Transcribed by Kevin Lin Lecture Date: July 17, 2014

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[General Pathology] [7] [Immunology V] by [Dr. McCutcheon]
[T Cell Development (last 4 slides), T Cell Activation (1/2)]

[Dr. McCutcheon] To start today, I have good news-good news & good-news
bad-news. Which do you want? Good news-bad news first? Well, so the good
news-bad news, I spent an hour in IT yesterday trying to get them to figure out why
my computer will not always play movies and so I played stump the IT guys and
the good news is I won. The bad news is I won. So their various suggestions were
that Windows 8 should kill itself or that I should get a different adapter. And I
pointed out (since Im a scientist) that if the adapter were the problem, it should
never work. And they tried to explain why it did and I said, but that doesnt make
any sense either. They said well, try that anyway because we cant think of anything
else. So my hypothesis at the moment is that if I leave the movie small, theyll play,
and if I make them big, they wont. So were going to try that today, were doing an
experiment.
The good news-good news is there are three lectures in this series that are
about concepts that are incredibly difficult. Were finishing the second of three. So
youre through the worst of it. The B cell development is also a concept lecture, but
B cell development has the same principles as T cell development. So its a variation
on the same lectures. So were almost through the most difficult part of this topic.
So have faith. So if I back up a couple of slides.


[20] [Positive selection of : T cells by cortical epithelial cells in the thymus]
[Dr. McCutcheon] So weve generated T cell receptors. The generation is
completely, totally, and entirely random. And because there are a limited number of
MHC molecules for any given individual, we have to find the T cell receptors that
bind to the MHC molecules that you got. That process is called positive selection. So
the T cell receptor, thats a double positive T cell. The T cell binds to both HLA Class
I and HLA Class II as it rolls along the arms of the dendritic cell. If the T cell receptor
can bind to an MHC Class I moleculeso its only binding to onethat gives us a
moderate affinity interaction. That T cell gets survival signals and it lives. Every T
cell that cant do that does not get survival signals and it dies through apoptosis.
And what other kind of cell is in the thymus besides thymocytes and T cells and
epithelial cells? Macrophages. So what are the macrophages there to do? Eat the
phagocytic bits.
So now weve picked out T cells that have receptors that recognize your HLA
molecules, but theyre your HLA molecules.

[21] [Double-positive thymocytes]
[Dr. McCutcheon] So we need to--. So our T cell binds, it gets positively selected, it
loses a receptor and now its asomewhere along in this process, it goes from a
double-positive to a single-positive.

[22] [Negative selection of : T cells by dendritic cells, macrophages, and other
cells in the thymus]
Transcribed by Kevin Lin Lecture Date: July 17, 2014

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[Dr. McCutcheon] And so now we need to make sure, since it is you that its binding
to, that it doesnt bind too well. And thats t-o-o well, and thats a high avidity
interaction. If the T cell binds with many receptors, binding to many HLA molecules,
it gets killed through apoptosis. That process is called negative selection. Okay? If
the T cell only binds to a few receptors, thats a low avidity interaction. It doesnt
get killed. So then it becomes a mature positive T cell. Single-positive T cell.
Alright? Failure to bind and get positively selected is not negative selection.
Negative selection is a second active process that occurs after positive selection.
The mechanism of cell death in negative selection is apoptosis.

[23] [Maturation Events]
[Dr. McCutcheon] And that is called central tolerance. So you get lots and lots and
lots of receptors binding and that is called crosslinking. An immature T cell and all
of the T cell in the thymus are immature T cells. Crosslinking causes apoptosis. And
this is the mechanism for central tolerance, apoptosis. And so any T cell that is
going to be self-reactive, meaning its going to bind to your host cells, gets killed off
in a central developmental organ, the thymus. And that is called central tolerance.
Okay? Mechanism of central tolerance is apoptosis.

