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Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 1
1. This is a continuation of what Dr. Peter Burrows was talking about last hour…
2. If you understand the study objectives, then you’ve got it.
3. Big picture. These MHC molecules are literally presenting antigens to T-cells and the T-cells are
making the decision via the T-cell receptor whether there is something foreign present or not. This
is depicted here.
4. So, what we’re really dealing with is a certain number of molecules. You’ve heard about some of
these. In our nomenclature, this is a cytotoxic T-cell (TC), and this is a helper T-cell (TH). In both
cases, the T-cell receptor on the T-cell is engaging a complex of the MHC molecule (in this case a
class I molecule) and a peptide antigen that is bound to it. One of the points he will emphasize is
that when the MHC molecule is made, one peptide is bound to it the whole time.
When it comes to the peptide being presented, it can originate from something foreign (like bacteria
or virus) or from self. If this ends up being a high affinity interaction, the T-cell will become
activated. If it’s a cytotoxic T-cell, the TC cell will kill the target cell. So it’s a biological response of
seeing something foreign.
In the case of the helper T-cell, it’s the same sort of engagement, except the TH -cell is encountering
an MHC class II. If that interaction is high affinity, the TH -cell becomes activated and secretes
cytokines such as interleukin II. It’s a different response.
So, low affinity interactions lead to no response. Most of the time T-cells are going around
surveying MHC molecules and making decisions as to whether or not they should become activated.
Most of the time, they encounter self antigen-containing MHCs, and so there is no activation.
5. If you’re looking at this, the macrophage is on the right and the T-cell on the left. When this TH cell is
engaging the macrophage, hundreds of T-cell receptors are engaging hundreds of MHCs on the
surface of the macrophage. There is a threshold for activation of the T-cell. Most people think that
the threshold is on the level of tens or hundreds. If you look at a textbook, it seems like only one
interaction is needed, but really it’s much more to exceed the threshold to see a response.
6. The types of T-cells. There are actually many more than this, but this gives us a general background.
Cytotoxic T-cells kill virally infected cells, cells containing cytosolic bacteria, and tumor cells. Helper
T-cells help activate macrophages to kill intercellular bacteria or activate B-cells to produce
antibodies. These are the cells that produce cytokines, and the cytokines activate macrophages or
B-cells.
The cytotoxic T-cells are the ones engaged via the class I MHCs. If they see something foreign, they
kill the target cells. In terms of our demarcations, cytotoxic T-cells recognize MHC class I, and helper
T-cells recognize MHC class II. Now there are exceptions, but for the purpose of this lecture, don’t
worry about it.
Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 2
7. This is probably a figure that you saw last hour, but I want to spend a little bit of time dealing with
this because it is important. So a class I molecule is made up or two subunits, an alpha chain which
contains the peptide binding site, and a beta-2 chain which has a completely invariant sequence.
The beta-2 microglobulin has the same sequence for all people. There is no variation.
The interesting thing about these MHC molecules (he means the alpha chains) is that they are
polymorphic. What that means is that there are sequence variations between individuals. We all
have different MHC sequences. In fact, MHC molecules were first characterized as transplant
antigens. It was clear that trying to do a skin transplant onto just anyone didn’t work because the
MHCs didn’t match. This is because all of our MHCs have polymorphic sequences.
OK, class II has two subunits, but in this case the class II molecule makes up the peptide binding site
using both subunits, not just the alpha.
8. If you were to look at the MCH class one molecule from the top down, what you would see is a sort
of platform with two alpha-helices sticking out like a mouth. It has a platform that has a sequence
where the peptide can sit into the “mouth.” The MHC molecules were first crystallized in the 80s by
Don Willey. The interesting thing about these molecules is that the peptide that binds in the mouth
of the MHC actually helps form the shape of the entire molecule. The MHC is not really folded until
the peptide sits in the binding site.
If you look at one particular MHC molecule, it has the ability to bind many different peptides. That is
a good thing because it allows us to be able to have many possibilities for presentation. If there is a
viral or bacterial infection, even though you have one particular sequence, each MHC could still
present a unique peptide sequence from the virus or bacteria. Each one of the receptors has the
ability to bind hundreds of different types of molecules. However, when it is being made, once it
acquires one of these peptide sequences, that peptide stays with the MHC for its entire lifetime.
Peptides do not bind and unbind to the MHC.
9. Now we start getting into the differences between class I and class II MHC. The class I peptide
binding site is made up of one alpha chain. The ends of the class I MHC are closed at both ends,
which means that small peptides can bind there. In class I MHCs, the size of the peptides that bind
in the peptide binding site is usually 8-10 amino acids. The peptides are not covalently attached to
the MHC molecule. They are bound via hydrogen and hydrophobic interactions, but they are strong
enough to keep the peptide in the peptide binding site.
