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, 2014

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[General Pathology] [3&4] [Innate Immunity I & II] by [Dr. Weil ]

The Innate Immune Response
[67] [Tissue Macrophages=
Dendritic Cells]
[Dr. Weil] Intracellular pathogens start their innate immune responses w/
dendritic cells. Remember all of the tissues that compact the outer environment
have dendritic cells in them. In the skin, Langerhans in the liver Kuffer. Dr. Wishe,
called them tissue macrophages. The real name now is dendritic cell. You can see
lots of arms. Their job is to phagocytosis cells.
[68] [A Dendritic Cell]
[Dr. Weil] Scanning ECM showing you arms of the cells.
[69] [Intracellular Pathogens]
[Dr. Weil] Dendritic cells are unique in that they can be infected by any kind of
virus. Most of the time they are usually tissue specific. A Rhinovirus would infect
nasal epithelium but not epithelium on the skin. So the dendritic cells in their
locations can be infected by any kind of virus. When they are infected, the first thing
they do is secrete cytokines. Normally when the virus infects, the virus takes over
machinery making more virus DNA and coat proteins. The virus then ruptures the
cell and goes into ECM and infects a new cell. In dendritic cells, they start making
interferons, named b/c they interfere w/ viral replication. It fights back and forms
INF-alpha/ beta. The virus has done some viral replication but it cant overwhelm
the cell like it would in another cell. It would shut down replication. The IL-1 would
cause the dendritic cells to mature and they pull in all of their arms and go off to the
lymph node. How are they going to go to the lymph node? Right. So which
lymphatic? Alphabetical. Afferent. You always have to go through the afferent
lymphatic into the nearest lymph node. Why didnt the dendritic cell go back into
circulation? Thats not why. Why cant the dendritic cell go into the veins/ veunules?
Why? TNF-alpha. Very good. It has closed off the veins and the only place to go is
into the lymphatic.
[70] [None]
[Dr. Weil] The virus infects, you get second messengers, notably NFkB and AP-1
they cause formation of INF-alpha/ beta and production of INF-alpha/ beta-
receptors. The cell puts up the receptors and they get secreted and bind to the
receptors on the cells that created them. It prevents viral replication. Even though
its infected it wont be destroyed by the virus. Its an unique trait to prevent being
killed by the virus. Other virally uninfected cells, the INF-alpha/beta cells can bind
to those cells and prevent those cells form being infected. The virus will infect them
but then cant replicate. If the cell is already infected it cant make receptors since its
too busy making virus and then its going to die. Its going to leave the site of
infection and go off to the lymph node.
[71] [None]
[Dr. Weil] So, you get increased resistance to viral replication to any cell that has a
receptor to INFs. The dendritic cells make the INFs. Increased expression for ligands
for receptors on NK cells making them potent to kill virally infected cells. You can
also activate NK cells to kill infected cells. NK cells work on certain viruses.
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[72] [None]
[Dr. Weil] So thats where we are w/ this. Complement, that really gets started
against the extracellular pathogen. You get C3b that gives you opsonization. C3a that
starts the vascular changes. Neutrophils come along and start eating everything in
sight. Macrophages are monocytes that migrate into the area and differentiate into
the area and secrete cytokines. The dendritic cells are phagocytic until they mature.
The intracellular pathogen starts w/ the dendritic cell. Compliment except for C3b
doesnt play any roll at all.

Antigen Processing and Presentation
[1] [Review]
[Dr. Weil] So what cells are involved in the innate immune response?
Macrophages. Neutrophils. Dendritic cells. Thats pretty much it. Whats the role of
complement? Its for extra cellular pathogens. Thats not really the role.
Opsonization and MAC. Whats the role of C3b? Opsonization and it does viruses for
the B cells. And? C3b is the point where you start getting that expansion of the rest
of the complement pathway. Once you have C3b you will get MAC b/c each
C3bC3bbb complex and C4bC2aC3b complex can cleave 1000 more C3bs and 1000
C5s. Where does the immune response begin for extra cellular pathogens?
Complement. Intracellular? Whats the role of the DC cell? To get infected by viruses
and then not be killed by them.
[2] [None]
[Dr. Weil] The next chapter in the book. They go from the innate immune
response and then they jump to what happens at the end of the immune response
w/ an adaptive immune response. They talk about how B cells and T cells work.
They then go back and explain how B cells are developed and T cells are developed. I
dont understand how you dont understand how B/T cells are developed you know
how they work. Rather then going to go in the order the book does, were going to go
in the order that it happens. For those of you that have looked at the study guides, I
have the readings posted on the study guides. For the next thing that happens in the
innate immune response is that the dendritic cells have to talk to T cells. W/ innate
immunity going, we have to get T cells activated before anything else can happen.
Well talk about what dendritic cells have to do to get T cells going. Well then talk
about what T cells are. Before you can understand how they work you need to know
how they are developed. We are doing innate, then doing antigen presentation and
then T cells and how they are developed. Well come back to our infection, activate
them, drop out to B cell development, well come back to B cell function and then
well shut it off.
