Hello.
I'm Michael Dustin
from the University of Oxford
and New York University School of Medicine.
Welcome to Part III of the immunological synapse,
focusing on extracellular vesicles.
This is an observation that we made recently,
in the interface between the T lymphocyte
and the antigen presenting cell
during this critical communication
that integrates
innate and adaptive immunity.
So, the communication
between the T cell and dendritic cell
takes place
through recognition of T cell receptors
on the surface of the T cell
with the MHC-peptide complexes
on the surface on the dendritic cell,
assisted by adhesion molecules
like LFA-1
interacting with its ligand ICAM-1.
This recognition process
generates a number of signaling outputs in the T cell
and also feeds back information
to the dendritic cells
or other types of antigen presenting cells,
such as macrophages for killing microbes
or B cells for production of antibodies.
Experimentally, we can recapitulate
some aspects of this communication
by replacing the antigen presenting cell
with a supported planar bilayer,
a supported lipid bilayer
containing purified adhesion ligands
such as ICAM-1 and the MHC-peptide complex
in a laterally mobile form.
The lipid film in this case is in
a liquid disordered state.
Transmembrane proteins in this system
become immobile,
but if you anchor the proteins
to the lipid bilayer directly,
for example through C-terminal histidine tag
in some of the protein extracellular domains
linked to chelating lipids in the bilayer,
the ligands are highly mobile
and allow formation of an immunological synapse.
So, we can use a quite varied microscopy tool kit
to interrogate these systems.
And for the supported bilayer system,
we can use widefield fluorescence microscopy
when we're focusing on molecules
that are in the bilayer itself,
since molecules that are in the cell
would be out of focus at the interface
and interfere with imaging the things in the interface.
We can, in those contexts,
switch to using total internal fluorescence microscopy,
which generates a very shallow evanescent wave
at the point where a laser beam
is reflected off of the interface.
So, this is a technology
that became available around 2000
in a commerical form
for high numerical aperture objectives,
and we use it very widely.
It also allows single molecule imaging
in the context of a cell bilayer interface,
which is methodology I'll describe later.
We also make extensive use of
reflection interference contrast microscopy,
which is based on interference
between light that's reflected off of the interface
between the cover slip and the media,
with reflections that are coming off of the interface
between the media and the cell.
So, those different reflections are phase shifted,
so when the cell gets very close to the substrate,
say, within 100 nanometers,
you start getting destructive inteference,
so the closer the cell becomes to the substrate
the darker the image will appear.
That's very useful for looking at contacts.
In this talk, I'll also highlight
the correlation of various types of fluorescence microscopy
with electron microscopy,
and particular with electron tomography,
which is a method where you take relatively thick sections
for transmission electron microscopy,
say, routinely you might use sections that are a few nanometers.
In the case of electron tomography,
you use sections that are up to 100 nanometers in thickness,
stain similarly for other transmission electron microscopy with heavy metals,
but tilt it in the electron beam
so that you collect a dataset
which can be used to deconvolve a three-dimensional image,
again, with very high,
you know, sub-nanometer resolution.
So this is a powerful method
for looking at structures in the interface.
It doesn't necessarily give you molecular identity,
but that can come from correlation
with fluorescence microscopy.
So, that's pretty much the tool kit
that I'll be using for the studies shown here.
So, the story I'm going to tell you
about extracellular vesicles enriched for T cell receptors,
kind of...
we started seeing indications of this phenomena
about 20 years ago now,
when we first started looking at T cells interacting
with these planar bilayer substrates,
and this is an example from a collaboration
with Emil Unanue when we were in St. Louis,
where we're seeing...
we're basically looking at a T cell forming an adhesion,
based on the interference reflection microscopy,
again, where the contact areas appear dark
and the accumulation of T cell receptor in this interface.
The substrate in this case is coated
with purified MHC-peptide complexes,
so this is a purely T cell receptor-driven adhesion.
Now, this is not the way we think things happen physiologically,
but as an experimental model
we were kind of interested in how this would...
basically, what the capabilities of the
T cell receptor/MHC-peptide complex are,
and this allowed us to basically see that
the T cell receptor would cluster in this interface
if you had sufficient densities of MHC-peptide complexes.
However, something that surprised us
is that when we removed the cell
by just basically flowing media over the surface
where the cell was adhering,
we actually found that we could knock the cell off,
but would leave a set of contact areas
and that these were positive for the T cell receptor.
So, we were somewhat surprised
to find that the T cell
would so easily release this T cell receptor,
which everyone was assuming
would be internalized and degraded
following its interaction with the MHC-peptide complex.