[extra slide] [Possible Diversity]

[Dr. McCutcheon] So we had cell death because the T cells couldnt make a beta
chain. We had cell death because the T cells couldnt make an alpha chain. We had
cell death because the T cell that made an alpha-beta chain, couldnt bind to your
HLA molecules. We had cell death because the T cells that made the alpha-beta
chain and were positively selected bound too well, t-o-o well, to your cortical, or
sorry, medullary thymic epithelium. And so theyre negatively selected. So we have
a lot death going on in the thymus, to the point that of the thymocytes that develop,
97% or so die. 97% of the cells that hit the thymus and try and become T cells die.
Why would we make such a wasteful system? This is the number of possible T cell
receptors that can be generated. 16 zeroes. I dont know what you call that.
Alright? So there are ten to the 16th possible T cell receptors that you can generate
through this process. Okay? So did anybody actually read the email that I sent? So
for everybody who is frustrated with me because I go through details too quickly, if I
think the details are important, there is a slide that says that. Okay? In order to
understand concepts, you have to provide details, but memorizing the details is not
going to teach you the concept. So what you should be doing rather than going
through the podcast and making sure that youve written down every single solitary
detail, you should be pulling out the study guides and answering the questions on
Transcribed by Kevin Lin Lecture Date: July 17, 2014

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the study guides because thats whats important. So if theres no question on the
study guide asking you about a detail, then the point of the detail is to help you
understand the concept. Okay? So use the study guide. Sir?
[student] Is there a zero missing? [screen was only showing 15 zeroes]
[Dr. McCutcheon] Sorry. Yes. Okay? So use the study guides. Because the point of
this is not to memorize every single solitary detail. That is not going to tell you how
the immune system works. You need to figure out the concepts. And the reason
youre going to do this is because if you cant figure out the concepts, you cant do
differential diagnosis. Okay? And if you want to be a competent dentist, you have to
be able to do differential diagnosis. Its not something that you going to get a list
and youre going to pick something off the list. You have to be able to put material
together from a number of sources to come up with the diagnosis that youre going
to do. And its the same process as figuring out the concepts for immunology. So
learning to do this is what is the foundation for learning to do a differential
diagnosis. So if youve never tried to think this way, and a lot of you have never
been challenged like this. Its new and its scary and its hard, but once you learn to
do this, then you can do it for everything else. So its worth putting in the time into
this. Alright. So thats T cell development. Now T cell development, most of it has
happened while you were still under 10. Remember because the thymus involutes.

[T CELL ACTIVATION ppt]

[1] [Review]
[Dr. McCutcheon] And so whats left, umm--. So T cells have decades long lifespans.
And by the time youre your age and really by the time youre my age, youre not
making very many T cells. Okay? So T cell development. The cells in the thymus,
they are immature thymocytes. When they exit the thymus, they exit the thymus as
mature single-positive T cells. These are the T cells that are participating in the
immune response that we have going on. Okay? Thymic development happens in a
different place. It happens in the thymus and it happens at a different time. So its
both spatially and temporally distinct from the immune response that were talking
about. If you ever see double-positive thymocytes in the bloodstream, you have
cancer. Okay? So mature, theyre mature, we call them mature, nave nave
meaning that they have not ever encountered antigen yet single-positive T cells.
And thats what exits the thymus. Thats what you find in the peripheral circulation.
Mature, nave single-positive T cells. So now that weve made our T cells, were
going to go back to the immune response. Okay? So we did something thats not a
part of the immune response. Were going to go back to the immune response.
Weve done antigen processing and presentation on either HLA Class I and HLA
Class II for an intracellular pathogen, or HLA Class II for an extracellular pathogen.
So now the mature nave T cells in the circulation are going to try and get going.
And thats what well pick up today.
So in what organ do T lymphocytes develop? Thymus. Whats the general
principle of making a T cell? Starts with a G, two words.
[student] Selection?
Transcribed by Kevin Lin Lecture Date: July 17, 2014

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[Dr. McCutcheon] Not yet. Gene rearrangement. Okay? The principle of making a
T cell is gene rearrangement. Whats the point of positive selection? To fish out the
T cells that bind to your MHC. Whats the point of negative selection? Get rid of T
cells that bind with high avidity. What exits the thymus? What do we call that?
Whats the phenotype of that? So its single-positive, but what does that mean? CD8
or CD4, but what else is on a T cell? T cell receptor and? T cell receptors dont have
peptides. HLA has peptides. So you have T cell receptor, you have to have
something else for the T cell receptor. CD3. Every T cell receptor has a CD3
molecule and CD4 or CD8. So TCR positive, CD3 positive, CD4 positive. Or TCR
positive, CD3, positive, CD8 positive. One or the other.

[2] [T cell Activation]
[Dr. McCutcheon] Okay, so our T cells have joined the immune system. Theyre in
the periphery.

