various cellular constituents like phagocytes, 09:27.0 that's I've mentioned, 09:28.1 in the context of those microglial cells in the brain; 09:30.0 chemical defenses; 09:31.2 various types of lymphoid cells; 09:33.1 all your tissue cells can be recruited into this 09:36.1 at some level during responses -- 09:38.2 and these cells would form a foundation 09:42.0 for these various types of lymphocytes 09:44.2 which engage in... 09:46.2 which are the components of adaptive immunity, 09:48.1 the cellular components. 09:49.3 So, B cells -- and the B, basically, 09:51.3 in this context stands for bursa, 09:53.1 which is the organ in birds in which they were first discovered -- 09:56.2 or T cells, 09:58.1 two different major types of T cells, which are 10:01.2 -- T is for thymus, in this case, 10:03.1 which is the organ that they develop in in both birds, 10:05.1 where this was maybe initially studied developmentally, 10:07.2 and in humans. 10:10.1 So, basically, if you also... 10:12.0 and then a way to remember B for B cell 10:13.3 has also been in vertebrates... 10:15.2 in other... well, in mammals, 10:17.1 they develop in the bone marrow. 10:19.0 Birds don't have bone marrow, 10:20.3 so they have to have a different organ, 10:22.1 but basically other types of vertebrates 10:23.3 use the bone marrow for this. 10:25.2 So, B and bone marrow also works. 10:28.1 So, these cells now have 10:31.1 to talk to these cells 10:32.3 and in order to to do 10:34.3 it seems that we had to evolve a different cell type, 10:36.2 and this is the dendritic cell, 10:38.1 that basically sits in this 10:40.1 kind of intermediate position, here. 10:42.1 It's kind of a bridge between the two systems. 10:44.0 And particularly the T cells 10:46.2 have a critical communication 10:48.1 with this dendritic cell. 10:50.2 Finally, there are a couple things 10:52.1 I want to mention about this. 10:53.2 So, there are several types of Helper T cells 10:55.1 that can essentially develop 10:58.2 in response to signals from the dendritic cell 11:01.1 that deal with different types of pathogens, 11:03.0 so, say, viruses, 11:04.3 extracellular bacteria, 11:06.3 fungi, 11:08.1 parasites, 11:09.3 all have different modes of Helper T cells 11:12.2 to deal with those, 11:13.2 and that's a very important thing. 11:14.2 If you make a mistake about that 11:16.1 you can end up with the wrong response for the pathogen 11:17.3 and that can lead to pathogen escape 11:20.1 and disease in some situations. 11:22.2 And the other thing that I want to point out 11:27.1 is that there's another, kind of, 11:29.1 a variation on a Helper T cell c 11:30.3 alled a regulatory T cell, or Treg. 11:32.2 These cells are very critically matched, 11:34.2 in some respects, to dendritic cells, 11:36.3 and they control the activity of the dendritic cells 11:40.1 in an antigen-specific way... 11:42.1 I'll get to the antigen in a moment, 11:43.3 but they essentially... 11:45.3 it's a type of cell, a similar type of adaptive receptor, 11:48.2 this pan-recognition process. 11:50.1 They tend to be actually self-reactive 11:52.0 and they suppress responses 11:53.2 in the context of self-recognition, 11:56.1 so they actually are critical in protecting us 11:58.1 from autoimmune disease. 12:00.1 If you lose these cells, say, 12:01.2 due to a primary genetic immunodeficiency, 12:03.2 you don't have a lack of immunity, 12:05.1 you have an excess of immunity, 12:07.0 and that's actually almost worse, 12:09.0 that can kill you faster 12:11.0 than the lack of immunity in some contexts, 12:12.2 and this is because it's your own immune system 12:14.1 attacking your body, 12:15.3 which, again, has devastating consequences. 12:17.1 Now, the other thing I wanted to point out 12:19.1 was Killer T cells 12:21.2 recognize components on host cells 12:23.2 that we'll talk about in a moment. 12:26.3 If these are subverted 12:29.0 by, say, viral or bacterial immune evasion mechanisms, 12:31.2 then you might think you would be vulnerable 12:34.2 to attack by those pathogens, 12:36.1 but in fact there's this Natural Killer cell type 12:38.2 that steps in and recognizes 12:41.0 the loss of those molecules 12:42.1 that are involved in that communication and kills those cells. 