Class II MHCs use both subunits to form the peptide binding site, and both ends of the mouth of the
binding site are open. The size of the peptides for class II MHCs can be much bigger than for class I
because of this open mouth configuration.
10. I want to give you a picture of what’s happening. This is human leukocyte antigen, that’s what HLA
stands for. For us, we have two different types each of HLA-DP, HLA-DQ and HLA-DR which all code
for class II MHCs. So these six sequences are usually unique to individuals. He says he might have
Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 3
one chain that matches somebody else, but for the most part everyone has different versions of
these molecules. HLA-A,HLA- B, andHLA-C are the class I molecules. Again, we have two different
As, Bs, and Cs, but they are probably unique to the individual (so we all have relatively unique MHC
class I and class II.)
11. One of the interesting things about the class I and class II is that, despite the differences between
them, the peptide binding sites look very similar. In spite of the fact that they are made completely
differently (the class I peptide binding site is made with one subunit, while the class II uses both
subunits), the two MHCs make a three dimensional structures that are very similar. That makes
sense because T-cell receptors have to engage these structures to be able to look at them. So the
class I and class II molecules look very similar.
12. If you were to look at a space filling molecule; again we’re looking straight down. Here is a class I
molecule. The amount of peptide that is being presented is very small. Remember that the peptide
is being presented by the class I molecule, and the T-cell receptor is trying to make the decision
based on the 8-10 amino acid sequence of the peptide. But the peptide is buried within the binding
site, only revealing three or four amino acids.
13. When we look at class II, we see the amount of peptide presented is much larger, but it still looks
like one continuous surface.
14. Once we had antibodies to these class I and class II MHCs, we wanted to figure out how the T-cell
receptor recognizes the antigen, and what is the nature of the antigen. One of the points I made
was that the peptides are not covalently attached to the MHC molecules. People used antibodies to
purify class I molecules, and then used high salt acidic conditions to elute the peptides out of the
peptide binding site. Then they took the peptides and performed mass spec to find out the
sequence of the peptides.
So from one particular group of class I molecules, they found that all of the peptides that came out
had nine residues. When sequenced, they found a glycine at position two, a proline at position
three, and a leucine or isoleucine (which are similar) at the last position. The last position for class I
MHC is often hydrophobic or basic, and it is an anchor position.
When they looked at a different class I MHCs, they found tyrosine as a constant position, and
isoleucine or valine (both hydrophobic nonaromatic) in the last position. All the other positions
didn’t seem to matter. What that said was that any particular class I molecule could bind any
peptide that had just those few residues that matched. So the specificity of binding is very low for
MHCs! Any class I molecule can bind hundreds of different types of peptides. Since we only have a
limited number of MHC molecules, it’s good that there are lots of possibilities in terms of peptides
that will bind to them. We can hence survey lots of different peptides with just a few MHC
molecules.
Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 4
15. The cartoon of this is shown. The anchor at position 2 seems to be critical for all class I MHCs, and
the last position seems to be an anchor as well. These keep the peptide embedded in the MHC
molecule. The peptide must be embedded at the ends because of the nature of the class I molecule.
If the peptide is a little bit big, it ends up bulging up because the ends have to be embedded.
(remember that class I MHCs have a closed mouth peptide binding site!)
16. An unusual aspect of this peptide binding is the fact that an MCH molecule has the peptide bound
deep within the MHC itself. Here is the surface of two class I molecules, with different peptides
bound. So the peptide only has eight or nine amino acids, and half of them are in fact buried! For
something to be foreign, it might only have one or two amino acid side chains that are different than
a self peptide. So you are asking the T-cell receptor to recognize something foreign even if it is
mostly buried! How can that happen based on only three out of nine residues present?
Until we had the structures, this didn’t make sense. The reality is that because the peptide is so
much a part of the structure of the MHC, the entire surface of the MHC changes with small changes
in the peptide. So the T-cell looks at the MHC with antigen bound as a whole, not just at the peptide
itself. The T-cell looks at the whole contour of the face of the MHC to decide if it is foreign or not.
The whole topology changes.
17. If we look at class II with the same scenario. We isolate class II molecules, elute the peptide with an
acid extract, and use mass spec to analyze the peptides. What we see is that now the peptide ends
do not matter (remember class II MHCs have an open mouth binding site). Here we see aspartic and
glutamic acid at a particular point, and some other points that seem sort of homologous. But even
the ones that are supposedly homologous aren’t that homologous. So class II exhibits remarkable
polymorphic ability to bind lots of different peptides. The positions at the ends don’t seem to
matter, and only a couple of residues within the sequence seem to matter. As long as the peptide
has reasonable affinity, it will bind to the MHC. This allows even more variability in the amount of
different peptides that can bind!