[3] [untitled picture of a leg]
[Dr. McCutcheon] We need to get them going. Okay? So, whats happening is
wherever the infection is, the dendritic cells have either phagocytosed or been
infected by the pathogen. They mature and when they mature, they pull up stakes
and they go off into the afferent lymphatic, so they can drain into the nearest lymph
node. And while theyre doing that, theyre doing antigen processing on HLA Class I
or HLA Class II.

[4] [Dendritic cells in peripheral tissues]
[Dr. McCutcheon] So, in the tissue, you can see the dendritic cell body and you can
see bits and pieces of the arms of the dendritic cell and theyre staining here for the
endocytic vesicles. So these things in the periphery what dendritic cells do is theyre
phagocytic. Their job is to eat up all the apoptotic bits of the tissue that theyre in.
Theyre not good antigen processing or presenting cells. They dont secrete
cytokines. Once you hit them with IL-1 and pathogen, they round up. So they pull in
all of their arms and they become this nice, round, fluffy thing. And thats whats
migrating through the afferent lymphatic. And at that time, the dendritic cell has
stopped phagocytosis entirely. Its put a bunch of HLA Class I and/or HLA Class II on
its cell surface. Its put a number of other proteins on its cell surface that are
necessary to get T cells activated. And when it gets into the lymphatic tissues, it
spreads out again. So it gets back to being a many-armed thing. And you can see
[bottom right picture], this is the dendritic cell and theyre huge compared to T cells.
So we see six T cells stuck on the arms of the dendritic cell. So the T cells are much
smaller than the dendritic cells. So the T cells are going to bind to the arms and the
body of the dendritic cell, looking for HLA Class I and HLA Class II.

[5] [DC Initiate Adaptive Immunity]
[Dr. McCutcheon] Everybody cross your fingers... [trying to make movie work]
Now this is supposed to work. Ahhh~. Okay. I give up. Well, alright, lets try one
more thing. No. Okay. Alright, so its doing the wrong thing. Alright, so my
hypothesis is wrong. Keeping it small does not do us any good. I give up, so What
Transcribed by Kevin Lin Lecture Date: July 17, 2014

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Ill do, these movies are apparently available on youtube. I will dig up the link and
so you can see the number of the movie in the slide and then you can just look at it
on youtube. Okay? And Im sorry about that, but as I said, I once stumped the IT
guys, so, Im kind of stuck.

[6] [Immature dendritic cells in peripheral tissues]
[Dr. McCutcheon] So in an immature dendritic cell, we have a little bit of HLA Class
I and a little bit of HLA Class II. We have some adhesion molecules. These are the
molecules that let dendritic cells move throughout the circulation. When the
dendritic cell matures, it gets lots of HLA Class I, lots of HLA Class II. It gets co-
receptors that well talk about in a minute, B7.1 and B7.2. It gets a protein called
DC-SIGN. It starts secreting a cytokine called CCL18, which recruits nave T cells to
the dendritic cell and it has adhesion molecules. So here were looking at the
dendritic cell and although theyre not all in the same plane, so it looks like theyre
detached. And we can see the some of the many arms of the dendritic cell. So it
changes-- it stops phagocytosis and it increases greatly the expression of HLA Class I
and Class II. It starts secreting DCCK and it has a number of other proteins that get
expressed. DC-SIGN, B7.1, B7.2.

[7] [Routes of antigen processing and presentation by dendritic cells]
[Dr. McCutcheon] So, pathogen can be presented on both HLA Class I and HLA Class
II. For bacteria, its always going to be receptor-mediated endocytosis. And thats
presented on HLA Class II. There are some kinds of other soluble antigens that can
be micropinocytosed [she said micro-, but slide says macro-]. So, if the bacteria
are secreting toxins, those can sometimes get micropinocytosed by the dendritic
cell. Sometimes, viruses can be micropinocytosed by the dendritic cell, not always.
And so those if theyre in a vesicle, they will end up being presented on HLA Class II.
Viruses will infect the dendritic cell. They end up in the cytosol that makes the
peptides go to the ER. They end up being presented on HLA Class I. Dendritic cells
also have a mechanism to bud off pieces of the ER and form vesicles. And what will
be in the vesicles are viral particles. So viral peptides, now its a vesicle. So if the
viral peptides are in the ER, they bind to HLA Class I. If the viral peptides are in a
vesicle, they bind to HLA Class II. And thats a shuttle mechanism and only dendritic
cells can do that. Other cells cant do that, just dendritic cells. So there are a number
of different ways that dendritic cells can get viruses into vesicles so that they bind to
HLA Class II. Viruses infected in the cell will bind to HLA Class I. So viral particles,
anything thats going to be an intracellular pathogen must be presented on both HLA
Class I and HLA Class II. Things that are living on the outside are only ever in a
vesicle. Theyre only ever presented on HLA Class II.

