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Transcribed by Sofiya Khazanovich July 23, 2014

General Pathology Lecture 10 B Cell Development by Dr. McCutcheon

Slide 1 Image
[McCutcheon]: Ok, lets go ahead and get started. All of you who are here from the conference, thank
you for showing up. As I am now into hour three of talking, I tend to start babbling, so if I babble get
your hand in the air so I could correct whatever I said that was wrong. Its just, I get tired. So weve
talked about T cell development and weve talked about what T cells do and so now were going to talk
about the other antigen specific cell, because the next thing that happens in our adaptive immune
response is that now that we have activated TH2 T cells, we can activate our B cells, but we dont have B
cells yet because we havent developed them. So were going to step out of the activated immune
response, were going to go to a different place, were going to go to the bone marrow, and were going
to go to something earlier in time. So, were going to talk about the B cells that are going to be
developed and once they're developed they will exit the bone marrow to the periphery and to the
lymph node where they can participate. So B cell development starts in the bone marrow, and whereas
the T cells migrate off to the thymus to develop, the B cells stay in the bone marrow. And what the B
cells are going to do is generate a B cell receptor. So were going to talk now about B cell receptors. So
like a T cell receptor, B cell receptors have constant and variable ends. And the variable end is away
from the membrane. Like a T cell receptor, a B cell receptor is made of two chains, but instead of being
two identically sized chains, alpha and beta, B cell receptors have a big chain which we call a heavy
chain, and a little chain which we call a light chain. So each B cell receptor is unlike a T cell, and unlike a
T cell, each B cell receptor is made up of a pair of identical chains. So you have two copies of identical
heavy chains and you have two copies of identical light chains. So the B cell receptor has a pair of heavy
and light chains, the two light chains are identical two each other, the two heavy chains are identical to
each other. The B cell receptor has a constant end and it has a variable end. Now, make note of the fact
that variable does not equal light, constant does not equal heavy. So the variable region you have, you
have variable on the light chain and variable on the heavy chain. You have a constant region, you have a
much bigger constant region on the heavy chain. So light chain, heavy chain, variable region, constant
region. And every B cell receptor is pair of identical chains.

Slide 2 Antibody binding to bacterium
[McCutcheon]: So we have all of these pictures or cartoons in the book and they show an antibody
binding to something. So heres an antibody binding to bacterium. So in reality, see the little red dot? If
the bacterium is drawn to scale that little red dot is ten times larger than the antibody would be .If I
made it smaller you couldnt see it. So the reality is that you can see bacteria through a microscope at
40x, if you want to look at an antibody you use a scanning transmission electron microscope, and youre
at 10^9 or 10^10 angstroms. So you know its 8 or 9 magnitudes so times ten smaller than the
bacterium. So antibodies are really little despite the fact that they have cartoons where they look like
theyre the same size as the bacterium. So just keep that in mind.

Slide 3 Image
[McCutcheon]: So one of the T cell receptor is a fairly rigid structure, although the amino acids of the
variable part can move around somewhat, the T cell receptor itself is a fairly rigid structure and it binds a
fairly rigid structure, it binds the HLA peptide complex. In contrast, antibodies are incredibly flexible, and
as a matter of fact they have what theyre showing here with these bands, thats called a hinge region,
so the two arms of the antibody can move around in space. And so that gives antibodies this huge
amount of flexibility and thats what theyre trying to show you in this picture. We have an antibody
Transcribed by Sofiya Khazanovich July 23, 2014

thats kind of binding something straight up and down, this is binding a different antigen and here weve
got you know some flexibility and here this antibody has hinged itself way open. So T cell receptors are
rigid structures and they bind to fairly rigid structures, in contrast the two arms of the antibody can
move about in space. So the antibody arms can bind.

