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.

00:07.3 Hello. I'm Michael Dustin,
00:09.3 from the University of Oxford
00:11.1 and New York University School of Medicine.
00:13.2 Welcome to the talk on immunological synapses,
00:16.1 Part 2 - Signaling and function.
00:20.1 So, this is a movie
00:22.1 of pre-immunological synapses forming
00:25.1 and you can see this
00:27.2 bullseye-like pattern
00:29.2 emerging from an initial less organized or even inverted pattern.
00:35.0 So, this is the process of immunological synapse formation.
00:38.1 We want to...
00:40.1 we'll define this a little bit more generally,
00:42.3 and then I will go on to talk about signaling,
00:45.1 effector function mediated by the immunological synapse,
00:48.0 signal amplification at the immunological synapse,
00:51.2 and applications to autoimmunity and cancer therapy.
00:56.2 So, in terms of definition,
00:58.2 the concept of a synaptic basis of T cell activation
01:02.1 basically went back to the 70s and early 1980s,
01:04.3 and there's a review by Mike Norcross in 1984
01:08.0 that describes a synaptic basis of T cell activation,
01:11.1 Bill Paul and Bob Seder
01:13.1 used the term in 1992 in a cell review,
01:15.2 but the first appearance in peer-reviewed publications
01:17.2 is in the late 1990s
01:19.1 with work from Avi Kupfer
01:21.3 describing supramolecular activation complexes
01:24.2 and our work describing...
01:26.2 you know, in reference to Kupfer
01:28.2 and our own work
01:30.3 referring to immunological synapses
01:32.2 and that final bullseye pattern
01:34.2 of a mature immunological synapse.
01:36.1 So, what wrote was:
01:37.2 The immunological synapse is characterized by a specific pattern of molecules
01:40.3 in the contact;
01:42.0 LFA-1 is localized to the periphery of the contact"...
01:45.2 what Kupfer described as the pSMAC,
01:47.1 or peripheral supramolecular activation complex...
01:49.1 while the T cell receptor is localized to the center of the contact...
01:53.1 or what Kupfer defined as
01:55.3 a central supramolecular activation complex.
01:59.2 This is described in these references from '98,
02:01.2 and later on,
02:04.2 in collaboration with David Colman,
02:06.0 who is a neuroscientist,
02:07.2 we basically thought about a few criteria
02:10.0 that were common to the neural and immunological synapses:
02:12.2 first, that cells remain individuals,
02:15.3 which is part of the neuronal doctrine;
02:18.0 that there bona fide adhesion molecules mediating these junctions,
02:21.2 basically cadherins in the neural synapse
02:24.1 and integrins in the immunological synapse;
02:26.1 that these are at least provisionally stable junctions
02:29.0 -- obviously, in the nervous system,
02:30.2 synapses can be stable for years,
02:32.2 in the immune system
02:34.2 the cells are extremely motile in the steady state,
02:36.3 so when they stop for a few hours
02:39.1 that's quite a stable...
02:41.2 quite a contrast in their behavior
02:43.2 and a relatively stable junction,
02:45.1 although it's much less stable than what you'd see in the nervous system
02:48.0 in many cases;
02:49.2 and you have directed secretion taking place
02:52.0 across this interface,
02:53.3 and I'll talk about that a little bit when we describe effector functions.
02:58.0 So, in terms of signaling mechanisms
03:00.2 or the actual triggering process,
03:02.1 the T cell receptor,
03:06.0 which is, again, at the heart of the immunological synapse,
03:08.1 provides the immunological specificity,
03:10.1 utilizes what's referred to as non-receptor tyrosine kinases,
03:12.0 so it utilizes a couple of...
03:14.2 several different kinases,
03:16.2 but the first two are the Src family kinase Lck
03:21.2 and the Syk family kinase ZAP70,
03:23.1 or zeta-associated protein 70,
03:24.2 as the major signal initiators
03:27.2 which are recruited to phosphorylated cytoplasmic domains
03:32.1 of this multisubunit complex.
03:36.0 This signaling process is modulated
03:38.1 by a number of other proteins,
03:39.3 and this paper published a couple of years ago
03:42.2 from James and Vale
03:45.2 reconstituted this process
03:48.1 in non-lymphocytes
03:50.1 using a combination of the T cell receptor,
03:52.1 the kinases I just described,
03:54.0 and then the modulators
03:56.1 CSK, a kinase that actually
03:59.1 inhibits Lck by phosphorylating it,
04:01.1 CD45, a phosphatase
04:04.1 that antagonizes the CSK-mediated inhibition,
04:07.3 and together this system
04:09.2 would basically set up a regulated basal state
04:11.3 from which T cell receptor engagement
04:13.2 would trigger signaling,
04:15.1 so this seemed to be a minimal
04:17.2 set of molecules required
04:20.0 for reconstituting T cell receptor signaling
04:21.3 in a non-T cell.
