Transcribed by David Landsman 7/25/14

General Pathology lectures 11 & 12 Immunology IX and X by Dr. McCutcheon

Continuing in the previous B-cell development lecture
Slide 32
[Dr. McCutcheon] - B lymphocytes and T lymphocytes develop their receptors by random gene
rearrangement. They pick fragments out and then they add or subtract nucleotides at random. So
because of that process, every single solitary b cell has a different receptor from every single
solitary other b cell. And every single solitary T cell has a different receptor from every single
solitary other T cell. Ok? Everybody with me on that? The original genomic DNA that the b cells and
the t cells used to make the b cell receptors and the t cell receptors is identical in every person on
the planet. The original genomic DNA that was used by the b cells and the t cells to make their
receptors is identical for every person on the planet. And although any of us in the room who have
the same HLA allele, for us northern European Caucasian descendants, HLA A2 is the most likely,
we may have some t cell receptors that are the same because they were selected out by the HLA A2.
And we all probably have some b cells with the same receptors, alright? So in HLA all of the cells in
one persons body are identical but everybody else is different. For B and T cells the starting
genomic dna is identical but every single solitary b cell and every single solitary t cell is different.
Although, in the population probably we all have some common or really similar b cell receptors.
And people with the same alleles may have some of the same t cell receptors. So we have 2
completely different kinds of variability in the immune system and its really important that you
understand the similarities and the differences between these two kinds of variability. You know,
HLA doesnt do gene rearrangement to make its receptors. And B and T cells dont have different
alleles. So completely different sets of variability that give us completely different kinds of
variability in completely different places. HLA, every cell is the same. B and T cells every cell is
different. HLA, Im different from everybody in the room who is different from everybody in the
room. We all probably have some B cell receptors that are really close and if we have the same
allele we probably have some t cell receptors that are really close. If you have completely different
HLA than I have we wont have any t cells in common. And thats why you have transplantation
problems, is because t cells only recognize what the MHC on that particular organ and if that
particular organ doesnt have the same MHC as the recipient then the T cells cant bind to it for
effector activities and as something you dont need to know, t cells are programmed to recognize
other HLA as foreign so they kill it. And thats graft versus host disease, because t cells recognize
foreign HLA as foreign, period, and decide they have to kill it. So every single solitary person starts
off with identical DNA for their b and t cell receptors, and its the random rearrangement and the
additional really random addition and subtraction of nucleotides at the junctions that gives you
each single solitary b cell is different from each single solitary t cell. But within a population there
probably are some b cell receptors that are fairly similar and if you have the same allele therell be
some t cell receptors that are fairly similar. So two completely different kinds of variability. OK. So
thats what I wanted to go through in this lecture again.


Lecture 11 and 12 - B-cell activation and function
Slide 1
[Dr. McCutcheon] - So now we are going to talk about B cells. So in the grand scheme of things, about
6 days ago somebody very kindly breathed all over me and gave me a virus. And so on Wednesday
afternoon after I talked for three hours I had a really scratchy throat and I didnt really feel very
well and I went home and my throat got a little bit less scratchy so I thought it was just because I
had talked for three hours and then in the middle of the night I woke up with a really scratchy
throat and a headache and a fever and I thought oh crap I have a cold! So at this point in time, that
Transcribed by David Landsman 7/25/14
was Wednesday, so the previous Wednesday somebody breathed on me. And up until now all that
virus has been replicating in my nasal and my respiratory epithelium, and my dendritic cells were
doing what they could, complement isnt really playing a role, neutrophils dont get recruited in
because the tissue is intact so its really only dendritic cells and interferon-alpha and interferon-
beta from the dendritic cells and since I drank a lot of green tea, and drinking green tea activates
your NK cells to secrete interferon-alpha and interferon-beta, the green tea that I drink has been
helping. So then on Wednesday, my CD8 T-cell but the dendritic cells were off in my lymph nodes
and my CD8 T cells got activated along with my CD4 t cells and so on Wednesday my CD4/8 t cells
hit the site of infection and they started lysing all the virally infected cells. And thats why my throat
started to get sore, because now theres tissue damage and I started to get a fever because with the
tissue damage you get IL-1, TNF-alpha and IL-6. OK and thats the headache and the woosyness and
the achy. And then the mucus starts to come up as youll discover today as I start having to blow my
nose all the time or I sneaze because I dont have intact epithelium anymore and so theres nothing
to get the mucous that is normally behind the intact epithelium from just kinda pouring out. And my
CD8 T cells are going to have to lyse all the virally infected cells before things can mend, so I have
another day of getting worse before I get better. Now in this point in time, by th1 t cells are also at
the site of infection and theyre directing the macrophages and the neutrophils to mop up all the
apoptocic lysed tissue bits from the cd8 t-cells. My b cells are still sitting in the lymph node. So
today is probably when my b cells are finally getting activated against this. So one of the big draw
backs to the adaptive immune response is its slow. It takes 5 to 7 days to get a t cell activated and
expanded and off to where it needs to be. And until then the only thing that you have going for you
are the innate immune cells. And with viruses, like cold and respiratory epithelium viruses, you
dont know you are sick until your CD8 t cells get there. It is when your CD8 T cells start lysing all of
the virally infected cells that the symptoms start to show up.

Slide 2
So heres the overview, TH2 t cells, their function is to activate b cells. So they stay in the lymph
node to do this. And this is through a combination of physical interactions, and we call the cognate
interactions (Ill explain why) and cytokines. And then the b cells get activated, they have 3 different
fates. One of their fates is to make antibodies and the cells that make antibodies go off to the site of
infection and make antibodies, and then the antibodies do all of the things that the cd8 t cells cant
do.

Slide 3
So and you know I wish they used a finger instead of a toe because its much more relevant to dental
students. So we have our infection, weve broken the physical barrier, our dendritic cells have
phagocytosed, micropinocytosed or been infected by whatever they are going to be infected by.
They move through the afferent lymphatic into the lymph node.

Slide 4
The DC also will move to part of the germinal center. So here we have radiolabelled the pathogen
and we see where are the radiolabelled cells are sitting and this is the follicle. Now the DC also are
holding onto antigen for B cells. They dont present it, it doesnt get taken inside of the cells, the DC
sit there and they have chunks of whole pathogen on the arms of the dendritic cells. So the DC in
addition to taking in pathogen inside the cell, they also grab hold of pathogen outside the cell. And if
the pathogen is a virus, does the DC have a molecule that can recognize a virus. Everybody who is
shaking their head no good job! But what does our virus have on its cell surface? Its on the slide
guys C3b. Right? Viruses have C3b on the virus. And DC have receptors for C3b but unlike other
receptors, normally when something binds a receptor what happens is the receptor that causes
receptor-mediated endocytosis and whatever that was bound to the receptor ends up in a vesicle
Transcribed by David Landsman 7/25/14
inside the cell. C3b doesnt do that. CR3 holds the C3b on the outside of the cell and so whatever is
the C3b is stuck to then sits on the outside of the dendritic cell. So we have a boat load of DC each
with lots and lots of copies of whatever pathogen it is. And it doesnt have to be a virus -- bacteria,
bacterial toxins, fungi, parasites, they will be held in place on the arms of the dendritic cell and
theyre held at the top of the germinal center. And whats the germinal center full of? Or primary
follicle. B cells. So this is how our b cells, one of the mechanisms that b cells can find pathogens.