[3] [Introducing Pathogen to T Cells]
[Dr. Weil] We have to introduce our T cells to our pathogen. Where are the T
cells? No they are not in the thymus. They are in the lymph nodes. Where else would
they be? Spleen and where else? They are in circulation. Where are the pathogens? I
heard circulation and thats wrong. If it is youre probably going to be dead. They are
in the tissues at the site of infection. We dont take the T cells to the pathogens. We
take the pathogens to the T cells via the dendritic cell. The DC which we forced out
of the site of the infection and has phagoctyosed bacteria which has been infected by
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viruses. That DC migrates off to the nearest lymph node. There are T cells in the
nearest lymph node. The DC cell needs to talk to the T cell.
[4] [T Cell Specificity]
[Dr. Weil] It talks to the T cell w/ specific proteins. A T cell by definition is any cell
that has a T cell receptor. Ok. So all T cells have T cell receptors. Any cells w/o any T
cell receptors is not a T cell. T cells have a T cell receptor. What T cell receptors
recognize is a fairly limited physical structure. Its a protein called MHC antigen. You
all know about MHC even if you dont know you know about them. These are the
transplantation antigens. The reason you cant transplant organs is b/c these
proteins are not the same form person to person. MHC is the broad name for these
antigens across all species. In humans we call MHC antigens HLA. B/c you guys are
going to be dentist I tend to use HLA & MHC interchangeably. In reality HLA is a
subset of MHC, but youre not going to be working on rats or mice so there is no
point of teaching you MHC names. HLA T cell receptors recognize one thing and that
is the peptide in every single HLA. They always have a peptide in it. If the cell is
healthy, the peptide came from host protein. If the cell is infected or if the cell has
been phagocytizing something then the peptides are going to come from the virus or
what was in the vesicle. There always have to be a peptide. No peptide means any
protein on the surface.
[5] [Location, Location, Location]
[Dr. Weil] This is where we go back to the dictomy. We have extracellular/
intracellular places to live. We need two kinds of T cells. The CD4 deals w/
extracellular pathogens and CD8 w/ intracellular.
[6] [Where the pathogen lives]
[Dr. Weil] Can complement get inside the cell to do anything to the virus? How
about antibodies? Can a neutrophil get inside the cell to do anything to it?
Macrophage or monocyte? How do you tell your immune system that youve got
something bad growing inside you. W/ my HLA protein. HLA protein acquire their
proteins from things originally in the cytosol. HLA class II on the other hand
acquires its peptides in vesicles. HLA Class II gets its peptides in vesicles. This
bacterium is sitting in ECM but some other bacterium was phagocytosed. The
phagolysome degrades the bacteria into peptides. One of them gets stuck on HLA.
Whats happens is that CD8T cells recognize HLA class 1 and the CD4 protein
recognizes Class II. What CD8 T cells do and they do it well. They induce apoptosis.
Whats the process for cell death? I know Dr. Kinnally just talked about this. The
membrane is last. Everything inside the cell including the virus proteins/ DNA/ RNA
gets degraded and then the membrane falls apart. Is there anything left to infect a
new cell? No. Apoptosis gets rid of pathogens and the infected cell. If the CD4 T cells
did the same thing have we gotten rid of extracellular infection? We need a T cell ,
CD4 do a whole host of other things that help get rid of pathogens. They dont
interact w/ human cells except for antigen presenting cells.
[7] [None]
[Dr. Weil] Skip
[8] [Extracellular and Intracellular Pathogens Must be Treated Differently]
[Dr. Weil] We have to deal w/ intra & extra cellular pathogens differently.
Extracellular pathogens get phagocytosed. They end up in vesicle and MHC Class II
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gets its peptides in vesicles. Intracellular pathogens in the cytosol, HLA Class 1 gets
peptides from the cytosol in dendritic cells. In DC there is a mechanism that lets you
take peptides cleaved in the cytosol and put the peptides in the vesicles. In ONLY the
DC there is a mechanism takes peptides cleaved in the cytosol and puts them in
vesicles. Now if you have peptides in vesicles they end up on HLA Class II. Bacteria
that live outside the cell are always presented in MHC Class II only. There is no point
of destroying a cell. Viruses are presented on both classes. In the end, it will become
apparent why that must be.
[9] [Generic Antigen Processing]
[Dr. Weil] So this is a generic antigen process. We call this process antigen
processing. This is generic. Heres our DC cell. We cant tell if thats infected or in
vesicles. They get cleaved and we cant tell if thats a phagolysome and enzymatic
cleaved or a proteasome whose job is chop proteins into peptides. Somewhere along
the way the MHC molecule get the peptide and it gets on the surface and the top part
binds to the T cell receptor. T receptors only recognize peptides plus MHC.