But this gave us an indication that T cells could,
in some way,
release their T cell receptors
towards an antigen-presenting substrate,
so essentially a surrogate antigen presenting cell.
So, for those of you who have seen some of the earlier talks,
you'll recall the work from Avi Kupfer,
demonstrating this formation of supramolecular activation complexes.
The imaging technology that was used for this
is basically widefield fluorescence microscopy,
optical sections through the cell-cell conjugate,
and then a computational deconvolution method
to reconstruct the 3-dimensional structure
with greater resolution than would be possible...
with greater clarity than would be possible
without the deconvolution,
and it revealed this segregation
of the T cell receptor,
shown in green,
from the adhesive interactions
with these two domains.
So, this is an example of combining the...
kind of, the adhesion system
and the antigen recognition process
as a normal cell-cell couple would do.
We could also then do this
in the supported planar bilayer system,
and again we saw something...
so, I guess in the movie on your left,
basically in the center there,
we're basically seeing the
T cell receptor/MHC-peptide complex interaction in green,
the LFA-1 adhesion interaction with ICAM-1 in red,
and essentially what you're seeing
is the formation of the pattern that Kupfer saw,
this bullseye-like pattern.
So, this was sort of...
it recapitulated what Kupfer had seen
and allowed us to see the dynamics.
That was very nice.
At the same time, we had other synapses form,
which were somewhat disordered in the early phases,
and then kind of completely decomposed,
like the cell actually moved on
and did not form a stable junction,
did not form this symmetric bullseye-like pattern,
but as you can see what's happening to the red signal,
which is the adhesion signal,
is actually moving off away from the
cluster of T cell receptor and MHC-peptide complexes,
and as you can see the cell moves away,
based on the red signal,
basically moving out of the frame,
these clusters of MHC-peptide complexes
presumably engaged by T cell receptors
are left behind in the substrate.
So, again, another indication that T cells
were leaving receptor behind with the antigen presenting cell,
which is something that, again, we hadn't expected.
We had expected internalization and degradation of the T cell receptor
to be the normal way to resolve these interactions.
So, this got us...
you know, so,
there was another observation related to this is,
is that if you look at the relationship between
the density of MHC-peptide complexes in the substrate
and the size of the cSMAC...
so, this is basically increasing MHC-peptide density
in the planar bilayers,
and the signal here is basically
the MHC-peptide complexes...
this is the T cell receptor...
so, you can see there's a very good correspondence between these,
and the intensity of this central structure, cSMAC,
as described by Kupfer,
is essentially linear with antigen increase,
and that's kind of quantified here.
So, in some ways,
this finding in itself was also surprising,
because if you look at the signaling
based on calcium mobilization in the T cell,
it basically is fully activated
at around 1 molecule per micron squared,
and then basically is saturated
at a few molecules per micron squared,
and then doesn't increase further,
whereas this basically...
signal that we were seeing in the cSMAC,
which initially we thought might be involved in signaling,
was completely,
you know, linear rather than having
this threshold-like behavior.
So, again,
this linearity with antigen input
was an interesting characteristic
that was very at odds
with the behavior that the T cell
would be displaying in terms of signaling.
So, again, it left us with the question
as to what the purpose of this central accumulation
of T cell receptor
in the immunological synapse actually is,
which initially we thought...
we had a hypothesis
-- we thought it was related to signaling --
but that seems to be incorrect,
so we had to think further about this.
So, one of the questions
that we could then ask is,
what is the molecular machinery
that creates this central cluster?
And a key experiment
in understanding this
was initiated by Santosha Vardhana,
who was working in my group
at New York University,
and what he did basically was
knock down a component of a machinery referred to as the
Endosomal Sorting Complex Required for Transport,
or ESCRT,
and in fact there are three major complexes
in the ESCRT machinery,
maybe four if you count ESCRT 0, 1, 2, and 3,
and this was basically...
he's knocking down ESCRT 1,
which is a protein in mammals
referred to as Tsg101,
or tranforming tumor suppressor gene 101,
and essentially what he found was that
when he knocked this down in T cells
he got two major phenotypes.
One was...
with the control siRNA,
you have cSMAC formation.
With the Tsg101 siRNA,
you basically have this ring of T cell receptor
with essentially very little T cell receptor
in that central region,
in the cSMAC region,
so failure to form the cSMAC,
and also if you look at the level of tyrosine phosphorylation
-- again, the T cell receptor
is linked to a tyrosine kinase cascade --
this is the physiological level of signaling
in the immunological synapse
through T cell receptor clusters.