Slide 4 Image
[McCutcheon]: So now here were looking at a crystal structure of the antibody and you can see how far
apart the two ends of the antibody can get. So heres our constant region, heres the constant region,
the red is the constant of the light chain, the blue is the constant region of the heavy chain. Heres our
variable region, here maybe its a little easier to see here. So that variable region is really formed up of
parts of the light and heavy chain. And in the three dimensional structure this looks kind of like a cup. So
thats whats going to sit down on top of the antigen. In the cartoon structure again, heres our variable
region, constant light, constant heavy. So antibodies theyre shaped like a Y and the two forks of the Y
can move anywhere in 3D space to be able to bind their pathogen.

Slide 5 Image
[McCutcheon]: So in other picture, so you get this kind of barrel structure and this is a really common
structure used in biology, and so youll see things that are called the IG gene family, so IG stands for
immunoglobulin, which is another name for antibody, and this barrel structure is fairly commonly used
by other proteins. So somebody may talk about the IG gene superfamily, it means it has this barrel
structure. So youve got beta pleated sheets running in one direction and beta pleated sheets running in
the other direction and loops in between. This part isnt flexible, so you dont have beta pleated sheet
move away from beta pleated sheet, that stays the same, but this beta pleated sheet can move away
from that beta pleated sheet, and then within that in between the beta pleated sheets you have these
loops and these loops are also very flexible. So not only can the arms of the antibody move, but the
loops in between the beta pleated sheets can move. So they can move around in space so they can bind
more appropriately to whatever amino acid sequence theyre binding to or carbohydrate or nucleotide
or lipid.

Slide 6 Image
[McCutcheon]: And if we sequence antibodies, do peptide sequencing, we have tremendous variability
in certain spots in the antibody and it turns out that that variability corresponds to the loop. So this
amino acid sequence is pretty much the same, when we hit loops we have tremendous variability. Well
correlate this to development in a minute.

Slide 7 Specificity
[McCutcheon]: I promised you back in May that we were going to talk about the word specificity a lot,
and today is the bulk of the discussion. So antibody binding is exquisitely specific. A single amino acid
change can prevent an antibody that binds to hen, chicken, albumin, and the chicken albumin has a
single amino acid difference from turkey albumin, and an antibody that can bind to chicken albumin
cannot bind to turkey albumin. So antibody binding can be exquisitely specific. One amino acid change,
antibody wont bind. What is not specific is the location of the antigen. So if you have an antibody that
binds to a carbohydrate residue on streptococcus molecules, that carbohydrate residue is going to be
found on many different streptococcus molecules, and so the antibody can bind to that carbohydrate
residue on whatever streptococcus molecule it comes across. Now the carbohydrate residues are
different from the carbohydrate residues on staphylococcus, so the antibody against the streptococcus
Transcribed by Sofiya Khazanovich July 23, 2014

carbohydrate residues is going to bind to streptococcus but not staphylococcus. An antibody against
staphylococcus will bind to all the carbohydrate residues on all different kinds of staphylococcus, but it
wont bind to the carbohydrate residues on the streptococcus. So you can have, even though the
antibody is highly specific, that doesnt mean that that particular antigen can only be found in one place.
So you can find the pathogens in many different locations and many different copies on a given, the
antigen in many different locations and many different copies on a given pathogen, the antibody will be
specific to that but it can bind that wherever it comes across that particular specificity, that particular
epitope. Antibodies are exquisitely specific, they bind to their thing and there maybe instances where
they can bind to something thats one or two amino acids away, but not always. They bind their thing,
thats what they bind to. That does not mean that their thing can only be found in one place. So
antibodies bind specifically to a given thing, T cell receptors bind specifically to a peptide within a certain
MHC. Antibodies bind specifically to an amino acid sequence and a shape, but that amino acid sequence
and shape can be found sometimes in many different places.

Slide 8 Antigenic determinants and epitopes
[McCutcheon]: The part of the pathogen that gets bound or the antigenic determinant is called an
epitope. So the part of the pathogen that the antibody actually binds to, the antigenic determinant is
called an epitope. And if you have a pathogen like a bacterium or even a virus, youre going to have
multiple copies of each epitope on the pathogen. And an antibody then one antibody thats specific for
that epitope can then bind multiple times; multiple antibodies can then bind that epitope on the
multiple copies of the antigen, of the pathogen. So the epitope is the part of the pathogen to which the
antibody binds.