[10] [None]
[Dr. Weil] What cells can be infected by a virus? DC cells. There is a 3-letter cell
that answers the question. All. If any cell can be infected by a virus which ones needs
a HLA class 1? All of them. All cells can express some degree of HLA class 1. Its
usually low and you can up regulate it f you need to . Class II will activate CD4 T cells
is really found on 3 cells: B, macrophages, and DCs. Class II is found on macrophages,
B cells, and DC cells. We call those 3 cells professional antigen-presenting cells
(APC). Their job is to talk to CD4 T cells. For purposes of this class, each one of these
cells talk to Cd4 T cells for different reasons. So DC present antigens on HLA Class I
&II if its a virus or HLA Class II if its an extracellular pathogen, and their job is to
take a nave T cell and activate it. Macrophages will express pathogens on HLA Class
II. Macrophages do this at the end of their life span (short lived cell, site of infection,
do their thing, and run out of enzymes). T cells can kill these macrophages at the end
of their lifespan.
[11] [None]
[Dr. Weil] B cells do endocytosis to put peptides on HLA Class II so that the B cells
can talk to the already activated T cells which then activates the B cells. First we
activate T cells and then the B cells. Once we activate B cells everything gets going
full blast and we get rid of the pathogen. For purposes of this class, DC are present
on HLA Class I & II. Macrophages are on Class II to be killed by B cells.
[12] [None]
[Dr. Weil] Here are cartons of the Class I/ II peptides. There are 4 proteins that I
expect you to know the structure of for this class. These are two of them. Class I is
composed of a heavy chain and a light chain. One of them is big and one of them is
smaller. The light change is B2 microglobulin. The heavy chain is called heavy chain.
Always a peptide. In an infected cell the peptide is from the infection and we do
something about it. Class II is composed of two polypeptides about the same size.
The biochemists in the 50s decided to use them the Greek alphabet (Alpha/ Beta). In
a healthy cell, the peptide comes from the host and the infected cell has a peptide
from the vesicle.
[13] [None]
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[Dr. Weil] This is the crystal structure of the Class I. I want you to put an eye in
there. And heres an ear, antler, ear, neck, tongue, nose, and this is a moose. The
peptides sit in b/w the antlers. Class II also looks like a very similar structure. They
both have to be bound by T cell receptors so they cant be that different.
[14] [None]
[Dr. Weil] Whats absolute in biology is that CD4 has a biding site on the B change
on the Class II. CD8 has a binding site on the neck of Class I.
[15] [None]
[Dr. Weil] The T cell receptor itself is going to bind to the top of Class I peptide
complex but the T cell receptor cant decide whether its binding Class 1 or 2. The
picking is always done by the co-receptor (CD8 for Class 1 and CD4 for 2). Alright.
[16] [None]
[Dr. Weil] Weve got domains in here that you dont have to know. CD8 is not
binding to the peptide binding part of the MHC complex. Its binding to the neck. The
CD4 is binding to the neck. The T cell receptor binds the peptide and the MHC and
the CD4/8 binds to a part closer to the membrane.
[17] [None]
[Dr. Weil] If I take the crystal structure and we roll it 90 degrees we are now
looking at the T cell landing site. Heres the alpha helices that form the ears. Here we
see a peptide. It has two sides to it. Theres the side thats buried in the HLA Class
molecule. The side thats sticking up is where the T cell receptors will bind. There
are amino acids that are going to bind deep within the peptide binding groove but
that leaves amino acids that are stuck up in space. Thats true for HLA Class 1&2.
HLA Class 1 we can see the peptide ends are buried within the peptide-binding
groove. The analogy for this, for HLA Class 1 you have a regular sized hot dog and a
regular sized hot dog bun. For Class II the peptides drape out over the binding
groups. Thats like putting a foot long over a regular bun. You still have to have a
couple residues that bury down into the binding groups.
[18] [None]
[Dr. Weil] Crystal structure again. Here we see peptide residue thats buried
within the binding groove. This one is sticking up and is in contact w/ the T cell
receptor. Here you can see again, those two residues are contacting the T cell
receptor. These are the peptide residues and these are other residues of the HLA
class 1 that are in contact. The T cell receptor has to recognize both the peptide and
the HLA molecule. Here to diagram the amount of surface area covered by the
surface receptors when they bind to HLA Class I or II.
[19] [None]
[Dr. Weil] Having talked about how the T cell binds, we will talk about how to
generate Class I and II molecules. We have to go back to basic bio. Where do you
start? Nucleus. What happens? Transcription and you make mRNA to the ribosomes
and then you have translation. Where does the protein end up first? In the ER. For
Class I and II they start by being transcribed in the nucleus (Class I). The mRNA goes
to the ribosomes. The heavy and light chain gets translated and it goes to the ER
Golgi secretory vesicle cell surface.
[20] [None]
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[Dr. Weil] Also going on is that a proteasome is cleaving proteins and turning
them into peptides. That happens all the time. In a healthy cells all the proteins
being cleaved are host proteins so the proteins generated are host peptides. If they
are virally infected, they are viral coat proteins. You gets lots of the same peptides.
The peptides get pumped into ER through TAP. Of all of the chaperone proteins in
Class I processing, this is the one you need to remember since next year you will
learn about what Herpes viruses does to TAP. TAP is involved w/ disease.
Proteasome cleaves proteins into peptides and gets pumped into ER by TAP.
