Session 4: Molecular View of Adaptive Immunity

Transcript of Part 2: The Immunological Synapse: Signaling and Function

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.