This session provides a molecular overview of T cell receptor (TCR) activation and signaling. It shows how TCRs bind to their corresponding peptide:MHC complexes to form the immunological synapse. As shown in this session, the stability and strength of the immunological synapse correlates with the activation of the TCR signaling cascade. TCR activation is mediated by the integration of convergent signals from antigen presenting cells, including the activation of Pattern-Recognition Receptors and the presentation of antigen via MHC molecules.
Review Questions for Session 4: Molecular View of Adaptive Immunity (Educators only)
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
21:26.1 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 WiskottÃ¢â‚¬â€œAldrich 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.
Egen JG, Allison JP. (2002) Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength. Immunity. 16(1):23-35.
- Michael Dustin iBioSeminar: The Immunological Synapse
Michael Dustin is Professor of Immunology and Director of Research at The Kennedy Institute of Rheumatology at the University of Oxford. Prior to joining the Kennedy Institute, Dustin was a faculty member at the Skirball Institute of Biomolecular Medicine at New York University from 2001-2013 and at Washington University School of Medicine from 1993-2000. Dustin received… Continue Reading