[21] [None]
[Dr. Weil] Using a bunch of chaperone proteins that you dont need to know, the
heavy and light chain get combined and go through the Golgi and then HLA Class I
peptide complex gets into the surface. It folds in the ER where it acquires a peptide
cleaved by the proteasome and pumped into the ER by TAP. The normal protein
processing and onto the cell surface.
[22] [None]
[Dr. Weil] HLA Class II starts off like Class I. Class II then ends up in the ER. Whats
also in the ER? Proteins and peptides. Ok. Where did the peptides come from? The
cytosol. If Class II puts peptides from the cytosol into the binding groove will it be
able to present peptides from the outside? No. It gets synthesized in the ER but then
we have to block the binding groove so that it cannot bind peptides in the ER done
by the invariant chain. The Class II alpha/ beta chain fold up around the invariant
chain so that the binding groove is blocked. Then it goes through the Golgi and
instead of going straight to the surface, the invariant chain causes the Class II to fuse
w/ another vesicle in the cell. Unlike Class I, the Class II gets detoured so that the
vesicle containing the Class II fuses w/ another vesicle and if it contains bacteria
degraded and then the Class II will bind to the peptide of the bacteria and then it
will go to the surface. Golgivesicle surface.
[23] [None]
[Dr. Weil] Here we see the Class II w/ the invariant chain blocking the peptides in
the ER. It gets through the Golgi and gets put into a vesicle. Along the way, the clip
looks like a candlesnuffer. The handle of snuffer gets cleaved on the way to the
vesicle containing the Class II binding to the vesicle w/ other things in it. The piece
thats left is CLIP. If its a healthy cell, what ends up on the surface is Class II and
CLIP. If its not a healthy cell, you have a protein named HLA-DM that removes the
clip and that allows the bacteria peptide bind into the groove. As an interesting
aside, HLA-DM was unusually discovered in humans. There was a family in
California that was sick all of the time. They found this protein and they figured out
what it did. I knew somebody who knew the scientist who had done these studies
and I called that person and asked can you find out what kind of dentition these
kids had? Since most dental pathogens are extracellular. They didnt have any
problems w/ their teeth. I said really? They said yea they had dentures. W/ a patient
w/ no HLA Class II you will have a patient w/ no teeth.
[24] [None]
[Dr. Weil] Do you want to try the movies? Fingers crossed.
[25] [How to maximize peptide presentation.]
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[Dr. Weil] Ok. Now, sorry about that. So now, in order to defend against every
possible pathogen that can ever evolve, you have to be able to present every
possible peptide from a pathogen. We can do this is one of the two diversity system
that the immune system has. You cant understand the immune response if you
dont know this. MHC molecules are polygenic. What does that mean? Multiple
genes. MHC molecules are polymorphic. Dr. Saxena talks about this. MHC molecules
are polymorphic. What does this mean?
[26] [None]
[Dr. Weil] Polygenic expressed on the surface are products of 3 different loci. HLA
A/B/C are all expressed during fetal development. All of your cells have 3 different
isotypes. For HLA E, Class II- DM/DO has a role in peptide processing so whats
expressed on the surface is HLA-DP/DQ/DR. 3 HLA molecules on the surface and the
presenting cells have 3 class I isotypes and 3 class II isotypes. Thats polygenic.
Polymorphism. Most of the time you have a locus (spot on chromosome for a
specific gene). You have a certain sequence and most of the time the sequence is the
same. Its called an allele. So normally most of the time you have the same amino
acid sequence from person to person. There are some systems that have 2 alleles.
You know both of them. One of them is a disease (African-Americanssickle cell).
Theres a normal hemoglobin allele and sickle celled hemoglobin. Theres another
system that you know. Look at your neighbors face. Eye color. There is an allele that
coats for melatonin. If you get the allele you get brown eyes..
[27] [None]
[Dr. Weil] HLA has hundreds. HLA has hundreds. Do we have any identical twins
in the class? You plug your ears. What you end up getting is a HLA-
A/B/C/DP/DQ/DR from your mother and father. Unless you come from somewhere
where you have interfamilial breeding for a long period of time, your mother and
father will have different HLA molecules. You will have different 2 different HLA-
A/B/C/DP/DQ/DR. Every cell in your body only has the two alleles that you got.
Unless you have an identical twin, your alleles are different from every single person
in this room. You are the same. All of your cells are identical. You are different from
everyone else. Thats why you cant transplant organs from person to person. This is
a population heterogeneity. The population varies. If you cant present a peptide on
one of your alleles, you may die, but the person next to you will live. This may not
protect you as an individual but keeps the population safe. As long as the population
has a wide array you can present something. You dont need to memorize numbers.
The probability of two different people having the same molecules (800
possibilities), its non-existent. Thats why paternity testing is so efficient. They look
at HLA molecules. Polymorphism means that there are hundreds of possible alleles.
You get two for each of the loci. You have 6 HLA Class I/ II unless you have a bunch
of inbreeding.