12:45.0 So, tumor cells or virally infected cells 12:47.0 that might lose molecules required for the communication 12:48.3 with the T cells are basically attacked by Natural Killer cells, 12:51.1 so you have this missing self-recognition 12:52.3 which is also critical in protecting yourselves. 12:54.3 So, that gives you kind of an overview 12:56.3 of the cells of adaptive and innate immunity. 13:00.2 So, a critical thing, 13:03.2 I've used the term antigen a couple times 13:05.2 and I think I need to define that at this point. 13:07.2 So, antigen... 13:09.1 the term comes from antibody generation, 13:11.2 but it also applies to T cells, 13:13.1 which don't use antibodies. 13:14.2 So, B cells, again, 13:15.3 make antibodies, 13:17.1 which, again, start out as a receptor 13:19.0 on the surface of the B cell 13:20.1 and are then eventually secreted 13:22.1 from a later developed form of B cell 13:25.0 called a plasma cell. 13:26.1 So, these antibodies 13:28.0 recognize intact forms of the antigens. 13:31.1 So essentially, this is... 13:33.1 the image here is a viral coat protein 13:35.1 called influenza hemagglutinin 13:37.1 with three antibody fragments, here in purple, 13:41.1 these three fragments here, 13:43.0 basically in kind of a... 13:45.0 it's a trimeric structure, the hemagglutinin, 13:47.3 so there are three copies of the antibody binding site 13:50.0 in the intact protein, 13:51.2 and that's the process you're seeing. 13:52.3 This antibody binding 13:54.2 would neutralize the function of that viral protein 13:57.1 and prevent further cycles of infection, 13:59.1 so this is a critical way the host defends itself 14:01.2 against viruses 14:03.1 and a critical... making these kinds of antibodies 14:04.2 is a critical target of vaccination, 14:06.1 so what you want to do when you're designing a vaccine 14:08.0 is make these neutralizing antibodies, 14:09.2 and for a highly mutable virus like influenza, 14:13.1 you want to make antibodies 14:14.3 that are broadly neutralizing. 14:16.1 That would be the holy grail at this point, 14:17.2 so, this would allow us to say... 14:20.1 now, we have these seasonal flu vaccines 14:22.3 because the antibodies are very specific, 14:24.1 are very strain specific. 14:25.1 If you could make vaccines 14:27.0 that generated these broadly neutralizing antibodies, 14:28.2 you could have broader coverage 14:30.1 and less need to vaccinate every year. 14:33.2 T cells, on the other hand... 14:35.1 so, the B cells see the intact proteins... 14:38.1 the T cells cannot see the intact proteins at all, 14:42.1 so they don't have any ability 14:44.2 to recognize a structure like this on a virus 14:47.0 or on any other type of pathogen. 14:48.3 What happens is 14:50.2 the dendritic cell that I mentioned before 14:53.0 will internalize the antigen, 14:55.0 often in viral particles or whole bacteria 14:58.2 -- they're a type of phagocyte, 15:00.1 they can take in large structures 15:02.0 that are almost as big as themselves in some contexts -- 15:04.1 they break them down, 15:07.3 digest those complex macromolecules into peptides, 15:09.3 and then bind these to histocompatibility proteins. 15:12.0 So, what you're seeing here in this structure 15:14.0 is the surface, the upper surface, 15:15.2 pretty much what the T cell would see, 15:17.1 with this... 15:18.3 the peptide binding groove, 15:20.2 it's almost like a hot dog bun in some ways, 15:24.0 holding this linear peptide, 15:26.1 which is derived from proteins 15:29.0 that are taken up by the dendritic cell. 15:31.0 These proteins can be from pathogens, 15:33.1 they can be from yourself, 15:34.3 they can be from harmless things 15:36.2 that you're breathing in or out, you know, 15:39.0 allergens, things that aren't really going to hurt you 15:41.0 but you might respond to. 15:42.1 So, all of these different 15:44.3 degradation products of these proteins 15:46.2 are binding to these MHC molecules. 15:48.3 So, this term MHC is 15:51.1 Major Histocompatibility Complex. 15:53.0 That terms comes from the fact that 15:55.1 these molecules also control transplantation. 15:57.2 So, if you look at skin transplantation 15:59.1 or organ transplantation, 16:00.2 there are differences between us, 16:02.2 in a population, 16:04.1 that reflect different types of these 16:06.1 peptide binding proteins. 