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Microbiology
8/25/2008
Antibodies and T cell Receptor Genetics: Dr. Peter Burrows
Transcriber: Kimberly Watkins
(Length of lecture)
Slide 1
Today I am going to talk about antibodies and T cell receptor genetics. You heard about
antibodies at the protein level from Dr. Mestecky but today we are going to talk about the way antibody
genes are generated and also how T cell receptor genes are created.
NEW SLIDE (revised version on website)
This figure is 1-10 from the Immunology book. The bottom is the immune system and these are
what the immune system has to recognize. We have foreign proteins, viruses, bacteria, parasites and
fungi. The immune system has evolved to be able to recognize all these different pathogenic
microorganisms and it does so by the receptors on lymphocytes. In a full-blown immune response you
are going to have the generation of cytotoxic T cells than can kill our own cells that are invaded by a
pathogen. You also get the generation of the helper T cells that will help B cells make antibodies. The B
cells have an antigen receptor, T cells have an antigen receptor and each lymphocyte has a unique
receptor so it can recognize a different antigen.
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 2
NEW SLIDE
Antibodies and T Cell Receptor Genetics Learning Objectives
• To understand mechanisms for creating diversity
– Be able to diagram changes at the DNA level required to produce a functional immunoglobulin gene
• To understand isotype switching at the molecular level
• To recognize basic differences between antigen receptors on B and T cells
You have to have a lot of diverse lymphocytes so you can recognize diverse pathogens. We are
going to focus mainly on the antigen receptor on B cells, so you should be able to diagram what happens
to the immunoglobulin genes during the differentiation of a B lymphocyte. We are also going to talk
about how you can make the different antibody isotypes. At the end we are going to talk about T cells
and how they differ from B cells in terms of their receptor and also how they recognize antigens.
Slide 2
Our survival requires that we have a diverse set of B and T cells. If you do not have either B or T
cells you are sick and if you do not have both B and T cells you are really sick. Immunodeficiencies point
to the fact that you would die without an immune system. Essentially, the immune system has to be
prepared to respond to all these different antigens. When you get an infection, like bacteria that can
replicate every 30 minutes, you cannot wait around and generate new lymphocytes that are able to
recognize that bacteria; they have to be there already. They will be in low frequency but you will have
lymphocytes that can react to any antigen we can think of. It has been estimated we can make one to
100 million different antigen receptors. The antigen receptors on B cells are called immunoglobulins
and are called T cell receptors (TCR) on T cells. We have to figure out a way to make one to 100 million
receptors on each lymphocyte.
Slide 3
The immune system has basically come up with two different ways to deal with the issue of how
to make a lot of diversity. One is the cellular solution. Each lymphocyte has a different receptor and one
way you can get a lot of diversity is to make a lot of lymphocytes. Every day you make million of
lymphocytes and each one has a different receptor and that gives you a pretty broad diversity. If you
produced a million lymphocytes every day from the day were born you would eventually be nothing but
lymphocytes. Essentially what happens is only the lymphocytes that eventually encounter antigens
survive and the rest of them die. There is a lot of wastage in the immune system. You are making
lymphocytes and most of them never see their antigen and die, but the ones you do are the ones you
need and that is how the system is set up. The antigen can select the right lymphocyte by finding one
that has a complementary receptor (which is shown on the following slide).
Slide 4
You have millions of B cells with different specificities. In this case we have a virus and it finds a
lymphocyte that has the right receptor that can bind to it. The B cell gets stimulated, divides and
differentiates into plasma cells that secrete antibodies that can neutralize the virus. At the same time
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 3
you have the generation of memory cells, which means you have expanded the population of cells that
can recognize the virus. Memory cells are very long-lived (they live as long as you do) and if that same
virus comes around again you can respond more quickly. This is the principal behind vaccination. For
example, you get vaccinated with tetanus toxoid to expand the population of B cells that can recognize
tetanus toxin and then if you happen to get infected by Clostridium tetani instead of getting locked jaw
you will be protected because you can make antibodies quickly to neutralize the toxin. This is how the
selection works and these other cells will disappear in a week or so unless they are stimulated. Also in
this process, you get isotype switching. You start out making IgM antibodies and depending on what
kind of stimulation, you can make IgA, IgG or IgE antibodies.
Slide 5
Dr. Mestecky talked about serum IgM, which is a huge pentamer with ~million kDA molecular
weight. This is a B cell with IgM on its surface and it is a monomer. It has a variable region of the light
chain and of the heavy chain and that is what is used to recognize antigen. Each individual lymphocyte
has IgM on its surface but each individual lymphocyte has a different variable region to bind antigen.