Slide 9 Image
[McCutcheon]: So this is actually the crystal structure of a virus, viruses are relatively simple compared
to bacterium, and this one has four different proteins. You have a blue, white, a red, and a yellow
protein. You have multiple copies of all of these proteins on the virus. So you have lots of different blue,
white, yellow, red. So we have four possible antigenic sites, antigenic determinants, and you can have
then an antibody against, one or more antibodies against each one of those antigenic determinants.
Now here, and so this is a virus right? Weve had to go to ten to the minus ninth or ten to the minus
tenth angstroms to be able to see this. So this is how big an antibody really is relative to the virus. And
this is an IgG molecule, it has two arms. And this particular one, the epitope, so this is the part of the
antibody that binds to the antigen on the virus, and so the white parts of the two antibodies, and so this
is the heavy chain and thats the light chain, will bind to the white protein. And because we have hinge
flexibility this could bind here and this part could bind here. Or it can bind up here, or it can bind over
there. So both arms of the antibody can bind because of the flexibility. And then we can have multiple
copies of this antibody binding to different parts of the virus as long as they're binding to the white part.
Then we can have an antibody against the blue part, and so it can bind to different parts of virus on the
blue part, we can have an antibody to yellow, it can bind to different parts of virus on the yellow part,
we can have an antibody to the pink part or red part that will bind to different spots on the virus in the
pink part. So each one of those proteins can have one or more antibodies generated against them
depending on your immune system and then these antibodies can bind in multiple copies on the
multiple epitopes on the pathogen. So pathogens always have more than one epitope, and they always
have more than one copy of every epitope. Pathogens always have more than one epitope; they always
have more than one copy of an epitope.

Transcribed by Sofiya Khazanovich July 23, 2014

Slide 10 Image
[McCutcheon]: So here multivalence, many different antigens, we have a red round thing, a green
triangle, a blue lumpy thing, and a green square. So each one of these is a different protein sequence, a
different protein sequence of antibody will be activated against this. So each one of these antibodies will
specifically bind to its epitope and not to any of the other epitopes. So thats many multivalent, many
different epitopes on the pathogen, and then there are always multiple copies of the epitope on the
pathogen. Of every epitope on the pathogen, so we have repeated epitopes and with the repeated
epitopes it means we can have higher copy numbers of antibody bound. So specificity for B cells, the B
cell will bind, the B cell receptor will bind its epitope, but the epitopes are always found in multiple
copies on whatever pathogen they're on, and they can be found pending on how specific the antibody
is, they can sometimes be found in many different places. So epitopes, B cells that recognize
carbohydrate residues, carbohydrate residues arent that complex, so youll have shared carbohydrate
residues by different species of bacteria. So a B cell that binds the carbohydrate residue of a
streptococcus is not going to bind to carbohydrate residues on E. coli, a B cell that binds to
lipopolysaccharide on E. coli can bind to lipopolysaccharide on different kinds of E. coli, but it will not
recognize the lipopolysaccharide on salmonella. And the B cell that recognizes the lipopolysaccharide on
salmonella is not going to recognize lipopolysaccharide on E. coli. So the antibodies are specific, that
does not mean that the epitopes are restricted. The epitopes are often found in many different