[8] [Unique Feature of DC]
[Dr. McCutcheon] So, dendritic cells are unique in their ability to activate T cells
because they can be infected by all viruses. Macrophages and B cells, which also
have HLA Class II, cannot be infected by any virus, except a macrophage or B cell
specific virus. The micropinocytosis is fairly unique to dendritic cells. CCL 18, used
to be called DC-CK, but its a chemokine specific for nave T cells. So the dendritic
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cell recruits the nave T cells to where the dendritic cell has gone to in the lymph
node. And then the shuttle mechanism is the cross presentation that lets things that
are inside a cell end up in a vesicle, so that you can present on HLA Class II. Alright?
So dendritic cells, their function is to get T cells started and they have a number of
ways to do this because they can present on HLA Class I and HLA Class II.

[9] [Lymph node]
[Dr. McCutcheon] So, the dendritic cells have come into the lymph node through
the? Afferent lymphatic. Now, the point of the lymph node, whats the major
function of the lymph node? Its a meeting place and whats the meeting of?
Dendritic cells and T cells. So how do the T cells get into the lymph node? Through
the bloodstream. And whats the structure they come in through? High endothelial
venule. So the dendritic cells are coming into here. The T cells are coming in from
here. Okay, and where do the T cells go in the lymph node? All this stuff in May, its
not fair. Pericortex. Okay? So the T cells end up in the pericortex, and if we want
the T cells to meet the dendritic cells, where do the dendritic cells have to go? They
go to the pericortex.

[10] [Naive T cells can enter lymph nodes in the afferent lymph as well as from the
blood]
[Dr. McCutcheon] So, we have our afferent lymphatics, into the nearest lymph
node. We have our T cells coming in through the HEV. T cells can also come from an
upstream lymph node through the bloodstream. And so, the T cells and the
dendritic cells end up in the pericortex of the lymph node.

[11] [Circulating T cell enters the high endothelial venule in the lymph node]
[Dr. McCutcheon] And there, theyre going to meet. Now, the way that the T cells
are moving through the blood is through this series of adhesion molecules. So, we
saw the movie where T cells roll. Okay? And with the T cells rolling, they have
adhesion molecules on the T cell that bind to receptors on endothelium. And that
causes this rolling interaction. As the T cells reach the site of infection or the lymph
node, the binding tightens so the T cell stops rolling. And that allows it diapedese
out of the circulation into the lymph node or out of the circulation to the site of
infection. Its the same process. So T cells always roll. So do B cells. Everything Im
saying about T cells is true about B cells. T cells always roll along. They dont just
get pushed. They have to have receptors binding ligands binding receptors binding
ligands. Its a loose interaction, so thats why they get pushed forward until you
reach the spot where theyre supposed to diapedese, and then you add receptors.
And that allows the T cell to diapedese from the vessel containing, from the
circulatory vessel its in, to the structure its going to. And that can be into a lymph
node if its nave T cells. It will be a site of infection if its an activated T cell.

[12] [untitled interaction between T cell and dendritic cell]
[Dr. McCutcheon] So of these interactions, the only one that matters to you is LFA-1
on the T cell, and thats for Lymphocyte Functional Antigen, binding to ICAM on the
endothelial or dendritic cell. So dendritic cells also have ICAM. Thats Immune Cell
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Adhesion Molecule. And there are a number of CAMs, so the first letter changes
depending on what tissue it is. We dont care about the others. So LFA-1 binds to
ICAM. Alright? LFA-1 on the T cell binds to ICAM on the dendritic cell. And thats
the first interaction. And thats a low affinity interaction, so the T cell is not going to
just stick in one spot on the dendritic cell. The T cells are going to roll along the
dendritic cell. And as the LFA-1 is binding to the ICAM and letting the T cell roll,