Slide 5
So heres our virus, our virus gets bound by C3b, some of it can be cleaved into a piece of C3b called
C3d. We have receptors both for C3b and C3d. Ok? On our follicular DC. And all of these little bumps
on the dendritic cell, those are called icoves. I-C-O-V-E. And all of those little bumps are where the
DC is holding pieces of the pathogen. So the arms of the dendritic cell, and its not DC in the lymph
node tend to have very long life spans. And they hold onto pieces of every single pathogen thats
ever passed through the lymph node. So these icoves arent all filled with this current pathogen.
Many of them have copies of old pathogens. And one of the functions is so that when memory cells
come around they can bind to that pathogen and get survival signals. So thats how we keep our
memory cells alive for decades and decades. So this DC has copies of every single solitary pathogen
that its ever had held on its cell surface. But of course it is going to have the most copies of the new
pathogen. Right? So one of the functions of C3b is that there are receptors on DC for both C3b and a
cleavage product of C3b, C3d, and that allows the DC to hold copies of intact pathogen be it intact
bacteria, virus, toxin, fungi on the surface of the dendritic cell.

Slide 6
So the b cell then also have receptors for c3b and c3d, and the names of the receptors are not
important. But the b cell can come along and it can bind its various c3b and c3d receptors to
particles of c3b and c3d. And this interaction is similar to the LFA and ICAM interaction between
the t cell and the DC. So the b cell is going to use these non-specific receptors to bind to the C3b and
the C3d of the pathogen.

Slide 7
And then the B cell receptor itself tries to bind to some antigen on the pathogen. And any b cell that
can only bind to the C3b/C3d receptors and cannot find something on the pathogen that its b cell
receptor is specific for, well that b cell goes away. So the C3d/C3b receptors on the b cell will come
and bind to the C3b/C3d on the pathogen on the dendritic cell. And the b cell receptors then try and
role around and bind to some antigen on that pathogen. So the b cell receptor is trying to bind the
antigen on the pathogen and if the only interaction is the c3d or c3b binding to one of those
receptors on the b cell, that b cell is not specific for that immune response, that b cell heads out the
door. Eventually, if we want to live, well have a b cell that has a receptor that will bind to something
on the pathogen. Ok? So we are going to talk about the b cell that actually bound because the ones
that leave dont really have a lot to do with the immune response. So this b cell is now going to bind
something on the pathogen, and it can be bacteria, virus, toxin. And remember that an antibody has
2 arms so it is going to bind to two copies of the antigen on the pathogen. What is the affinity of this
interaction? Give me a number. Whats the definition of affinity? The sum of binding of one
molecule. So whats the affinity? One. The affinity of this interaction is one. Whats the avidity? Two.
Ok, so the avidity of this is two.

Slide 8
Alright, so now in immature b cells and immature t cells, when we have high avidity interactions.
We have numerous receptors on the t cell binding to numerous HLA class I, or numerous receptors
on the b cell binding to particulate or soluble antigens - thats negative selection and we do
Transcribed by David Landsman 7/25/14
something to stop that cell. In mature cells, we want high avidity interactions. So in an immature t
cell, high avidity, negative selection, apoptosis. In mature t cell, high affinity, activation. Immature b
cell, high avidity, apoptosis or anergy. Mature T cell, high avidity, activation. And the avidity in a b
cell, because you can have multiple copies of the b cell arms binding to antigens on the same
pathogen, this is called cross-linking. And the way to do this for everybody whos sitting next to
each other. So everybody grab hold of your pencil and hold it up like this. So for those of you who
have a neighbour, hold your hands like this. No, so when youre holding your hands like this what
are you connected to? If your feet are the cell membrane how many things are you touching? Now if
you hold this to your neighbour and they grab hold of your imaginary pen, how many things are you
touching? So for those of you who are a group, you guys are cross-linked. You guys are all cross-
linked. This is not cross-linking if you are only touching one molecule. But if you are bound to the
people next to you, now you are cross-linked. Ok? So the b-cell can tell when it has multiple copies
of receptor in the same place that are all stuck to the same antigen. And that interaction of cross-
linking is what allows the b-cell to get the signals that it needs.

Slide 9
And the signals that it needs are friends and what are all these things whose names you dont need
to know are doing? Theyre phosphorylating. So with cross-linking you get phosphorylation. And
phosphorylation ends up with changes in gene expression in the nucleus.

Back to slide 7
Ok, so the reason that C3b is critical for viral activation. It is critical for bacterial activation but it is
critical for other things than bacteria too. Im sorry, it is critical for b cell activation. Is because
c3b/c3d lowers the threshold for cross-linking. So in real numbers, if you had a viral particle and
there was no c3b or d on it, you would have to have a thousand b cell receptors bound to a
thousand antigens on the virus. How often do you think that happens? It never. You put a single,
real number, single molecule of c3d or b actually c3d, on the virus you lower the threshold ten-
fold so now you only need a hundred antibodies bound to something on the virus. You put two
molecules of C3d on the virus, now you lower the threshold to ten antibodies on the virus. So in the
absence of that complement piece C3b, because you need the breakdown product C3d, the
threshold to get a b cell activated is biologically impossible. So by putting C3b so it can break down
to C3d on a virus you bring the threshold down to ten.

Slide 8
And now the b cell can cross-link...

Slide 9
that causes phosphorylation

Slide 10
and you end up with activation. So the b cell uses the complement receptors on complement
pieces on something that stuck to complement receptors on the dendritic cell to roll along and find
a pathogen to which its antibodies will bind. Its b cell receptor will bind. And now because the b cell
receptor bound, we know that b cells can make antibodies that will bind to the pathogen so we
want to activate that b cell. We have to have enough copies of the receptor bound so that the b cell
can do cross-linking. Cross-linking causes phosphorylation, cross-linking also causes receptor
mediated endocytosis. So the pathogen now gets yanked off the dendritic cell and gets sucked into
the b cell through receptor mediated endocytosis. And after receptor mediated endocytosis where
is the pathogen in the b cell? It is in a vesicle. What happens to things in vesicles? They end up on
MHC class II. And what does a TH2 t-cell recognize? What kind of a t cell is a th2 t cell? So what does
Transcribed by David Landsman 7/25/14
it recognize? Peptides on mhc class II. So the reason we call this a cognate interaction is because
something on the b cell the b cell receptor recognized some part of this pathogen and the bacterial
peptide that it presents on its MHC class II from this pathogen is recognized by the already
activated t cell. So theres a link between what the b cell recognizes and what the t cell recognizes.
So the b cell recognizes pathogen with its b cell receptor plus C3d and the receptor against that,
enough copies of the receptor binding and you get cross-linking, cross-linking sends
phosphorylation signals and you get receptor mediated endocytosis. Anything in a vesicle gets
chopped into pieces and those pieces end up on HLA class II back on the surface of the b cell. HLA
class II plus peptide is bound by CD4 t cells and in this case our t cells are activated CD4 t cells so
the CD4 t cell binds its t cell receptor to the peptide MHC class II complex on the b cell. CD4 t cells
have CD40 ligand, CD40 ligand binds to the CD40 on the b cell and thats the co-stimulatory signal
like CD28 on the t cell binding to B7 on the APC. So the first signal comes through the receptor
mediated binding, the second signal comes through the CD40 ligand t cell binding to CD40. That
causes the b cell to put up receptors for cytokines. Alright? So the b cell binds to pieces of
complement on the pathogen on the dendritic cell with its complement binding receptors. If the b
cell receptor itself can bind to the pathogen, that yanks the pathogen off the dendritic cell, the b cell
does receptor mediated endocytosis. The pathogen is in a vesicle, pieces of it get put on HLA class II
and the HLA class II goes back up to the surface of the cell. That allows a t cell thats already been
activated against this peptide on HLA class II to bind the t cell receptor to the B cell and that
receptor mediated endocytosis caused phosphorylation and signalling. The T cell now binds if the t
cell has cd40 ligand, which it does, it binds to CD40 on the b cell, thats the costimulatory signal. If
there is no costimulation that b cell now becomes anergized. No co-stimulation through CD40, b cell
becomes anergized. If the b cell gets costimulation through CD40 ligand binding to CD40 on the b
cell, the b cell puts up cytokine receptors.