16:08.0 It's important the population have that diversity 16:10.2 because you could imagine with this peptide binding process, 16:13.0 there's some specificity here. 16:14.2 Some individuals may not be able to bind 16:17.2 peptides from some pathogens, 16:19.1 then they'd have a hole in their repertoire. 16:20.2 So, this... individuals, then, 16:23.3 may be susceptible to that particular pathogen, 16:25.2 but in the population, 16:27.0 because there's more diversity in the population of these molecules, 16:29.2 it makes you able to defend yourself against a wide array of pathogens. 16:32.1 However, it also prevents transplantation, 16:34.1 or at least makes transplantation challenging 16:37.0 and requires immunosuppression, 16:39.0 sometimes for the life of the individual. 16:41.2 Of course, inducing transplant tolerance, 16:43.1 then, is sometimes experimentally 16:45.0 or, you know, therapeutically, 16:46.2 that we'd like to be able to achieve. 16:49.2 Okay, so now what I want to describe 16:52.1 is how immune cells come in contact 16:55.0 with antigen in the body. 16:58.1 So, if you imagine an infection in the skin, 16:59.3 you have a break in the skin, 17:01.2 some microbes have entered and started to replicate, 17:04.2 innate immunity has tried to deal with this, but failed, 17:07.2 the organism is increasing in numbers, 17:09.1 so now you have an increasing amount of 17:11.2 particulate material or small molecules, 17:13.2 proteins and things, 17:14.3 being released by the growing pathogens, 17:17.2 and these are draining, now, 17:19.3 through lymphatics to structures 17:22.1 referred to as lymph nodes, 17:23.2 which are basically filters 17:25.1 which are packed with T and B lymphocytes, 17:28.0 and also sites where dendritic cells congregate 17:30.1 to show antigens to T cells, 17:32.1 and B cells basically become exposed 17:35.1 to materials that are draining to the lymph node 17:37.3 from these tissue sites. 17:39.2 So, this set of movies 17:41.2 from Facundo Batista's lab 17:43.0 basically show how the B cells, 17:45.2 which are these antigen-specific B cells, 17:47.1 which are these red cells, 17:49.2 so they have a particular antibody on their surface 17:51.1 that recognizes the antigen 17:53.2 that they are using in these experiments, 17:55.0 which is green. 17:56.2 So, what you see here is the filter capturing the... 18:00.2 filter at the outside of the lymph nodes, 18:02.1 which is cellular actually, it's phagocytes, 18:04.2 capturing the antigen, 18:05.2 and then the B cells, 18:07.0 it's kind of looping between these three views 18:08.2 -- the large view and then two detailed views, 18:10.1 one at the filter boundary 18:12.0 and then one at the place where the T cells are -- 18:13.3 and what you can see basically is the B cells, 18:16.1 at this edge where these phagocytic cells 18:20.1 are capturing the antigen, 18:22.1 displaying it in a way that the B cells 18:23.3 can test if their antigen receptor 18:25.2 has the right specificity to capture and concentrate that antigen, 18:29.0 then they will process that, 18:30.2 make the MHC-peptide complex 18:32.1 as I described before, 18:33.2 and then they very quickly 18:35.1 go to this zone where the T cells are... 18:37.2 so basically there's a boundary 18:39.0 between the place where most of the T cells stay 18:40.3 and most of the B cells stay, 18:42.0 they're usually segregated, kept apart, 18:43.3 but under the conditions where antigen comes into the system 18:46.2 they come together at that junction 18:48.1 and have a chance to test... 18:50.1 the T cells test their antigen receptor, 18:52.1 to determine if it recognizes any of the MHC-peptide complexes 18:54.2 being presented by those B cells 18:56.2 and if they get a match, 18:57.3 that is a situation where you start to get 18:59.2 help for the B cell 19:01.1 to make high-affinity antibodies against that pathogen, 19:05.2 starting with the receptor that they used to capture the antigen 19:08.0 and then trying to improve it 19:09.2 by mutating it and reselecting it, again, 19:12.2 with continual advice from the T cells. 19:14.0 The T cells, in that situation, 19:15.1 have already received instructions from the dendritic cells, 19:17.1 which are also looking at the same pathogen 19:19.0 and helping the T cell identify 19:21.2 what kind of response is needed. 