04:23.3 But when we go back to T cells
04:25.1 and basically look at these molecules,
04:26.3 we can see that they're very highly organized
04:29.1 in the immunological synapse,
04:30.3 so a number of the experiments that I'm going to describe
04:34.0 utilize a reconstituted system
04:36.2 in which the antigen presenting cell
04:37.2 is replaced by a supported planar bilayer,
04:40.1 kind of schematized here,
04:43.0 which... again, a technology developed in Harden McConnell's lab
04:45.1 at Stanford,
04:46.2 where an artificial bilayer
04:49.1 is deposited on a glass substrate.
04:51.1 This is a very optically ideal system.
04:53.1 We can put purified molecules
04:55.1 in a laterally mobile form in the substrate
04:57.3 and T cells can assemble an immunological synapse.
05:00.2 We can then use optical methods
05:03.1 that are only applicable to this type of interface,
05:05.2 like total internal reflection fluorescence microscopy,
05:08.1 in which you're bouncing a laser beam
05:10.2 off of the interface
05:12.0 and creating this very shallow evanescent wave
05:13.3 that excites fluorescence
05:15.2 within about 200 nanometers of that substrate,
05:17.1 so it's very synapse-specific in this case
05:19.1 and, for example,
05:20.2 we can then look at signaling processes
05:22.1 like the recruitment of ZAP-70
05:24.0 to these T cell receptor clusters,
05:25.3 which are actually the active component
05:27.2 in the signaling immunological synapse.
05:29.2 So, you have this large central cluster,
05:31.2 which is actually not active in signaling,
05:34.0 but these small peripheral clusters
05:35.2 are the places where most the
05:38.2 ZAP-70 is being recruited,
05:40.0 and most of the other evidence of this tyrosine kinase cascade
05:43.1 is localized.
05:44.3 We can also see that these microclusters,
05:46.3 based on this work from Rajat Varma,
05:48.3 which was done in my lab,
05:50.2 where the...
05:52.3 if you look at CD45,
05:55.1 the signal here,
05:56.2 in relation to the T cell receptor
05:58.1 on these planar substrates,
06:00.0 with an MHC-peptide complex
06:01.2 that triggers this T cell,
06:03.2 that you, very early on in the contact,
06:05.1 or later, once you have this mature
06:08.1 bullseye-like immunological synapse,
06:09.3 that you have CD45 exclusion from these microclusters,
06:14.2 and this is where the ZAP-70 is being recruited.
06:16.2 So, while one hypothesis
06:20.0 for triggering through the T cell receptor
06:21.2 is that the local exclusion of these phosphatases
06:24.2 like CD45
06:26.1 could be critical to this,
06:28.1 and this is an idea that was first floated by Tim Springer
06:30.1 and has been expanded on extensively
06:32.2 by Simon Davis and Anton van der Merwe, but...
06:35.1 and certainly supported experimentally,
06:36.2 at least as a kind of a...
06:40.2 in a descriptive fashion, here.
06:45.1 So, another remarkable thing about
06:48.2 both the immunological synapse
06:50.1 and this total internal fluorescence microscopy
06:52.2 as a method to study it
06:54.2 is that we can detect single MHC-peptide complexes
06:57.0 and we can actually basically start to the study
06:59.2 the sensitivity of the T cell
07:01.2 and directly demonstrate this single molecule recognition process.
07:04.2 So, these are single MHC-peptide complexes
07:06.2 that are being captured,
07:08.0 they're diffusing around in this planar bilayer substrate,
07:10.1 and the ones that are immobile here
07:12.1 in this red area,
07:13.2 which is actually the T cell receptor,
07:15.2 are effectively being captured by the T cell receptor.
07:17.3 The T cell has about 50,000 receptors on its surface,
07:21.2 maybe over 10,000 of those are in this interface,
07:24.2 and those are there waiting
07:26.3 in a proper environment defined by the adhesion molecules
07:30.2 to very efficiently capture these molecules.
07:32.1 So, to point out that the LFA-1/ICAM-1 adhesion system
07:34.3 is in place in this system,
07:36.2 it's just not labeled in this context.
07:37.1 So, you'd only achieve this very high sensitivity
07:39.3 with the adhesion system in place
07:42.0 and actually, if we looked at it,
07:44.1 it would be forming this ring-like pSMAC.