Slide 11
And the b cell is now activated so when the cytokines that have been secreted by the t cell for
several days now hit the cytokine receptors on the b cell, and its IL-4, IL-5 and IL-6, that drives
clonal proliferation. And now if you think about it, this poor lymph node, youve had a massive
number of (well since I have a virus) I have a massive number of CD4 t cells that are dividing. Half
of which are staying in the lymph node. I have a massive number of CD8 t cells which have divided
off. Now I have a massive number of b cells that are dividing. So what happens to that lymph node?
It gets really swollen. So the reason the lymph nodes get really swollen is you have all this b and t
cell division. So the b cells divide, now at this point daughter b cells do something different than t
cells. At this point, t cells are ready to go. 2 of them go off to the site of infection and do their thing
and the other ones stay in the lymph nodes and does its thing. B cells have further differentiation to
go through. So there are 3 fates for b cell differentiation.

Slide 12
So here if we look again, we have our th2 t cell. CD40 ligand on the t cell binding to the cd40 on the
b cell. MHC class II on the t cell binding to the b cell. That has the t cell moving its cytokines to this
interface, so the cytokines get dropped directly and the b cell which has put up b cell receptors - you
can see these little patches in immunofluorescence. So the cytokines are falling directly on the b cell
thats going to be activated and that causes the b cell to divide.

Slide 13
So now we start to get clonal proliferation of the b cell. And here we are seeing b cells coming in. t
cells coming in. the t cells react with the dendritic cell, they get activated, clonal proliferation. Here
our b cells have picked up copies of the antigen, mind you this is not to scale, the t cells can now
interact cognately with any b cell that got a hold of the antigen. They are not going to interact with
Transcribed by David Landsman 7/25/14
any b cells that dont have that antigen because those b cells dont have the right peptides on their
HLA class 2. So all of the other b cells in the area, even though there are b cells in the area, the t cell
receptors cant bind the pathogen cause those b cells have what protein in their HLA class 2? CLIP.
Right? There is nothing to pull the CLIP out of the HLA class II in the vesicle because the b cell only
has host. So all the b cells that didnt get a hold of peptide, they cant bid to the t cell so nothing
happens to them. They dont get killed they are just left alone. So its only the b cell that got a hold of
the pathogen that gets to divide. So now the b cells go through clonal proliferation and they start to
form secondary follicles, or secondary germinal centers, and Im gonna skip the movie because Ill
lose the battle with the computer but Ill show it at the break.

Slide 15
So we have a schematic representation of the germinal center. So these dividing b cells are called
centroblasts and the ones that are going to do something next differentiate are called centrocytes.
The mantle zone, we have t cells along. What are these things? Dendritic cells. What else could they
be? Macrophages. Stick macrophages in your head.

Slide 16
So our cloned daughter b cells, they can go through three fates. You know about two of them. One of
them is a plasma cell. Thats a workhorse cell that goes to the site of infection and secretes antibody.
The second one is a memory cell, that cell sits out of this immune response but its ready for the
next time you get infected so Ill never get this particular version of the virus again. And the third
one is a process called somatic hypermutation. The third fate is a process called somatic
hypermutation. And what happens is the original antibody produced by a b cell is probably a very
low affinity. So there are very few amino acid interactions on that antibody that have it stuck to the
pathogen. And if you go back to your basic chemistry, low affinity interactions what happens is the
molecules stick together and the molecules fall apart. The molecules stick together and the
molecules fall apart. So the antibody binds the pathogen, the antibody falls off. The antibody binds
the pathogen, the antibody falls off. If the antibody has fallen off is it going to do anything? So low
affinity antibodies arent very useful. So this process does through this process of somatic
hypermutation you create antibodies that have a really high affinity. And up to 30,000 times higher
than the original parent b cell affinity. And so that antibody will bind and it will stay bound literally
for days. And by then youll have something that can take care of whatever the antibody is bound to.
So this process is going to create waves of antibodies with successively higher affinity, which means
that they can stay on the pathogen for successively longer periods of time. This process is not gene
rearrangement. It is random changes in the V region gene. So the gene rearrangement is done, it is
fixed, it is not going to be changed. Here you just come in and make random mutations in the V
segment of that variable region gene. So somatic hypermutation is accomplished through random
mutations in the variable region of the already fixed part of the heavy and light change genes. Its
not gene rearrangement, thats done, those proteins are long gone. So thats done, and when you do
random rearrangement, so this is happening in the centrocyte. So some of the cytokines cause those
centrocytes to undergo somatic hypermutation. And any b cell thats undergone somatic
hypermutation now must go through that same process that the original b cell went through, it has
to bind to a follicular dendritic cell and get a hold of pathogen with the b cell receptor. Now if you
are making random changes in a gene, whats the probability that making the random change is
going to have no effect whatsoever on binding? Whats the probability that its going to make
binding worse? Moderate to high. Whats the probability that its going to make binding better?
Low, right. So most of the daughter b cells that went through somatic hypermutation theyre going
to try and bind to the follicaular dendritic cell and get pathogen, but their antibody doesnt work
very well. So they dont bind to the follicular dendritic cell and they cant bind to the pathogen on
the follicular DC, and if they cant bind to the pathogen on the follicular DC can they talk to the TH2 t
Transcribed by David Landsman 7/25/14
cell? No. So they fail to get survival signals and they die by apoptosis. And what cell did I just tell
you to remember? Macrophages. What are the macropahges there for? To eat all of those apoptotic
cells. So you know, one in a hundred, one in a thousand. One in a hundred, one in a thousand, that b
cell actually makes a receptor that has a higer affinity for the pathogen. It takes the pathogen from
the follicular dendritic cell, it goes through receptor mediated endocytosis, it puts the peptide on
mhc class 2, the mhc class 2 on the b cell can talk to the T cell receptor on the T cells. CD40 ligand
binds to CD40, that b cell goes through another round of proliferation. Now in the beginning, 99%
of the b cells go through somatic hypermutation. Right? They have a really low affinity antibody so
having tons of really low affinity antibody isnt going to do our immune response a lot of good. Now
a few of them will go off and become plasma cells. They go to the site of infection because low
affinity antibody is better than no antibody. Especially in a virus where the IgM thing didnt work.
Ok? You want me to go through somatic hypermutation again? I thought so.