Slide 11 Image
[McCutcheon]: So there are three different primary ways an antibody can bind and the first is this lock
and key mechanism, and this is where always against a protein, and so if you have one change in the
amino acid sequence of this protein, that antibody will no longer bind. And this plays a role when you
have diseases where you have fairly high mutation rates like flu. So the flu mutates, and the antibody
you made against the previous form of the flu that bound like this, they dont work anymore. This is
called the lock and key and you have a very narrow set of binding, and this is frequently driven by
covalent or ionic bonds. So you have a positively charged amino acid on the antibody and it binds to a
negatively charged amino acid on the protein and if you take away that negatively charged amino acid
the binding falls apart. You can also have binding sites that are more broadly based, and so this is usually
against the secondary sequence of the amino acids together and the shape that they form, this is against
a secondary and tertiary sequence, so its not only the amino acids themselves but how they line up.
And then you can have these really broadly based antibody binding sites so this is very small, this is
larger, and these are protein to protein interactions, so Van der Waals forces, ionic and covalent bonds,
hydrogen charges, all the things that make amino acid bind to each other, thats whats causing this to
bind. So some antibody binding sites are exquisitely specific for the amino acid sequence, if you mutated
amino acid in here it probably isnt going to matter. So some antibodies can be exquisitely specific, other
antibodies have a broader specificity. But that doesnt mean just because it has a broader specificity it
doesnt mean an antibody against a protein on a streptococcus will bind proteins on staphylococcus,
unless its the same protein. So antibodies can have exquisitely specific binding thats down to single
amino acid changes and you lose the binding, antibodies can have broader binding that also takes into
account tertiary structure, and then antibodies can have a really broad binding. The broader and
broadest binding are not nearly so dependent on amino acid sequence. And sometimes theyre not
nearly as dependent on tertiary structure. So an antibody like this will bind probably to the protein when
its in its natural state, and if you denature it, because they really are only looking for secondary
sequence, youll still have antibody binding. If you have an antibody that binds something like this,
Transcribed by Sofiya Khazanovich July 23, 2014

where all of those amino acids have to be next to each other, if you denature that protein and they're
scattered across the linear form of the protein, this kind of an antibody wont bind.

Slide 12 Image
[McCutcheon]: So linear epitope, so this is based on the secondary sequence. Tertiary discontinuous
epitope, based on tertiary sequence in the native protein, these two amino acids next to each other, if
you denature that and stretch them out the antibody wouldnt bind. Sometimes shape matters and
sometimes it doesnt. And again this is in complete contrast to T cell receptors that only recognize the
main form of the MHC complex and peptide.

Slide 13 Review
[McCutcheon]: Antibodies are formed from two pairs of chains, you have two identical heavy chains and
two identical light chains. Antibodies have constant regions and they have highly variable antigen
binding regions. The thing that the antigen binding region binds is an epitope.

Slide 14 Compare and contrast
[McCutcheon]: So B cells make antibodies and when the antibody membrane bound, we call it a B cell
receptor. It can bind any kind of molecule, proteins, carbohydrates, lipids, nucleic acids. It can have
different shapes of epitopes some of which are dependent on tertiary confirmation. T cell receptors,
they only bind peptide, and they only bind peptide on the correct MHC molecule. And you have to have
the primary confirmation of the peptide MHC molecule for the T cell receptor to bind.

Slide 15 Antibody Formation
[McCutcheon]: Antibody formation, B cell receptor formation, works the same way as T cell receptor
formation. So we have gene segments, we do gene rearrangement, and its a developmental step. So in
the bone marrow, the B cells that are undergoing gene rearrangement are immature B cells. There can
be mature B cells in the bone marrow too, cause B cells hang out in the bone marrow, but the ones that
are undergoing gene rearrangement are immature B cells, theyre not out in the periphery, they're not
participating in immune response and you have to make the receptor before the B cell can exit bone
marrow. So gene rearrangement occurs first, then the B cell can exit the bone marrow and can go off
into peripheral circulation where it can participate in adaptive immune responses.

Slide 16 Image
[McCutcheon]: Gene rearrangement, what were going to do is concentrate the variability in the antigen
binding sites.