19:23.0 So, this is a highly coordinated process and I just wanted to point out the... 19:26.0 use this movie to point out the dynamics of this process. 19:29.2 So, this is another... 19:31.2 a static electron micrograph of a T cell 19:33.2 and a dendritic cell. 19:35.1 Now, you know this is not the way things actually happen in vivo, 19:37.1 the system would be much more dynamic 19:39.1 than the still image conveys, 19:41.1 but I just want to basically use this image 19:43.2 to say a little bit about this interface, 19:45.1 the immunological synapse 19:47.1 between the T cell and the dendritic cell. 19:49.1 So, this is... 19:51.2 again, T cells only see these MHC-associated peptide fragments, 19:56.0 which are on the surface of the dendritic cell or the B cell, 19:58.2 as I just mentioned. 19:59.2 The dendritic cell and the B cell take them up differently, 20:01.2 but they're basically... 20:03.2 eventually the T cell would recognize the same structure 20:05.2 on either cell type. 20:07.2 The T cell receptor is also only on the surface, 20:11.1 there's no soluble T cell receptor, 20:13.1 so basically the T cell and the antigen presenting cell, 20:16.2 whether it's a dendritic cell or a B cell, 20:19.0 are always going to be dealing with this... 20:21.2 the dimensions of these molecules, 20:23.1 which will only span about 13 nanometers (nm) 20:25.2 between the two cells, 20:27.0 and this is a structure, 20:29.1 an X-ray crystallography-based structure, 20:30.2 of a T cell receptor, 20:32.0 kind of the specific part of the T cell receptor, 20:34.1 then the MHC-peptide complex, 20:36.1 there's the peptide, 20:37.2 this is the histocompatibility antigen... 20:40.1 you know, so essentially 20:42.2 this is only 13 nm long 20:44.1 and these cells are about 10 microns or so across, 20:47.1 which is 10,000 nm. 20:49.0 So, basically the gap between these cells 20:51.1 is very small compared to the cells 20:52.3 and the cells have to get very close to each other 20:55.1 to achieve this recognition. 20:57.1 I guess the other aspect that I've already touched on 21:00.0 is that the dendritic cells are these very dynamic cells in the tissues, 21:03.2 they're part of... 21:05.2 this motility is involved in 21:07.2 essentially allowing them to drink up 21:09.2 large amounts of fluid 21:11.3 and engulf particles, 21:13.1 which are basically... 21:14.2 could be either derived from the host, 21:16.0 other host cells, 21:17.1 or from a pathogen... 21:18.3 takes them into a lysosomal compartment, 21:22.0 degrades them partially 21:23.2 #NAME? 21:26.1 Those peptides come into contact 21:27.3 with the MHC molecules 21:30.0 that have been recently synthesized, 21:33.0 those molecules become receptive to the peptide, 21:35.1 bind the peptide, 21:36.1 and then go to the surface 21:37.3 as the dendritic cells move to a lymph node. 21:41.1 Once these dendritic cells get to the lymph node, 21:44.3 they basically distribute in the T cell zones 21:49.1 and essentially take up a position in a network, 21:52.1 and then continue to undergo a very high level of surface dynamics. 21:56.2 They have a very large surface area, 21:58.1 so they'll come in contact 21:59.2 with about 1,000 T cells per hour, 22:01.3 and in this movie there's some antigen-specific T cells, and control T cells that are light or dark blue. 22:07.1 This is an image in an experimental setting 22:09.1 where we kind of knew the specificity of these cells 22:11.3 and we were looking at their interactions with the dendritic cells, 22:13.2 but if you looked at all the T cells in this tissue 22:16.3 the image would just be packed with T cells. 22:19.1 So they're, you know, really, 22:20.3 this image would contain tens of thousands of T cells, 22:23.0 and those T cells would be moving around, 22:25.2 coming in contact with these dendritic cells, 22:27.2 again, looking for a fit between the antigen receptor 22:28.3 and the MHC-peptide complexes. 22:31.3 So, the initial encounter 22:33.2 for any kind of antigen with a T cell 22:35.1 would be on th dendritic cells, 22:36.2 they are the best cell for initiating T cell responses. 22:39.2 The activated T cells, 22:41.0 which are relatively... 