So, the b cells start to go through clonal proliferation except its not really clonal. In this first round
of b cell proliferation, most of the daughter b cells are going to go through a process called somatic
hypermutation. Theyre making random changes in the nucleic acid sequence of the V region gene
and thats going to change the amino acid sequence and that can either increase the affinity, leave
the affinity alone or decrease the affinity. Right? Increase the affinity, leave the affinity alone
because it happened in a part thats not actually binding or decrease the affinity.

Slide 17
Those b cells that went through somatic hypermutation must interact with the follicular DC or the
free pathogen thats in the lymph node and try and bind to it. If you havent increased the affinity, or
if you have decreased the affinity, those b cells will not be able to put peptide on HLA class 2, they
will not be able to talk to helper t cells, they will not get survival signals and they will die by
apoptosis. The one or two b cells that manage to make a receptor with high affinity get pathogen
from the follicular DC, they do receptor mediated endocytosis, they put peptide on HLA class 2, the
HLA class 2 peptide on the b cell binds to the t cell receptor, CD40 on the b cell binds to CD40 ligand
on the t cell, the b cell gets survival signals, it now goes through another round of proliferation with
a higher affinity antibody. Most of these b cells undergo somatic hypermutation, they have to repeat
the whole process over. If you have 10,000 daughter b cells (not a real number) and 9999 go
through somatic hypermutation the first time youre gonna get 3 that had a higher affinity antibody.
So those 3 b cells will go through another round of proliferation, youll get 30,000 daughter b cells
(10,000 from each of those) and 10 of them 9990 of them will go through somatic hypermutation
the second time through. So you keep this process going and every round of clonal proliferation the
cells that go through somatic hypermutation have to go through this process but the one or two or
three daughter cells that get picked out their affinity goes up. And the longer the immune response
goes on, the higher the affinity gets. And what happens is over time you run out of pathogen. Right?
You now have a really good immune response going on, youve sucked up all the free pathogen
thats floating around in the extracellular space. Youve gotten most the pathogen off the follicular
dendritic cells, so eventually even when you make a really high affinity antibody there isnt any
pathogen for it to bind so all those cells die by apoptosis. But with each successive round you start
to change the proportions. So in the beginning, made up numbers, you know 9995 of the b cells did
somatic hypermutation, 5 of them went off to become plasma cells and secrete low affinity
antibody. As the affinity of the antibody improves you want to have more cells becoming plasma
cells to secrete higher affinity antibody and now you are gonna start to make some memory cells so
that they are going to have high enough affinity antibody so that they are worth having around. So
each successive generation the proportions of the cells that go through somatic hypermutation or
become plasma cells or become memory cells. And its an OR, it can only do one of those three
Transcribed by David Landsman 7/25/14
things. It cant become a plasma cell and a memory cell, it cant go through somatic hypermutation
and do any more differentiation without going through this interaction.

Slide 18
So then what happens once the cells that dont go through somatic hypermutation, theyll either
have IL-10 and IL-4 receptors. If they are bound by IL-10 they differentiate into plasma cells.
Plasma cells are terminal stage cells, they live a month and all they do is secrete antibody. And if
you were to take a plasma cell out of the tissue and stick it in a laboratory dish and let it secrete
antibody for a month, at the end of the month if you took that antibody and purified it and dried it
down, it would have actually secreted enough antibody so that you could see that protein without a
microscope. You have a little lump of protein about the size of a grain of salt. And thats a boat-load
of protein, to be visible by the naked eye. So plasma cells secrete massive amounts of antibody.

And in my viral infection whats going to happen in another day or so is my high enough affinity
antibodies are going to get to the site of infection and they are going to prevent the infection of new
cells, so eventually no new cells will be infected and once my CD8 t cells have killed off all of my
virally infected cells, the snot will go away, as will the cytokines that are giving me all of the other
symptoms. If the b cell runs into IL-4 instead of IL-10, it differentiates into a memory cell, memory
cells go off into the bone marrow and do nothing, at some point in time after this infection is over,
ok because for the next 30 days Im going to have really high levels of antibody. The memory cells
will come out, theyll go back to the lymph node, theyll bind to the residual pathogen that is sitting
on the arms of the dendritic cells in the icoves. Theyll get survival signals and theyll secrete a
round of antibody. And therell be memory cells that do this in my body for however many years.
Another 30 or 40 years. So as long as I have those memory cells that keep secreting antibody, if I
come into contact with this particular virus the antibody will bind to it before it infects cells and I
will be sick but Ill never have symptoms. So it will get rid of the pathogen before I can become
symptomatic. So this is why you have to have antibodies against viruses, because antibodies are
what prevent new cells from becoming infected, antibodies are what prevent infection, you have
memory CD4 and memory CD8 t cells but whats the good of having memory CD8 t cells because
they can only kill things after theyre infected. It would be much better to prevent any cells from
being infected in the first place. So you have to have antibodies in order to prevent reinfection of
viruses and bacteria. And if you have to have antibodies what kind of cell do you have to have to get
the b cells activated? TH2 t cells. And what kind of a t cell is a TH2 t cell? CD4. So for a viral infection
you have to activate CD4 t cells or you dont get TH2 t cells and then you dont get antibodies and
then you cant prevent the new infection. And you cant prevent yourself from getting reinfected
and your cd8 t cells just keep killing things. So it is crucial for a viral intracellular infection to have
both CD8 t cells and CD4 t cells because without the CD4 t cells you cant get the antibodies. And
without the antibodies youre not going to be able to keep cells from being infected and youre not
going to be able to prevent reinfection. So later on in the response, by Monday or Tuesday, Ill have
had multiple rounds of somatic hypermutation, that will give me very high affinity antibodies and
that process is called affinity maturation. And once I have the high affinity antibodies they will stay
stuck for days, I will start making lots of plasma cells that will come to the site of infection and
secrete antibodies. I will be making memory cells that are going off to the bone marrow to sit
through the rest of this response but then in a month or so theyll start secreting antibodies to keep
me from being reinfected by this pathogen.

Slide 19
Ok, so in addition to IL-4, 5 and 6, and hes very inconsistent on the cytokines that are secreted. So
the TH2 T cells they secrete IL-4, IL-5, IL-6, IL-10, IL-13 and TGF-beta. IL-4, 5, 6, 10, 13, TGF-beta. As
we saw a couple of slides ago IL-4, 5, and 6 are a part of what gets the b cell activated so they are
Transcribed by David Landsman 7/25/14
necessary for b cell activation. They do not cause b cell activation. They are necessary for b cell
activation but they are not sufficient for b cell activation. If you drop IL-4, 5 and 6 on a b cell that
has not come into contact with a T cell or its pathogen, nothing will happen. So the cytokines, in and
of themselves do not activate B cells. They are necessary for B cell activation but they can only act
on B cells that have already gone through cross-linking receptor mediated endocytosis and T cell
cognate interactions. So Il-12 can activate an NK cell. And IL-1 can activate a dendritic cell. But B
and T lymphocytes cant be activated by cytokines, they have to have those physical interactions
before the cytokines can do what they can do, you still have to have the cytokines but by themselves
cytokines are not sufficient.