[28] [None]
[Dr. Weil] The variability is found in the peptide-binding grove. HLA Class I
variability concentrates here. Class II is found in the beta chain. Alpha chains are
less polymorphic. If you change the amino acid sequence what do you change? You
change the kinds of peptides that combine. Each allele binds a different kind of
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peptide based on the fact that youve change the nucleic acids that change the amino
acids.
[29] [None]
[Dr. Weil] The way you pick a peptide is through a peptide-binding motif. 4 given
alleles. HLA-A2 .You have to have a small hydrophobic amino acid at the end. A leu,
meth, valine. The ends bury in into the peptide. The rest that are sticking up. It
doesnt matter what they are. So if you think about it. IF you have 9 possible amino
acids, you can have hundreds of different possibilities all of which will have that
motif but then the other 7 amino acids dont matter. That gives you the hundreds of
possibilities. These are called anchor residues. Each HLA molecule has an anchor
motif, but the rest of the amino acids dont matter. HLA Class II can be up to 25. The
peptides bind based on the 2-3 amino acids. The rest of them doesnt matter what
they are. The rest are up and contact the T cell receptor. The other thing to
recognize, you dont have 1 HLA A molecule on the cell surface. In real numbers
you probably have about 20,000 HLA- A/B mother and father; 10,000 HLA- C
mother and father. In a healthy cell where you have lots of different peptides being
cleaved, each one of those 20,000 has one peptide and they are all different. In a
virally infected cell w/ few proteins to cleave up, youll have lots of the same peptide
copy. Ok. And, why dont we take a break.
Ok. So, just in case you got lost in the information I gave you. I had a couple people
that said I talk fast. You will note that I repeat things over and over and over. If you
need me to, I will repeat myself. One of the reasons you have these big spaces b/w
the lectures is so you have time to go back and learn this. Its not reading repetition
but its talking repetition. Explain it to the person next to you. When it makes sense
to them youve got it figured out. This is why were going through the same slide
again. This is one of the difficult concepts. Its hard. Understanding the diversity of
B/T cells is a difficult subject. Its not you, its hard. If you get it figured out, its going
to make the rest of your career easier. Im always happy to do extra tutoring
sessions. We can do some extra sessions if that will help. We have 100,000 HLA
Class I molecules on the surface of most B/T/ APC cells. B/c we have 3 different
genes (A/B/C) and two types of genes (mother/father), you have 200,000 of each
allele on the surface. DP/DQ/DR doesnt work the same way. They are on the
surface of the APC. Each one of the proteins has a peptide and it was selected b/c it
had specific residues at specific points called anchor residues. If you have specific
residues at specific points, it means you can have any other residue at any other
space. Peptides are selected based on their anchor residues. The remaining residues
can be anything. That gives you a repertoire of large group of peptides that any
allele can bind. One protein binds one peptide but you have 20,000 copies of
proteins so you can have 20,000 peptides binding. Over your 6 HLA Class I
molecules, you can present about 60,000 different peptides. Thats the reason when
new diseases come along, most of the population lives. Class I is a bit easier since the
peptides are shorter.
[30] [None]
[Dr. Weil] So if we go back and look at the 3D picture again, you have anchor
residues here and there you have these other residues that can be anything. These
are the residues that contact the amino acids of the T cell receptor. The other
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residues that are different are the residues on the alpha helices. They have a
different sequence then a different allele. This T cell receptor is only going to
recognize this one kind of allele. Thats called MHC restriction. A T cell receptor will
only recognize one of the MHC alleles you have. Thats called MHC restriction. Its
only going to recognize a certain number of amino acids on the peptide called
peptide restriction. T cell receptors are peptide specific MHC restricted binding. So a
T cell receptor contacts amino acids on the peptide making it peptide specific and
its only going to bind one of the alleles so its MHC restricted binding. Peptide
specific MHC specific binding. The T cell receptor will recognize 1-3 A.A. of the
peptide and it will recognize A.A. on the amino helices. T cell receptor that
recognizes HLA- II will not recognize B27 at all. This is why you cant transplant
organs. The T cells wont recognize the peptides and will kill it. Everybody in this
room has different HLA alleles except for my twin back there. If youre like me and
are a mutt of northern European descent, some of my alleles will be shared. Asians
have alleles that are different from non-Asians. HLA is actually non-politically
correct. People from different regions have different groups of alleles.
[31] [None]
[Dr. Weil] Here were looking at restriction. Here we have a T cell receptor for
HLA- A. Its specific for this peptide so you get antigen presenting so the T cell
recognizes this and the T cell will go off to do its things. The cell receptor cant
recognize the molecule so nothing happens. Here we have the right molecule but its
presenting the wrong peptide so nothing happens. The T cell will recognize the
right peptide on the right HLA molecule. The anchor residues are on the peptides.
The anchor residues are the part that are buried in the peptide-binding group. The T
cell receptors are recognizing the parts of the peptide in the middle that are sticking
up.
[32] [None]
[Dr. Weil] Heres our last slide. It took me a while on how to read this. Its read left
to right, top to bottom, top to bottom. Here we have a population w/ 4 different
alleles. It explains why its good to have heterogeneity and what happens w/
population selection. These are homozygous. Disease comes along. Everybody only
green dies off. The other people survive and they go off and reproduce for a while.