22:42.2 the antigen-specific T cells, which are relatively rare, 22:45.0 become activated and undergo a proliferative burst, 22:47.0 which greatly increases their numbers, 22:48.3 so a T cell can go from being, 22:51.1 you know, 1 in 100,000 22:53.0 to being about 5 or 10% of your total number of T cells 22:56.1 in about 5 days during an antiviral response, 22:59.2 so this proliferative burst can be quite dramatic. 23:02.2 And then these effector T cells 23:05.2 will then exit the lymph node 23:07.0 or move to the B cell follicles, 23:08.3 and once they exit the lymph node 23:11.1 they'll go to sites of inflammation via the blood, 23:14.1 and once they're entered those sites of inflammation 23:16.1 they'll be prepared to kill virally infected cells, 23:18.2 for the Killer T cells, 23:20.1 or help other cells in the system 23:21.3 basically coordinate their response 23:23.3 to the pathogen, those are the Helper T cells, 23:26.0 and, again, because the dendritic cells 23:28.1 have instructed those two cells to take on certain attributes, 23:31.2 they should be well-equipped to deal with the type of pathogen 23:34.2 that they encounter once they get to the site of inflammation in the body. 23:37.2 So, this is, again, a very well-coordinated system, 23:40.2 but the recognition process underlying this 23:43.1 then faces a lot of challenges related to working within this... 23:46.1 working with these constraints in the system. 23:49.3 So, again, just to summarize these challenges, 23:52.2 the T cell receptor (TCR) and the MHC-peptide complex (pMHC) are small; 23:55.3 the MHC-peptide complexes are rare 23:57.2 because they're competing with all of these self proteins 23:59.3 and other types of proteins 24:01.1 that are essentially present in the tissues 24:03.2 that are in addition to the proteins from the pathogens; 24:06.3 the affinity of this interaction is low, 24:08.2 I haven't really touched on that very much 24:10.2 but this is... compared to antibodies, 24:12.1 the affinity of the T cell receptor interaction 24:14.1 with an MHC-peptide complex 24:15.3 is about three orders of magnitude 24:18.1 lower than what you typically see 24:21.1 for antibodies binding to their intact antigens; 24:23.2 and the T cell and the dendritic cell are moving, 24:25.2 so you have this, you know, 24:27.0 kind of search going on, 24:28.1 so the cells really have relatively little time 24:30.1 to decide whether they have a fit or not, 24:32.0 they have to do that in a few minutes, basically, 24:34.1 in a response that may go for u 24:37.0 p to a couple of weeks overall. 24:39.2 So, how do you deal with these challenges? 24:41.3 So it turned out in maybe around the mid-1980s 24:44.3 that we didn't know very much about this. 24:46.3 We knew that there was this antigen recognition process, 24:50.0 we were beginning to understand the T cell receptor 24:52.1 in the late 80s, 24:53.2 this picture of the MHC-peptide complex 24:55.1 became more clear, 24:56.2 and at the same time 24:58.2 investigators started to explore 25:01.2 this issue of how this recognition process works. 25:06.1 And basically one of the key things that this transmission electron micrograph shows 25:11.2 is this very close interface between a target cell 25:14.2 and a cytotoxic T cell. 25:16.2 So, the cytotoxic T cell will kill the target cell, 25:19.2 in this case based on allorecognition, 25:20.3 which is the mode of recognition you have in transplantation, 25:23.2 so basically seeing foreign MHC proteins. 25:25.2 This is a very strong type of recognition, 25:29.1 but it's clear that the antigen recognition process itself 25:32.3 can't account for this very tight interface, 25:34.2 this very extensive interface. 25:36.1 It would seem like you'd need something else to do this, 25:38.1 so investigators started immunizing mice 25:41.3 with the T cells 25:43.2 and then trying to screen for monoclonal antibodies, 25:46.2 so basically individuals antibodies 25:48.2 -- so, using the immune system to study the immune system -- 25:50.2 that would essentially block this recognition process, 25:53.2 and they found a number of antigens, 25:56.1 essentially in this case, 25:57.2 functional molecules of the T cell, 26:00.1 that were involved in this process. 26:01.2 So, here we have a little schematic 26:03.2 that introduces a few of these. 