Slide 17 Image
[McCutcheon]: And we have segments, and so like the T cell receptor, the heavy chain has three
segments, a V, D, and J. So like the T cell beta chain, the heavy chain for the B cell has three segments,
V,D and J. Like the T cell alpha chain, the light chain has V and J segments. Like the T cell, where we start
development on the beta chain, we start development on the heavy chain for the B cell. Were going to
hook together V, D and J, were going to transcribe through a constant region, splice the constant region
Transcribed by Sofiya Khazanovich July 23, 2014

on, send the protein off, send the messenger RNA off to the ribosomes, if you can translate a protein,
you get a beta chain or heavy chain. If you cant translate it, apoptosis. And like the T cell, theres more
than one try.

Slide 18 Image
[McCutcheon]: We translate the heavy chain, we get a heavy chain, this point in time we can try and
translate, so heavy chain gene rearrangement shuts off, we start to try and translate a light chain. The
light chain arranges its V and J segments and unlike the T cell which only has one set of alpha genes, the
B cell has two sets of light chain genes. So one of the sets is called the lambda, and other set is called the
kappa. The B cell, it can try and rearrange on lambda 1, and if it cant make a productive rearrangement
on lambda 1, it can try on the second lambda, and if it cant make a productive rearrangement on the
second lambda, then it can try on the kappa. If it cant make a rearrangement on the kappa, it can try on
the second kappa, and if it cant do a rearrangement there, then it dies. But because it has twice as
many light chain genes, we have twice as many B cells surviving as T cells because you have more
chances to make the light chain versus the alpha chain. So while theres a lot of death by apoptosis in
bone marrow while B cells are developing, its less because you have twice as many chances to make a
light chain compared with a T cell. Unlike T cells, which do most of their developing while you are under
ten, B cells are generated throughout life. And whereas T cells have a life span of around 70 years, B
cells last for two or three days. Nave B cells, so youre constantly making new B cells. But youre making
the B cells in the bone marrow and they dont participate in an immune response until they're already
mature. And so youre not having a B cell go off to the lymph node and then try to rearrange the
receptor to fit the antigen. B cells come out of the bone marrow with the receptor that they have and if
they happen to encounter the antigen in the three days theyre alive whoopee, and if not they die. But
were making more so it doesnt matter. B cell, you start off rearrangement on heavy chain V D and J, if
you make a heavy chain which the B cell knows because its translated and gone to the cell membrane
and given the B cell survival signals, you shut off heavy chain gene rearrangement and start lambda
chain rearrangement, if you cant make a lambda chain you try a kappa chain, if you cant make a kappa
light chain, the B cell dies, we dont care about those, they're not participating in immune response. If
the B cell successfully makes one of the two light chains you wind up with an antibody on the surface of
the B cell.

Slide 19 Image
[McCutcheon]: This process is the same for B and T cells. The proteins that do the gene rearrangement
are the RAG proteins. Does everybody know what SCIDs kids are? Severe combined immunodeficiency.
Did anybody see the old movie the boy in the plastic bubble? No? Ok so, SCIDs is a disease primarily,
there are several causes but one of them is that the RAG proteins have mutations and so you cant do
gene rearrangement, and if you cant do gene rearrangement you have no T cells and you have no B
cells and the kids who have that die in infancy. I think it was in the 70s they managed to keep a kid who
had SCIDs alive by keeping him in a sterile environment. So he basically lived in a plastic bubble. Then in
the 90s the University of Pennsylvania tried gene therapy to replace the RAG proteins and it was
successful in that the child that they planted the genes in started making B and T cells, but the vector
that they used was a viral vector and not long after he started making B and T cells, he got leukemia
from the virus and he died, and thats Jesse Gelsinger, and that was a huge controversy in the media
about the fact that they did this gene therapy trial on a human and it cost him his life. In addition to this
random selecting whatever V segment that the RAG proteins select and binding it to whatever J
segment that the RAG proteins select.