18. So we’ve talked about the MHC molecules, we’ve talked about the peptides, but now we have to
talk about where the peptides come from. What’s the origin of the peptides? Here’s our antigen
presenting cell and a class II MHC. We take an antigen presenting cell and just add an antigen like
cytochrome C and let them incubate for an hour. We then fix the cells with paraformaldehyde. This
freezes the surface of the cell. Anything that was on the surface can’t go away, and anything that
wasn’t on the surface can’t come out. So you fix the cells and then add a helper T-cell to the cells.
Can the T-cell become activated to proliferate or produce cytokines like IL-2? YES. So by incubating
an intact antigen that you added from the outside, the cell was able to process the antigen and
present it to the helper T-cell.
Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 5
If you do the experiment the other way, and fix the cell before adding antigen, the helper T-cell is
unable to activate. So a fixed antigen presenting cell that’s incubated with antigen can’t process the
intact antigen to a peptide that the T-cell can recognize. You must have a viable surface or the cell!
In this case, you fix the cells, and then add antigen that has already been fragmented by trypsin to
the fixed cells. Now the T-cell can be activated. What this says is that the cell must cleave the
antigen into peptides inside the cell, but if you cleave the antigen, the fragments can be presented
to the helper T-cell.
So processing is simply clipping the antigen into fragments.
19. One of the points I probably should have mentioned earlier is that class II MHCs are mainly found on
dendritic cells, macrophages, and B cells. Sometimes you can find them on other cells, but under
normal conditions, these are the only three types.
Class I, on the other hand, are found on all nucleated cells.
20. So the HLA A, B, and C are all on nucleated cells.
21. So now we’re going to get into the processing part. How do we generate these peptides? When
you listen to this, make sure you keep track of which process is associated with MHC class I and MHC
class II.
We’re going to start with degradation of intracellular proteins. All intracellular proteins are
degraded by a proteosome, which is a large proteolytic cellular machinery made up of lots of
different subunits. It’s based on the idea that proteins that become ubiquitin modified are
destroyed. Ubiquitin is like a red flag that demarks proteins for proteolysis by the proteosome. This
is the normal cellular machinery that is used by the cell to degrade old proteins. So all proteins
undergo this ubiquitin modification and degredation. Some proteins are short lived (minutes), and
other long lived (hours). All the cytosolic proteins are degraded by this ubiquitin system.
The immune system has taken advantage of this to use the ubiquitination process to degrade and
capture some of these cytosolic fragments. For class I, the class I peptides are literally coming from
this pool of ubiquitinated proteins that get clipped into fragments.
Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 6
22. Topology becomes an issue here, and I have a cartoon that illustrates the whole process. When we
degrade a cytosolic protein, it gets degraded into fragments of peptides which become associated
with class I MHCs. What I will show in the next slide is that the class I molecule is being made in the
endoplasmic reticulum, and the acquisition of the peptide occurs in the lumen of the ER.
We have to get the cleaved peptides from the cytosol into the lumen of the ER. This is done by a
transporter associated with androgen processing (TAP transporter). It is made up of two subunits
and uses ATP hydrolysis to transport peptides from the cytosol into the lumen of the ER.
We have to get the peptides to where the newly synthesized class I is being made.
23. Here’s a better topology picture. Here’s our cytosol, and the majority of substrates for the
proteosome (not shown with ubiquinin, but imagine it’s there) become fragments that are 8-10
residues. Other peptidases basically chew these up to individual amino acids. 99.9% of the
degradation by proteosome is used to generate new amino acids for new protein synthesis. A tiny
fraction end up being substrates for the TAP transporter to bring them to the lumen of the rough ER.
A number of chaperons, such as calnexin, calreticulin, tapasin (which is TAP associated protein)
make sure that the peptide becomes associated with the class I MHC. So it’s a sequence of events,
with the whole purpose being “let’s get the peptide associated with the class I molecule.”
Once the peptide is associated, the MHC can go to the surface to present the peptide antigen.
24. Here’s the overview, and what I’ll do is deal with the class I pathway, and then the class II pathway.
This is the big picture. With class I, we consider it an endogenous pathway. This means that the
antigens are being synthesized within the cell. The easiest way to remember this is to consider what
happens when a virus infects a cell. When a virus infects, it takes over the machinery of the cell.
The cell is normally making lots of different proteins, but when it becomes virally infected, almost all
the protein that gets made is viral protein. So that is endogenous synthesis. So a cytotoxic T-cell is
literally looking for virally infected cells. If a virus takes over the machinery, then some of the
peptides associated with the virus will end up being bound to Class I MHCs, which will migrate to the
surface. The cytotoxic T-cell will recognize the foreign peptides on the class I MHCs and will kill the
infected cell. This shuts down a virus factory.