07:47.1 There's not a large cSMAC in this case
07:49.2 because the cSMAC size is linearly related
07:51.3 to the MHC-peptide density
07:53.1 and at very low MHC-peptide densities you don't see much.
07:55.2 We'll talk more about that in talk 3.
07:58.1 So, with this single molecule,
08:00.2 you know, recognition process,
08:02.0 we looked for CD45 exclusion
08:04.2 to see if this CD45 movement from those sites
08:07.2 was seen even when the T cell receptor
08:09.1 was just seeing a single MHC-peptide complex,
08:11.0 and what you can see from this plot is that we did the...
08:14.2 well, we did these measurements
08:16.2 of where the single MHC-peptide complex was engaged,
08:19.0 and then at the point of engagement,
08:21.2 and then around that point,
08:23.0 and then basically did a local comparison,
08:25.1 set that to 1,
08:26.3 and then basically looked at basically
08:28.2 what was there...
08:30.1 err, the surrounding area was set to 1,
08:31.2 and then we'd look at what was happening in that central region.
08:33.1 And what you can see is that there's a lot of noise in these measurements,
08:35.1 but we can still see exclusion of CD45,
08:38.0 on the order of 10%
08:40.3 from these sites of single MHC-peptide engagement.
08:43.1 So, again, from a...
08:45.1 just kind of a phenomenological standpoint,
08:47.0 this CD45 exclusion
08:48.3 is happening at points where the T cell receptor is engaged,
08:51.2 even by single molecules.
08:53.0 We know, again, that signaling is being initiated, based on ZAP-70 recruitment.
08:57.2 So, one model for T cell receptor triggering
09:00.1 that accounts for the single-molecule sensitivity
09:03.0 is this CD45/phosphatase exclusion model,
09:05.3 again van der Merwe and Davis would be sources
09:09.0 to look for more information on that, if you're interested.
09:11.1 There's also... there are other ways to do this, though.
09:15.1 So, Art Weiss has demonstrated recently that
09:17.1 Csk inhibition can also trigger T cell receptor signaling
09:20.0 using a pharmacological agent.
09:22.1 So, either CD45,
09:26.3 as the sort of negative regulator of local tyrosine phosphorylation,
09:28.3 or CSK to regulate Lck
09:31.2 will achieve this rapid activation.
09:34.3 Balbino Alarcon and colleagues
09:37.1 basically have been studying
09:39.1 conformational changes in the T cell receptor
09:40.3 and evidence of that.
09:42.0 This is, again,
09:44.2 an ongoing area and no one has a structure
09:46.2 for a complete T cell receptor
09:48.1 with the signal transduction components,
09:49.2 so we don't know at this point if this is right,
09:52.3 but he has signatures of these conformational changes.
09:55.2 Kai Wucherpfennig and earlier, even, Larry Stern,
09:58.2 have described the...
10:00.3 kind of a conformation of the cytoplasmic domains
10:03.0 attached to membrane lipids,
10:04.2 and this modification of the sequestration by lipids
10:07.2 in the inner leaflet of the membrane
10:09.1 can also be used as a triggering mechanism.
10:10.3 And finally, Cheng Zhu
10:12.2 has come up with some very interesting data
10:15.0 suggesting that the T cell receptor, again,
10:16.2 in a kind of a conformation change mode,
10:18.3 acts as a catch bond
10:20.1 that in response to force
10:22.1 undergoes conformational changes
10:23.3 that both increase the strength of its interaction
10:25.3 with the MHC-peptide complex
10:27.1 and contribute to the signaling process.
10:30.2 So, again, there are a number of things going on in this area
10:33.2 and we don't...
10:35.0 really, the jury is still out on
10:37.0 what combination of those mechanisms,
10:39.0 or even unknown mechanisms
10:40.2 that people haven't thought of,
10:42.0 are actually acting to account for this high sensitivity.
10:44.2 So, this centripetal movement,
10:46.2 again, of the MHC-peptide complex,
10:49.3 the T cell receptor,
10:51.1 and adhesion molecules is very dramatic,
10:53.2 and this movie from Ron Vale's group
10:55.2 generated by Adam Douglas
10:58.2 using the Jurkat cell line
11:00.1 expressing fluorescent GFP-actin
11:04.0 beautifully illustrates this centripetal movement
11:07.1 using a method of speckle microscopy,
11:09.0 which is based on spinning disk confocal microscopy.
11:13.0 So, this is clearly an engine
11:16.0 for the formation of these centripetal patterns,
11:18.2 and the differential association of the T cell receptor
11:22.0 and the integrins with actin
11:23.3 probably then account for this bullseye-like patterning.