We know this from crystallographic studies. Crystallographers isolate the Fab part of the molecule,
crystallize it and look at the structure of that part of the antibody together with its antigen.
Slide 6
This shows different Fabs from different immunoglobulins; they have different shapes in terms
of what they recognize. The first hapten (looks like a ball) is sitting directly down in the antigen binding
site formed the variable regions of the light and heavy chains. The second is like a “hotdog and bun”; it
covers the entire the antigen binding site. The last one covers the entire variable region. We have
millions of different B cells capable of recognizing different shapes.
Slide 7
There is a problem that we come to in making antibodies. Remember, there are nine different
isotypes. If you were to use typical gene expression, you would need somewhere between 9 and 900
million genes to make that many antibodies. Similar numbers for the T cell receptors. If you take a
conservative estimate (10 million genes) and make them small as possible (10^3 base pairs DNA/gene)
you will need 10x10^9 bp of DNA, but will only have three million base pairs. The problem is that you
can not use up your entire DNA to make antibodies. So obviously the immune system cannot use
standard approaches in the way it makes genes for antibodies and T cell receptors.
Slide 8
Before we get into the genetics, I wanted to show the anatomy of an ordinary gene. In
eukaryotes, nearly all of our genes are composed of exons, which are expressed and introns, which are
non-coding sequences in between the exons. It also has a leader sequence and this particular gene will
make a protein that is secreted or goes into the plasma membrane. During the expression of this gene,
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 4
you transcribe the whole thing, splice out all of these introns and then translate it and make the protein.
That is how all genes in us operate and that it what is illustrated here (next slide)
Slide 9
Here is a B lymphocyte and you are looking at its DNA. Points out the light chain protein being
made (left side), the mRNA and the DNA. It has a leader sequence and an exon to encode the variable
region and also an exon to encode the constant region. You make the mRNA splice out the introns and
you make protein. You have the same thing with the heavy chain. It has a leader sequence, exons to
encode variable region and exons to encode the constant region. So this is how the gene looks for the
light chain and for the heavy chain in a B cell. DNA in the liver cell would look like this.
Slide 10
What you inherit are not functional genes unlike most other genes. In the case of lymphocytes,
the genes for these receptors do not exist until these cells develop them. Where all this business
happens is in the variable region exons. For example, this B cell splices together bits and pieces to make
a full complete variable region exon. For B cells, we call this immunoglobulin gene rearrangement and
for T cells it is called T cell receptor gene rearrangement. Basically instead of using up 3 times more DNA
than we have to make antibodies, we can make a lot of antibodies and TCR without monopolizing the
whole genome.
Slide 11
Let’s look at the light chain. Now if we look in the germ cells of the same individual we have our
constant region exon (that is still there) but the variable region exon is now split into two parts. It is
called a V and another called a J, which is called a joining gene segment. During the differentiation of a
B cell, we have to join up this B gene segment with that J gene segment to make a functional light chain
gene. The same thing happens in the heavy chain. We have a functional gene and if we look in the
germline there are actually 3 different segments (J, D and V) that have to be joined together to make a
functional gene. What does it do for variability and diversity to join these two gene segments together?
If we only have one section of each we would not be generating much diversity. In fact there are
multiple copies of these gene segments.
Slide 12
We are not going to talk about the λ (lambda) light chain only the κ (kappa) light chain and the
heavy chain. You can see the constant region of the κ light chain and upstream you have five J kappa
gene segments, and then upstream from there (depending on the species) you can have about 40 V
kappa gene segments. That is what your light chain locus looks like in your liver cells but again you
cannot make any light chains from that. What you have to do is join one of these V to one of these J to
make a functional variable region exon.
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 5
Slide 13
During B cell development in the bone marrow, this V joins with this J and actually kicks this part
out. This can be a huge piece of DNA that is tossed out of your chromosome and this is not the usual
way. Typically your cell wants to maintain its DNA. So you join a V to a J and now you have a functional
variable region. The mixing and matching generates a lot of diversity.
Slide 14
The heavy chain locus is what we are talking about here. They have three segments to join
together, the VH, the DH (which stands for diversity of small segment) and JH. This is a two-step
process. During the development of B cells, the first thing the B cell does is start to rearrange its heavy
chain gene by sticking one D segment to one J. Now you have kicked out everything in between and you
have a DJ rearrangement. However, we are not finished because you cannot make a heavy chain yet
(only halfway there). The next step is to pick one of those V segments and join to that DJ and now we
have a functional variable region exon. The developing B cell can start making a new heavy chain and
this is the first step in B cell development. You can recognize these as being B cells because they make a
new heavy chain.