Eventually you end up w/ some more homozygous for green and yellow. New
disease comes along. Everyone who isnt blue dies off. This has happened and weve
studied it. The most recent example was SARS. There was this big thing about SARS
since 50% of the people who ended up in the hospital died. We eventually
developed an antibody test for it. 10,000 people had gotten SARS but didnt get sick
enough to go to the hospital. What was happening that the people who died didnt
have an allele that could prevent this virus. No B/T cell activation. The other
instance this happened, was when the Europeans showed up in this country,
smallpox and diphtheria occurred. 90% of purebred N.A. died in the first 100 years.
The ones that survived had intermingling w/ people to have a broader range of
haplotypes. This is a reason why you have a taboo when you marry a family
member. Ok? Questions? So youve got two kinds of variability going on here. This is
a population-based variability. You have half of the same as one parent and half the
same as another parent. Every single person is different from everyone else.
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10
[33] [None]
[Dr. Weil] The DC is doing all of the processing while travelling to the lymph node.
It will hit the node and it now has pathogens for Class II and intracellular pathogens
it has Class I/ II. DC can put pathogens can put Class I & II for an intracellular
pathogen. Where is it going to meet up w/ T cells? Where is the T cell area? Para
cortex. The DC will go to the Para cortex and it will meet mature T cells. Now were
going to go back in time and off to the thymus and talk about how T cells get
developed. Im told all of these movies are on YouTube. The ones that my computer
keep changing the settings. The movies are published w/ his (Janeway) textbook
and same publisher.

Thymic Education
[1] [Review]
[Dr. Weil] Next topic. Why do we need two kinds of MHC molecules? Intracellular
and extracellular. What cell is activated by each type of molecule? CD8 for Class1
and CD4 for Class II. So. Intracellular and extracellular and what cells are activated
by each type of molecule. Whats the source of peptide binding to HLA Class II?
Vesicle. Whats the source of binding for Class 1?
[2] [Review]
[Dr. Weil] How are peptides selected? Peptide binding motif, anchor residues.
Explain the diversity of MHC, cellular and population. Polygenic and polymorphic.
What are the cells in your body? Whats the polymorphism within your body? And
so are your cells the same or different? Same. Whats the polymorphism on a
population basis? Different. The diversity in HLA, every single cell in you is identical.
You are different for everyone else. For HLA, the diversity means your cells are
identical but different from everyone else. So if we want to be able to defend against
all possible pathogens, you have to have T cells that recognize all possible peptides.
Were going to talk about how T cells develop. This is the second kind of diversity
that we will discuss.
[3] [Title]
[Dr. Weil] T cells develop in the thymus. The purpose of the thymus is to get
generate receptors.
[4] [Thymic Education]
[Dr. Weil] We decide if the T cell is CD8/ CD4 and then we make sure they dont
bind to host cells.
[5] [None]
[Dr. Weil] Heres our receptor again. There are two different kinds of T cells. They
are selected based on what sort of genes they use to generate the T cell receptor. We
have the A and the B (alpha/beta) but also gamma and delta. You can have a T cells
that use alpha beta to make a:B T cell or make a gamma: delta T cell. 97% of
circulating T cells are alpha: beta T cells. You have conventional T cells (90% are
alpha beta), a few cells use gamma: delta and they are found in the tissue. We will be
talking about alpha: beta. Does that mean its the same thing as a HLA Class II? No.
Why not? Youre using different genes. Ok. So we have two proteins called alpha/
beta and they are not the same thing. Its not the same thing in the receptor and
MHC molecule. The very outer most part of the receptor is called the variable region.
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The amino acid sequence of most of this is identical but the rest has a very variable
region. This is the part thats going to bind the HLA Class I/ II molecule and the
peptide. The outer most part has a variable sequence to it. Thats the reason
receptors bind different HLA molecules and peptides?
[6] [None]
[Dr. Weil] How do we generate a variable region? Before we get there. T cell
receptors like all other proteins act through second messengers. The cytoplasmic
domain has 1 amino acid. Is it enough to activate a second messenger? Every single
T cell receptor is surrounded by another protein called CD3. The function of it is for
antigen recognition. It does the signal transduction. The T cell receptor binds and
changes the confirmation and that allows the second messenger to get going. CD3
signals, CD4/8 pick whether or not the T cell receptor will bind to a Class 1 or II
molecule. T cell receptors job is to bind. CD3- signal.
[7] [Thymic Education]
[Dr. Weil] We need to generate a population of T cells w/ different receptors. If
they all have the same receptors how would they recognize different things. We
need a large amount of variability. We need the T cell receptors that can recognize
the HLA molecules you have and not someone elses molecules. How do we generate
variability? One way is to have a gene for every receptor. Im going to use an analogy
for an associate professor in the city. If you have a tiny closet and need a different
outfit everyday, youve got a problem. Youd have this massive amount of DNA used
by T cells that couldnt be used by any other cell. Whats the other way to assemble a
wardrobe. You buy suits. Wear it over outfit to create different outfits. Thats how
T/B cells make receptors. There are segments of genes. The T cells hook these
segments together and every time they select out the segments you make a protein
out of it. T cells make a different protein by hooking together different segments of
genes in order to end up w/ a different nucleic acid sequence different A. A.
sequence.