26:05.2 So, the antigen receptor and the MHC complex 26:07.2 provide the specificity, 26:09.2 but a set of non-polymorphic molecules 26:11.2 were defined in these studies 26:13.1 for which antibodies binding to those proteins 26:17.0 would inhibit the functional process, 26:19.2 and these included LFA-1, 26:21.2 or lymphocyte function-associated 1, 26:23.2 which is a member of the integrin family; 26:25.2 ICAM-1, which is actually a member of the immunoglobulin superfamily, 26:28.3 so it's related to antibodies; 26:30.2 and CD2 and LFA-3, 26:32.3 also sometimes referred to as as CD58, 26:34.1 which are also members of the immunoglobulin superfamily, 26:37.1 which interact across these gaps. 26:39.1 So, these molecules 26:41.2 are all present in around 26:43.1 something on the order of 50,000-100,000 copies per cell, 26:45.2 but all of these molecules are capable of interaction, 26:48.1 whereas maybe 26:51.2 only a very small fraction of the MHC molecules 26:53.1 have the appropriate peptide. 26:54.3 So, these molecules effectively 26:56.2 give the T cell the ability 26:58.1 to make these short, these tight interfaces, 27:00.0 but this then posed somewhat of a problem, 27:01.3 which is that if the T cell 27:03.1 is going to survey all these different cells and has this ability to stick to then, 27:07.2 how is that regulated? 27:08.2 And it turned out that you needed another layer of understanding 27:11.2 in this to kind of start 27:15.2 to understand the whole process, 27:17.1 and actually this really comes into, 27:18.2 what is the immunological synapse? 27:20.1 How does it work? 27:21.2 So, the T cell receptor itself 27:23.2 is a signaling molecule. 27:25.1 So, this is basically a schematic of the T cell receptor 27:27.1 -- these parts here are involved in antigen recognition, 27:30.2 these parts here are involved in signal transduction, 27:34.2 they're non-covalently associated with each other, 27:36.2 so it's quite a complicated feat 27:38.2 to basically build this complex, 27:40.2 that was studied quite a bit -- 27:43.1 but the key to the signaling process 27:45.1 is that these cytoplasmic motifs 27:47.2 contain tyrosine residues 27:49.0 and they're phosphorylated by kinases, 27:51.1 and this is a kind of a schematic of this process 27:53.2 from Art Weiss' lab. 27:55.0 So, Art Weiss described this ZAP-70 kinase, 27:58.1 there's also this so-called Lck, or lymphocyte kinase, 28:01.2 that's a Src family kinase, 28:03.2 it's associated with a co-receptor, CD4, 28:05.3 that also interacts with the MHC proteins 28:08.2 that are involved in Helper T cell function. 28:10.2 So, when you have recognition between the T cell receptor 28:14.1 and the MHC-peptide complex 28:15.2 and, again, in this 13 nanometer or so gap, 28:17.1 you have CD4 that comes in, 28:19.1 binding the MHC molecule, 28:22.1 and this is a non-antigen-specific process, 28:24.1 so the antigen specificity just comes from this interaction, 28:26.0 and then you have the Lck that phosphorylates 28:28.1 the cytoplasmic domains of the complex, 28:30.2 and that recruits ZAP-70, 28:33.2 then ZAP-70 starts hitting other substrates 28:35.1 and this becomes an amplified 28:38.0 phosphotyrosine cascade, 28:40.2 leading to things like 28:42.1 phospholipase C-gamma activation, 28:44.2 which leads to calcium and Ras-MAP kinase activation, 28:47.1 and basically a whole cascade 28:49.0 controlling both immediate behavior of the cell 28:51.1 and transcriptional effects, 28:53.2 and proliferation 28:55.0 -- cell cycle control gives you that proliferative burst -- 28:57.1 cytokine production -- 28:58.3 diffusible molecules that allow the cells to communicate... 29:01.1 so this is basically the heart of the recognition process. 29:04.3 So, this also talks to the adhesion systems, 29:08.1 and this was discovered through experiments 29:10.3 that actually I was involved in, 29:12.1 so I'll describe them a little bit. 29:13.3 So, basically, we radiolabeled T cells that were taken from peripheral blood of a human, 29:17.1 and we had substrates that we could coat with 29:20.1 adhesion molecules like ICAM-1, 29:22.1 and then we would incubate these radiolabeled cells 29:25.3 on the adhesion molecule-coated substrates, 29:27.2 and what we found is that 29:29.1 if you took cells right out of human peripheral blood 29:30.3 they did not stick to ICAM-1, 29:32.1 so the adhesion molecules were inactive, 29:35.1 as kind of illustrated here in timelines, 29:37.0 but if you engaged the T cell receptor with antibodies, 29:40.1 and also this works 29:42.1 with eventually the MHC-peptide complexes, 29:44.3 you dramatically increase the level of adhesion, 29:47.3 and then this is transient. 