This is what we're seeing essentially
with Tsg101 knockdown,
which is about a 10-fold increase in tyrosine phosphorylation.
So, you had hyper-signaling
through the T cell receptor and failure to form the cSMAC.
So, in some respects,
this phenotype with the ESCRT machinery
fit with some other things that were known about
the way this ESCRT machinery
deals with growth factor receptors,
in terms of signal termination,
but the next step in that process,
which would be degradation in lysosomes,
clearly was not happening in this particular context
of the immunological synapse.
We were ending up instead with
a transport process, which was resulting in the formation of a cSMAC
when this system was functioning normally.
So... and this was, again,
not an expected phenotype
for this ESCRT machinery,
which would otherwise not be involved, usually,
in sorting out molecules at the cell surface.
So, again, we need to think about this a little bit more.
So, just to kind of introduce you
to the ESCRT machinery a little bit more...
again, I mentioned that
there's basically four complexes.
We're looking at Tsg101, here,
and what Tsg101 is doing
is recognizing ubiquinated cell surface receptors,
which are recognized usually
in endocytic compartments
and results in...
this is the cytoplasmic side,
this is the extracellular side,
so this would, say, be the extracellular part of the T cell receptor,
this would be the cytoplasmic side on this side,
and you're essentially looking at the
formation of a bud
into the lumen of an endosome,
which would result in the degradation
of that protein
after the endosome fused to a lysosome.
So, this system is also used
for viral budding, though,
and that, for T cells,
can happen certainly at the plasma membrane
like in the context of human immunodeficiency virus (HIV),
which buds from the plasma membrane in T cells.
So, we became kind of interested in the idea
that perhaps Tsg101 in this situation
is acting with the T cell receptor
the way it would with, say,
viral glycoproteins
in the formation of HIV particles,
and actually creating a bud into the extracellular space.
So, to test that,
we basically needed to use
another set of methods.
Now, another observation that we had
that essentially fit with this was
work from Rajat Varma published in 2006,
which showed that, in some contexts,
we would see T cell receptor-positive particles
outside of the immunological synapse
that were basically interacting
with the supported lipid bilayer
and essentially diffusing around
in 2 dimensions on that surface.
So, this again fit with the idea that
although many of the T cell receptors
appeared to be confined with the cSMAC region,
under certain conditions a lot of these receptors
seemed to be getting loose outside of the cell,
in the extracellular space,
and at the same time
continued interacting with the supported bilayer,
where the MHC molecules are,
so, again, fitting with
potentially a budding process which would generate
T cell receptor-positive particles
that were actually in some cases then
moving out of the cSMAC region in this model system,
where there's no antigen presenting cell
that could further process those.
So, to test this, we started using
this electron microscopy approach
with David Stoakes at NYU,
and Kaushik Choudhuri in my group,
and Jaime Llodrá in David's lab, basically,
along with Lance Kam at Columbia,
set up a system where they could
form supported planar bilayers
on a grid, a chrome grid,
which would basically give us an imprint,
in the electron microscopy experiment,
that we could then use to orient our fluorescence imaging,
shown here,
to electron micrographs
acquired after fixation and processing of the sample
for electron microscopy.
And due to skills of a very talented technician
who could take these en face sections
off the face of these blocks,
which would basically be, in this case,
maybe 100 nanometer or so sections,
essentially lined up manually by eye,
which was quite a significant skill,
we were able to basically correlate...
obtain these correlative images where you have,
in this case,
an actin signal that tells you where the cell is,
the T cell receptor that tells you
where the cSMAC is,
that... again, using Kupfer's nomenclature...
and essentially when you look at the
electron micrograph
from that overlay in more detail,
just focusing in on this region
where the T cell receptor has accumulated,
what you'd see was basically these little rings,
these little circles in any kind of level in the tomograms,
or in the thin sections,
and the variability of the diameter of these circles
is consistent with that of small vesicles.
And in fact serial tomograms
that were taken on the vertical axis
through the interfaces...
and this is basically just a movie
that shows the results of four of these tomograms,
indicated that you have
small vesicles on the outside of the cell.
So, this line down here at the bottom
is the supported planar bilayer...
this is the plasma membrane, is here...
and the structures in the interface
between the T cell and the planar bilayer
are these extracellular vesicles.
And because this tomogram
allows these 3-dimensional reconstructions,
and that's basically the end of the fourth tomogram,
that information is a little bit hard to digest,
so Kaushik spent quite a bit of time
going in and tracing the relevant structure,
the vesicles and some of the cytoplasmic organelles,
and generating this model,
which we can then play back as a movie,
and basically makes it a little bit easier to focus
on the various elements
that we found of interest.