11:26.0 In talk 3, we'll also discuss how the ESCRT machinery
11:29.2 may be important for actually the final deposition
11:31.3 of the T cell receptor in the center.
11:34.2 So, effector function of immuno...
11:37.0 so, there are two kinds of...
11:38.1 I've talked about signal integration
11:39.3 and the signal integration
11:41.2 can accomplish transcriptional changes
11:43.1 and cell cycle regulation
11:45.0 that leads to the proliferative burst,
11:46.3 but once that proliferative burst is over
11:49.1 the T cells become effector cells
11:51.1 and they have to do things,
11:52.1 they actually can't just proliferate and things,
11:54.1 they actually have to kill targets or help B cells,
11:56.0 and this synapse also helps in this stage.
11:59.1 And this is a fantastic movie
12:01.1 done by a method called
12:03.1 laser lattice light sheet microscopy,
12:06.1 which was developed by Eric Betzig at Janelia Farms,
12:10.2 and this Ritter et al paper with Gillian Griffiths
12:12.1 and Jennifer Lippincott-Schwartz
12:14.0 beautifully shows T cells
12:16.1 with a green fluorescent actin probe
12:18.1 and orange markers of their cytolytic granules,
12:21.1 which contain agents that will kill this target cell,
12:25.0 and also the centriole
12:26.2 -- the microtubule organizing center --
12:28.1 marked with this little dot here.
12:29.2 So now, if you watch this movie,
12:31.3 this T cell really goes after this target cell,
12:35.2 forms an immunological synapse...
12:37.3 you can see, with the clearing of actin...
12:40.1 kind of a bright actin structure at the periphery
12:42.2 and then a clearing of actin towards the center,
12:44.1 where those granules and the microtubule organizing center
12:46.1 dock right up to that interface
12:48.3 and deliver cytolytic agents that will result in, again,
12:52.1 death of the target cell.
12:54.1 So, this is a beautiful example of effector function.
12:55.2 Again, this was published this past year.
12:58.1 So, in terms of thinking about...
13:00.2 like, how important is it that you do that to kill a target?
13:04.0 So, we were able to do some experience
13:05.3 in collaboration with Yuri Sykulev
13:07.2 where we basically could kind of measure
13:09.3 the efficiency of the cytolytic T cell
13:12.0 by comparing how many granules it released,
13:15.1 which can be measured through a serine esterase release assay,
13:18.0 and how efficiently it killed the target.
13:20.2 So... and you can kind of use target killing
13:22.3 over granule release
13:24.0 as kind of the measure of efficiency,
13:25.2 and then set up situations
13:27.2 where the T cell either formed an immunological synapse
13:30.0 or had a broken immunological synapse,
13:31.2 so it could basically...
13:32.3 you know, pharmacologically repair or break the synapse
13:35.3 depending upon, you know...
13:37.3 experimentally, and what we found is that
13:40.3 basically forming an intact immunological synapse
13:42.2 improved killing about 3-fold.
13:44.0 And you can say, "Well, that's not that much...".
13:45.3 Some biologists have a cut-off at 5-fold,
13:47.3 but we actually think that
13:49.2 this could be very important in the context of,
13:52.2 say, killing in vivo, particularly tumor cells,
13:54.1 where it can take up to 6 hours for a T cell
13:56.3 to kill a tumor cell based on work from [unknown],
13:59.0 so a 3-fold change in efficiency
14:01.0 would take things from 6 hours to 18 hours,
14:03.1 and then, you know,
14:05.0 you're actually starting to deal with timeframes
14:07.1 that are relevant to the replication rate of the tumor cell.
14:10.1 So, if you want to keep up, basically,
14:12.1 it's probably good to have an intact synapse,
14:14.0 and that seems to be the case,
14:15.1 as I'll show you in a moment.
14:17.0 So, you know...
14:18.2 the other thing I wanted to talk about
14:20.3 is the actin, you know,
14:22.1 which again highlights
14:24.1 both this bullseye-like formation,
14:25.3 the formation of this junction
14:29.1 that the cytotoxic T cell uses to grab onto the target cell,
14:33.1 also amplifies the signal, it turns out,
14:36.0 from the T cell receptor
14:37.2 downstream of that early tyrosine kinase cascade,
14:40.1 and this is work from Sudha Kumari in my group
14:44.1 that kind of uses a similar kind of actin probe
14:47.1 to that used by Ritter et al
14:49.0 to follow T cell receptor microclusters
14:51.1 and then look at the, you know,
14:53.1 very specifically at the amount of filamentous actin at these sites,
14:57.1 and one way to basically
15:00.0 take these kinds of movies,
15:01.1 which can be a little bit difficult to deconvolve,
15:03.1 and illustrate events that are happening over time...