[8] [None]
[Dr. Weil] Here is the thymus. The reticular cells, the T cells supported by the
thymic epithelial cells.
[9] [None]
[Dr. Weil] So the T cells migrate from the bone marrow and enter they thymus at
the sub capsular space. They know they are going to become T cells but dont have T
cell proteins. The first thing that they do is begin the process for the different
segments. Its called gene rearrangement. The first thing occurs under the sub
capsular space. It will take pieces of these segments and hook them together. It spits
out the intervening DNA. This process starts on the Beta chain and the gamma and
delta chain. Gene rearrangement begins and you start to hook on segments on Beta
chain. There are 3 segments (D, V, J). This is happening on the beta chain. VDJ, Beta
chain. The T cell starts to rearrange these segments on the gene. Its not occurring in
the RNA. Its a random process. Eventually you transcribe on the rest of the gene
(constant region). What happens after youve transcribed a gene? mRNA and what
happens? Translation. If youre just randomly hooking ends together are you always
going to get translatable codons? No. The mRNA and tries to translate into a protein
which may or may not work. It doesnt work more than it does. If the cell is unable
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to translate the protein you have a non-productive rearrangement and the cell dies
via apoptosis. The T cell that wants to become one starts to do gene rearrangement
on the B chain w/ VDJ segments. It tries to hook together one of the Vs to a D to a J.
That protein then has to translate. If the protein can translate, the cell wins. If the
protein cant translate the cell dies. The dead cells are never going to participate in
an immune response so well leave the cells. At this point living cells gets a prize.
The live and multiply. You get a whole bunch of copies w/ the same T cell and B
chain. You then shut off gene rearrangement for the beta chain. That turns on
several proteins (CD4/8) and then the T cell starts to rearrange the alpha chain. In
the subscapular space, it didnt have any active parts. We call that a double negative
T cell. There is no CD4/8 and no T cell receptor or CD3. Its the most immature T
cell. If we end up w/ a beta chain, the T cell has migrated into the middle of the
cortex. The T cell puts up a Beta chain which has to be surrounded by CD3 and
becomes double positive turning on CD4 and CD8 genes. If the T cell can successfully
do gene rearrangement on the VDJ and can translate, it is rewarded by living and
expands to become a double positive cell by expressing CD 3/4/&8. Double negative
double positive phenotype. They are still immature T cells since they are in the
thymus. In the thymus you also have a ton of macrophages. The phages are there to
eat the apoptotic bits. The T cells in the beginning doesnt express any of the T cell
specific markers. That T cell has yet to express CD4/8. Once you successfully a beta
protein on a surface, you start to have expression of all 4 components and that is
called a double positive cell (middle of the cortex). Its trying to now do
rearrangement on the two segment alpha chain (D & V). You stopped trying to do it
on the beta chain and start to do rearrangement on the alpha chain. Again,
sometimes youre successful and sometimes you arent. The ones that arent get
eaten by macrophages. At this time we have a conventional alpha/beta receptor and
these T cells are at the corticomedulary junction. We have a lot of T cells w/ the
same beta chain but a different recognition since the alpha chain is different. The
combination of alpha/ beta which recognizes the HLA molecule peptide and that
will change the specificity. So every T cell will have a different receptor. So you start
off w/ the same exactly identical gene segments. Every T cell and every single
solitary person on the planet has the same variable diversity for joining segments
for the beta and alpha chain. Every T cell starts off w/ identical segments. The
random process of rearrangement means that every single solitary T cell will have a
different amino acid sequence. HLA diversity, we all start off different HLA but all of
our cells are the same. In T/B cells everyone starts off w/ identical segments but
after rearrangement, the proteins generated are different. Every T cell has a
different receptor than every other T cell in my body. The diversity of the T/B cells
are such that through these gene rearrangement process for both polypeptides, you
get a different nucleic acid and amino acid sequence for every kind of solitary
segment. For HLA we have different alleles that we arranged from out parent but
the cells are identical. In T cells we start off with the same cell but we hook together
differently so every T cell is different from every other T cell. This random
combination gives you a different nucleic acid and amino acid sequence for the T cell
receptor. Every T cell you make will recognize something different. We started w/
double negative in the sub capsular space. If the cell can produce a beta chain, the
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cell starts to express CD4/8 it then has to rearranged a beta chain. If it doesnt, it
does. If it does, it is now at the junction and has to do the next step of its evolution.
[10] [None]
[Dr. Weil] Here they are showing a T Cell that doesnt know it wants to be a T cell.
It starts to do rearrangement the beta, gamma, and delta chain. Gamma delta is
found in the alpha chain. Until you rearrange alpha chain, they can become gamma:
delta cell. Start to rearrange a beta chain and it becomes a double positive. Then you
do alpha.