They do not make a light chain yet. What actually happens is the cell does this rearrangement
and divides after each step and then the cell can rearrange a light chain gene. It can generate a lot of
different cells with a lot of different antibodies specificity because the progeny cell can pick a different
light chain gene. Now we have a regular gene, we transcribe it, splice out the introns and we make a
new heavy chain. It is all an issue of taking gene segments and sticking them together and tossing out
everything in between and in this case it is a two-step process. You do DJ and then bring in a V and
you now have a functional gene where you can make a new heavy chain. It is the same deal with light
chains only here you have three segments instead of two.
Slide 15
You can look at the numerology and figure out that this process is a really good way to do this.
 40 V kappa segments X 5 J kappa segments  200 kappa V regions by picking randomly
 We did not talk about lambda but you have 30 V lambda X 3 J lambda  90 lambda V regions
 The heavy chain locus by having 3 different gene segments gives you more probability and
different combinations. (65 VH X 27 DH X 6 JH 10, 530 heavy V exons by this process)
The question now becomes how many antibodies you can make. The B cell developing in the bone
marrow first makes a heavy chain rearrangement, the cell divides, and then each one of those cells can
pick a light chain to rearrange. We have ~10,000 different possible VH and 200 different kappa light
chains so you can make 2 million different antibodies as the cell differentiates and randomly picks gene
segments to rearrange. Lambda is a little bit smaller by almost a million. We get 3 million genes (3
million different B cell receptors) just from a total of 176 gene segments.
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 6
His example: Restaurant says they have 1000 different meals. Look at the one sheet menu and you see
10 appetizers, 10 entrees, and 10 desserts and you can go 1000 times and eat a different meal. This is
the same thing the immune system does with random mixing of gene segments. Each B cell only makes
one antibody and each B cell goes through this process and makes a heavy chain and then it makes
either a kappa or a lambda light chain (NOT both) and in the end you have a B cell with a single
receptor. It is the only cell type in the body that does this type of gene rearrangement besides T cells
because it not a good thing to be doing. There are a lot of potential problems when you start cutting
your chromosomes and re-ligating them.
Slide 16
Now we have made a B gene. The original B cell was making IgM as an antigen receptor but then
we have to worry about how to make the different isotypes. The isotypes have different functions: IgA
for mucosal surfaces, IgE for allergies, and so on. What we have done so far is make this variable region
exon, the cell making IgM. If we look at this DNA in that B cell and we look downstream we see other
constant regions. This is actually the heavy chain locus in a mouse and he has IgG3, IgG1, IgG2b, and
IgG2a, IgE and IgA .They are all sitting downstream from this functional B gene not being expressed. If
this B cell meets its antigen and in this case it has a signal to switch to IgG3, you cut out everything
upstream of IgG3 constant region. It no longer makes IgM because the mu constant region. It now
makes IgG3. It is a matter of somatically changing the DNA in an individual B or T cell; first to make a
variable exon and in the case of B cells to switch isotypes. Isotype switching only occurs in B cells
because T cells do not have isotypes. This allows you to start with an individual B cell making IgM, and
then once it sees antigen and especially if it gets T cell help then it can switch to the different IgG
subclasses, IgA, or IgE depending on what kind of cytokine it gets hit with.
Slide 17
The switch recombination is good because otherwise you would have to make a VDJ
rearrangement for each different isotype and that would be a waste of cells because most of the B cells
die. If you had to make each different isotype, each different B cell, you would have to have 9 times as
many B cells. This way they start out making IgM and if they get stimulated by antigen (which is the
minority of cells) then they can switch. Only the cells that are responding to antigen do this isotype
switching. It is irreversible because you have kicked out whatever is upstream of the isotype you have
switched to.
For example[goes back to previous slide], if you switch to IgA you would kick out all the gamma
subclasses and the IgE constant region and you would only be left with the IgA constant region. Each B
cell makes only one specificity: starts out making IgM, divides during antigen exposure and then each
one of its daughter cell can make a different isotype. ONE B CELL MAKES ONE ISOTYPE AND ONE
SPECIFICITY. The immune system goes through a lot of trouble to keep each lymphocyte mono-specific.
You do not want a B cell with two specificities because then you might make antibodies to the wrong
thing if you stimulated through one receptor and made two antibodies.