[13] [T cell initially binds dendritic cell through low-affinity LFA-1 : ICAM-1
interactions]
[Dr. McCutcheon] we have the T cell receptor trying to check out peptide on MHC.
Okay? So the T cell receptor is checking out peptide on MHC as the LFA-1:ICAM
interactions are letting T cell roll. And whether its checking out peptide on MHC
Class I or whether its checking peptide on MHC Class II depends on whether the T
cell is a CD4 T cell or a CD8 T cell. So if its a CD8 T cell, then the T cell receptor is
binding to peptide on MHC Class I. If its a CD4 T cell, the T cell is binding to peptide
on MHC Class II. So the T cell receptors on the T cell are binding, or trying to bind to
the MHC Class I peptide complex, or the MHC Class II peptide complex. Were going
to stick with Class II now for the minute just because I cant keep saying both of
them all the time. So 99.9% of the time, or maybe even less than that, the T cell
receptor for that particular T cell is not going to recognize a peptide on MHC. Okay?
Because every T cell has a different receptor. So most of the T cells, they come, they
check out the dendritic cell, they leave. Alright? And they dont participate in this
particular immune response, so we dont care about them. Nothing happens to
them. I mean, they just go back out into the circulation through the HEV. Alright?
What were going to have is per infection, were going to have one and three T cells
that actually recognize the peptide. So it only takes one T cell to give you an
immune response. So between one and three is sort of the real number. It only
takes one. So that one T cell, where the peptide MHC complex is recognized by the T
cell receptor. So the T cell, you know, one receptor on the T cell binds to one
peptide MHC complex. And that causes signaling, and the signaling results in a
change in conformation in the LFA on the T cell. So if you happen to be the one T
cell that happens to have a T cell receptor that can bind this particular peptide
presented by the MHC, that sends a signal through CD3, that changes the
conformation of LFA. Okay? So, T cell receptor binds, signal through CD3, change
conformation of LFA. LFA tightens binding. Okay? That means the T cell cant roll
anymore.

[14] [The co-stimulatory molecule B7 on the dendritic cell binds CD28 on the
naive T cell]
[Dr. McCutcheon] So the T cell, we see our signal here. The T cell binds through the
T cell receptor. Signals through CD3. LFA changes conformation that tightens
binding and the T cell cant get away now. Its stuck there. And because its stuck
there, it gives it time to move other T cell receptors to the point of contact. So now,
other T cell receptors are going to try and bind other MHC molecules in the area.
And it turns out, that as soon as you get 80, a real number 80, T cell receptors bound
to 80 MHC peptides that is the first signal of activation of the T cell. So it doesnt
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take very many T cell receptors, right? You had a hundred thousand Class I
molecules, you need 80. Doesnt take very many T cell receptors. So the first signal
is through the T cell receptor, CD3. The T cell receptor itself, remember, cant do
any signaling. And then the dendritic cell has a co- stimulatory protein. The
dendritic cell has a co- stimulatory protein called CD28 or B7. Or Im sorry, called
B7. B7 on the dendritic cell binds to CD28 on the T cell. So the first interaction of
the T cell has to be the T cell receptor binding. And if the T cell receptor doesnt
bind, those T cells are out of there. They have nothing to do whatsoever with the
immune response. If that T cell receptor for the one T cell that we have binds, then
you get more T cell receptors binding to more MHC peptide complexes on that
dendritic cell. And then we get a second signal, and this is B7 on the dendritic cell
binding to CD28 on the T cell. And thats called a co-stimulatory signal. And that is a
critical signal because without it, the T cell gets turned off. Okay? So the second
signal B7 through CD28 is called the co-stimulatory signal. That is a critical signal
because without it, the T cell gets turned off. Alright? So this is--. We had central
tolerance where we killed off T cells. This is the mechanism of peripheral tolerance,
where T cells that can bind, a receptor to a cell, but the cell doesnt have the co-
stimulatory protein, those T cells get shut off. So the only cells that have B7 are
dendritic cells, B cells, and macrophages. None of the other cells in your body have
the co-stimulatory proteins.
So lets say you had an infection in the pancreas. And so you send a bunch of
CD4 T cells off to the pancreas. And there, theyre binding to proteins on islets of
Langerhans cells, and what do you think the most common peptide in an HLA
molecule on the islet of Langerhans cells would be? What do the islets of
Langerhans cells make? So what do you think the most common peptides on the
islets of Langerhans cells is? Insulin. Okay? Do you want our T cells to bind insulin
on the islets of Langerhans cells? No. So the islets of Langerhans cells dont have co-
stimulatory molecules. So if the T cell binds to the insulin peptide on the islets of
Langerhans cells, and doesnt not get co-stimulation, it gets anergized. Its not killed,
but its no longer able to function. And that is called peripheral tolerance because
its happening in the periphery, its not happening in a central developmental organ.
And that is the mechanism that prevents us from type I diabetes. And when you get
type I diabetes, you somehow or other break the tolerance, and those T cells now go
off and killed your islets of Langerhans cells. Without islets of Langerhans cells, you
dont make insulin. If you dont make insulin, you have type I diabetes.