So, typicallly, immunological synapses
have a high degree of cellular polarity.
You have the centrioles, or microtubule organizing centers,
indicated here in purple,
microtubules in lighter purple...
the Golgi apparatus is basically up here...
and some of these other structures...
there are endocomes, mitochondria in kind of orange...
the blue structure up here is the nuclear envelope,
and, again,
the gold structures in the interface
between the supported planar bilayer,
in light blue,
and the plasma membrane, in green,
are these extracellular vesicles
that were being released into the cSMAC structure.
Again, quite an unexpected finding
to see this, really,
almost complete correlation of the T cell receptor signal
with these extracellular vesicles,
which before we had thought basically were
T cell receptors that were still in the T cell plasma membrane
interacting with these
MHC-peptide complexes on the antigen presenting cell.
But this explained a lot to us in terms of how we would see,
you know, termination of signaling
and this prior evidence of the T cell receptor
being left behind on the substrate
because the T cell receptor in these vesicles
is no longer coupled to the cells,
it's now released to this
surrogate antigen presenting cell,
but because the planar bilayer is a passive system,
it basically can't do anything with it.
So, what we see when we basically
then go back to our cell-cell conjugation systems
with T cells and B cells
and look at what's happening
in the interface between those cells,
we can actually see evidence of transfer of T cell receptor into the B cell,
which becomes, you know, particular visible...
so, here's the T cell,
here's the B cell,
and basically the...
after around 15 or 20 minutes,
we'll see these T cell receptor-positive punctae within the B cell,
and this is inhibited when we don't have antigen,
so there's no...
basically, it's antigen-dependent,
and we can also show that if we knock down Tsg101 in the T cell
we can also prevent this transfer.
And furthermore, if you look by electron microscopy
in situations where the cytoplasm of the T cell is labeled
with this... in this case,
this kind of sulfur-enhanced,
you know, gold labeling,
these dark particles that are in the T cell cytoplasm...
the B cell in this case, which is here,
doesn't have these,
doesn't have that basically genetically-encoded tag
that basically lets us tell the T cell from the B cell,
but we see within the B cell
vesicles that have double-walled, essentially,
compartments within the B cell
that contain T cell cytoplasm,
suggesting that the B cells
take these particles up from the T cells
during the process of immunological synapse formation,
and this was a form of information transfer
that we hadn't previously appreciated.
So, what we think is happening here
is that you have these complexes
of T cell receptor and MHC-peptide complexes
at the interface between the T cell
and the antigen presenting cell,
and our suspicion is that these complexes,
when you have enough MHC-peptide complexes,
become effectively irreversible,
and certain B cells can take up a lot of antigen due to their
high affinity B cell receptors
to concentrate the antigen,
so we think these are the situations
where you have these high-density MHC-peptide complexes
that create these essentially irreversible interactions
and then the T cell can either...
the system between the T cell and the B cell
have to negotiate what to do, then,
for them to be able to separate.
So, in this situation, we think there are two ways you can go.
One way, basically, is for
the T cell to essentially pull off a patch of membrane
from the B cell
through an endocytic process
where it would basically use molecules
like clathrin and dynamin
to essentially create this invagination
and pull off a bit of the B cell membrane
with the MHC-peptide complexes,
or it can basically go the other direction
and it can basically use the ESCRT machinery
to release a vesicle
towards the B cell
using the ESCRT-1 proteins,
like Tsg101 to initiate the sorting,
the ESCRT-3 polymers
to basically create the vesicle,
and then VPS4, this ATPase,
to actually scission the vesicle off
and freely release it to the B cell,
which can then internalize that fragment
from the T cell.
And we have evidence, in looking at the T cell/B cell conjugates,
for both phenomena happening.
So, basically the T cells transferring vesicles to the B cells
and pulling vesicles off the B cells...
and so we're still studying, basically,
what regulates the balance
between those two phenomena,
but there's definitely always a prominent component
of this ESCRT-dependent vesicle release.
So, as I showed you in those very early movies
of the immunological synapse
on the planar bilayer system, T cells,
when the break symmetry and leave a particular site,
will leave behind trails of T cell receptor
interacting with MHC-peptide complexes
and we can also see this in the electron micrographs
as the release of trails of vesicles
that the correlative light-electron microscopy
shows are red, or T cell receptor-rich,
and...
you know, an example here,
and kind of a blowup, here...
so, this is a very common phenomena in our preparations,
and gave us the opportunity to basically, then,
determine what happens if we take B cells
with appropriate MHC-peptide complexes
and bring them into contact with these trails
of T cell receptor-positive vesicles
that were released by the T cells.