15:06.1 this view is called a kymogram,
15:08.0 which gives you essentially this region here,
15:11.1 this line effectively is the top,
15:14.0 for the T cell receptor and for the actin,
15:15.3 of this graph,
15:18.0 and then you basically just run that same region
15:20.1 at different times for both signals,
15:23.1 and what you can see is that within this very complicated pattern in the actin
15:27.1 you have very steep diagonals,
15:30.0 which means things are moving very fast,
15:31.3 they're doing a lot of movement in a short time,
15:34.1 but then these kind of more shallow...
15:36.1 these kind of somewhat larger angles
15:38.2 going down in this direction...
15:40.1 they basically reflect slower movement,
15:43.3 and those are tracking perfectly with the T cell receptors.
15:45.3 So you have these
15:48.0 foci of actin that are following the T cell receptor very closely
15:50.2 and what Sudha was able to show
15:53.1 is that these actin foci
15:55.2 were dependent upon a protein called
15:57.1 Wiskott–Aldrich Syndrome protein,
15:58.2 which is deficient in a primary immunodeficiency,
16:00.1 where you have B cell and T cell dysfunction,
16:02.2 and also platelet dysfunction.
16:04.2 It's basically an actin regulator
16:08.1 that regulates the so-called Arp2/3 complex
16:10.0 that makes branched actin networks,
16:12.1 and if you don't have WASP,
16:14.1 you don't basically form these actin foci
16:17.1 at the T cell receptors.
16:18.1 But a lot of your other actin polymerization is fine
16:20.2 and you can actually form a synapse just fine,
16:22.1 so that was a bit confusing initially,
16:24.1 but looking at that again,
16:26.0 at just that actin at those T cell receptor foci
16:28.1 was critical for understanding this.
16:29.2 And Sudha went on to show
16:31.1 basically that phospholipase C-gamma recruitment
16:33.1 is critically dependent upon these actin foci,
16:35.1 and phospholipase C-gamma
16:37.1 is a critical sort of, you know,
16:40.3 nexus or focal point for T cell receptor signaling.
16:43.0 So, activating PLC-gamma
16:44.3 gives you both calcium and Ras activation,
16:46.2 so it's both important for acute signaling
16:49.2 and also for some of the sustained signaling
16:51.1 that leads to transcription,
16:52.3 and this is critically dependent upon these actin foci.
16:55.1 So, these actin foci
16:57.0 are not just forming the whole synapse...
16:58.2 or F-actin is not just forming the whole synapse,
17:01.0 but there are subtypes of actin in the synapse
17:03.1 that are driving this signal amplification process.
17:06.1 And this is just a kind of a movie that kind of illustrates, again,
17:11.1 the remarkable things that are happening in the synapse with actin.
17:13.2 These little dots are basically
17:16.0 microcontact-printed regions of a T cell receptor ligand
17:19.3 and the system is back-filled
17:22.1 with the adhesion molecule ICAM-1,
17:23.2 so the cell is spreading on the ICAM-1,
17:25.2 actin is polymerized at the T cell receptor,
17:27.0 and then it's forming these intricate kind of spirals,
17:30.0 which basically, with the actin,
17:31.3 kind of distributing to the integrins,
17:33.3 and the integrin is kind of taking this actin
17:36.0 that's being generated at the T cell receptor
17:37.2 and then kind of modifying it.
17:39.0 So, there's a, you know,
17:40.3 quite a bit to learn about the specifics of this,
17:43.0 but at this point one thing we feel that we know
17:46.2 is that this is very important for signal amplification.
17:50.2 So, I want to talk a little
17:52.1 about the applications of the immunological synapse
17:53.2 at this point.
17:55.0 So, having told you some things about the way it's working...
17:57.1 so, what is the significance of this?
18:00.0 So, we think that there are a couple of particular things...
18:02.1 I mean, one recent work
18:04.2 with Kai Wucherpfennig's group on autoimmune disease I want to point out,
18:07.2 and also a recent collaboration we did
18:10.1 with a group of cancer immunologists who use radiation therapy
18:12.2 in combination with a new type of biologic
18:15.0 called a checkpoint blockade drug
18:18.3 that essentially...
18:21.1 again, and these checkpoint blockade antibodies
18:23.3 have really revolutionized melanoma therapy at this point,
18:27.1 so kind of really changed the game in a lot of ways.
18:30.3 So... but then first i want to basically
18:34.0 define one other term
18:36.1 or at least mode of interaction
18:38.1 between T cells and substrates,
18:39.2 and this is...