[15] [untitled movies: 7-1_TCR_Signaling.mov & 7-3_CD28_Costimulation.mov]
[Dr. McCutcheon] Okay, were going to try--, Ugh. Im being stubborn, I know. And
were on duplicate And you cant see [still doesnt work]
Alright. Im going to give up, sorry. So, Ill put the link on there, and youre going to
have to look at the movies on your own.

[16] [untitled dendritic cell (yellow) in contact with Lymphocytes (blue)]
[Dr. McCutcheon] So to help you understand that this is really a 3-D thing, and not
a 2-dimensional activity. Here we have a dendritic cell, okay? So this is all dendritic
cell. And you can see how much surface area the lymphocyte has in contact with the
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dendritic cell in a 2-dimensional. And there is a movie that has a beautiful 3-
dimensional view of the contact between lymphocytes and dendritic cells, then
between lymphocytes and other cells. But you can see the 3-dimensional and the
amount of surface area thats covered, that I cant show you because the movies are
now acting up. Ugh.

[17] [Co-stimulatory signal and specific signal]
[Dr. McCutcheon] So, you get the signal through the T cell receptor and if you get a
signal through the co-stimulation, which is B7 on the APC or dendritic cell to CD28
on the T cell, that activates the T cell. If you signal through the T cell receptor, but
theres no B7 on the cell that has the MHC with the peptide, you dont signal through
the CD28, and that permanently shuts off the T cell. Hopefully permanently. And
thats anergy. If the T cell just has CD28 binding to the B7 on the APC, and the T cell
receptor doesnt bind at all, nothing happens. That T cell doesnt get activated, it
doesnt get anergized, it just leaves. Okay, so three things can happen. If you have
both T cell receptor binding and co-stimulation, you activate the T cell. If you have
only T cell receptor binding and no co-stimulation, you anergize the T cell. If no T
cell receptor binds and only theres co-stimulation, then the T cell is left alone and it
leaves the lymph node to go look for something else to bind to. And 99.9-some% of
the T cells, thats what happens, that last pathway. So its only the one T cell that
binds through both peptide MHC binding to the T cell receptor and co-stimulation
that gets activated.
[student] What does anergy mean again?
[Dr. McCutcheon] Anergy is a permanent shutting off of the T cell. And theres
actually a movie that I was going to show that shows how that happens. The T cell
starts--. So all of this happens through phosphorylating second messengers. Okay?
And the second messenger starts to phosphorylate and they form this structure.
And then if theres no co-stimulatory signal, the next step in the structure,
recruitment doesnt happen. And the T cell just shuts off. Alright?

[18] [untitled movies: 9-4_TCR-APC-Interaction & 9-
3_Visualizing_T_Cell_Activation,mov]
[Dr. McCutcheon] So all of this is happening through second messengers. So thats
the movie that has the 3-D image that Im not going to try and show because itll just
shut things off.