29:49.0 So, why is it transient? 29:50.1 So, what we think is that you have the adhesion molecules, 29:52.2 which we've kind of illustrated as these little closed hands at this point, 29:55.3 because they're not functional, 29:57.2 and then these receptors, 29:59.3 which I just showed you the schematic of before, 30:01.1 much more complicated, 30:02.2 but just very simply schematized. 30:05.2 So, the antibody that we're putting in 30:07.2 is crosslinking the antigen receptors 30:10.0 and triggering signals in the T cell 30:12.1 that activate the adhesion molecules, 30:14.3 and now the hands are opening, 30:16.2 they're ready to grab the ICAM-1 on the substrate, 30:18.2 and that's when you see this peak of adhesion. 30:21.1 And then once these 30:24.1 T cell receptor complexes 30:26.1 that are crosslinked get internalized and degraded, 30:28.2 that terminates the signal 30:30.1 and the adhesion molecules go back to being inactive. 30:32.2 So, you have this kind of power steering for the immune system, 30:35.2 that antigen recognition 30:37.3 is linked to the adhesion molecule function 30:39.1 that allows the T cells to tune their interaction 30:41.1 with antigen presenting cells. 30:42.2 If they see something that has a good antigen, 30:44.1 they latch onto it. 30:46.0 Otherwise, they could have very transient, casual interactions. 30:49.2 So, if we look at this by time-lapse microscopy, 30:52.0 we can basically see that the... 30:54.2 using substrates that have two different components on them, 30:57.1 one coated with the adhesion molecule 30:59.1 and MHC-peptide complex, 31:00.2 and then another, kind of a backfill, 31:02.1 with just the adhesion molecule. 31:03.2 If we then look at the T cells, 31:05.1 so these are individual T cells in time-lapse imaging, 31:08.1 the T cells on the adhesion molecule alone crawl very rapidly, 31:12.2 because they have weak adhesion. 31:14.1 Then, when you go across this line, 31:16.0 now you're in an area with the appropriate MHC-peptide complex 31:18.3 for these T cells, 31:21.0 and the T cells, basically, that are crossing that line stop moving, 31:24.2 accumulate along this edge, 31:25.3 and the T cells that have basically fallen onto this part of the substrate 31:29.1 show much less motility than the T cells out here. 31:31.0 So, this is basically the search strategy. 31:33.3 Search and, then once it's found its 31:35.2 cognate antigen presenting cell, 31:37.0 it'll stop for a while, not forever, 31:38.2 but just for a few hours, 31:40.1 exchange information, 31:41.2 initiate its proliferative burst 31:43.2 or execute an effector function, 31:45.1 and then eventually move on and go on to other... 31:48.1 so this is, again, a highly motile system, 31:49.3 so this would be a transient stopping effect, 31:52.1 that then would be related to the antigen receptor signaling dynamics. 31:56.1 So, this gets us to the immunological synapse. 31:59.2 So, the coordination 32:01.1 between the adhesion molecule 32:02.3 and the antigen receptor. 32:05.0 It's not just timing, as I just showed you, 32:07.2 but also spatial, 32:09.2 so this was kind of a breakthrough in the mid-1990s 32:12.0 based on deconvolution microscopy, 32:14.1 this technology that was developed 32:15.3 by Agard and Sedat 32:18.2 basically for looking at chromosomes, 32:20.3 applied by Avi Kupfer, 32:23.0 who was then at the University of Colorado, 32:24.1 now he's at Johns Hopkins, 32:26.0 to essentially look at the... 32:28.1 used fixed conjugates between T cells and B cells 32:31.0 that are antigen specific, 32:32.2 and look at where the T cell receptor, 32:34.2 and the adhesion molecules, 32:35.3 and LFA-1 are sitting, 32:37.1 and what you see from the side 32:39.1 is that there's this cluster of T cell receptors 32:41.1 in this optical section, 32:43.3 there's a hole in the adhesion molecules, 32:45.1 but now if you take this three-dimensional reconstruction 32:47.3 of this conjugate 32:49.2 and rotate it so that now you're looking at the... 32:52.1 maybe the T cell's view of this process, 32:54.2 you can now see this bullseye-like organization. 