Slide 20
So, TH1 T cells, site of infection, go off and kill foamy macrophages. TH2 T cells stay in the lymph
node and get b cells going to make antibodies.

Slide 21
And well take a break a little bit early, Ill show the movie and well pick it up with cytokine
functions.

TH2 cytokines actually have 2 separate sets of functions. One of them has to do with b cell
activation and one of them has to do with shutting off the immune response and I decided to
separate them out. So well talk about the other functions on Monday. So necessary for b cell
activation, not sufficient. Cytokines in and of themselves cannot activate adaptive immune cells, not
b cells not t cells, or classical b cells or classical t cells. They have to be there, theyre necessary but
by themselves they cant do it. Cytokines. IL-4 isotypes is a growth factor for TH2 t cells. Thats how
we got a TH2 t cell is IL-4 bound the IL-4 receptor that eventually gave you GATA3 which gave you
the cassette of IL-4, 5, 6, 10, 13 and TGF-beta. IL-4 isotype switches to IgG and IgE and it causes
differentiation to memory cells. IL-5 is necessary for B cell activation. IL-5 is also necessary for
eosinophil activation and for those of you who have asthma, this is the culprit, the asthma activates
the eosinophils, the eosinophils activates the IL-5, Il-5 makes more eosinophils, eosinophils
activates IL-5, IL-5 makes more eosinophils. IL-10 with TGF-beta causes isotype switching to IgA
and causes differentiation to plasma cells. IL-13 is a growth factor for B cells. I put IL-6 on here
whered it go? Alright, Ill update this because I did put IL-6 on it.

Slide 22
TGF-beta with IL-10 isotype switches to IgA and activates neutrophils. CD40 ligand induces
cytokine receptor production in activated B cells, drives b cell proliferation, drives B cell
differentiation and causes isotype switching. IL-6 in the lymph node helps, is necessary for B cell
activation and it increases antibody production. Why doesnt CD40 ligand on a TH2 t cell activate a
macrophage? Why doesnt CD40 ligand on a TH2 t cell activate a macrophage? What do you think
TH2 T cells do at the site of infection? They activate macrophages. So why doesnt CD40 ligand on a
TH2 t cell activate a macrophage? It is not at the site of infection. Why doesnt CD40 ligand on a TH1
t cell activate a b cell? It is the TH1 T cell is at the site of infection and the b cell is in the lymph node.

Slide 23
So function can very definitely be location dependent. Especially for membrane bound cytokines. So
b cells bind antigen with their B cell receptor, that causes receptor mediated endocytosis and you
get class II processing and presentation. B cells find an already activated and differentiated TH2 T
cell, the MHC class II on the b cell binds to the T cell receptor. CD40 ligand on the B cell binds to the
CD40 on the T cell receptor and thats the costimulatory signal. Lack of CD40 ligand binding to
CD40 gives you an anergized B cell and you get peripheral tolerance.
Transcribed by David Landsman 7/25/14

[Student] - What about the macrophages in the lymph node that

[Dr. McCutcheon] - They are in a different state because they are not activated by gamma interferon
and CD40 ligand on the TH1 t cell because the gamma interferon is all off at the site of infection. So
they are really there to eat those thousands and thousands and thousands of apoptotic b cells.

And then you have to have cytokines. And the TH2 t cells are activated before the b cell so youve
got a cytokine soup in the lymph node at this time. Youve got lots of IL-4, 5, and 6. So once the b
cells put up the 4, 5 and 6 receptors theres plenty of cytokine for them to bind.

Slide 24
B cells proliferate, the daughter cells have one of three mutually exclusive fates. They can go
through somatic hypermutation OR they can become plasma cells OR they can become memory
cells. They cannot do more than one. And the proportion of cells that go through each fate changes
with successive rounds of proliferation so early on you want lots of somatic hypermutation, you
dont need any memory cells, you want a few plasma cells. Middle of the infection, you are gonna
have about half somatic hypermutation youre gonna be making a lot of plasma cells and some
memory cells. End of the infection youre gonna have very little somatic hypermutation, youre
gonna be making lots and lots of plasma cells and youre gonna be making a lot of memory cells.
And every successive round the cells that get fished out have a higher affinity, affinity maturation,
and the end result is that you if you have a long enough infection, you end up with an affinity of
the daughter cell antibodies that is around 30,000 times greater than the affinity of the parent B
cell.

Slide 25
So, IL-4 so antibodies, as we are gonna talk about next, you have different isotypes of antibodies
which means the binding end is set, the functional end changes. Oh, weve already talked about that
sorry. So we have IgM, IgG, IgA, IgE. IgG has different subclasses, for purposes of this class we lump
them together. So IL-4 you get IgG and IgE. IL-5 you get some IgA and I thought in humans you got
IgE so this may be in mice. Gamma-interferon you get IgG. TGF-beta you get IgG and IgA. Alright?

Slide 26
So what do these antibodies do, because this is really what a b cell does. The b cell in and of itself
isnt particularly active in the immune response, it produces the antibodies and the antibodies are
the molecule that is what we need. So were gonna look at these different functions and we are
gonna lump all of the IgGs together. So we are gonna start with the two simplest antibodies. And the
book is out of date. We used to think that IgG was important for b cell activation and it had no other
function. And in 2010 and or 2012, I cant remember which, somebody final showed that IgD has a
function and thats why Ive put the two plus signs over the minus sign in the table. And what IgD
does is that there are receptors on mast cells, basophils and eosinophils for IgD. There are receptors
for mast cells, basophils and eosinophils for IgD. And if you have binding of an antigen to the IgD on
the mast cell, basophil or eosinophil, you suppress the function of the mast cell, basophil or
eosinophil. So binding through IgD suppresses mast cells, basophils and eosinophils. IgE also only
has one function, it binds to mast cells, basophils and eosinophils but in contrast to IgD, if you have
binding through the IgE receptor you get activation and degranulation of the mast cells, basophils
and eosinophils and for anybody who has hay fever, you know what happens. So IgD and weve only
just begun to figure out IgD so we dont really understand this really well, but IgD apparently is
necessary for suppression of mast cells, basophils and eosinophils and IgE is necessary for
activation of mast cells, basophils and eosinophils. So thats why mast cells, basophils and
Transcribed by David Landsman 7/25/14
eosinophils can be suppressive or cause disease. It depends on which receptor you go through. The
simplest function and the only function that antibodies can do by themselves is neutralization and
this is primarily done by the IgGs and the IgA. And as Ill show you in the next slide. Neutralization
is the only function that an antibody can do by itself. And neutralization is when the antibody binds
to the spot on the pathogen that the pathogen needs to be able to cause disease. So if you bind to the
spot on the pathogen that can cause disease and its a high affinity antibody and it stays bound for
days and days and days, the pathogen cant cause disease. Neutralization, the only thing the
antibody can do by itself, everything else has to happen through receptors. So opsonization like
C3b, IgG is an opsonin. IgG is necessary for NK cells to be able to recognize any kind of target and
thats called antibody-dependent cell-mediated cytotoxicity. So antibody binds to something, NK
cells can come along and kill it. Antibody-dependent cell-mediated cytotoxicity or ADCC. Antibodies
can sensitize mast cells or desensitize mast cells, and as we talked about earlier antibodies can
activate complement. IgA is the antibody that gets out of the tissues, so IgA gets transported into
fluids, namely saliva sweat and tears. So as dentists, IgA is the antibody that will get into saliva that
will prevent bacteria from attaching to teeth. It doesnt happen, which is why strep mutans attaches
to teeth. IgGs get transported into the placenta so thats what protects the fetus and they get stuck
into extravascular sites, so you have IgG in the tissue, youll have IgG in saliva and it is not because it
is transported there it is because it leaks there from the effluent around people and so the more
periodontal disease the more IgG you have in saliva. And if you look at how much IgE you find in
serum it is almost none and that is because IgE is always stuck on to mast cells, basophils and
eosinophils. You dont find it free in serum. The other antibodies are in serum. IgE is not, it is always
stuck on a receptor.