[19] [Summary]
[Dr. McCutcheon] So in summary, T cell activation. First LFA binds to ICAM. Then
the CD4/CD8 determines whether the T cell is going to bind to HLA Class I or HLA
Class II. The T cell receptor has to bind to the MHC Class II peptide, and that is the
critical, that is the most important step. If the T cell receptor doesnt bind, that T cell
is not going to play a role in this infection, and it will just go off into the next lymph
node. So were only talking about one to three T cells that are actually going to do
something. LFA tightens binding. CD28 binds to B7 on the dendritic cell. If theres
no CD28 binding to B7, then you get peripheral tolerance, the mechanism of which is
anergy. And then. So the important parts are the MHC Class II binding to the T cell
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receptor. And if I can get somebody to come up here and hold hands with me, Ill
give you a visual demonstration. Cmon.
[student] For exam purposes, will you use just the B7 or are you going to
interchange the CD80 and B7?
[Dr. McCutcheon] Uhh, Im probably. I will use B7. For all of the chemokines that
have changed their name from IL-whatever. Those you have to use the new name or
it will be wrong. Okay?
[student goes up on stage] So youre T cell and Im a dendritic cell and youre
going to hold up your hand like this. So that the first interaction--. No, actually. Im
going to be this; youre going to be that. This is LFA binding to ICAM. Okay? So its a
light interaction, right? And you can move around, right? Yes. So now, with LFA
binding to ICAM, with this loose interaction, now the T cell receptor is going to try
and bind. And were a T cell receptor that has a match, right? So theres binding
here. And at this point, the LFA tightens and changes conformation. Now move
around. [class laughs] N-not the T cell receptor. [class laughs] but the point is once
the LFA tightens binding, the T cell isnt going anywhere. So its going to have to
stay there and get all of the other T cell receptors to bind to all of the other HLAs.
[*clap clap clap*] So thats a critical step. The LFA tightening binding, so the T cell
stays on the dendritic cell and gets the time to have the T cell receptors bind to HLA
molecules on the dendritic cell. Now, the importance of CD28 binding to B7 is
because once that first interaction has occurred, okay. If you get the second signal
that T cell is activated, and its being at the top of the roller coaster when the carts
lets go of the chain, and youre going to go off down the pathway, do you think you
can stop that when youre half way down? Once the T cell hits that point, it is going
to do what it is going to do, and there aint a dayum thing that you can do to stop it.
Okay? So once you get that signal through B7 binding to CD28, the T cell is going to
be activated. It can do everything else it needs to do on its own, and you cant stop
it. So this is the critical point. Because without it--. If you dont have the CD28
binding to B7, you get peripheral tolerance, the mechanism of which is anergy.

[20] [In the resting T cell the ITAMs are not phosphorylated]
[Dr. McCutcheon] Okay? So this happens, you signal through CD3. This all occurs
because youre doing phosphorylation of second messengers. You notice I have not
said the names of any of the second messengers. Okay? You dont need to know
their names. You do need to know that its all done through phosphorylation.
Alright? So, T cell activation, phosphorylation of second messengers, they go off into
the nucleus, they start transcribing things. What do they transcribe?

[21] [Naive T-cell recognition of specific antigen presented by a dendritic cell
initiates pathways of signal transduction that lead to clonal expansion and
differentiation]
[Dr. McCutcheon] So heres the pathway you turn on all of these things and
eventually you end up in the nucleus. And you start with transcription factors and
you change the pattern of gene expression. The names of all of these things you do
NOT need to know. You do need to know that youre going to turn on some
transcription factors and thats going to change the pattern of gene expression.
Transcribed by Kevin Lin Lecture Date: July 17, 2014

11

[22] [IL-2]
[Dr. McCutcheon] And so, heres what happens. We have several forms of the IL-2
receptor and nave T cells have IL-2 receptors. And the form of the IL-2 receptor
that nave T cells have is the beta-gamma c chain. And by IL-2, and theres always a
low level of IL-2 around in lymph nodes. IL-2 binding to this IL-2 receptor on nave
T cells gives you survival signals. So that keeps the T cell alive. Alright? When the T
cell becomes activated, okay, you got binding through both T cell receptor and CD28
binding to CD80 or B7, one of the genes that gets turned on is the IL-2 receptor
alpha chain. And so now the IL-2 receptor changes form. It goes from this beta-
gamma c form, which is a moderate affinity for IL-2 that gives you survival signals.
This [top right picture, alpha-beta-gamma] form of the IL-2 receptor is high affinity.
And when IL-2 binds to the high affinity form of the IL-2 receptor, that causes cell
proliferation. So the first thing that the T cell does is it turns on the expression of
the IL-2 receptor alpha chain. Now you get an alpha-beta-gamma IL-2 receptor.
And that form of the IL-2 receptor causes proliferation when IL-2 binds it. The T cell
also turns on the gene for IL-2. So the change in gene expression. One, it turns on
the gene for IL-2 receptor, the high affinity form of the IL-2 receptor. Two, it starts
secreting IL-2. So the IL-2 gets secreted by the T cell. It binds to the IL-2 high
affinity form of the receptor on the T cell. That T cell is not going to proliferate. And
every daughter T cell of that T cell will have the identical receptor to the parent. We
call that clonal proliferation. Say that fast three times. Alright? So, the moderate
affinity form of the IL-2 receptor, always on a T cell, gives the T cell survival signals.
The activated T cells, and only the activated T cell is going to make the high affinity
form of the IL-2 receptor, and its going to secrete IL-2. So when IL-2 binds to the
high affinity form of the IL-2 receptor, that causes clonal proliferation. That
daughter T cell is now going to divide and its not going to two. Its going to give you
about ten thousand T cells. And thats the reason we only need one. One activated T
cell will give you somewhere in the vicinity of at least ten thousand daughter T cells.
And theyre going to what theyre going to do, and you cant stop them at this point
in time. Which is why we have such heavy regulation on making sure we dont get T
cells activated against the wrong thing because once they are, you cant stop them.
So, the change in gene expression is adds an additional chain for the IL-2 receptor
that gives you a high affinity form of the IL-2 receptor. You transcribe and then
secrete IL-2. IL-2 from the T cell binds to the IL-2 receptors on the T cell. So this is
an autologous loops, self-binding. And that causes clonal proliferation.