Transcribed by Sofiya Khazanovich July 23, 2014

Slide 20 Image
[McCutcheon]: You have enzymes that can randomly add and subtract nucleotides at the end of the
cleavage sites, and this is also true for T cells. So not only is selecting the segments random, but then
you can add and subtract nucleotides that were or were not in the genome. So you can add a couple
nucleotides that were not encoded you can subtract some that were, so its this completely random
process at the joining site, and thats why you can get such high variability. Because not only do you do
the random selection, but you can add and subtract nucleotides, so you change whats genetically
encoded. So here they're showing you this complicated diagram but you can add nucleotides with TdT,
you can subtract nucleotides with endonucleases, then you join the two strands of the nucleic acids
together, you fill them in so you have double stranded DNA, and that gets translated and goes off to be
translated in the message. So those random selections of the V D and J and V and J segments, and in
addition at every junction you can randomly add or subtract nucleotides. This is also true for T cells. Im
adding it here cause I figured you had enough to learn the first time you went through this for T cells. So
B and T cells, random rearrangement plus random additions and subtractions.

Slide 21 Outcomes of rearrangement
[McCutcheon]: If you end up with a coding protein, the B cell survives and goes on to try and make the
light chain or if this was the light chain it survives and goes to the next step. If you end up with
noncoding proteins the B cell dies by apoptosis, failure to get survival signals.

Slide 22 Image
[McCutcheon]: So now I said that wed look at the variability. It turns out that this variability which is on
the most flexible of the flexible antibody this is the junction. So the junctions between the segments are
where all of the variability occurs which makes sense. Youre randomly adding and subtracting
nucleotides to randomly generated segments. So this is called the hypervariable region. There are
hypervariable regions on both the heavy and the light chain despite what the cartoon looks like. Cause
the light chain has one random junction so the heavy chain has more but the light chain has one. And so
its the combination of the hypervariable regions on the heavy and the light chain that creates the part
of the antibody that binds to the epitope. So all of the variability gets concentrated in the junctions
between the different regions and they happen to be loops that stick out of the beta pleated sheets, and
these loops themselves because they're loops they're also able to move around in space so they can
deform themselves to better be able to bind the antigen. And so you have three hypervariable regions
on the heavy chain because you have the V and the D and the J junctions, and one hypervariable region
on the light chain from the V and J junction. And its the combination of the hypervariable regions that
gives you the part of the B cell that actually binds to whatever its going to bind to.

Slide 23 Summary
[McCutcheon]: So the antigen binding end that is generated by a random rearrangement of segments,
the antigen binding loops are generated by the random addition or subtraction of nucleotides. So within
this variable structure you have really variable spots which we call hypervariable regions.

Slide 24 Constant region
Transcribed by Sofiya Khazanovich July 23, 2014

[McCutcheon]: Unlike T cells, T cells are membrane bound proteins and they stay stuck on the
membranes. Nave B cells, the antibody the B cell receptor which is the antibody but its membrane
bound, so the B cell receptor is antibody, its the same protein, but when the Bb cell is nave the B cell
receptor is always membrane bound. And once youve activated the B cell then you can start producing
the B cell receptor thats not membrane bound, it gets secreted and thats when we call it antibody. So B
cell receptors are the membrane bound form of the antibody, you dont get antibody secreted until that
B cell has been activated which well talk about on Friday. Secreted antibodies have two ends, theres
the end that binds the pathogen, the epitope on the pathogen but the reality is that antibody can only
do one thing, and that one thing, if they bind to the part of the pathogen thats the part the pathogen
needs to infect or to adhere or to cause disease, then they can neutralize that. If they bind somewhere
else on the pathogen and the protein on the pathogen that binds to infect a cell is still bare then that
virus can go ahead and infect a cell, the fact that its got antibody stuck in other places wont make a
difference. So the antibodies in and of themselves can only do one thing and thats neutralize, and that
means they're blocking a protein that the pathogen needs to be pathogenic. Antibody that are binding
to other proteins, because remember there are going to be many more proteins especially on bacteria
than the one used to infect, those dont do anything. So the end that actually does something is the
constant region. So the variable region binds to the pathogen but what happens to the antibody and
whatever its stuck to is determined by what constant region is on the antibody. So unlike T cell
receptors which are membrane bound and signal and thats all they do, B cell receptors have different
functions and you get different functions by changing the constant region. Youre not changing the
variable region. The constant region got spliced on during transcription, so what you do is the variable
region is fixed and then you start swapping out different constant regions to get different functions.