Slide 27
So we are going to look at how neutralization works. And here we have a toxin and lets give
ourselves botulism toxin. So heres our toxin, it has evolved to bind to a protein on the surface of
mammalian cells. If it binds to the surface of mammalian cells it causes receptor mediated
endocytosis it then releases the toxic subunit and the cell dies. And you suffer painfully and horribly
and probably die. If you neutralize it, so if the antibody is bound to here, can this bind to there?
Youve neutralized it. Now if this is a low affinity antibody and it falls off, can this bind to there? So
you need high affinity antibodies so that they stay stuck on for days. And so this is how you prevent
viruses from infecting cells, you bind to the part of the virus that was going to bind to the part of the
host cell, the protein on the host cell that would cause receptor mediated endocytosis and allow the
virus to get inside the cell. Its how you prevent toxins from getting in cells, its how you bind to
spots on the bacteria that allow them to attach to cells and that prevents the bacteria from being
able to attach to cells. Neutralization. Youre not doing anything to whatever the antibody is sticking
on but you are preventing that pathogen from being able to do what it needs to do to be infectious
or cause cell death.

Slide 28
Everything else has to go through a receptor. How many people in the room think you should
memorize all this stuff about receptors? There are no hands up, good call. So you need to know that
there are receptors and they are specific about isotypes, and you have gamma-receptors, you have
epsilon receptors, well these are all gamma receptors, but you have a receptor for IgG, you have a
different receptor for IgE, you have a different receptor for IgD. You dont have receptors for IgA or
IgM so their activities are not going to happen through receptors.

Back to slide 26
If we go back, look at what IgA does. It neutralizes, and where do we find IgA? In fluids. So what its
job is to do is to bind things in fluids and prevent them from getting inside the body. Part of your
Transcribed by David Landsman 7/25/14
chemical barrier. And it neutralizes because there arent any cells on the other side of the barrier to
interact with the IgA. For purposes of this class we dont care about the one pluses. K so IgA
neutralizes, everything else IgG and IgE and IgD have receptors and they work through
phosphorylation.

Slide 29
So here we see opsonization, the bacteria are bound by antibody. Antibody, the receptor for the IgG,
the bacteria are bound by an IgG, the receptor for IgG has a much higher affinity than a toll receptor.
So much higher affinity, the macrophage can stay stuck to the bacteria for longer and its easier to
swallow. Opsonization. And again, if the antibodies have all fallen off the bacteria because theyre
really low affinity, can the macrophage bind to the antibodies that are not bound to the bacteria?
No. so you have to have the high affinity antibodies so that the receptors on the cells that have
receptors can do their thing. So opsonization we end up with peptides and a phagolysozome.

Slide 30
Here we have antibodies that are bound to pathogen. Our natural killer cell comes along, it has a
receptor for antibodies that are bound. Now, IgE binds to antibody and then the pathogen binds to
the IgE thats already bound to the mast cell. Sorry. IgE binds to the receptor on the mast cell and
then the pathogen binds to the IgE thats already bound to the receptor on the mast cell. IgG
receptors work the other way. So IgG has to bind first and only after the IgG is bound does the other
end of the IgE molecule have the right conformation to bind to the receptor. So receptors cannot
bind to empty IgG, they have to bind to IgG thats already attached to its pathogen.

Back to slide 29
So here, if we had IgG in space, these IgG receptors could not bind to that IgG. They could only bind
to that IgG thats stuck on the pathogen.

Slide 30
Here we have the IgG bound to the pathogen then the IgG receptors on the natural killer cell can
bind to the IgG on the pathogen, the natural killer cell then induces apoptosis of the target cell. So
NK cells dont have toll receptors, they cannot act against bacteria until you put an IgG molecule on
it. Once youve put the IgGs on the bacteria, then the NK cells can come along and kill it. And this is
what kind of killing? That thing that I just gave you. Antibody-dependent cell mediated cytotoxicity.
OK.

Slide 31
Here we have our mast cells, our mast cells are already bound to IgE and IgD. So this is free IgE and
free IgD, and if the mast cell comes along and binds to if the IgE on the mast cell is then binds to its
antigen, that causes degranulation. All those histamines and other wonderful things go off into your
tissues and you suffer. If the mast cell binds through IgD, and were not clear if the IgD is already
bound to the receptor on the mast cell or if IgD binds to the pathogen and then binds to the IgD
receptor, so we dont know that yet. But if it goes through the IgD receptor then you suppress the
mast cell function.

Slide 32
So IgD is found in mucosal associated tissues and it seems to help suppress immune activation
against gut pathogens. And thats actually a good thing. And the whole reason we figured this out is
because someone managed to make an IgD knockout mouse and they were expecting that you
simply wouldnt get B cell activation but there were these other things that happened, you started
getting disease and the only thing that was missing was the IgD and so if you take IgD away and
Transcribed by David Landsman 7/25/14
suddenly you start seeing food allergies and things then that tells you the IgD must be doing
something. And so, then once they knew to look for it they started looking for receptors. So, I mean,
and thats only been a couple years so we arent that far along. So IgD is doing suppressive things
and we know that at least in mice it blocks IgE mediated activation of cells. So for purposes of this
class you have to know that IgD has a function and thats to suppress mast cells. The other stuff we
dont really know well enough to understand.

Slide 33
IgM does one thing and it does it really well: it fixes complement. Some of the IgGs also fix
complement, we dont care about the one pluses.

Slide 34
So again, back to the beginning. Here we have our IgM, IgM is a pentamer. Right? Whats the affinity
of IgM? Affinity F.F. Right. One. Whats the avidity? Nope. Ten! Right? Five pairs, one, two, three,
four, five, six, seven, eight, nine, ten. Ok. So the single IgM molecule sits down here and it forms this
platform, and a single platform of IgM is big enough that a C1 can bind to it. So it only takes one IgM
stuck to a bacterium to get C1 bound and activated. And thats why that even that really low affinity
IgM secreted by the B1B cells is effective at activating complement. Doesnt have to be there for
really long, and once you get the C1 to start cleaving the C2 and C4, everything goes.