And what we see in this context
is signaling in the B cell.
So, if the B cell...
basically, with the appropriate MHC-peptide complexes
engages T cell receptors on these released vesicles,
we see an increase in cytoplasmic calcium in the B cells,
suggesting that B cells are getting activated
by the vesicles,
distinct from any interaction with the T cells.
So, one idea would be that T cells
could transfer material to the B cells,
and in fact in in vitro systems
we can quantify this transfer
by a method like flow cytometry,
where we can basically look at the T cell receptor
fluorescent signal in this axis,
and essentially see that,
based on gating on B cells in the other axis,
that we can see B cells
with increasing amounts of MHC-peptide complexes
when we increase the level of,
essentially, surrogate antigen molecules,
in this case so-called staphylococcal superantigen
that bridges the T cell receptor
and the MHC-peptide complexes,
and allows us to experimentally, essentially,
dose in higher levels of T cell receptor engagement,
which generates transfer of T cell receptors to the B cells.
And in fact other signals
seem to be moving through a similar conduit,
and we see molecules like CD40 ligand,
which is an important non-antigen specific signal
that can activate and increase the proliferation of B cells
and their differentiation,
really a critical signal in T cell and B cell communication...
so, the molecule itself is not antigen specific,
it's essentially like a cytokine,
but its specificity comes from the fact
that it's being conveyed through this immunological synapse
and we think it's being conveyed
in the form of these vesicles
released along with the T cell receptor
to the B cell,
so this would help us understand
how the T cell receptor-positive vesicles
and their transfer
could be linked to other signals the B cells
need to differentiate and, for example,
to undergo processes like
antibody class switching,
to make the appropriate type of antibody,
and affinity maturation,
to make higher affinity antibodies in processes in lymph nodes.
And one model or hypothesis that we have for this
is that the transfer of these vesicles
into a B cell
could help distinguish high affinity and low affinity B cells
based on the high affinity B cells,
or B cells with high affinity antigen receptors,
collecting more antigen,
making high densities of MHC-peptide complexes,
B cells with lower affinity B cell receptors
making lower numbers of MHC-peptide complexes,
and then essentially
the amount of the MHC-peptide complexes
then being translated into a number of these vesicles,
and B cells that basically collected more of these vesicles,
through having more MHC-peptide complexes,
would basically then undergo more rounds of division,
and this would then, you know,
based on the uptake and effectively
serial dilution of these vesicles from the T cell,
would be able to effectively count
the number of times that they would divide
before they would stop dividing,
and then potentially could then differentiate
into antibody producing cells
or further mutate their receptor
for more rounds of selection.
So, this would be, kind of, a bit of a token economy
that the T cell would be using
to basically convey to B cells they want to help
an ability to undergo a larger proliferative burst
and compete better in this process
of making high affinity antibodies.
And then the lower affinity B cells
would be less well rewarded and would proliferate less,
and would eventually...
could be outcompeted by such a process,
allowing the higher affinity B cells
to essentially dominate the response.
So, in conclusion,
we have discovered that there are these T cell receptor-enriched extracellular vesicles
that are generated in the immunological synapse,
and effectively make up a large part
of the cSMAC originally describe by Avi Kupfer,
and that could be recapitulated
in the supported bilayer-based reconstitution experiments
that I've shown you earlier.
ESCRT machinery, including Tsg101,
is required to terminate signaling
and sort these T cell receptors into these vesicles,
which then are released to the B cell,
and this VPS4 protein
can be shown to be critical for the actual scission of the vesicles
and the final transfer.
So, these microvesicles activate B cells
in an antigen-dependent manner,
and we've also found critical signals like CD40 ligand
mixed in with these vesicles in the cSMAC,
and this suggests that this vesicle transfer process
may have a critical role in T cell help from B cells,
and this is something that, really,
we feel was discovered through this reconstitution approach,
and really kind of demonstrates
the value of, you know,
some kind of biochemical approaches
when applied to live cells like the T cells in the immune system.
So, there are a number of colleagues
who basically contributed to this work,
both at NYU,
Harvard Medical School,
Columbia.
I basically mentioned all of the names during the presentation,
but I just want to leave them up here,
and I also want to thank the National Institutes of Health
and the Wellcome Trust
and The Kennedy Trust in the UK
for supporting our work in this area.
Thank you.