[24] [T cell Proliferation]
[Dr. McCutcheon] Okay, change affinity. Notice my slide does not have the names
of the chains of the IL-2 receptor. So do you think those are important details? No.
Okay? Knowing that it changes affinity, yes. Okay? IL-2 secreted by the T cell. IL-2
binding to the IL-2 receptor causes clonal proliferation. This T cell is now going to
make huge quantities of IL-2. The lymph node is filled with nave T cells. Is the IL-2
from the activated T cell binding to the IL-2 receptors on the nave T cells going to
activate them? Will it make them proliferation? No. Why not? They have the wrong
form of the receptor. So only the activated T cell with the high affinity form of the
Transcribed by Kevin Lin Lecture Date: July 17, 2014

12
receptor can proliferate when the IL-2 binds to the IL-2 receptor. All the other T
cells just get survival signals.

[25] [Differentiation pathways]
[Dr. McCutcheon] And at this point, the T cell is now going to run into other
cytokines, and its going to differentiate into one of three kinds of T cells. These are
CD4 T cells. If theres TGF-beta. the T cell will differentiate, will activate the FoxP3
transcription factor, and it will become a iT-reg. That stands for inducible T-
regulatory cell. So TGF-beta, you induce the transcription factor, FoxP3, you end up
with an iT-regulatory cell.
If the T cell runs into IL-12 or gamma-Interferon (IFN-gamma), you induce
the transcription factor T-bet. You end up with a TH1. CD4-positive TH1 T cell. IL-
12 or gamma-interferon, you induce T-bet, you end up differentiating into a TH1 T
cell. And FoxP3 T cells secrete TGF-beta and IL-10. TH1 T cells secrete a lot of IL-2
and Interferon-gamma.
If the T cell runs into IL-4, it turns on the transcription factor, GATA-3. You
end up differentiating off a TH2 T cell. And those secrete IL-4 and IL-5 and some
other cytokines. Alright?
So depending on the next cytokines the T cell runs into, tells you whether
youre going to get a TH1 or a TH2 T cell, or further in the infection, if youre going to
differentiate off an iT-regulatory cell. So most of the daughter T cells become either
TH1 or TH2. iT-reg show up a little bit later. And the function of an iT-reg cell is to
suppress the TH1 T cells.
Yes?
[student] Where did these IL-4 and TGF-beta, where did they come from?
[Dr. McCutcheon] Okay, so cytokines can be secreted by anything. So in the lymph
node, there are other cells that can be secreting either the pro-inflammatory
cytokines, which are the IL-12, IFN-gamma, IL-1, TNF-alpha. Or anti-inflammatory
cytokines, TGF-beta, IL-4, IL-5, IL-6. So they can come from other T cells that were
activated earlier. They can come from gamma-delta T cells, if the T cells are
migrating off to the site of infection as they differentiate. The gamma-delta T cells
can switch cassettes, so they can secrete pro-inflammatory, then they can secrete
anti-inflammatory, then they can secrete pro-inflammatory. So, and other cells can
secrete cytokines. So, there are always cytokines around. Alright?
So primarily at the beginning of the infection, we end up with TH1 and TH2 T
cells. So our T cell gets activated. It causes itself to divide, and you cant stop that.
And once it divides, you end up producing this army of daughter T cells.

[26] [Alternate CD4
+
T Cells]
[Dr. McCutcheon] And we will pick it up from here. Okay? So I will see you
tomorrow morning. The conferences, were going to go over the study guides. We
still do the study guides through T cell activation, which we will finish tomorrow
morning, for next week. And Ill put that up on Classes today.