Slide 35
Now IgA has to get out of the plasma and into the things on the other side of epithelium. And it does
this through a process of transcytosis. So theres a polyimmunoglobulin, immunoglobulin is another
word for antibody. So Ig is immunoglobulin. Theres a poly-Ig receptor on the basal surface of
epithelial cells. We have plasma cells that are secreting IgA and IgA is dimeric so whats the affinity
of IgA? Affinity is always [one]. Whats the avidity? Four. OK? One, two, three, four. And it has to be
the dimeric form of IgA. So the dimeric form of IgA binds to the Ig receptor. You get receptor
mediated endocytosis and this transcytosis, so it just goes from one end of the epithelial cell to the
other end of the epithelial cell. So it transits through the epithelial cell and it gets released on the
other side into the lumen of whatever. Sweat glands, saliva or tears. And when it does that, a piece
of the immunoglobulin receptor breaks off and stays stuck to the IgA. And that piece that breaks off
is called the secretory component and the secretory component is actually very resistant to
proteases. So it helps prevent bacterial proteases from cleaving the IgA. And one of the reasons that
Porphyromonas gingivalis is such a potent pathogen is because it has a protease that can cleave the
secretory component so the IgA is then susceptible to degradation. So you get the IgA in saliva
against the Porphyromonas gingivalis and then the Porphyromonas gingivalis secretes proteases that
can destroy the IgA which then lets it infect. Remember? Because the function of IgA is to
neutralize, and thats an aside by the way.

So IgA goes through this process of transcytosis. Poly-Ig receptor on the basal layer. Receptor
mediated endocytosis transits through the cell and gets spit out the other side. And when it gets spit
out the other side you have the secretory component. Ok.

Slide 36
So IgG is in the capillaries. We need to get it into the extracellular spaces because thats usually
where the pathogens first come in and if we dont want to get a reinfection we want to get hold of
the pathogen before it has a chance to do anything. So IgG, theres also a receptor and it gets put
from one side of the endothelial cell to the other side of the endothelial cell in the extracellular
space. And it dissociates. So we have IgG and all of our extracellular fluids, so that when we get
reinfected with whichever pathogen I mean some of this will be neutralizing IgG so it can prevent
Transcribed by David Landsman 7/25/14
the pathogen from binding, others will be high affinity so its gonna stay stuck for days, others will
be IgG, and there are receptors on the immature dendritic cells for IgG that can then cause receptor
mediated endocytosis and the dendritic cell fuses phagolysosome, pathogen is destroyed. Doesnt
matter if it is a bacteria or virus at that point because it has the IgG bound to it.

Slide 37
So neutralization, done by IgA. IgG, primary function is to stop it binds to whatever the pathogen
is going to need to be able to be infective or cause disease and if you bind that the pathogen isnt
going to be able to do its thing, and if you bind to that with a high affinity antibody the pathogen
isnt going to have a chance to do its thing. Opsonization: coat with butter, it makes it really easy for
phagocytic cells to be phagocytic. So thats IgG. Complement activation is IgM and IgG. You get C1q
bind-- C1 binding and activation of the C1 convertase, off the classical pathways goes. Antibody
dependent cell mediated cytotoxicity, IgG binds, natural killer cells come along and bind to the IgG
and they kill whatever the IgG is bound to. Mast cell degranulation is IgE. Prevention of mast cell
degranulation is IgD.

Slide 38
Alright, so those are what antibodies do during our adaptive immune response. And, one of the
things you need to understand is even if you only have one solitary B cell that responds to a
particular pathogen. As you undergo through all of those rounds of clonal proliferation and somatic
hypermutation and affinity maturation, you end up with a species of B cells, or a species of antibo
well species of B cells and a species of antibodies. So you get this, its not just one antibody. So in a T
cell you can only have one receptor thats gonna do all of the work but when you get B cells you get
a range of antibodies, some are going to bind with higher affinities, some are will bind with lower
affinity. They're all binding to the same bacteria but theyre not identical, at the end of that immune
response. And honestly in a b cell, the b cell response you never have just one b cell, you dont have
hundreds but you have more than T cells. So youll have antibodies that will bind to a lock and key
sort of binding, youll have antibodies that will bind to a sort of broader area and this is called a
polyclonal response because you have many clones. And even if you only started out with one b cell,
because it goes through somatic hypermutation at the end you still have a polyclonal response.
Those antibodies will be closer to each other than antibodies from a different b cell, but theyre still
different. OK? And that is good for some things and part of that is because if you have a whole bunch
of different antibodies against a pathogen and the pathogen mutates you are going to lose
antibodies from one family but youll have all of the other antibodies. So even though the pathogen
mutates you still have an antibody response that is going to work. And it will take a long time for
the pathogen to mutate enough so that all of the antibodies that you made against that response
dont work anymore. So naturally occurring immune response gives you this really broad set of
antibody coverage. So youll have multiple different kinds of epitopes, the really broad epitopes, the
lock and key epitopes, and all of the antibodies that do that will have slightly different antibody
binding sites because of that somatic hypermutation process. So your naturally occurring immune
response is a polyclonal response.

Slide 39
Back in 1991, a graduate student named George Kohler working in a lab in Italy for a guy named
or England. Working in a lab in England for a lab named Cesar Milstein figured out that you could
do this thing with B cells. So, myeloma cells are the cancer form of a B cell. And cancer forms of B
cells will grow forever in tissue culture. You can take B cells from a person and infect them with
Epstein-barr virus and turn them into cancer cells and theyll grow forever. Quite happily. If you
take normal B or T cells out of the animal, be it human or mouse, and you put them in a tissue
culture dish theyll die after a while and theres nothing you can do to keep them alive. So we had
Transcribed by David Landsman 7/25/14
these cells that would grow forever, and what he did is he went and they immunized mice with
specific things. OK? So, Im making this up but lets say albumin. They immunized the mice with lots
and lots of albumin. Chicken albumin. And so youre gonna get a B cell response eventually, right,
from this immunization with the chicken albumin. So youre gonna have antibodies, youre gonna
have B cells that are making antibodies against the chicken albumin. So after youve done the
immunization and youve reimmunized them a couple times so you have lots and lots of B cells
making antibodies against the chicken albumin, they killed the mouse and they took all of the B cells
out of it. They took all the blood out of it and then they got the B cells out of the blood. And now one
of the things they did is they made these tumor cells missing an enzyme. Ok? So if you grow the
tumor cells in the medium that contains the substance that they dont have the enzyme for the
tumor cells grow forever. So they took the tumor cells, they took the b cells that they got out of the
mouse, and they put them in with some polyethylene glycol, you know the antifreeze stuff? Which
causes membranes to dissolve. So what you got was immortal myeloma cells fusing with the B cells.
And then they put them in media that did not contain the stuff that the immortalized cells did not
have the enzyme for. Now the B cells had the enzyme but the immortal cells didnt have the enzyme.
So all of the immortal cells no longer have everything they need to grow and they all die. So any cell
that didnt fuse died. And these B cells, even though they didnt fuse, they were gonna die anyway
because we cant keep them alive in culture. So the only thing were left with are fusions of the
immortal B cell and a single other B cell from the mouse. And just so you know, the details of this
technique are not something I am gonna ask you. Im explaining it so that you understand where
this comes from. So now each B cell that fused only produces a single kind of antibody right? So
what we end up with is a bunch of B cells that can now grow forever in culture, they secrete
antibody but each one of those B cells only secretes a single antibody. And you can fish out the B
cells that are secreting the antibody you want through some techniques and then you can expand
that population and now you have a whole bunch of B cells that are all secreting a single antibody,
and if it is single it is what clonal response? Monoclonal response. And monoclonal antibodies are
the tool that really changed the face of biology. Because before if you wanted to purify T cells out of
a mouse, you would start at 6 oclock in the morning and you would kill the mouse and youd collect
out the lymph nodes or the spleen, and then youd mush up everything and youd run it through a
sieve and get all the cells out. And youd put the cells in a test tube and youd spit it out and that
would get rid of all the red blood cells and youd suck off the buffy coat, and then youd pour these
percol [?] gradients and youd have to have uber steady hands and youd pour these gradients that
had slightly different densities and that would have all the macrophages and neutrophils be heavier
than the lymphocytes and then youd suck the lymphocyte coat off and if you wanted T cells youd
run that bunch of lymphocytes over nylon wool and the B cells would not stick to the nylon would
and the T cells would stick to the nylon wool. So then you take the nylon wool and you wash it out
and 16 hours later youd have a population of cells that was approximately 70% pure T cell.