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 7
Slide 18
If we look at serum immunoglobulins after you have been immunized with tetanus toxoid every
few days, you will first see an IgM response which is called the primary response. Then later you will see
an IgG response. Then if the real tetanus antigens got introduced into your system, you would get a
small IgM response again but you would get a much higher IgG response. If you think about this in terms
of the B cells and what is going on with their genes, then the original B cell coming out of the bone
marrow has IgM as a receptor for antigen, it gets stimulated and differentiates into plasma cells that
secrete IgM antibodies. But during that stimulation process, you can switch isotypes so that you will
eventually start making IgG antibodies. This is in serum; if you look elsewhere you might find other
isotypes. These eventually drop down but during this whole antigen stimulation event you have
generated memory cells that are more easily stimulated than the primary B cells. A lot of them have
already switched isotypes and this means that when you stick the antigen back in again you can get a
more robust IgG response in a much shorter time (they can differentiate into plasma cells more quickly
and can make a lot of IgG antibodies). That is how this serum antibody response fits in with what is
going on at the DNA and cellular level.
Slide 19
We are going to briefly touch on T cells. The T cells undergo development in the thymus and
during that process they have to develop a T cell receptor gene. So, the B cells are doing it in the bone
marrow and the T cells are doing it in the thymus.
**SLIDE 23**
If we look and compare the B cell receptor (immunoglobulin) and the T cell receptor we can see that the
immunoglobulin has light chains and heavy chains and each one has a variable region. The T cell
receptor has an alpha (α) chain and beta (β) chain but each one of them has a variable region. Again the
similarity here is that the regions are encoded by rearranging genes; the genes are different. Here we
have immunoglobulin genes and the T cell uses T cell receptor genes but the process is very similar.
Slide 20
This is just a different way of looking at the T cell receptor. It has an alpha chain and beat chain
and they are both transmembrane whereas for the B cell receptor only the heavy chain is stuck in the
membrane. They have both variable and constant regions.
Slide 21
The T cell receptor is a heterodimer (two different chains: alpha and beta). The TCR is never
secreted. The B cells start with a membrane protein as a receptor but their plasma cell progeny secretes
that receptor. The TCRs do not secrete their receptor because they have to function by direct cell
contact. A cytotoxic T cell has to come right up on the cell that it has to kill and stick some granules to
kill the cell. And the same way a helper T cell has to come up on the B cell and secrete some cytokines to
stimulate things like switching. The variable regions of the TCR are generated by somatic gene
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 8
recombination as the T cells develop in the thymus and it is the same enzymes that are used for Ig gene
rearrangement but different gene segments.
Slide 22
Not going to go into detail. Here is our alpha chain and beta chain of the TCR. The alpha chains
are similar to the immunoglobulin light chain genes in that they only have two segments. You have a V
alpha and a J alpha and during T cell development you pick one V and one J to join together and kick out
what is in between. Again, it is a very similar process but different genes. The beta chain is similar to the
heavy chain in the immunoglobulin having three gene segments (you have J beta, D beta, and V beta).
During the development of the T cell in the thymus, first it rearranges a D to a J and then a V to the DJ to
make this functional beta chain gene.
Slide 24
The reason we have B and T cells is because they recognize antigens in different ways. This is a
crystal structure of the antibody Fab. It consists of the variable region of the heavy chain and light chain
binding to this small lysozyme protein. The antibodies are essentially recognizing different bumps and
grooves on the surface of these proteins. That is called the epitope and that is what the variable region
of this particular antibody is recognizing. Another antibody might recognize some other protrusion or
indentation. This small protein has a lot of different possible epitopes, so you might be able to make
several hundred different antibodies to this protein.
Slide 25
T cells do not recognize intact antigens; they have to look at degraded antigens. This is a T cell
that has V alpha and V beta TCR that will only recognize degraded peptides in association with these
MHC molecules on the cell that it is recognizing.
Slide 26
This diversity is not without problems. The first thing to mention is that this is a random process.
During development you are going to make autoreactive T cells and autoreactive B cells and there are
experimental proof and also autoimmune diseases that are caused by antibodies or T cells attacking self
antigens. You have to get rid of them and most of the time it works but sometimes it does not. So you
have myasthenia gravis, multiple sclerosis, SLE (systemic lupus erythematosus), diabetes and
autoimmune diseases where your immune system did not quite get rid of the T or B cell it recognizes.
The other problem is when you start breaking DNA sometimes you get what is called translocation
where instead of kicking out that circle of DNA and joining the chromosome back together, you actually
join a piece of another chromosome to your chromosome. A lot of times when translocation happens
you introduce an oncogene into the heavy chain locus and once it is in the heavy chain locus it gets
regulated like the heavy chain (which is not the usual way it is regulated) and this can lead to lymphoid
malignancies. A lot of the leukemias and solid tumor lymphomas are due to mistakes in VDJ
recombination or mistakes made during isotype switching. MICK
Microbiology: Antibodies and T cell Receptor Genetics
Kimberly Watkins
pg. 9