32:57.0 So, this is what we refer to as 32:59.2 a mature immunological synapse, 33:01.0 so you have this segregation of the T cell receptor 33:03.1 from the adhesion molecule, 33:05.0 again suggesting another layer of organization, 33:08.0 both of this interface as a communication medium for the T cell 33:14.3 and, in this case, a B cell, 33:17.0 but could also be applied to a dendritic cell, 33:18.2 and essentially these images 33:21.2 evoked many hypotheses about how this was working. 33:24.2 So, one of our contributions 33:26.2 to the study of the immunological synapse 33:28.1 was to set up this reconstitution system 33:31.2 where we have a supported lipid bilayer, 33:33.2 this is a technology developed in Harden McConnnell's lab at Stanford, 33:37.0 presenting purified ICAM-1 and MHC-peptide complexes 33:40.3 in a laterally mobile form with a live T cell. 33:44.3 So, when the T cell comes in contact with the substrate, 33:46.2 the T cell is activated by these molecules, 33:49.0 and because these molecules are laterally mobile, 33:51.1 the T cell is capable of reorganizing 33:55.1 these purified proteins 33:58.1 into the pattern of the immunological synapse 34:00.1 described by Kupfer in the cell-cell junction model. 34:03.1 So, basically this is a functional reconstitution of the synapse 34:06.1 and the optics of this system 34:08.1 allowed us to study the dynamics of the immunological synapse. 34:10.3 So, this is one of the original 34:13.2 movies of the initial engagement of the T cell receptors, 34:17.1 which surprisingly was in the more periphery of the junction, 34:20.0 and then it's centripetal movement 34:22.3 into that central cluster. 34:24.1 So, this illustrated for us the dynamics 34:26.0 of the immunological synapse 34:27.2 and the idea that the membrane cytoskeleton complex of the T cell 34:32.0 was able to sort of cell-autonomously 34:34.2 assemble this junction, 34:36.1 as long as the molecules were presented 34:38.0 by the antigen presenting cell in a laterally mobile form. 34:42.1 So, this system also allowed us 34:44.3 to determine that this T cell 34:46.3 has single-molecule sensitivity 34:48.2 for these MHC-peptide complexes, 34:50.1 so it really started to allow us to solve many of the problems 34:53.2 that we encountered in thinking about 34:55.3 how the T cell would accomplish this, 34:57.1 even without using the dendritic cells, 34:59.1 by using these artificial systems 35:01.2 and then taking these questions or hypotheses 35:03.0 from this system back into the in vivo setting, 35:05.0 with live cells. 35:06.3 So, we now know how we can 35:09.1 use the immunological synapse 35:10.2 to overcome many of these challenges, 35:12.1 but there are still many questions 35:13.3 about, say, how this, say, single-molecule sensitivity is achieved. 35:16.1 One of these is basically, 35:19.0 how do you coordinate this cytoskeletal machinery 35:22.1 and the membrane of the T cell to accomplish this? 35:25.1 How does the cytoskeleton of the antigen presenting cell 35:28.3 modify this? 35:31.0 Essentially, how do these different components, 35:33.1 the different central clusters, 35:36.1 the ring of adhesion molecules, 35:38.1 smaller elements that are involved, 35:39.2 how do they actually function? 35:42.2 And what goes wrong when the system fails, 35:45.1 like when you have pathogen or tumor escape, 35:47.1 or autoimmunity? 35:48.2 What's going wrong and can we fix it? 35:51.2 So, these are all very important questions 35:53.2 that we're trying to deal with, 35:55.1 using both these artificial platforms, 35:57.1 in vivo imaging approaches, 35:58.3 and, you know, 36:01.1 trying to develop new ways to study this process 36:04.2 in vitro and in vivo. 36:07.0 So, I just want to acknowledge 36:09.2 my colleagues who contributed to this work, 36:11.0 starting at Washington University, 36:14.2 Harvard Medical School, 36:16.2 New York University, 36:18.1 and now Oxford. 36:19.2 And I think... 36:20.3 obviously I've reviewed a lot of work from many other colleagues 36:23.2 in the field, 36:25.1 and basically there are citations 36:27.1 in the talk that basically point those out, 36:29.2 and lots of additional other reading that could be pursued. 36:32.2 And I hope you'll rejoin me for Part 2 and Part 3 of this series. 36:37.0 So, thank you. Bye-bye.