Slide 25 Isotypes
[McCutcheon]: So the different consent regions are called isotypes, and in gene its the Greek letter and
in protein its the English letter, so the mu constant region gives us IgM. The delta constant region gives
us IgD, the gamma constant region gives us IgG, the alpha gives us IgA, and the epsilon gives is IgE. So in
the gene its the Greek, in the protein its the English. So you have five different isotypes. These are not
in nave B cells, this is not in a developing B cell, this happens after the B cell has been activated and its
secreting the antibody rather than having them membrane bound, and then you can start switching out
constant regions to give you different functions of the antibody.

Slide 26 Image
[McCutcheon]: So here we see the C-mu, so which isotype is that going to be? IgM. So here we, and
thats the first isotype that comes along after the variable region gets formed, so the first antibody that
you always produce are IgM antibodies but in the membrane bound form theres simply that pair of
heavy and light chains.

Slide 27 Image
[McCutcheon]: So here we see weve done our gene rearrangement so weve created a VDJ region, we
translate through the C mu region and when we splice that through we create an IgM protein on the cell
surface. B cells can also do an alternative splicing so the B cell thats successfully rearranged its heavy
and light chain variable regions, it can also skip transcribing through the C mu region and it transcribes
through the C delta region to give us IgD. So then instead of making an M protein we make an IgD
protein, so this newly developed B cell thats still in the bone marrow ends up having both IgM and IgD
Transcribed by Sofiya Khazanovich July 23, 2014

on the cell surface. So the antibody binding region, thats identical. Thats already done its gene
rearrangement and youre not messing with that. Youre splicing on different constant regions, so you
have an identical antigen binding region on every single solitary antibody or B cell receptor thats on the
cell surface, but you can have two different constant regions. The antibody can be IgM or IgD.

Slide 28 Image
[McCutcheon]: So a cartoon, you dont have to know the specifics of this. So IgG, IgM, IgD, IgA, IgE. And
what it is is the antigen binding site is the same, you have changed the constant region on the heavy
chain and you do that by differential transcription. On the nave B cell thats still in the bone marrow,
the immature nave B cell the only two isotypes you have are IgM and IgD, you dont get IgG, IgA, or IgE
until the B cell has been activated and is producing secreted antibody. So IgM and IgD on the immature
and mature nave B cell, the IgA, IgG, and IgE happen after the B cell has been activated and the
antibody is being secreted.

Slide 29 Image
[McCutcheon]: So heres our B cell receptor, like the T cell has CD3, the B cell has a costimulatory
molecule that does most of the signaling, thats what you need to know about that. (So the T cell
receptor, the T cell receptor has CD3 as a coreceptor, right? The B cell receptor has this thing as a
coreceptor, and like the CD3 it does signaling. Thats what you have to know about it. So each B cell
receptor has a coreceptor the coreceptor does signaling. I dont even need you to know the name of the

Slide 30 Nave B Cells and activated B cells
[McCutcheon]: So nave B cells we have IgM and IgD and thats the B cell receptor. Activated B cells is
where we get isotype switching to IgG, IgE, or IgA. [Question asking her to go back to last slide,
answered above in parenthesis]

Slide 31 Summary
[McCutcheon]: So we start with gene rearrangement and the gene rearrangement gets translated into
the variable part of the antibody binding pair, then we splice on a constant region in the heavy chain,
and that splicing can be differential in the immature nave B cell and the mature nave B cell, the two
constant regions that are used are IgM and IgD, in the activated B cell that is now secreting antibody we
can have IgM and IgD but we can also have IgG, IgA, and IgE, and the acronym for remembering the
names of the isotypes is GAMED.