So you would hope that all endogenous protein produced would be self. If it is not (if the cell is
tumor derived or virally infected), the cell will be recognized and destroyed by cytotoxic T-cells.
Recap: so proteosomes degrade protein to peptide fragments, which go to the TAP to be brought to
the lumen of the ER. Tapasin is a chaperone that puts the class I molecule near the TAP transporter
carrying the peptides to receive the peptide. Calreticulin and calnexin (not shown) are two
chaperones that are literally there to make sure that the MHC class I molecule is properly folded
with the peptide. Heavy chain + light chain + peptide = complete MHC class I. From there the class
Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 7
I goes to the golgi apparatus and then the cell surface. The peptide acquired in the ER is the peptide
is retains for its entire life (about ten hours). The MHC sits on the cell surface and waits to be
surveyed by the T-cells. The T-cells continually survey the cells, looking for aberrations that come in
the form of high energy interactions.
Class II: the class II has another chaperone called the invariant chain. It’s called this because the
sequence is invariant. The class II alpha and beta chains may have different sequences, but the
invariant chain is always the same, and performs two functions. It blocks the peptide binding site of
the class II. This is necessary because if the binding site wasn’t covered, the class two would bind
peptides intended for class I MHCs. The invariant chain also has targeting information that carries
the class II to the lysosome from the ER.
So, class II does not acquire antigens in the lumen of the ER, but rather is carried to the lysosome. In
the lysosome, the class II acquires exogenous antigens that have been brought in from the outside
via endocytosis or phagocytosis. The proteases (lysosomal hydrolases) that are in the lysosome
chew up the exogenous antigens to peptides, which can bind with the class II MHCs. Interestingly,
the invariant chain is susceptible to proteases, but the alpha and beta chains of the MHC class II are
not. So, inside the lysosome, the invariant chain is chewed up, allowing space for the peptide to
bind to the MHC. Another chaperone called HLA-DM facilitates removal of the last part of the
invariant (called the CLIP) and attachment of the peptide inside the lysosome. After assembly in the
lysosome, the class II molecule is transported to the surface of the cell.
Again, the peptide acquired in the lysosome is the only peptide that particular MHC will ever bind
to, and it is kept for the entire life of the MHC class II.
25. So putting it into a schematic. What you want to remember is that endogenous antigens and the
cytosolic pathway mean we are talking about MHC class I. Dealing with the endocytic pathway, we
have exogenous antigens that come in from the outside via endocytosis or phagocytosis and end up
in lysosomes, where lysosomal hydrolases generate peptides from the antigens. These peptides
associate with MHC class II and are presented on the cell surface.
Microbiology
08/27/2008
Antigen Presentation – Dr. Jim Collawn
Transcriber: Luke Powell
43:22
Page 8
26. If we look at this in terms of the HLA-DM, which I didn’t have in the other slide, here’s the class II
complex with the invariant chain covering it. When the class II gets to the lysosomal compartment,
the proteases chew up all of the invariant chain except the CLIP, which sits in the mouth of the
peptide binding site on the class II MHC. The HLA-DM is a chaperone that’s in the lysosome, and it
facilitates the exchange of peptide with the CLIP at the MHC binding site. If a cell lacks HLA-DM,
only MHCs with the CLIP still intact will be presented on the cell surface.
The name Human Leukocyte Antigen –DM makes it look like it’s a class II molecule just by the
nomenclature. The structure does look like a class II, but what we know is that it doesn’t bind
peptides. We don’t know how it facilitates the exchange.
HLA-DO is another chaperone that’s found in B-cells and he doesn’t care what we know about it. It
seems to modulate the effect of HLA-DM.
27. What do you need to know? What I tried to emphasize was:
Where do the antigens come from for class I MHCs (self proteins, endogenous)
What are the proteases involved for class I (proteosome)
What are the sizes of the peptides and how did they become associated with class I (8-10 amino
acids, association by tapasin in the lumen of the ER)
Know about the chaperones (tapasin, calnexin, calreticulin, invariant chain, HLA-DM
The source of the antigens for class II (exogenous by endo or phagocytosis)
Antigen processing requirements (viable cell membrane, lysosomal hydrolases in lysosomes)
The types of T-cells involved (class I is cytotoxic T-cells, class II is helper T-cells)
What’s the role of the invariant chain? (to keep peptides from binding class II MHCs until they get to
the lysosome)
HLA-DM facilitates the exchange of CLIP with peptide in the lysosomal compartment for class II
MHCs.