Monoclonal antibodies, you inject the mice with CD3, you inject the mice with CD3, you inject the
mice with CD3. Which it took you 5 weeks to purify ok? Hehe, or maybe three months. And so now
you inject the mice with CD3, you get a bunch of B cells that are making antibodies against CD3, you
fuse them with the myeloma cells, you kill off everything that is not a fused cell, you put purified
CD3 protein, you attach it to a bead, so now you have a bunch of beads that have CD3 protein. You
take your spleen cells from you take your cells that youve collected from a different mouse, you
get rid of the red blood cells. You dont have to do anything else, no percol gradients, you pour all of
the white blood cells over the column that has the beads that have the CD3 antibody, or the CD3
protein, all of the B cells, all of the cells that have CD3 on them, so all of the T cells are gonna stick to
the beads, the antibody on the beads, everything else washes off. Four hours later you have a
population of 90% pure T cells. Ok? So it was that tool that let us pull out cells that had specific
proteins that let us really start understanding how what proteins did and how cells were identified.
Transcribed by David Landsman 7/25/14
Those guys won a nobel prize for that. So monoclonal antibodies are a tool in laboratories to be able
to identify specific proteins and then figure out where they are and what they do.

Slide 40
We also use them diagnostically, so heres a machine called a flow cytometer. So here weve stuck
ourselves and weve got antibodies with different kinds of fluorescence. And you can use this just
for analysis, so you spread the cells out so they pass a laser one at a time and if theres an antibody
with a specific wavelength of fluorescence you get a signal. Ok? So cells that have no antibodies that
fluoresce give no signal, cells that have antibodies that fluoresce give a signal. Now one of the things
you can do is if you have the signal you are looking for, you can have the machine put a charge on
that cell and then that cell goes off into a different pot. So now you take your spleen and your lymph
nodes from your mouse, you mush them all up, spin out the red blood cells, you stain them with
antibodies, you run this machine and two hours later you have a 98.9 to 99.5% pure population of
the cell that you wanted. So weve gone from a 16 hour, 70% pure population to a 3 hour, 98.8 to
99.5 pure population. So its made life a lot easier. But you can also use this diagnostically. So here
what weve done is weve stained cells with an anti-IgM antibody, so weve made an antibody
against the antibody IgM, and weve stained them with an antibody against T cell receptor. And so
what we see here are we see 60% of the cells from this persons blood are binding to the anti IgM
antibody. 8% of the cells from this persons blood arent binding to any antibody and 31% of this
persons blood are binding to the anti T cell antibody. So if we change the labels on these and we
were using so we are gonna do 3 colours now. A colour that binds to CD3, and then were going to
have a different colour that binds to CD8 and a third colour that binds to CD4. So the CD3 is going to
tell us which cells are T cells. And so we can look at our population of T cells and then we can tell
how many T cells have CD4 versus how many T cells have CD8. And what disease am I going to
diagnose if I do that? Looking at how many T cells are CD4 versus how many T cells are CD8. Were
gonna see if people have HIV or if theyve gone to full blown AIDS. And thats how you do it. Thats
the test that you use. You put it through a flow cytometer.

OK. So Ive gotten us now to about 2004-2005. Now, were using monoclonal antibodies as drugs.
OK. You have rheumatoid arthritis? Whats the cytokine thats driving rheumatoid arthritis? Whats
the cytokine that activates osteoclasts? IL-1, or TNF-alpha that is making lots of IL-1. So you have
rheumatoid arthritis you have way too many osteoclasts, you want to get rid of them. Easy way to
get rid of them? Get rid of the IL-1. How do you get rid of the IL-1 without getting rid of anything
else? You make an antibody thats exquisitely specific for IL-1. Or you make an antibody thats
exquisitely specific for TNF-alpha. Or you make an antibody thats exquisitely specific for IL-6, thats
Humira. OK? Remicade is TNF-alpha. And so patients with Crohns disease, inflammatory bowel
disease, rheumatoid arthritis, psoriasis, theyre being put on monoclonal antibodies to get rid of
specific cytokines. And the reason you have to know cytokine function is because your patients are
going to be taking these monoclonal antibodies, and theyre gonna come into your office and youre
gonna need to know what will happen to them if you do treatment and theyve just had their
antibody infusion treatment and they have no IL-1 or TNF-alpha. So its really important to know
that because your patients are going to be on these and I suspect that some point in time, although I
dont know if dentists are going to be prescribing them because they are tricky to give, we may start
using some of these against severe periodontal disease that doesnt seem to come under control in
other ways. And it may not be an anticytokine antibody but it will be an antibody against
something. So this is called biologicals and it is the hot new area of medicine because you are
specifically preventing one thing from happening without preventing everything else. So unlike
methotrexate and steroids you are not suppressing the entire immune system, youre getting rid of
the one thing that is causing disease.

Transcribed by David Landsman 7/25/14
So antibodies in a natural occurring immune response, you have a polyclonal response which is
very good, because it means when the pathogen mutates youll lose one set of antibodies but
everything else is going to work. We can use antibodies as tool by making monoclonal antibodies.
You do not have to know the details of how you make monoclonal antibodies, you do need to
understand that you have a single specificity. Ok? So you get these single specificities and that lets
us do tools in the laboratory, we can fish out specific molecules very easily, we can fish out specific
cells, we can make a neutralizing antibody against something and find out what it does, if you block
its function. You can make antibodies against receptors so we can find out what happens when we
bind to that receptor in either an antagonist or agonist fashion. We can use antibodies
diagnostically. And then we use antibodies as drugs. So the moral of the story is antibodies are
good.

Ok and it is Friday afternoon on a gorgeous day, thank you all for coming and Im gonna let you go
early.