Slide 32 Final step of B cell development
[McCutcheon]: So we have the B cell thats made a B cell receptor and its in the bone marrow. Its still
immature. We have both IgM and IgD and IgD is critical for B cell activation.

Slide 33 Image
[McCutcheon]: And the B cell has to do one final thing. B cell receptors recognize anything, right? I know
youre tired, Im tired. B cell receptors recognize anything, yes or no? Maybe wasnt a choice. Who says
Transcribed by Sofiya Khazanovich July 23, 2014

yes? Who says no? Youre all wrong. So B cell receptors can recognize any structure. They can recognize
lipids, proteins, carbohydrates, nucleic acids. B cell receptors recognizing nucleic acids is what gives you
the symptoms of systemic lupus erythematosus. So B cell receptors can recognize anything. If B cell
receptors can recognize anything, do we need positive selection? No we dont. B cell receptors can
recognize anything, therefore we dont care what the receptor recognizes as long as it isnt host. If we
want to make sure that B cell receptors are not going to start binding to host, what do we need to do?
So unlike T cells, B cells do not go through positive selection. They dont need it. They have to go
through negative selection. This is still the immature B cell in bone marrow, it must go through negative
selection. It actually has two different forms of negative selection. So B cells that bind to particulate
antigens, so proteins on cells on the surface in the bone marrow. B cells that bind to particulate
antigens, so proteins on host cells in the bone marrow, do we want our B cells to bind to proteins on our
cells? No. So we kill them, apoptosis. But B cells can bind to anything and so there are soluble factors in
the bone marrow too, albumin, transferrin, B cells that bind to soluble factors, do we want B cells to be
able to bind to transferrin or insulin? No. But we dont have to kill them, what we do is we make them
anergic. So that they cant act again, and we can do that because B cells die in three days, right? You
cant risk that with T cells because they live for so long. So against particulate antigens you get
apoptosis, so the p in particulate and the p in apoptosis. If its a soluble antigen you have a lower copy
number, and that ends up anergizing the B cell, so its still there it just shouldnt do anything for the
three days its alive. And so if the B cell undergoes apoptosis its dead and its not going to participate in
an immune response. If the B cell undergoes anergy, it will migrate to periphery but its not going to be
activated. And what we did is we down regulated the amount of IgM, and that for some reason, you
cant activate a B cell without enough IgM. B cells that do not get killed or anergized, they migrate to
periphery and now they are a mature nave B cell.

Slide 34 Tolerance
[McCutcheon]: And I think I changed this three times and managed to post the wrong version. This is
tolerance in a primary organ. So this is central tolerance. And in B cells central tolerance has two
mechanisms. There can be apoptosis against what kind of antigens? Particulate. And if its a soluble
antigen you have central tolerance through anergy. So two mechanisms of central tolerance, its central
because its in a primary organ. Apoptosis against particulate, anergy against soluble, those anergized B
cells can leave the bone marrow and they can go off into the circulation and migrate to lymph nodes but
they dont do anything, and since they're short lived theyre going to die.

Slide 35 Types of B cells
[McCutcheon]: In addition we have the CD5 B cells like gamma delta T cells, CD5 B cells live in the
tissues. Like gamma delta T cells, CD5 B cells do not need T cells help to get started, they can recognize
their pathogen and go. They do not secrete any kind of antibody except IgM. They cant isotype switch.
The IgM antibodies they make are primarily directed against carbohydrate residues, so these tend to be
antibodies that bind to bacterial carbohydrate residues. They do not develop memory. So you have a
different set of CD5 B cells secreting antibody against carbohydrate residues as you age. They do their
thing and then they die. But since they dont need T cell help, once you have a CD5 B cell activated
against a carbohydrate residue, that means you always have IgM antibody against carbohydrate
residues, and what does IgM do? It activates complement. So you know, shortly after youre born you
have the ability to activate complement against common carbohydrate residues of bacteria through the
classical pathway. In the event that a carbohydrate residue you havent made IgM antibody against, you
still have the alternate pathway.