18:41.2 essentially to compare a synapse
18:43.1 and what we refer to as a kinapse.
18:44.3 So, just to illustrate what this is...
18:46.1 so, we have a synapse on this side,
18:48.2 which is... kind of, maybe a movie you've seen before.
18:51.1 It's radially symmetric, the T cell receptor goes towards the center.
18:54.1 This is showing the T cell receptor signal in this case.
18:56.1 But you also see cells
18:59.2 that essentially start to form a symmetric synapse,
19:01.1 but then they break symmetry
19:03.0 and they start to move,
19:04.1 and this one actually is moving kind of down and towards me,
19:07.2 so what you'll basically start to see
19:10.0 is that it starts to have a trailing edge and a leading edge.
19:12.2 And we can describe the cytoskeletal dynamics
19:17.2 of Kupfer's supramolecular activation complex
19:20.1 as having characteristics like a lamellipodium,
19:22.2 a lamella,
19:24.3 which is basically a site of adhesion molecule
19:27.0 concentration for the pSMAC,
19:28.3 and then the cSMAC being sort of like
19:31.1 the uropod in this migrating cell.
19:32.2 So, we think that the machinery
19:34.1 is actually quite similar,
19:35.2 so when a cell is basically moving
19:37.1 or engaged in this,
19:38.2 what we refer to as a kinapse, a moving junction,
19:41.2 it's actually integrating signal in much the same way
19:43.3 as the cell that's forming the radially symmetric synapse,
19:46.1 but this cell will basically
19:48.2 move away from the place where it started out,
19:50.2 whereas this cell will pretty much stay in place
19:52.2 because the radially symmetry balances all the forces
19:54.2 and the cell will stay.
19:56.1 So essentially, this is an issue of, you know,
19:58.1 Should I stay or should I go?
19:59.3 The cells do these different behaviors in different contexts in vivo,
20:03.0 and we've found that this is
20:06.2 potentially useful in both the autoimmune and cancer immunotherapy context,
20:09.2 so we think about it.
20:10.2 And we know genetically,
20:12.1 based on a paper by Tasha Sims in 2007,
20:16.2 again, a postdoc in my lab,
20:19.1 that essentially the symmetry breaking
20:22.0 is driven by a protein kinase called protein kinase theta,
20:25.1 and the reformation of a stable synapse,
20:29.0 from this state going to a stable synapse,
20:31.2 requires WASP,
20:34.0 the kinase I described before.
20:36.0 And again, it's probably in synergy with the integrin system
20:38.2 to basically, essentially,
20:40.3 repair the broken pSMAC
20:42.1 and basically get you back to a symmetric state.
20:44.2 So, this heatmap...
20:47.1 it's complicated, but the punchline is that basically
20:50.1 autoreactive T cells, and these are T cells
20:52.1 from patients that recognize bona fide autoantigens
20:54.2 in multiple sclerosis and type I diabetes,
20:58.0 and comparing these to T cells
21:00.1 that are responsible for killing
21:02.3 flu-infected T cells in influenza...
21:04.2 or sorry, influenza-infected target cells,
21:07.2 you essentially...
21:10.2 these form fantastic synapses,
21:11.2 so the red and orange all say that these are,
21:14.0 you know, attributes of stable synapse formation,
21:17.1 and then as you basically go into these different clones,
21:20.1 which are indicated up here,
21:21.3 in the multiple sclerosis
21:23.2 or type I diabetes setting,
21:25.0 you see, you know,
21:27.2 progressive essentially
21:30.1 defects in this immunological synapse formation process.
21:33.2 So, I guess... and this is just kind of illustrated here...
21:35.2 you have essentially kinapses
21:37.2 forming with these
21:40.2 MS-specific T cells
21:41.3 and type I diabetes-associated
21:44.0 autoreactive helper T cells.
21:45.3 So, this is... and again,
21:47.3 this is a characteristic that appears
21:50.0 to come from the antigen recognition process,
21:52.1 so self-antigen recognition in this context,
21:54.0 driving a weaker synapse,
21:56.1 and we think one possibility is that
21:58.1 this failure of stable synapse formation
22:00.2 may make these more difficult to regulate.
22:02.0 That's our hypothesis at this point
22:04.1 and I think that this is something that we're looking to test in the future.
22:08.0 So... cancer...
22:10.2 so, uhh, immune evasion
22:13.0 has recently been made by cancer biologists,
22:16.0 this review by Hannahan and Weinberg,
22:18.2 who kind of revisited the hallmarks of cancer...