[11] [None]
[Dr. Weil] Here we are looking at our genes. There are 50-70 variable region
sequences. Here we are hooking a variable to a diversity. Eventually you hook on a
constant region and translate that protein. We hook on a constant region and
translate the protein. The cell dies if its not translatable. If the cell can translate, you
shut off beta, gamma and delta keep going, and start alpha. Unsuccessful alpha =
gamma: delta or death. If you successfully hook together VJ for alpha chain now you
have the double positive receptor and expression of CD4/8
[12] [None]
[Dr. Weil] So again here we are showing you, you start rearrangement on al 3 loci.
If you do it on gamma delta, it becomes gamma: delta and it goes to tissue. If you do
beta, you do beta: alpha and it stays in the circulation.
[13] [None]
[Dr. Weil] This is not a one shot deal. It can try more than once to make beta and
alpha chains. Most rearrangements are not productive.
[14] [None]
[Dr. Weil] Before you have a heart failure, thats the part you need to know.
[15] [None]
[Dr. Weil] Here we are doing the DJ rearrangements of the beta chain. Then we
hook on the V and now we have a double positive proliferative chain. We try to
rearrange the alpha chain. If the alpha is rearranged you had a CD4/8 double
positive cell.
[16] [None]
[Dr. Weil] Skip
[17] [Fate of T cells]
[Dr. Weil] If you get a productive rearrangement, determined by the fact that
protein gets synthesized and sent to the surface. You prevent apoptosis. If a protein
does not get to the surface, the cell dies.
[18] [None]
[Dr. Weil] We have macrophages b/c we have lots of apoptotic cells. This is a stain
for that. The next thing the T cell has to do (still at the corticomedullary junction a T
cell receptor positive CD3, double positive CD4/8) is to find out if the receptor binds
your MHC molecules. You only have 12 right? Theres no thinking in this. Its
random. If it binds to one of the hundreds of alleles you dont have, it can never be
activated so whats the point of keeping the T cell around.
[19] [Definitions]
[Dr. Weil] In this random process they now have to figure out if they bind one of
your 12 HLA molecules. There are two principles that cause this. The first is affinity-
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the strength of binding on one molecule to another at a single site. Its driven by Van
der Waals forces, hydrogen bonds. A.A. A.A. binding. Single molecule and its
ligand. Avidity on the other hand is the sum of binding. All of the molecules on one
cell that can bind to another cell. Affinity is single while Avidity is all.
[20] [None]
[Dr. Weil] Affinity is the principle that guides the first step. Double positive T cell
that has to go through positive selection. Its the fishing out of the T cells that made
receptors that actually recognize your MHC molecules. If the T cell has a receptor
binding one of your MHC molecules, it doesnt matter if its binding Class I or II. If
you can bind a single molecule, it can get survival molecule and lives. If it cant bind
to the Class I or II molecules on the thymus epithelial cell, it doesnt get survival
signals and dies. Positive selection is fishing out those T cells that can bind one
receptor on the T cell to one of the MHC molecules on the epithelial cells. It gets
survival signals and lives Positive selection is the ability to bind one receptor to its
ligand w/ moderate to strong binding. That cell lives. The T cells that cannot bind to
any of the molecules that you have, that cell dies. Positive selection is fishing out the
T cells that can bind to your HLA molecules. You only have 12 of the hundreds of the
possibilities. You randomly fish out the receptors that can bind to 1 MHC molecule
w/ moderate strong binding. They get survival signals and they live.
[21] [None]
[Dr. Weil] Weve just picked out T cells that have bound to MHC molecules. In the
corticoepithelium what kinds of peptides are on those molecules? Self. Weve just
picked up a bunch of T cells that can bind to you. Is that a good thing? Its an OK cell
as long as there arent lots of receptors that can bind to the self-peptides. In a
healthy cell you have a low copy number of a solitary peptide. You have lots of
different proteins in a normal cell. Youll have 3-4 copies of a single peptide. The
positivity that the T cell will come into contact w/ a MHC molecule w/ the same
peptide is slim. It will only contact one. That high affinity, single receptor is enough
to give you positive selection and survival signals.
[22] [None]
[Dr. Weil] If the T cell then goes ahead and binds w/ high avidity (more than one)
which bind to the MHC w/ the same peptides will allow it to bind to any cell in the
body and kill it. That gives you autoimmune disease. We delete these cells through
negative selection. This is an active process where binding of multiple copies of
receptors, induces apoptosis. Negative selection. The T cells that go through positive
selection then go through negative selection to make sure they dont bind w/ high
avidity. Low avidity= good. Those T cells are then actively killed. Thats negative
selection. Failure to be positively selected is not negative selection. Positive
selection gets survival signals. Negative selection kills cells that bind. The difference
is moderate to high affinity vs. low to high avidity. When the sum of binding is low,
the T cell is fine. When the avidity is high, the T cell gets killed. Along the way, the T
cell drops the CD4/8 receptor. It becomes a single positive receptor. We will start
next week on positive selection so Ill go through that process again. In the mean
time start going over diversity b/w HLA and T/B cells.
[23] [Maturation Events]
[Dr. Weil] Skip
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