22:21.1 so, cancer immune evasion
22:24.1 is now considered a hallmark of cancer.
22:26.0 So, this is...
22:27.1 and this is a big change over the past decade
22:29.1 in how people think about this,
22:30.3 and a lot of this change
22:32.2 has been driven by a couple of advances
22:34.2 based on these therapeutic agents
22:36.3 that basically remove inhibitors
22:40.2 of the immune system
22:42.1 or neutralize inhibitory pathways
22:43.3 that are probably normally protecting us from autoimmunity
22:46.2 or immunopathology,
22:47.2 but in the context of cancer
22:49.2 can be allowing the cancer to evade the immune system.
22:51.2 You block these and the immune system
22:54.0 suddenly will see the tumors
22:56.1 much more clearly and destroy them.
22:58.0 So, again, a change in opinion.
22:59.3 And this is one of those systems,
23:01.1 so, an antibody called Ipilimumab
23:03.2 that's targeting a protein called CTLA-4,
23:06.1 which is cytotoxic T lymphocyte antigen-4,
23:10.1 and this is an image from work by
23:13.2 Jim Allison and Jackson Egan,
23:16.2 basically showing a fluorescent CTLA-4
23:19.2 moving to the immunological synapse.
23:21.2 So, this is a synaptic marker,
23:25.1 it's a regulator of effector T cells,
23:27.1 it's generally thought of as an inhibitor
23:29.2 -- when it's blocked by antibodies
23:31.3 it then increases activation --
23:33.2 and essentially also it's used by the regulatory T cells
23:38.1 I mentioned in part 1,
23:40.2 which buffer responses and therefore,
23:43.0 because CTLA-4 is an effector molecule of the regulators,
23:45.3 inhibiting it actually...
23:47.2 it neutralizes some of the function of the regulatory cells too.
23:50.2 And finally, CTLA-4 cross-linking,
23:53.1 though, has kind of a side effect.
23:55.1 I mean, all drugs have side effects,
23:56.2 and in this case it's synapse destabilization.
23:59.1 So, again, CTLA-4,
24:01.2 perhaps because it's involved in an axis
24:04.1 that regulates protein kinase C-theta,
24:05.3 which I mentioned before is a synapse breaker,
24:07.2 may enhance the activation of PKC-theta
24:09.2 and in doing so essentially
24:12.2 destabilize these synapses.
24:14.1 And we've actually seen this is in a model of
24:18.1 cancer therapy in the mouse,
24:19.3 where we're setting up a model
24:22.0 of a breast carcinoma
24:23.2 implanted in the flank of a rodent
24:25.1 that then grows over a period of
24:27.2 about 12 days and metastasizes to the lung,
24:30.2 so it's a model that basically
24:33.1 recapitulates key events
24:36.2 in the development of breast carcinoma,
24:37.2 although it doesn't start from the orthotopic site
24:40.2 like the ducts of the mammary glands.
24:43.1 It basically is...
24:44.3 we're taking a cell line
24:46.2 and putting it into the animal.
24:48.1 So, this tumor is susceptible...
24:50.1 and we can then view the T cells within these tumors
24:54.2 using a method called two-photon intravital microscopy,
24:57.1 in which an anesthetized animal
24:59.1 is basically placed on the microscope stage,
25:01.1 kept warm,
25:03.2 kept alive,
25:05.0 but under anesthesia,
25:06.2 and the tumor can be surgically exposed and imaged
25:10.2 using pulsed infrared lasers
25:12.1 that basically allow you to image deep into the tissue
25:14.1 and do timelapse imaging in real time.
25:17.1 And these are some images
25:19.0 and this is basically just illustrating
25:21.0 our therapeutic model.
25:22.2 So, if we put in anti-CTLA-4, again,
25:25.1 an anti-mouse version of Ipilimumab,
25:28.1 what we find is that, as in some patients, the therapy fails,
25:32.3 so the tumor continues to grow, you have high metastases.
25:35.3 The blue cells here are the tumor cells,
25:37.2 the red are some tumor vasculature,
25:39.3 which, again, is famously abnormal,
25:42.1 but nonetheless the T cells are getting in,
25:44.1 but what you can see the T cells are doing
25:46.2 is they're highly motile.
25:47.2 Even in the tumor, the T cells are moving.
25:50.1 This means they're forming kinapses;
25:51.2 they're not forming synapses.
25:54.0 However, if we combine the anti-CTLA-4
25:56.3 with radiation therapy,
25:57.3 which was the therapeutic modality
26:00.0 that Sandra Demaria and Silvia Formenti
26:03.0 were focusing on in this case,
26:05.3 now we see that the T cells
26:07.2 -- the green cell, the T cells --
26:09.1 within the tumor are arrested on the tumor cells,
26:12.0 so they're now forming synapses,
26:13.1 and this is correlated with tumor shrinkage
26:15.1 and low metastasis.
26:17.1 So, what we went on and then showed...
26:19.2 and these are basically... these just illustrate the two modes,
26:22.2 so the low motility in the tumor is associated with the,
26:25.1 again, effective combination therapy,
26:28.0 whereas the high motility is associated with the anti-CTLA-4,
26:31.2 again, kind of side effect.
26:32.3 We found that we could basically recapitulate,
26:35.1 even in the combination with radiation therapy,
26:38.1 we could recapitulate this increase in motility
26:41.2 by inhibiting another receptor called NKG2D,
26:45.0 and this is...
26:46.2 you know, I don't want to...
26:48.1 obviously the details of this are important,
26:49.2 but this is a, kind of,
26:51.0 what's referred to as a co-stimulatory receptor.
26:53.2 So, CTLA-4 antagonizes the activity of CD28,
26:56.3 which is one type of co-stimulator.
26:58.2 NKG2D is in another class of co-stimulators
27:02.1 on the CD8 T cells,
27:03.2 and what appears to happen is that
27:05.3 when you irradiate the tumors they upregulate ligands for NKG2D
27:09.1 and that additional co-stimulatory pathway
27:12.1 stabilizes the synapse,
27:14.1 so unlike CD28 that activates PKC-theta
27:16.2 and inhibits synapse formation,
27:17.3 NKG2D is delivering activating signals
27:20.3 that are stabilizing the synapse without activating protein kinase C-theta,
27:24.1 we believe,
27:26.1 and this is leading to essentially a reversal
27:28.3 of the therapeutic effect.
27:30.2 So we think that what we're essentially...
27:32.2 and this is just illustrated here.
27:34.1 So, if you block the NGK2D,
27:36.1 you kind of increase the lung metastasis
27:38.2 versus the effect of ionizing radiation 9H10,
27:42.1 which is the anti-CTLA-4 antibody...
27:44.1 so, no lung mets,
27:46.1 and basically the antibody to NKG2D, you get the lung mets back.
27:50.2 So, essentially the behavior of the T cell in the tumor
27:54.2 correlates very strongly with our clinical outcome
27:58.0 in this rodent model,
27:59.3 and this approach is in human trials
28:03.1 and, you know,
28:06.0 is showing promise in conjunction with Ipilimumab.
28:08.2 So, this is sort of an illustration
28:11.3 of how we can use information
28:13.2 about the immunological synapse
28:15.1 to think about what's happening
28:17.2 in the context of immunotherapy,
28:20.1 and this is a movie of the situation
28:22.2 where the combination therapy
28:24.2 has been very successful in eradicating the tumor,
28:26.2 no tumor cells, lots of T cells.
28:28.2 So, at least in the immediate post-tumor site period,
28:32.0 this is probably what we want to see.
28:33.2 So, there are a number of factors
28:35.2 that will basically be controlling this,
28:37.1 but I think one of the key things that we're,
28:40.1 you know, kind of coming back to
28:42.1 is that the radiation therapy
28:44.1 is basically triggering an innate injury response,
28:47.1 it's upregulating NGK2D ligands on the tumor cells,
28:51.0 and this is allowing the immune system
28:54.1 to get back on target in the presence of the therapy
28:57.1 with anti-CTLA-4
28:59.1 to eradicate or at least significantly slow down the growth of the tumor.
29:03.2 So, this anti-CTLA-4 side effect,
29:05.1 its synapse destabilization,
29:06.3 can be counteracted in this case by irradiation therapy,
29:09.2 but we think the more specific thing that's doing
29:12.1 is engaging this other signaling pathway
29:14.1 that stabilizes the synapse and, you know,
29:17.3 essentially this what's referred to as a stress induced molecule,
29:19.2 the NGK2D ligand, called RAE-1.
29:24.0 If you're interested in the detail, it's in this paper.
29:27.0 So again, that's an example of using
29:29.2 information about the immunological synapse
29:31.1 to think about mechanisms in disease and disease treatment.
29:36.2 I'd like to acknowledge many investigators
29:39.1 who contributed to this at both...
29:42.3 particularly NYU and more recently Oxford,
29:45.0 where we've moved in the last two years.
29:47.0 Again, there are many other things
29:50.2 that you can follow up on
29:52.2 in terms of citations in the talk
29:54.1 and, you know, I guess, Happy Explorations.
29:57.2 So, thank you. Bye-bye.